When your immune cells detect the presence of a foreign pathogen, it would immediately begin the process of destroying copies of the same pathogen as part of the innate immune system. However, this may not be enough to eliminate every single copy of the pathogen because the infection may have spread throughout the body. Your immune system would need specialised immune cells located through the body that can identify and specifically target that particular pathogen for destruction, which would take some time to occur. This is known as adaptive or acquired immunity.
3. Adaptive Immune System
- Also known as the acquired immune system, the adaptive immune system features an army of specialised, systemic cells and processes that eliminate the presence or prevent the growth of pathogens.
- It comprises of both humoral immunity and cell-mediated immunity components that aim to disintegrate invasive agents. However, the system is highly specific to each particular pathogen it interacts with.
- Robert Good was the first to describe this immune response as "adaptive", referring to antibody responses in frogs as a synonym for "acquired immune response" in 1964.
- He theorised antibodies were plastic and could adapt themselves to the molecular shape of antigens, and/or to the concept of "adaptive enzymes" in bacteria, whose expression may be stimulated by their substrates.
- This phrase was used more or less exclusively by Good and his student and a number of other immunologies investigating marginal organisms until the 1990s when its usage increased in tandem with the phrase "innate immunity".
- A majority of immunology and biology textbooks today mostly use the term "adaptive" and mention the term "acquired" as a synonym in glossaries.
- Since Tonegawa's discovery, the definition of "acquired immunity" was classically "antigen-specific immunity mediated by somatic gene rearrangements that create clone-defining antigen receptors".
- Since the 1990s, the term "adaptive" was increasingly used to describe another class of immune response closely associated with somatic gene rearrangements.
- When pathogens avoids the innate immune system and reproduces antigens towards a threshold level, it emanates "stranger" or "danger" signals to activate dendritic cells, which stimulates the adaptive immune response.
Main functions of the adaptive immune response include:
- Specific "non-self" antigens being distinguished from the "self" antigens, during antigen presentation step.
- Immune responses adapts to maximally eliminate specific pathogens or pathogen-infected cells.
- Immune system develops immunological memory, which features memory B cells and memory T cells remembering the pathogens.
- Human adaptive immune response takes between 4 and 7 days to launch.
i. Lymphocytes
Making up about 18-42% of circulating white blood cells, lymphocytes are the primary immune cells found in lymph that consists of natural killer cells (for cell-mediated, cytotoxic innate immunity), T cells (for cell-mediated, cytotoxic adaptive immunity), and B cells (for humoral, antibody-driven adaptive immunity).
How do lymphocytes develop?
- Haematopoiesis begins within the bone marrow, where mammalian stem cells differentiate into various blood cell lines. All lymphocytes differentiate from a common lymphoid progenitor before differentiating into their distinct lymphocyte types.
- Lymphopoiesis is defined as the differentiation of lymphocytes undergoes distinct pathways in a hierarchical manner.
- Mammalian B cells mainly mature in the marrow, whereas avian B cells mature in a lymphoid organ called the bursa of Fabricius.
- Naive T cells migrate to the blood stream and mature in the thymus. Subsequently, mature T cells enter circulation and peripheral lymphoid organs (such as the spleen and lymph nodes) where they scavenge for invasive agents and/or tumour cells.
- After B and T lymphocytes interact with an antigen, they differentiate further to form effector and memory lymphocytes.
- Effector lymphocytes release antibodies (referring to B cells), cytotoxic granules (CD8+ T cells), or signal other immune cells (helper T cells) in order to eliminate the antigen.
- On the other hand, memory T cells remain in the peripheral tissues and circulation for an extended time poised to react with the same antigen upon future exposure.
What are the characteristics of lymphocytes?
- Under a microscope, in a Wright's stained peripheral blood smear, lymphocytes have a big, dark-staining nucleus with virtually no eosinophilic cytoplasm. Under normal circumstances, this nucleus is approximately the size of a red blood cell (~ 7μm in diameter).
- Some lymphocytes were discovered to contain a clear perinuclear zone (or halo) around the nucleus.
- When lymphocytes are observed under an electron microscope, polyribosomes can be detected, which are organelles involved in protein synthesis, hence the production of cytokines and immunoglobulins.
a. T-Cells
How do T cells develop?
- All T cells derive from c-kit+Sca1+ haematopoietic stem cells (HSC) located in the bone marrow, occasionally in the foetal liver during embryonic development.
- HCS subsequently differentiates into multipotent progenitors (MPP), retaining the potential to transform into both myeloid and lymphoid cells.
- It differentiates into a common lymphoid progenitor (CLP), which can only differentiate into T, B or NK cells.
- CLP cells then migrate via the bloodstream to the thymus, where they engraft. There they become an immature T cell called a thymocyte.
- The earliest cells reaching the thymus lack the expression of CD4 and CD8 co-receptors (double negative), therefore they are labelled as CD4−CD8−CD44+CD25−ckit+ cells, better known as early thymic progenitor (ETP) cells.
- Subsequently they divide and downregulate c-kit to become double-negative (DN1) cells. Thymocytes experience a multitude of DN stages, as well as positive selection and negative selection to become T cells.
- Double negative thymocytes express CD1, CD2, CD5 and CD7 on their surface, except CD34.
- On the other hand, double positive thymocytes express both CD4 and CD8, and mature into either CD4+ or CD8+ cells.
How does the T cell receptor develop?
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| The T-cell receptor complex with TCR-α and TCR-β chains, CD3 and ζ-chain (CD247) accessory molecules. |
- Each mature T cell expresses a unique T cell receptor (TCR) that responds to a randomly particular pattern, which gives the immune system the ability to recognise many possible types of pathogens. This allows the immune system to develop immunity to never-before-seen pathogens resulting from random variation.
- When a thymocyte survives the process of functional TCR development, it transforms into an active T cell.
- The TCR comprises of 2 major parts: the α (alpha) chain and β (beta) chain. Variations in these chains lead to a wide array of unique TCRs, which require rigorous testing.
- Firstly, the thymocytes attempt to produce a functional β chain, and test it against a "mock" α chain. Next, they attempt to produce a functional α chain to complete the TCR structure.
- Every newly created TCR is tested to ensure it detects non-self pathogens correctly by recognising its MHC in a process known as 'positive selection'.
- If the thymocyte is confirmed to not respond adversely to "self" antigens, this process is known as 'negative selection'.
- Once the TCR successfully completes both positive and negative selection processes, it becomes operational and thymocyte matures into a T cell.
i. TCR β-chain selection
- At the DN2 stage (CD44+CD25+), thymocytes upregulate the recombination genes RAG1 and RAG2 and re-arrange the TCRβ locus, as well as amalgamates V-D-J recombination and constant region genes in order to produce a functional TCRβ chain.
- As the maturing thymocyte progresses through the DN3 stage (CD44−CD25+), it expresses an invariant α-chain called pre-Tα alongside the TCRβ gene.
- Charles Janeway (2012) found successful pairing of the rearranged β-chain with the invariant α-chain leads to the creation of signals that result in cessation of the β-chain rearrangement and suppression of the alternate allele.
- These signals need the pre-TCR at the cell surface, which are independent of ligand binding to the pre-TCR.
- If the chains successfully combine, this creates a pre-TCR, which subsequently leads to downregulation of CD25, forming a DN4 cell (CD25−CD44−). By that stage, thymocytes experience a round of proliferation, before re-arranging the TCRα locus during the double-positive stage.
ii. Positive selection
- Double-positive thymocytes (CD4+/CD8+) migrate into the cortex of the thymus, where they undergo a process called positive selection. Thymic cortical cortical epithelial cells present self-antigens on MHC molecules to the double-positive thymocytes.
- If thymocytes strongly bind to MHC-I or MHC-II, they receive a "survival signal". That means the remaining thymocytes that weakly bind to the same molecules don't receive any signal and ultimately die from neglect.
- This ensures the surviving thymocytes have an 'MHC affinity' in order to elicit useful functions in the body, such as reacting to MHC in the immune response.
- Double-positive thymocytes (CD4+/CD8+) that strongly interact with MHC-II will become CD4+ "helper" T cells, whereas thymocytes that strongly interact with MHC-I will become CD8+ "killer" T cells.
- A thymocyte fated to mature into a CD4+ T cell downregulates expression of the CD8 cell surface receptor until it becomes a single positive T cell, and vice versa.
- Note that positive selection does not filter for thymocytes that may trigger autoimmune responses.
iii. Negative selection
- Thymocytes that survive positive selection migrate towards the cortex and medulla boundary in the thymus, where they are presented with self-antigens by the MHC complex of medullary thymic epithelial cells (mTECs).
- In order to properly express self-antigens from all tissues of the body on their MHC-I peptides, mTECs are required to be Autoimmune regulator positive (AIRE+).
- If mTECs are phagocytosed by thymic dendritic cells, they become AIRE- antigen presenting cells (APCs), which gives them the ability to present self-antigens on MHC-II molecules.
- Note that positively selected CD4+ cells have to interact with MHC-II expressed on APCs, which need to be present for CD4+ T-cell negative selection.
- If thymocytes bind too strongly with the self-antigen, it will receive an apoptotic signal that results in their death.
- If the thymocytes binds weakly to self-antigens, they become either regulatory T-cells or mature naive T cells as they exit the thymus to become recent thymic emigrants. Typical naive T cells that exit the thymus (via the corticomedullary junction) are described as self-restricted, self-tolerant, and single positive.
- This process plays a crucial role in establishing central tolerance and avoid the creation of self-reactive T cells that could potentially trigger autoimmune responses.
What is a TCR?
- The T-cell receptor (TCR) is a protein complex expressed on the surface of T cells (T lymphocytes) that serves to recognise antigen fragments as peptides bound to major histocompatibility complex (MHC) molecules.
- Since the interaction between TCR and antigen peptides is of relatively low affinity, numerous TCRs recognise the same antigen peptide and many antigen peptides are recognised by the same TCR.
- It was first discovered by Nobel laureate James P. Allison in 1982, before Tak Wah Mak and Mark M. Davis identified the cDNA clones encoding the human and mouse TCR respectively 2 years later.
Describe the structure of the TCR complex
- The TCR is a heterodimeric protein complex anchored to the cell membrane and connected via disulfide bond, which typically comprises of the highly variable alpha (α) and beta (β) chains expressed as part of a complex with the invariant CD3 chain molecules.
- This receptor belongs to a large group of proteins responsible for binding, recognising, and adhering to antigens called the immunoglobulin superfamily.
- In humans, about 95% of T cells contain TCRs composed of an alpha (α) chain and a beta (β) chain (encoded by TRA and TRB, respectively), thus about 5% of T cells contain TCR composed of gamma and delta (γ/δ) chains (encoded by TRG and TRD, respectively).
- Each chain contains 2 extracellular domains: Variable (V) regions and a Constant (C) region, both of Immunoglobulin superfamily (IgSF) domain forming antiparallel β-sheets.
- In addition, the Constant region is proximal to the cell membrane, which is followed by a transmembrane region and a short cytoplasmic tail, whereas the Variable region interacts with the peptide / MHC complex.
- The variable domain of both the TCR α-chain and β-chain each contain 3 hypervariable or complementarity-determining regions (CDRs).
- The residue amino acids in these variable domains are situated in 2 segments of the TCR, specifically at the interface of the α- and β-chains and in the β-chain framework region, which may be proximal to the CD3 signal-transduction complex.
- Although CDR3 principally recognises the processed antigen, CDR1 of the alpa chain binds to the N-terminal portion of the antigenic peptide, whereas CDR1 of the β-chain binds to the C-terminal portion of the peptide.
- The Constant region of the TCR contains short connecting sequences in which a cysteine residue creates disulfide bonds to establish a connection between the 2 chains.
- In the plasma membrane, the TCR α and β chains link with 6 additional adaptor proteins to create an octameric complex. Therefore, the complex consists of both α and β chains, forming the binding site for ligand to interact with, as well as the signalling modules CD3δ, CD3γ, CD3ε and CD3ζ.
- Call et al. (2002) found charged residues in the transmembrane domain of each subunit produce polar links, which results in stability of the complex's assembly.
- Since the TCR's cytoplasmic tail is short, CD3 adaptor proteins contain the signalling motifs required for propagating the signal from the stimulated TCR into the T cell.
- The signalling motifs involved in TCR signalling are tyrosine residues in the cytoplasmic tail of the adaptor proteins, which become phosphorylated when mMHC binds to TCR.
- Those residues situate in a specific amino acid sequence Yxx(L/I)x6-8Yxx(L/I), where Y, L, I indicate tyrosine, leucine and isoleucine residues respectively, x denotes any amino acids, the subscript 6-8 indicates a sequence of 6 to 8 amino acids in length.
- Dusket et al. (2012) referred this motif as the immunoreceptor tyrosine-based activation motif (ITAM), which are common in activator receptors of the non-catalytic tyrosine-phosphorylated receptor (NTR) family.
- In addition, CD3δ, CD3γ and CD3ε each contain a single ITAM, whereas CD3ζ contains 3 ITAMs, hence the TCR complex contains a total of 10 ITAMs. When ITAMs are phosphorylated, they serve as binding sites for SH2-domains of recruited proteins.
How is TCR diversity generated?
- TCR diversity is generated by genetic recombination of the DNA-encoded segments in individual somatic T cells by somatic V(D)J recombination via RAG1 and RAG2 recombinases.
- Note that TCR genes don't experience somatic hypermutation, and T cells don't express activation-induced cytidine deaminase (AID).
- Janeway et al. (2001) found each recombined TCR demonstrated unique antigen specificity, which is determined by the antigen-binding site structure created by the α and β chains in the case of αβ T cells or γ and δ chains in the case of γδ T cells.
- The TCR α chain is created by VJ recombination, whereas the β chain is created by VDJ recombination. Furthermore, TCR γ chains are generated by VJ recombination, whereas TCR δ chains are generated by VDJ recombination.
- The diversity of TCR specificity for processed antigenic peptides is further increased by unique combination of the variable regions, as well as palindromic and random nucleotide additions (respectively termed "P-" and "N-").
- Its antigenic specificity can be altered by TCR revision via reactivation of recombinases later in development, which involves re-editing of individual CDR loops of TCR in the periphery outside thymus.
How does TCR distinguish between self-antigens and foreign antigens?
- Each T cell expresses clonal TCRs that recognise a specific peptide presented by a MHC molecule (pMHC), either on MHC class II on the antigen-presenting cell (APC) surface or MHC class I on the surface of any other cell type.
- Feinerman et al. (2008) stated T cells are able to distinguish peptides produced by healthy, endogenous cells from peptides produced by foreign or abnormal (e.g. infected or cancerous) cells in the body.
- On the contrary, APCs don't possess such capability to distinguish self peptides from foreign peptides, because they usually express numerous self-derived pMHCs on their cell surface and only a few copies of any foreign pMHC. Yang et al. (2016) found T cells infected with HIV possess only 8-46 HIV-specific pMHCs, compared with 100000 total pMHCs, per T cell.
- Since T cells experience positive selection in the thymus, there is a non-negligible affinity between self-pMHC and the TCR. However, TCR signalling ideally shouldn't be activated by self-pMHC so T cells can ignore endogenous, healthy cells.
- If these cells do contain a minuscule trace of pathogen-derived pMHC, T cells would be activated and trigger immune responses.
- The phenomenon of T Cells ignoring healthy cells but reacting to the same cells expressing foreign pMHCs is referred to as "antigen discrimination".
- Donermeyer et al. (2006) explained T cells require a high degree of antigen specificity in order to demonstrate antigen discrimination, in spite of relatively low affinity to the peptide / MHC ligand.
- Cole et al. (2007) estimated the affinity, given as the dissociation constant (Kd), between a TCR and a pMHC was determined by surface plasmon resonance (SPR) to be between 1 and 100 μM, with an association rate (kon) between 1000 and 10,000 M−1 s−1 and a dissociation rate (koff) between 0.01 and 0.1 s−1.
- Altan-Bonnet & Germain (2005) demonstrated a single amino acid alteration in the presented peptide would change the affinity of the pMHC to the TCR, hence decrease the T cell response, which can't be compensated by a higher pMHC concentration.
- Dushek et al. (2011) discovered a negative relationship between the dissociation rate of the pMHC-TCR complex and the strength of the T cell response. Huang et al. (2013) posited pMHC interacting with TCR for an extended period time would trigger a stronger activation of the T cell. This demonstrates T cell's high sensitivity to a single pMHC, which can result in activation.
- T cells were observed to rapidly scan pMHC on APCs in order to increase the likelihood of detecting a specific pMHC. Miller et al. (2004) calculated a T cell encounters an average 20 APCs per hour.
The molecular mechanisms behind this specific and sensitive process of antigen discrimination is not well understood, therefore researchers have proposed a number of theories.
- The most probable model is the TCR's role in kinetic proofreading. This model suggests a signal isn't directly produced upon interaction but rather a series of intermediate steps prior to ensure a time delay between binding and signal output.
- Such intermediate "proofreading" steps may involve tyrosine phosphorylation, which require high energy expenditure and thus can't occur spontaneously, unless a ligand binds to the receptor.
- This ensures a signal can only be produced when a ligand with high affinity interacts with TCR for an adequately long time.
- McKeithan (1995) stated all intermediate steps are reversible, which implies the receptor can revert to its original unphosphorylated state after a ligand removes the binding site.
- The model predicts the maximal T cell response reduces for pMHC with shorter lifespans, which has been confirmed experimentally by Dushek et al. (2011). Nevertheless, the basic kinetic proofreading model has a trade-off between sensitivity and specificity.
- If additional proofreading steps are added, the specificity of the receptor increases but the sensitivity of the receptor decreases. It is argued the model doesn't sufficiently explain the high sensitivity and specificity of TCRs observed in experiments.
- It is known T cells that have interacted with many different antigens have higher antigen sensitivity than naive T cells. von Essen et al. (2012) suggested the sensitivity of effector and memory T cells are less dependent on costimulatory signals and higher antigen concentration compared to naive T cells.
Describe the signalling pathway of the TCR
- When TCR binds to pMHC, it triggers a signalling cascade that activates transcription factors and remodels the cytoskeleton, which activates the T cell.
- Active T cells release cytokines, experience rapid proliferations, induce cytotoxicity and differentiate into effector and memory cells.
- Murphy & Weaver (2016) found T cells subsequently create an immunological synapse to consistently communicate with the APC for hours.
- On a population level, the dose-response curve of ligand to cytokine production is sigmoidal, which indicates T cell activation associates with the strength of TCR stimulation.
- If the stimulus exceeds a certain threshold, T cell activation reaches maximum levels with no intermediate state.
To achieve full activation, T cells require 3 signals:
- Signal 1 = TCR recognises specific antigen on a MHC molecule.
- Signal 2 = Co-stimulatory receptors, such as CD28, ICOS, CD80 or CD86 (to make B7 protein; B7.1 and B7.2 respectively), presented on the surface of other immune cells to signify a detected infection by the innate immune system
- Signal 3 = Cytokines modifying the differentiation of T cells into different subsets of effector T cells.
i. Receptor activation
- When the TCR interacts with a specific pMHC, the tyrosine residues of the immunoreceptor tyrosine-based activation motifs (ITAMs) in its CD3 adaptor proteins become phosphorylated. van der Merwe & Dushek (2011) found the residues function as docking sites for downstream signalling molecules, which helps propagate the signal.
- ITAM phosphorylation is regulated by the Src kinase Lck, which is anchored to the plasma membrane linked to the co-receptor CD4 or CD8, depending on the T cell subtype. Helper T cells and regulatory T cells express CD4 that is specific for MHC class II, whereas cytotoxic T cells express CD8 that is specific for MHC class I.
- When the co-receptor interacts with the MHC, it recruits Lck to the CD3 ITAMs. Nika et al. (2010) demonstrated around 40% of Lck activity occurs before the interaction between TCR and pMHC, thus it is able to persistently phosphorylate the TCR.
- Phosphatase CD45 halts phosphorylation of tyrosine residues and inhibits signal initiation, which prevents tonic TCR signalling.
- When phosphatase C45 binds, it disrupts the balance between kinase activity and phosphatase activity, which results in increased phosphorylation and triggers a signal. Salmond et al. (2009) suggested Tyrosine kinase Fyn plays a role in ITAM phosphorylation but not a major role in TCR signalling.
ii. Proximal TCR signalling
- Phosphorylated ITAMs located in the cytoplasmic tails of CD3 recruit protein tyrosine kinase Zap70 to bind to phosphorylated tyrosine residues with its SH2 domain. This allows Zap70 to approach Lck, which leads to Lck phosphorylising and activating it.
- Activated Zap70 then phosphorylates multiple tyrosine residues of the transmembrane protein LAT, which is a scaffold protein associated with the membrane. Although it can't catalyse anything, it provides binding sites for signalling molecules via phosphorylated tyrosine residues.
- LAT interacts with another scaffolding protein Slp-76 via the Grap2 adaptor protein, which provides more binding sites. The LAT/Slp-76 protein complex creates a platform for the recruitment of numerous downstream signalling molecules.
- When these signalling molecules approach these binding sites, Lck, Zap70 and other kinases subsequently activate them. Thus, the LAT/Slp-76 complex serves as a cooperative signalosome. Huse (2009) listed the signalling molecules binding the LAT/Slp-76 complex include phospholipase Cγ1 (PLCγ1), SOS via a Grb2 adaptor, Itk, Vav, Nck1 and Fyb.
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| This is a simple diagram of the T lymphocyte activation pathway. It shows T cells contributing to immune defences in two major ways; some direct and regulate immune responses; others directly attack infected or cancerous cells. |
iii. Signal transduction to the nucleus
- This pathway begins with an enzyme called PLCγ being activated by the tyrosine kinase ltk, which is recruited to the cell membrane by binding to Phosphatidylinositol (3,4,5)-trisphosphate (PIP3).
- Phosphoinositide 3-kinase(PI-3K) phosphorylates Phosphatidylinositol 4,5-bisphosphate (PIP2), which in turn produces PIP3. However, it isn't clear whether PI-3K is activated by the TCR itself or not. Nonetheless, a co-stimulatory receptor called CD28 is known to provide the second signal, which activates PI-3K.
- When both signals 1 and 2 are received, PLCγ becomes activated by phosphorylation, which subsequently hydrolyses PIP2 into 2 secondary messenger molecules known as diacyl glycerol(DAG) and inositol 1,4,5-trisphosphate (IP3).
- DAG and IP3 strengthen the TCR signal and propagate the prior localised activation to the entire T cell and activate protein pascades that result in the activation of transcription factors such as NFAT, NF-κB and AP1, as well as a heterodimer of proteins Fos and Jun.
- When all 3 aforementioned transcription factors are activated, it stimulates the transcription of interleukin-2(IL2) gene.
i. NFAT
- When IP3 ceases to bind to the membrane, it diffuses rapidly in the T cell.
- IP3 binds to calcium channel receptors on the endoplasmic reticulum (ER) to stimulate the release of calcium (Ca2+) into the cytosol).
- As the Ca(2+) level decreases in the ER, it results in STIM1 clustering on the ER membrane.
- This activates cell membrane CRAC channels that permits more calcium entry from the extracellular space to the cytosol of the T cell.
- This cytosolic calcium binds calmodulin, which triggers a conformational change of the protein that subsequently binds and activates calcineurin.
- Calcineurin dephosphorylates NFAT, which deactivates it. This means deactivated NFAT is unable to enter the nucleus because nucelar transporters can't recognise its nuclear localisation sequence (NLS) due to phosphorylation by GSK-3.
- When NFAT is dephosphorylated by Calcineurin, it can translocate into the nucleus.
- Huse (2009) found PI-3K recruits the protein kinase AKT to the cell membrane via signalling molecules. This suggests AKT deactivates GSK3 and inhibits phosphorylation of NFAT, which may play a role in NFAT activation.
ii. NF-κB
- DAG first binds and recruits Protein kinase C θ (PKCθ) to the cell membrane, which it activates the membrane bound scaffold protein CARMA1.
- CARMA1 subsequently experiences a conformational change in order to be able to oligomerise and bind the adapter proteins BCL10, CARD domain and MALT1.
- The multisubunit complex comprised of CARMA1,BCL10, CARD domain and MALT1 binds the Ubiquitin ligase TRAF6.
- When TRAF6 ubiquinates, it functions as a scaffold to recruit NEMO, IκB kinase (IKK) and TAK1.
- TAK1 phosphorylates IKK, which sequentially phosphorylates NF-κB inhibitor I-κB. This results in the ubiquitination and subsequent degradation of I-κB.
- Meanwhile, I-κB blocks the NLS of NF-κB, which prevents the translocation of NF-κB to the nucleus.
- When I-κB degrades, it is unable to bind to NF-κB and the NLS of NF-κB then becomes accessible for nuclear translocation.
iii. AP1
- This pathway involves a phosphorylation cascade of 3 successive acting protein kinases to transmit a signal. The 3 MAPK pathways in T cells feature 3 different kinase families: MAPK, MAP2K, and MAP3K.
- Initially, GTPase Ras or Rac phosphorylates the MAP3K, which triggers a molecular cascade.
- The cascade involves the enzymes Raf, MEK1, ERK, which leads to the phosphorylation of Jun.
- This triggers a conformational change that allows Jun to bind to Fos, which produces a transcription factor called AP-1.
- Next, Raf is activated via second messengers DAG, SOS, and Ras. DAG recruits a guanine nucleotide exchange factor (GEF) called RAS guanyl nucleotide-releasing protein (RasGRP) to the membrane.
- RasGRP then activates the small GTPase Ras by exchanging Guanosine diphosphate (GDP) bound to Ras with Guanosine triphosphate (GTP).
- Ras is also activated by the guanine nucleotide exchange factor SOS, which interacts with the LAT signalosom.
- Next, Ras triggering the MAPK cascade involves MEKK1, JNKK and JNK stimulating protein expression of Jun.
- Another cascade involving MEKK1 as MAPK3 activates MKK3 /6 and p38 that stimulates Fos transcription.
- When MEKK1 is activated by Slp-76 and (by Ras), it recruits the GEF Vav to the LAT signalosom. This subsequently activates the GTPase Rac.
- Finally Rac and Ras activates MEKK1, therefore initiates the MAPK cascade.
What are the different types of T-Cells?
1. CD4+ Th (T-Helper) Cells
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| This diagram illustrates the activation of macrophage or B cell by T helper cell. |
- Known as CD4+ (CD4-positive) cells, T helper (Th) cells play an important role in assisting other cells by releasing cytokines, which influence the behaviour of target cells that express receptors for these specific cytokines. It allows the cells to polarise the immune response depending on the nature of the immunological threat.
- Mature Th cells express a surface protein called CD4, thus are named CD4+ T cells. Examples of these cells helping other immune cells include a combination of inter-cell interactions (e.g. CD40 (protein) and CD40L) and cytokines between the CD4+ T cell and an APC presenting a peptide antigen on MHC class II protein.
How are naive helper T Cells activated?
- Naive T cells that leave the thymus are labelled recent thymic emigrants (RTE) and move to secondary lymphoid organs (SLO) such as spleen and lymph nodes, where they mature.
- This produces mature naive T cells, but they lack or have downregulated expression of the RTE-related surface markers, such as CD31, PTK7, Complement Receptor 1 and 2 (CR1 and CD2) and the production of interleukin 8 (IL-8).
- CD4+ T cells express the TCR-CD3 complex, with the TCRs having an affinity for MHC Class II, as well as the CD4 receptor determining MHC affinity during maturation in the thymus.
- When a Th cell interacts and recognises the antigen on an APC, the TCR-CD3 complex binds to the peptide-MHC Class II complex on the surface of professional APCs. The co-receptor of the TCR complex, CD4, binds to a different section of the MHC molecule.
- Roughly 50 of these molecular interactions are required to activate the Th cell and assemblies known as microclusters, which are created between the TCR-CD3-CD4 complexes of the Th cell and the MHC Class II proteins of the dendritic cell at the zone of contact.
- This allows CD4 to recruit the Lck kinase, which phosphorylates immunotyrosine activation motifs (ITAMs) situated on the CD3 gamma (γ), delta (δ), epsilon (ε), and zeta (ζ) chains.
- The protein Zap-70 binds these phosphorylated ITAMs via its SH2 domain and subsequently itself becomes phosphorylated, in which it coordinates the downstream signalling responsible for T cell activation.
- CD45 activates Lck by dephosphorylating a tyrosine in its C-terminal tail, while Csk phosphorylates Lck at the same position. This indicates CD45 and Csk activities mediates Lck activation.
- If CD45 levels reduce, it results in a form of SCID since lack of Lck activation prevents appropriate T cell signalling. Courtney et al. (2019) found memory T cells utilise the same pathway and express higher levels of Lck, thus inhibits Csk function in these cells.
- There is little understanding about the role of a bulky extracellular region of CD45 during cell interactions, as CD45 contains a number of isoforms that vary in size depending on the Th cell's activation and maturation status.
- e.g. CD45 truncates following Th cell activation (CD45RA+ to CD45RO+), however it is unknown whether this shortening impacts activation.
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| This diagram illustrates antigen presentation of naïve CD8+ and CD4+ T cells to respectively become mature "cytotoxic" CD8+ cells and "helper" CD4+ cells. |
ii. Signal 2 = Survival
- If the naive T cell receives signal 1, it activates a second independent biochemical pathway, better known as signal 2. This ensures a T cell responds correctly to a foreign antigen. Without signal 2 during initial exposure to antigen, the T cell presumes it to be auto-reactive.
- This leads to anergy, which is generated from the unprotected biochemical changes of signal 1. Anergic cells won't react with antigen, even in the presence of signals 1 and 2. Elmore (2007) suggested anergic cells circulate throughout the body with little usefulness until they undergo apoptosis.
- Communication of signal 2 requires the interaction between CD28 on the CD4+ T cell and proteins CD80 (B7.1) or CD86 (B7.2) on the professional APCs, known as co-stimulation.
iii. Signal 3 = Differentiation
- When the 2-signal activation is complete, the T helper cell subsequently undergoes proliferation. It releases a potent T cell growth factor called interleukin 2 (IL-2), which acts upon itself in an autocrine fashion.
- Autocrine or paracrine secretion of IL-2 can interact with the same Th cell or other Th cells via the IL-2R, hence stimulating proliferation and clonal expansion of Th cells.
- If Th cells receives both signals of activation and proliferation, it subsequently become Th-0 (T helper zero) cells that releases IL-2, IL-4 and interferon gamma (IFN-γ).
- Next, Th0 cells differentiates into Th1 or Th2 cells depending on the cytokine environment.
- IFN-γ and IL-4 drives Th1 and Th2 production respectively, whereas IL10 / IL-4 and IFN-γ inhibits Th1 cell and Th2 cell production respectively.
What determines the effector T helper cell response?
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| This is the Th1/Th2 Model for helper T cells. When an antigen is ingested and processed by an APC, its fragments are presented to T cells via MHC Class II. IFN-γ = interferon gamma TGF-β = transforming growth factor beta mø = macrophage IL-2 = interleukin 2 IL-4 = interleukin 4 |
What are T helper 3 (Th3) cells?
https://en.wikipedia.org/wiki/T_helper_3_cell
- Th3 cells are a subset of T lymphocytes that play important roles in immunoregulation and immunosuppression, which may be triggered by administration of foreign oral antigen.
- They primarily act through the release of anti-inflammatory cytokine transforming growth factor beta (TGF-β).
- Chien & Chiang (2017) depicted Th3 cells as CD4+FOXP3− regulatory T cells in both mice and human.
- Weiner et al. (2011) first reported the existence of Th3 cells when researching oral tolerance in the experimental autoimmune encephalitis (EAE) mouse model and later described them as CD4+CD25−FOXP3−LAP+ cells, which were stimulated by oral antigen through T cell receptor (TCR) signalling in the gut.
- Their main roles are in mucosal immunity and protection of mucosal surfaces in the gut from non-pathogenic non-self antigens, as well as regulation of the non-inflammatory environment by releasing TGF-β and IL-10.
- TGF-β triggers the class switch to a non-inflammatory antibody called IgA, which doesn't typically activate the complement system nor plays a role in phagocytosis.
- Unlike CD25+CD4+ Treg cells, Th3 cells isn't dependent upon IL-2 for survival. In addition, in vitro differentiation of Th3 cells is promoted by TGF-β, IL-4, and IL-10.
- Studies hypothesised Th3 cells have a different lineage from naturally arising CD25+CD4+ Treg cells. However, it is still uncertain whether Th3 cells are identical to induced Treg cells due to the lack of a specific marker for Th3 cells.
- Iwasaki (2007) demonstrated TGF-β was created by intestinal dendritic cells, which may be the source of cytokines that induces Th3 cells in the intestine.
- Chen et al. (2003) reported naturally arising Treg cells constitutively expressed cytotoxic T-lymphocyte antigen 4 (CTLA-4) that stimulated production of TGF-β. There is a theory that this phenomenon may induce the differentiation of both stimulated Treg cells and Th3 cells.
What molecules do Th3 cells express or secrete?
- The phenotype of Th3 cells is currently known as CD4+CD25−CD69+FOXP3-LAP+, however they lack expression of the transcription factor FOXP3.
- Chien & Chiang (2017) reported a lack of a certain transcription factor for complete and reliable recognition of the Th3 cell population.
- Cibrián & Sánchez-Madrid (2017) reported type II-lectin receptor CD69 isn't specific for Th3 cells, since it is usually expressed on other lymphocytes.
- Boswell et al. (2011) found the latency-associated peptide (LAP) noncovalently bounds TGF-β and is expressed by numerous cells of the immune system.
- In the case of tumours, Th3 cells express lymphocyte activation gene-3 (LAG3), create an abundance of TGF-β and a low amount of anti-inflammatory cytokine interleukin 10 (IL-10).
- Scurr et al. (2014) discovered, in colorectal cancer, Th3 cells were about 50 times more potent immune suppressors than the classical regulatory FOXP3+ T lymphocytes and their functions were primarily regulated by secretion of suppressive cytokines.
- Andrews et al. (2017) stated LAG3 is expressed on NK cells and other T cells that negatively regulated T cell activation and function. This molecule can bind MHC class II molecules due to its structural similarity to CD4.
- Jørgensen et al. (2019) found Th3 cells are activated by TCR stimulation they recognise an antigen or are stimulated by TGF-β from CD4+ T lymphocytes in the presence of IL-10 and IL-4 cytokines.
- It is currently known Th3 cells are involved in regulating the immune response via mechanisms independent of intercellular contact. They release anti-inflammatory cytokine TGF-β to maintain homeostasis in the gut and suppress amplified inflammatory and autoimmune responses in the body.
- Weiner et al. (2011) explained TGF-β is an essential cytokine for maintaining the naturally occurring Treg cells that inhibit Th1 and Th2 immune functions.
- Gol-Ara et al. (2012) stated Th3 cells release TGF-β to directly suppress Th1 and Th2 cells and provide support to B cells towards IgA production.
i. Th17 Helper Cells
- T helper 17 cells (Th17) are a subset of pro-inflammatory T helper cells that produce interleukin 17 (IL-17). They associate with T regulatory (Treg) cells and the signals triggering Th17 differentiation actually inhibit Treg differentation.
Describe the differentiation pathway of Th17 cells
- In mice and humans, the cytokines that play a role in Th17 formation include TGF-β, IL-6, IL-21 and IL-23. They are produced by activated antigen presenting cells (APCs) after interacting with pathogens.
- Ivanov et al. (2006) identified the key factors involved in Th17 differentiation include signal transducer and activatory of transcription 3 (Stat3) and retinoic acid receptor-related orphan receptors gamma (RORγ) and alpha (RORα).
- Th17 cells can change their differentiation program, which result in either protective or pro-inflammatory pathogenic cells. The protective and non-pathogenic Th17 cells stimulated by IL-6 and TGF-β are known as Treg17 cells, whereas the pathogenic Th17 cells are stimulated by IL-23 and IL-1β.
- Korn et al. (2007) found a cytokine produced by Th17 cells called IL-21 triggers an alternative pathway for the activation of Th17 populations.
What are the functions of Th17 cells?
- Th17 cells are known to induce adaptive immunity against pathogens, but Vautier et al. (2010) pointed out its role in anti-fungal immunity is restricted to particular sites with detrimental effects.
- Their main effector cytokines include IL-17A, IL-17F, IL-21, IL-22 and granulocyte-macrophage colony-stimulating factor (GM-CSF).
- IL-17A and IL-17F target innate immune cells and epithelial cells to stimulate production of G-CSF and IL-8 (CXCL8), which results in the production and recruitment of neutrophils.
- Weaver al. (2013) summarised that Th17 cells regulate neutrophils, Th2 cells regulate eosinophils, basophils and mast cells, and Th1 cells regulate macrophages and monocytes. This indicates these 3 T helper cell subsets impact the myeloid aspect of the immune system.
- Esplugues et al. (2011) found Treg17 cells containing in vivo immune-suppressive properties in the gut, described them as rTh17 cells.
- Treg17 cells produce IL-17 and IL-10, as well as low levels of Il-22 to suppress autoimmune and other immuen responses.
- Bellemore et al. (2015) found CD4+ T cells polarised with IL-23 and IL-6 are pathogenic upon adoptive transfer in type 1 diabetes, whereas T cells polarised with TGF-β and IL-6 are non-pathogenic.
- Stockinger et al. (2014) found Treg17 cells express the intracellular aryl hydrocarbon receptor (AhR), which is activated by a number of aromatic compounds. They are regulated by IL-23 and TGFβ.
- IL-22 production by Th17 cells is regulated by AhR and Treg17 depending on the activation of the transcription factor Stat3.
- In a steady state, TGF-β and AhR ligands decrease IL-22 expression and increase expression of AhR, c-MAF, IL-10, and IL-21, which may offer protection in cell regeneration and host microbiome homeostasis.
- In mice, Th17 cells were understood to moderate tumour regression, as well as promote tumour formation induced by colonic inflammation.
- Crome et al. (2010) stated Th17 cells play a role in recruiting B cells via CXCL13 chemokine signalling, as well as promote the production of antibodies.
- When Stat3 was selectively deleted, it triggered spontaneous severe colitis due to the lack of Treg17 cells and increase in pathogenic Th17 cells.
- Treg17 cells express a chemokine receptor called CCR6, which facilitates trafficking into areas of Th17 inflammation. Gagliani et al. (2015) found conversion of pathogenic Th17 cells in vivo at the later stages of an inflammatory diseases process by TGF-β leads to the generation of cells similar to Treg17.
ii. Thαβ Cells
- Thαβ helper cells play a fundamental role in host immunity against viruses, whose differentiation is stimulated by IFN α/β or IL-10.
- Their key effector cytokine is IL-10, and their main effector cells are NK cells, CD8 T cells, IgG B cells, and IL-10 CD4 T cells. Their key transcription factors include STAT1, STAT3 and IRFs.
- IL-10 released by CD4 T cells activate the ADCC on NK cells in order to trigger apoptosis of virus-infected cells and stimulate both host and viral DNA fragmentation.
- To prevent virus replication and transmission, IFN-α/β act to suppress transcription of viral DNA.
- If THαβ cells overactivate after interacting with autoantigens, it leads to type 2 antibody-dependent cytotoxic hypersensitivity, a common symptom in myasthenia gravis or Graves' disease.
2. CD8+ cytotoxic T-Cells
- A cytotoxic T cell (or TC, cytotoxic T lymphocyte, CTL, T-killer cell, cytolytic T cell, CD8+ T-cell or killer T cell) is a T lymphocyte that serves to kill cancer cells, or cells infected by intracellular pathogens (e.g. viruses or bacteria), or cells damaged by other methods.
How do CB8+ T-cells develop?
- These T cells undergo the same developmental processes as any other naive T cell. They initially create a variety of TCRs comprised of an alpha chain and a beta chain, or a gamma chain and a delta chain.
- Haematopoietic stem cells (HSCs) in the bone marrow migrate into the thymus, where they experience V(D)J recombination of their beta-chain TCR DNA to generate a pre-TCR.
- Upon successful rearrangement of the beta chain, the alpha-chain TCR DNA subsequently undergoes rearrangment to produce a functional alpha-beta TCR complex.
- The highly-variable genetic rearrangement of TCR genes produces a considerable number of unique T cells with unique TCRs, which gives the body's immune system the capacity to respond to virtually any invader.
- A majority of the T cell population express alpha-beta TCRs (αβ T cells), with a minority of T cells in gut epithelial tissues express gamma-delta TCRs (γδ T cells) that recognise non-protein antigens.
- Deseke & Prince (2020) discovered γδ TCRs are able to both recognise antigens not presented to them and microbial toxic shock proteins and self-cell stress proteins.
- γδ T-Cells demonstrate a broad functional plasticity after they recognise infected or transformed cells, because they can create cytokines (e.g. IFN-γ, TNF-α, IL-17) and chemokines (e.g. IP-10, lymphotactin), as well as trigger cytolysis of target cells with perforins and granzymes, interact with other cells (e.g. epithelial cells, monocytes, dendritic cells, neutrophils and B cells).
- Tuengel et al. (2021) pointed out a number of infections (e.g. human cytomegalovirus) triggers clonal expansion of peripheral γδ T cells with specific TCRs, which suggests these cells modulate the adaptive nature of the immune response.
T cells with functionally stable TCRs express both the CD4 and CD8 co-receptors to become "double-positive" (DP) T cells (CD4+ CD8+). They process to the next stage of exposure to a variety of self-antigens in the thymus and experience 2 types of selection trials.
- Positive selection = Double-positive T cells bind to foreign antigen in the presence of self-MHC. They differentiate into either CD4+ or CD8+ T cells dependning on which MHC is associated with the antigen presented (MHC1 for CD8, MHC2 for CD4). In this case, CD8+ T cells are positively selected because their TCRs are capable of recognising self MHC-I molecules.
- Negative selection = Double-positive T cells binding too strongly to MHC-presented self antigens are signalled to undergo apoptosis because they are deemed autoreactive, resulting to autoimmunity.
- Therefore, CD8 T cells that bind weakly to the MHC-self-antigen complexes are positively selected, and subsequently differentiate into single-positive CD8+ T cells. They mature and proceed to become cytotoxic T cells following their activation with a MHC class I-restricted antigen.
How are CD8+ T-cells activated?
Cytotoxic T cells require a number of simultaneous interactions between molecules expressed on its surface and molecules expressed on APC's surface.
- Signal 1 = TCR interacts with a peptide antigen presented on MHC-I molecule. Another molecule called CD8 coreceptor interacts with the MHC-I molecule as well to stabilised the signal.
- Signal 2 = CD28 expressed by T-cell interacts with either CD80 or CD86 (also called B7-1 or B7-2 respectively) expressed by APC. CD80 and CD86 are costimulators for T cell activation, which is augmented or replcaed by stimulating the CD8+ T cell with cytokines released from T helper cells.
- Bennett et al. (1998) found CD40 released by infected cells modulates maturation of CD8+ T cells after naive CD8+ T cells binds to infected cells.
- Activated CD8+ T cells experience clonal expansion stimulated by a growth and differentiation factor called IL-2. This expands the T cell population specific for the target antigen that subsequently spread throughout the body in search of somatic cells sharing the same antigen.
What are the effector functions of CD8+ T-cells?
- When CD8+ T cells are exposed to infected somatic cells, they release the cytotoxins perforin, granzymes, and granulysin. Perforin allows the entry of granzymes into the cytoplasm of the target cell, followed by their serine protease function triggering the caspase cascade, a series of cysteine proteases that eventually lead to apoptosis. Chang et al. (2017) described it as a "lethal hit", meaning a wave-like death of the target cells.
- Rudd-Schmidt et al. (2019) found cytotoxic cells are resistant to the effects of their perforin and granzyme cytotoxins due to their high lipid order and negatively charged phosphatidylserine in their plasma membrane.
- When a CD8+ T cell is activated, it expresses a surface protein called FAS ligand (FasL)(Apo1L)(CD95L), which binds Fas (Apo1)(CD95) molecules expressed on the target cell.
- Bakshi et al. (2014) found the Fas-FasL interaction leads to recruitment of the death-induced signalling complex (DISC). Then the Fas-associated death domain (FADD) translocates with the DISC, which promotes procaspases 8 and 10.
- These caspases subsequently activate the effector caspases 3, 6, and 7, which lead to cleavage of death substrates such as lamin A, lamin B1, lamin B2, PARP (poly ADP ribose polymerase), and DNA-PKcs (DNA-activated protein kiase). This ultimately leads to apoptosis of cells expressing Fas.
- Pearce et al. (2003) hypothesised the transcription factor Eomesodermin associates with CD8+ T cell function, functioning as a regulatory gene in the adaptive immune response. Moreover, they discovered reduced expression of Eomesodermin reduces the amount of perforin produced by CD8+ T cells.
3. γ/δ T-Cells
i. Laboratory mice (Mus musculus)
-- Mouse Vγ chains
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| Mouse Vγ locus for C57BL/6 genome that is drawn to scale. Chromosome 13: 1.927 to 1.440 Megabp Heilig notation |
ii. Human forms
-- Vγ9/Vδ2 T cells
- In humans and primates, Vγ9/Vδ2 T cells constitute about 0.5 - 5% of the leukocyte population in peripheral blood. It is suggested they play an important role in detecting invading pathogens early as they expand dramatically in many acute infections.
- Vγ9/Vδ2 T cells are known to recognise a natural intermediate of the non-mevalonate pathway of isopentenyl pyrophosphate (IPP) biosynthesis called (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP). HMB-PP is known to be a metabolite in most most pathogenic bacteria including Mycobacterium tuberculosis and malaria parasites.
- Bacterial species lacking the non-mevalonate pathway actually synthesise IPP via the classical mevalonate pathway, such as Streptococcus, Staphylococcus, and Borrelia. This indicates they can't produce HMB-PP, hence Vγ9/Vδ2 T cells won't be activated.
- It is known the structure of IPP is closely related to HMB-PP and ubiquitously present in all living cells (including human cells), but it potency in vitro is substantially decreased. However, it isn't apparent whether IPP serves as a physiological 'danger' signal of stressed or transformed cells.
- Hewitt et al. (2005) found evidence of Vγ9/Vδ2 T cells not directly recognising aminobisphosphonate 'antigens' (e.g. zoledronate (Zometa), pamidronate (Aredia)), and those antigens indirectly acting on the mevalonate biosynthetic pathway, resulting in IPP accumulation.
- Vγ9/Vδ2 T cells are known to be activated by a number of alkylated amines in vitro, but only at minute concentrations.
- A question that remains unanswered is whether non-peptide antigens bind directly to the Vγ9/Vδ2 TCR or if a presenting element exists. Since none of the known APC molecules (e.g. MHC-I and MHC-II or CD1) are required to activate γδ T cells, it indicates a new presenting element may exist.
- Human Vγ9Vδ2 T cells express multiple receptors for inflammatory chemokines (CXCR3, CCR1, CCR2 and CCR5), which is evidence of an inflammatory migration program characteristic of professional antigen-presenting cells (APC). This suggests IPP or HMB-PP stimulation triggers migration of Vγ9Vδ2 T cells to the lymphatic tissues, specifically the T cell region of lymph nodes.
- When Vγ9Vδ2 T cells are stimulated by phosphoantigens, they express several markers associated with APC such as MHC-I and MHC-II, co-stimulatory molecules (CD80, CD86) and adhesion receptors (CD11a, CD18, CD54). Therefore it suggests activated Vγ9Vδ2 T cells act like APCs (γδ T-APC) and present antigens to αβ T cells, which transforms naïve CD4+ and CD8+ αβ T cells into effector cells.
- Differentiated effector T cells resulted in T helper cell response, which is usually pro-inflammatory Th1 response with subsequent release of IFN-γ and TNF-α. If γδ T-APC: CD4+ ratio is low, this results in differentiation of ome naïve αβ T cells into Th2 (IL-4) or Th0 (IL-4 plus IFN-γ) cells.
- Researchers suggested human Vγ9Vδ2 T cells demonstrate antigen cross-presentation activity, which involves uptaking exogenous antigen and its dispatch to the MHC I pathway to induce CD8+ cytotoxic T cells. This allows activated cytotoxic T cells to effectively kill infected or tumour cells.
-- Non-Vδ2+ T cells
- The complex structural diversity of Vδ1 and Vδ3 TCRs and the action of Vδ1+ clones against MHC, MHC-like, or non-MHC molecules imply non-Vδ2 cells recognise highly diverse and heterogeneous set of antigens.
- There is debate whether MHC class-I-chain-related gene A (MICA) or MHC class-I-chain-related gene B (MICB) plays the role of a tumour antigen recognised by Vδ1+ T cells. However, surface plasmon resonance analyses estimated MICA–Vδ1 TCR interactions demonstrate significantly low affinity.
- Upregulated endogenous gene products during infection are thought to trigger expansion of Vδ1 cells in a majority of cases.
- On the other hand, non-Vδ2 γδ T cells expansion occurs when exposed to intracellular bacteria (e.g. Mycobacteria and Listeria), extracellular bacteria (e.g. Borrelia burgdorferi) and viruses (e.g. HIV, cytomegalovirus).
- Since Vδ1+ T-cell responses aren't inhibited by monoclonal antibody directed against known classical or non-classical MHC molecules, it is hypothesised they recognise a new class of conserved antigens triggered by stress.
- Tuengel et al. (2021) found primary cytomegalovirus infection in infants increased population of Vδ1 T cells expressing NK cell associated markers NKG2C and CD57.
- von Lilienfeld-Toal et al. (2006) discovered a subset of Vδ1 IELs (intraepithelial lymphocytes) situated in the gut that express high levels of a natural cytotoxic receptor (NCR) called NKp46. Moreover, they found NKp46 are expressed almost exclusively by natural killer (NK) cells and play a fundamental role in its activation, which is similarly found in γδ T cells.
- It is concluded NKp46+/Vδ1 IELs may serve as a prognostic marker in the clinical diagnosis of colorectal cancer (CRC) in order to follow up its progression.
- Mikulak et al. (2019) found reduced levels of NKp46+/Vδ1 IELs in healthy intestinal tissues surrounding the tumoir mass correlates with faster tumour progression and metastasis.
4. Regulatory (Suppressor) T-Cells
- Formerly known as suppressor T cells, Treg cells are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and suppress autoimmune responses.
- Treg cells act as the immune system's "self-check" to prevent harmful overreactions. Each form can be distinguished by the receptor expressed on its surface, which includes CD4, CD25 and FOXP3 (CD4+CD25+).
- CD4+ FOXP3+ CD25(high) Treg cells are labelled "naturally occurring" Treg cells, which is distinct from suppressor T cells generated in vivo.
- Other Treg populations include Treg17, Tr1, Th3, CD8+CD28-, and Qa-1 restricted T cells.
How do Treg cells develop?
- Treg cells are derived from progenitor cells in the bone marrow, which are committed to their lineage in the thymus.
- It begins as CD4-CD8-TCR- cells at the DN (double-negative) stage, which it rearranges its TCR genes to create a unique, functional molecule. Subsequently, this new molecule is tested against cells in the thymic cortex for a minimal level of interaction with self-MHC.
- If they receive the appropriate signals to proceed to the next stage, they subsequently proliferate and express both CD4 and CD8, transforming into double-positive cells.
- Treg cells are selected by either radio-resistent haematopoietically-derived MHC class- II-expressing cells in the medulla or Hassall's corpuscles in the thymus.
- At the double-positive (DP) stage, they are selected by their interaction with cells within the thymus. This initiates the transcription of Foxp3, which transforms the immature T cells into Treg cells.
- Since Treg cells don't have restricted TCR expression like in NKT or γδ T cells, they exhibit larger TCR diversity than effector T cells, which are skewed towards self-peptides.
- If a T cell receives a strong signal, it undergoes induces apoptotic death. If a T cell receives a weak signal, it survives the selection process and transforms into an effector T cell. If the T cell receives an intermediate signal, it subsequently transforms into a Treg cell.
- Because of the stochastic nature of T cell activation, all T cell populations with a particular TCR will eventually consist of both effector and regulatory T cells.
- Foxp3+ Treg cell generation in the thymus takes several days to initiate compared to effector T cells, thus doesn't approach adult levels in either the thymys or periphery until around 3 weeks post-partum.
- Since Treg cells require CD28 co-stimulation and B7.2 expression is primarily limited to the medulla, Treg cell development parallels that of Foxp3+ cells.
i. Thymic Recirculation
- Thiault et al. (2015) found some FOXP3+ Treg cells recirculate back to the thymus, where they develop and usually the main site of Treg cell differentiation.
- These Treg cells in the thymus, as well as the foetal thymic tissue culture, inhibit development of novel Treg cells by 34-60%, but spares Tconv cells. This suggests Treg cells recirculate to the thymus inhibit only de novo development of Treg cells.
- Pandiyan et al. (2007) explained Treg cells adsorb IL-2 from its microenvironments, which triggers apoptosis of other T cells lacking IL-2 (its main growth factor).
- Recirculating Treg cells in the thymus express a high concentration of high-affinity-IL-receptor-α chain (CD25) encoded by IL-2ra gene, which collects IL-2 from thymic medulla, and reduces its concentration. However, newly generated FOXP3+ Treg cells in thymus don't express as high amount of IL-2ra on their surface.
- Thiault et al. (2015) discovered a population of CD31- Treg cells in the human thymus, which may be used as a marker of newly generated Treg cells.
- Toker et al. (2013) and Nikolouli et al. (2021) discovered a population of CD24 low FOXP3+ Treg cells in the thymys with increased expression of IL-1R2 compared to peripaheral Treg cells. Furthermore, inflammation increases IL-1β levels, which reduces de novo development of Treg cells in the thymus.
- Recirculating Treg cells with high IL-1R2 expression situated in the thymus during inflammation aids in uptaking IL-1β and decreasing its levels in the medulla microenvironment, therefore augmenting development of de novo Treg cells.
- Peters et al. (2013) found binding of IL-1β to IL1R2 on the surface of Treg cells don't trigger signal transduction due to lack of Intracelluar (TIR) Toll interleukin-1 receptor domain, which is usually found in innate immune cells.
What are the functions of Treg cells?
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| Diagram of Treg cell, effector T cells and dendritic cell illustrating putative mechanisms of suppression by Treg cells. |
The molecular mechanisms by which Treg cells demonstrate to suppress or regulate the discrimination between self and non-self isn't well understood. A number of functions have been proposed based on experimental findings:
- Generation of inhibitory cytokines such as TGF-β, IL-35, and IL-10, as well as inducing other cell types to express IL-10.
- Production of Granzyme B, which triggers apoptosis of effector T cells.
- Reverse signalling by directly interacting with dendritic cells and stimulating immunosuppressive molecule indoleamine 2,3-dioxygenase.
- Signalling via ectoenzymes CD39 and CD73 by producing the immunosuppressive molecule adenosine.
- Direct communication with dendritic cells via LAG3 and TIGIT molecules.
- Involvement in the IL-2 feedback loop = When T cells are activated by antigen, it releases IL2 ligand that bind to IL-2 receptors on Treg cells. This signals Treg cells of high T cell activity occurring in a particular area, which triggers a suppressory response against them. This negative feedback loop plays an essential role in preventing overreacting T cells. If the feedback loop is disrupted, it results in immune hyperreactivity.
- Prevents co-stimulation through CD28 on effector T cells by the molecule CTLA-4.
What are induced Treg cells?
- Induced regulatory T (iTreg) cells (CD4+ CD25+ FOXP3+) suppress T cell poliferation and experimental autoimmune diseases, as well as play an important in tolerance. They develop from mature CD4+ conventional T cells outside of the thymus, rather than inside of it.
- Haribhai et al. (2011) found iTreg cells are an essential non-redundant regulatory subset of Treg cells that supports natural regulatory T (nTreg) cells, partially due to expansion of TCR diversity within regulatory responses.
- In mouse models, acute reduction of iTreg cell pool lead to inflammation and weight loss. However, it's unknown how much iTreg cells contributes to maintenance of tolerance compared to nTreg cells.
- Sun et al. (2007) stated the environment in the small intestine contain high levels of vitamin A and a site for producing retinoic acid. Dendritic cells within this region produce retinoic acid and TGF-β to signal the generation of Treg cells.
- Mucida et al. (2007) found vitamin A and TGF-β stimulates T-cell differentiation into Treg cells rather than Th17 cells, even when IL-6 is present.
- Ziegler & Buckner (2009) found the intestinal environment results in iTreg cells with TGF-β and retinoic acid. Povoleri et al. (2018) found some iTreg cells express lectin-like receptor (CD161) that serve to maintain barrier integrity by promoting wound healing.
- Coombes et al. (2007) observed Treg cells within the gut differentiate from naïve T cells upon the introduction of antigen.
- Cook et al. (2021) demonstrated human Treg cells are stimulated from both naive and pre-committed Th1 cells and Th17 with a parasite-dervied TGF-β mimic, secreted by Heligmosomoides polygyrus and termed Hp-TGM (H. polygyrus TGF-β mimic).
- White et al. (2021) found Hp-TGM stimulated stabile murine FOXP3 expressing Treg cells in the context of inflammation in vivo.
- Moreover, Cook et al. (2021) discovered Hp-TGM-induced human FOXP3+ Treg cells were stabile during inflammation and elevated levels of CD25, CTLA4 and reduced methylation in the FOXP3 Treg-Specific demethylated region compared to TGF-β-induced Treg cells.
What is the role of Treg cells in disease?
- Hypotheses suggest the Treg cell activity downregulate upon interaction with infectious microorganisms, either directly or indirectly, by other cells to facilitate removal of the infection.
- Mouse models demonstrate a number of pathogens may have evolved to manipulate Treg cells to immunosuppress the host and thus potentiate their own survival. For example, Treg cell activity increased in several infectious scenarios, such as retroviral infectious (e.g. HIV), mycobacterial infections (e.g. tuberculosis), and various parasitic infections (e.g. Leishmania and malaria).
- During HIV infection, Treg cells suppress the immune system, therefore limit target cells and decreased inflammation. However, this impedes the clearance of virus by the cell-mediated immune response and augments the reservoir by pushing CD4+ T cells to a resting state, including infected cells. Furthermore, HIV can infect Treg cells, which directly expands the HIV reservoir.
- Sivanandham et al. (2020) experimented a number of Treg cell depletion strategies in SIV infected non-human primates, and discovered they triggered viral reactivation and promoted SIV specific CD8+ T cell responses.
- Treg cells are associated with the pathology of visceral leishmaniasis and prevention of excess inflammation in patients cured of such disease.
- Dranoff (2005) found CD4+ Treg cell levels increase in cancers such as breast, colorectal and ovarian cancers, which correlate with a poorer prognosis.
- Yang et al. (2007) observed CD70+ non-Hodgkin lymphoma B cells trigger FOXP3 expression and regulatory function in intratumoral CD4+CD25− T cells.
- Beers et al. (2017) stated Treg cells demonstrate dysfunction and induce neuroinflammation in amyotrophic lateral sclerosis due to reduced FOXP3 expression.
- Tsuda et al. (2019) found Treg cell levels increase via polyclonal expansion both systemically and locally during healthy pregnancies in order to protect the foetus from the maternal immune response (i.e. maternal immune tolerance). Moreover, preeclamptic mothers and their offspring demonstrate impaired polyclonal expansion.
- Hu et al. (2019) hypothesised decreased production and development of Treg cells during preeclampsia may deteriorate maternal immune tolerance, which result in the hyperactive immune response characteristic of preeclampsia.
-- Cancer
- The presence of tumour antigens expressed on cancerous (tumour) cells triggers an immune response in the host, which increases the concentration of tumour-infiltrating lymphocytes (TILs) in the tumour microenvironment.
- TIL are hypothesised to target cancerous cells and inhibit or halt the development of tumour cells. However, the process is not well understood because Treg cells prefer to be trafficked to the tumour microenvironment. Oleinika et al. (2015) found 20-30% of the total CD4+ population around the tumor microenvironment consist of Treg cells, compared to about 4% of CD4+ T cells.
- Plitas & Rudensky (2020) identified a correlation between an abundance of Treg cells in the tumour microenvironment and poor prognosis in numerous cancers, such as breast, ovarian, renal, and pancreatic cancer.
- This suggests Treg cells inhibit effector T cells and hamper the body's immune response against cancer. In some types of cancers (e.g. colorectal carcinoma and follicular lymphoma), an abundance of Treg cells associated with a positive prognosis, nonetheless.
- Since Treg cells can inhibit general inflammation, it can induce cell proliferation and metastasis. The opposite effects suggest Treg cells play an important role in cancer development depending on both type and location of the tumour.
- Lippitz (2013) stated Treg cells bind to ligand CCL22 (secreted by tumour cells) via its chemokine receptor CCR4 in order to infiltrate the tumour microenvironment. Subsequently, a molecule produced by tumour cells called TGF-β cytokine triggers differentiation and expansion of Treg cells.
- Ezzeddini et al. (2021) identified Forkhead box protein 3 (FOXP3) polymorphism (rs3761548) may play a role in gastric cancer progression by modifying Treg function and secreting immunomodulatory cytokines such as IL-10, IL-35, and TGF-β.
- Curiel (2007) found reducing Treg cell levels in animal models lead to an increased efficacy of immunotherapy treatments, which resulted in future immunotherapy treatments incorporating the principle of Treg cell depletion.
What characteristic molecules are expressed by Treg cells?
- A majority of Treg cells expressing forkhead family transcription factor FOXP3 (forkhead box p3) are situated within the major histocompatibility complex (MHC) class II restricted CD4-expressing (CD4+) population, which also express an abundance of interleukin-2 receptor alpha chain (CD25). Furthermore, there is a small population of MHC class I restricted CD8+ FOXP3-expressing Treg cells.
- Ellis et al. (2014) reported that although these FOXP3-expressing CD8+ T cells aren't active in healthy individuals, they are recruited during autoimmune disease by T cell receptor stimulation to suppress IL-17-mediated immune responses.
- The gold standard surface marker combination expressed on Treg cells within unactivated CD3+CD4+ T cells is elevated expression CD25 combined with the reduced or scarce expression of CD127 (IL-7RA) surface protein.
- A number of additional markers expressed on Treg cells that have been mentioned include CTLA-4 (cytotoxic T-lymphocyte associated molecule-4) and GITR (glucocorticoid-induced TNF receptor), but their functional role requires further investigation.
- Experimental studies by Seddiki et al. (2014) and Zaunders et al. (2009) used an activation-induced marker assay by CD39 expression in combination with co-expression of CD25 and OX40(CD134) to detect Treg cells, which define antigen-specific cells after being stimulated with antigen for 24 to 48 hours.
- Wieczorek et al. (2009) discovered a particular region within the FOXP3 gene (TSDR, Treg -specific-demethylated region) being demethylated only in Treg cells, which allows monitoring of Treg cells through a PCR reaction or other DNA-based analysis method.
-- Epitopes
- In 2008, studies discovered Treg epitopes consisting of linear sequences of amino acids contained within monoclonal antibodies and immunoglobulin G (IgG). Researchers hypothesised Treg cell epitopes play an important role in the activation of natural Treg cells.
- Numerous studies posited a number of potential applications of Treg cell epitopes, which includes tolerance of transplants, protein drugs, blood transfer therapies, type I diabetes, and moderation of the immune response for the treatment of allergies.
5. Natural killer T-Cells
- Natural Killer T (NKT) cells are a heterogeneous group of T cells that demonstrate traits of both T cells and NK cells. A majority of NKT cells recognise the non-polymorphic CD1d molecule, an antigen-presenting molecule that binds self and foreign lipids and glycolipids.
What are invariant NKT cells?
- This subset of CD1d-dependent NKT cells expresses an invariant T-cell receptor (TCR) α chain. They are known as type I or invariant NKT cells (iNKT) cells.
- They develop in the thymus, and spread throughout the periphery, as well as liver, spleen, peripheral blood, bone marrow and fat tissue.
- They are rapidly activated by danger signals and pro-inflammatory cytokines, which lead to effector functions, such as NK transactivation, T cell activation and differentiation, B cell activation, dendritic cell activation and cross-presentation activity, and macrophage activation.
- iNKT cells recognise lipid antigens presented by CD1d, a non-polymorphic major histocompatibility complex class I-like antigen presenting molecule.
- Brennan et al. (2013) found the highly conserved TCR consists of Va24-Ja18 paired with Vb11 in humans, specific for glycolipid antigens.
- Kawano et al. (1997) identified the best known antigen of iNKT cells is alpha-galactosylceramide (αGalCer), a synthetic form of a chemical purified from the deep sea sponge Agelas mauritianus.
Based on the cytokines produced, the 5 major distinct iNKT cell subsets include:
- iNKT1
- iNKT2
- iNKT17
- iNKT cells specialised in T follicular helper-like function
- iNKT cells specialised in IL-10 dependent regulatory functions
- iNKT1, iNKT2 and iNKT17 cells produce the same cytokines as Th cell subsets.
- Berzins et al. (2011) stated iNKT cells are activated when they engage with other immune cells, such as dendritic cells, neutrophils and lymphocytes, via their invariant TCR.
What are the functions of NKT cells?
- Activated NKT cells generate abundant levels of IFN-γ, IL-4, and granulocyte-macrophage colony-stimulating factor, as well as other cytokines and chemokines (e.g. IL-2, IL-13, IL-17, IL-21, and TNF-α).
- Recognises protected microbial lipid agents presented by CD1d-expressing antigen presenting cells, as part of a pathway to fight against infections and strengthen humoral immunity.
- Bai et al. (2013) found NKT cells supports B cells in its microbial defence and assists in targeting for B-cell vaccines.
6. Memory T cells
What are the sub-populations of memory T cells?
All memory T cell subtypes are known to be long-lasting and can rapidly proliferate to abundant levels of effector T cells upon re-exposure to their cognate antigen, which provides immunological memory against previously encountered pathogens. Akbar et al. (1988) stated memory T cells may be either CD4+ or CD8+ and typically display CD45RO and simultaneously lack CD45RA.
i. Central memory T cells (TCM cells)
They express CD45RO, C-C chemokine receptor type 7 (CCR7), and L-selectin (CD62L), as well as CD4. They can be found in the lymph nodes and in the peripheral circulation.
ii. Effector memory T cells (TEM cells)
- They express CD45RO and CD44 but not CCR7 and L-selectin. Since they lack the CCR7 lymph node-homing receptors, they are located in the peripheral circulation and tissues.
- TEMRA stands for terminally differentiated effector memory cells re-expressing CD45RA, which is a marker typically found on naive T cells.
iii. Peripheral memory T cells (TPM cells)
These cells are known to express intermediate levels of CX3CR1. They migrate to the tissues from blood and traffic to the lymph nodes in CD62L-independent manner to search for antigen in the tissues.
iv. Tissue-resident memory T cells (TRM cells)
- These cells populate the tissues throughout the body such as the skin, lung, gastrointestinal tract, etc. without recirculating.
- Mueller & Mackay (2016) found a number of TRM cell surface markers include CD69 and integrin αeβ7 (CD103). However, they highlight that TRM cells situate in various tissues that express different sets of cell surface markers.
- Steinert et al. (2015) found CD103+ TRM cells are confined to epithelial and neuronal tissues, while the TRM cells confined in salivary glands, pancreas and female reproductive tracts in mice don't express either CD69 or CD103.
- Shin & Iwasaki (2013) suggested TRM cells play an important role in protective immunity against pathogens.
- Kumar et al. (2018) implied TRM cells are involved in both protection and regulation. This is based on the finding that TRM cells release increased levels of protective-immunity-related cytokines and express reduced levels of he proliferation marker Ki67, compared to TEM cells.
- Kumar et al. (2018) conjectured these characteristics may support long-term maintenance of TRM cells, and maintaining balance between the rapid response to antigen invasion and evasion of unnecessary tissue damage.
- A 2017 study by Brigham and Women's Hospital found an association between dysfunctional TRM cells in autoimmune diseases, such as psoriasis, rheumatoid arthritis, inflammatory bowel disease. In addition, they estimated genes in TRM lymphocytes involved in lipid metabolism are approximately 20- to 30-fold more active than in other types of T-cells.
v. Virtual Memory T cells (TVM)
- Although TVM cells are quite abundant within the peripheral circulation, individual TVM cell clones situate at relatively low levels.
- Studies hypothesised homeostatic proliferation produces populations of CD8 TVM cells and CD4 TVM cells.
What are the functions of memory T cells?
- Memory T cells specific to antigen such has viruses or other microbial molecules are located in both central memory T cells (TCM) and effector memory T cells (TEM) subsets.
- Memory T cells were observed to exist not only in the cytotoxic T cells (CD8-positive) subset, but also in both the helper T cells (CD4-positive) and the cytotoxic T cells subset.
- The main function of memory cells is to support immune response after reactivation of those cells by reintroducing pathogen into the body.
- Central memory T cells (TCM) = They share a number of attributes with stem cells, such as self-renewal due to high levels of phosphorylation on key transcription factor STAT5. Experimental studies found TCM lymphocytes provide more potent immunity against bacteria, cancer cells and viruses, compared to TEM lymphocytes.
- Effector memory T cells (TEM) = TEM and TEMRA lymphocytes mainly express CD8, which are typically responsible for cytotoxic functions against pathogens.
- Tissue-resident memory T cell (TRM) = Since TRM lymphocytes survive for longer periods in tissues, they play an important role in inducing a rapid response to any breach of the cellular barrier as well as any relevant pathogen present. Gebhardt et al. (2009) found TRM release granzyme B to limit pathogens.
- Stem cell memory T cells (TSCM) = Their main functions include self-renewal, production of both the TCM and TEM subpopulations.
- Virtual memory T cell (TVM) = It's known TVM cells generate various cytokines, but more research is required to understand their role in suppressing unnecessary immunological states and their operation in treating autoimmune disorders.
i. Homeostatic maintenance
- Farber et al. (2014) theorised continuous replication and replacement of old memory T cells is important in the maintenance process because they have shorter half-lives than naïve T cells. However, the mechanism behind memory T cell maintenance isn't well-known and requires research to better understand it.
- There is a suggestion that memory T cells occasionally respond to novel antigens, induced by intrinsic diversity and the range of the T cell receptor binding targets.
- When these T cells cross-react to environmental or resident antigens in our bodies and proliferate, it may maintain the memory T cell population.
ii. Lifetime overview
- Kumar et al. (2018) stated T cells in the peripheral blood are primarly naïve T cells during birth and early childhood.
- When memory T cells are frequently exposed to antigen, it undergoes proliferation and, thus, its population increases.
- This stage of memory generation lasts from birth to about 20-25 years old, a period our immune system encounters the most amount of novel antigens.
- During the memory homeostasis stage, memory T cell levels plateau and becomes stabilised by homeostatic maintenance. Moreover, the immune response transitions to maintaining homeostasis as memory T cells don't encounter many novel antigens, as well as tumour surveillance.
- When a person reaches 65-70 years of age, they experience the immunosenescence stage (i.e. immune dysregulation), which involves dwindling T cell functionality and elevated susceptibility to pathogens.
Describe the proposed models of lineage
 | |
| On-Off-On model: 1. When the naive T cell (N) encounters an antigen, it activates and starts to proliferate (divide) into numerous clones or daughter cells. 2. A few T cell clones then differentiate into effector T cells (E). 3. A few cells also form memory T cells (M) that survive in an inactive state for a long period of time until they re-encounter the same antigen and reactivate. |
- When naïve T cells are activated by TCR binding to antigen and its downstream signalling pathway, they subsequently proliferate and produce a copious amount of effector cells. Effector cells then undergo a number of effector activities such as release cytokines.
- Restifo & Gattinoni (2013) found several of these cells form memory T cells after eliminating antigen, either in a randomly determined fashion or are selected based on their level of specificity.
- Youngblood et al. (2013) found these cells can switch from the active effector role back to an inactive state reminiscent of naïve T cells, and require "turning on" again upon the next exposure to antigen.
- This model conjectures that effector T cells can transit into memory T cells and survive, which retains the ability to proliferate. In addition, it also conjectures that a particular gene expression profile would result from the on-off-on pattern during naive, effector and memory stages.
- Youngblood et al. (2013) discovered the genes associated with survival and homing that arise from the on-off-on expression pattern, in including interleukin-7 receptor alpha (IL-7Rα), Bcl-2, CD26L and others.
 | ||
| Developmental differentiation model: In this model, memory T cells generate effector T cells, not the other way around. |
- This model asserts that effector cells created by activated naïve T cells would all experience apoptosis after eliminating antigen. It suggests memory T cells are created by activated naive T cells, but it hasn't fully matured into the effector stage.
- Since the progeny of memory T cells aren't specific to the antigen as the expanding effector T cells, they aren't fully activated.
- Restifo & Gattinoni (2013) discovered the telomere length and telomerase activity decreased in effector T cells comparing to memory T cells, which indicated that cell division of memory T cells was less frequent than effector T cells, which conflicts with the On-Off-On model.
- Henning et al. (2018) found repeated antigenic stimulation of T cells, such as HIV infection, triggered elevated effector functions but decreased memory.
- Moreover, substantially proliferated T cells were observed to likely produce transient effector cells, whereas shortly proliferated T cells would produce lasting cells.
What are the epigenetic modifications?
- Schmidl et al. (2018) found epigenetic modifications in CD4+ memory T cells lead to up-regulation of key cytokine genes during secondary immune response, which include IFNγ, IL4, and IL17A.
- It was found a number of these epigenetic modifications remain after antigen clearance, which manifest an epigenetic memory that provides a quicker activation upon re-encounter with the same antigen.
- Furthermore, enhanced expression of certain genes is dependent on the strength of the initial TCR signalling for the progeny of memory T cells. This correlates with the the regulatory element activation that directly alters the level of gene expression.
What is TCR-independent (bystander) activation?
- During the early stages of infection, T cells specific for a separate antigen are activated only by inflammation. This means T cells are able to be activated independently of their cognate antigen stimulation, i.e. without TCR stimulation.
- Several studies concluded this occurs locally and systematically in the inflammatory milieu caused by microbial infection, cancer or autoimmunity in both mice and humans.
- Lee et al. (2022) discovered bystander activated T cells migrate to the infection site, due to increased CCR5 expression. Whiteside et al. (2018) observed this phenomenon in memory CD8+ T cells that have reduced sensitivity to cytokine stimulation, compared to their naive counterparts and become activated in this manner more efficiently.
- Lee et al. (2022) discovered virtual memory CD8+ T cells exhibited elevated sensitivity to cytokine-induced activation in mouse models, but this finding wasn't directly replicated in humans.
- Bystander activated CD8+ T cells were discovered in human cancerous tissues that were specific for virus but not tumour, which suggests it plays a crucial role in the anti-tumour immunity and eliminating cancer cells.
i. Drivers of bystander activation
- Studies stated the major drivers of bystander activation are cytokines, such as IL-15, IL-18, IL-12 or type I IFNs, often acting with synergy.
- It is known IL-15 is involved in cytotoxic activity of bystander-activated T cells. Studies also discovered IL-15 stimulates expression of NKG2D (a receptor typically expressed on NK cells) on memory CD8+ T cells, which results in innate-like cytotoxicity, i.e. recognition of NKG2D ligands as indicators of infection, cell stress and cell transformation, and destruction of transformed cells.
- Kim & Shin (2019) demonstrated TCR activation suppresses IL-15-mediated NKG2D expression on T cells. Moreover, IL-15 triggers expression of cytolytic molecules, cell expansion and elevates the cell response to IL-18.
- Studies stated IL-18 is another cytokine involved in bystander activation of memory CD8+ T cells that mainly act in synergy with IL-12, which result in augmentation of the differentiation of memory T cells into effector cells i.e. induces IFN-γ production and cell proliferation.
- Whiteside et al. (2018) associated toll-like receptors (TLRs), such as TLR2, with TCR-independent activation of CD8+ T cells upon bacterial infection too.
ii. Bystander activation of CD4+ T cells
- In 2010, Onur Boyman stated the process of bystander activation of memory CD4+ T cells is less efficient, possibly due to reduced CD122 (IL2RB or IL15RB) expression.
- Lee et al. (2020) found memory and effector CD4+ T cells display increased sensitivity to TCR-independent activation. Furthermore, they found IL-1β synergies with IL-12 and IL-23 to induce memory CD4+ T cells and stimulate Th17 response.
- IL-12, IL-18 and Il-27 are found to stimulate effector and memory CD4+ T cells to release cytokines. IL-2 was regarded as an activation stimulator of CD4+ T cells that can supersede TCR stimulation even in naïve cells.
- In addition, TLR2 was observed to exist on memory CD4+ T cells, which react to their agonist by IFNγ production, even without TCR stimulation.
iii. Role in pathogenicity
- Despite its benefits, studies identified bystander activation may result in a deleterious outcome, particularly in chronic infections and autoimmune diseases.
- Lee et al. (2022) found non-HBV-specific CD8+ T cell infiltration into the tissue lead to liver injury during chronic Hepatitis B virus infection, as well as during the acute Hepatitis A virus infection.
- Furthermore, they discovered activated virus unrelated CD4+ T cells resulted in ocular lesions in Herpes Simplex Virus infections.
- Whiteside et al. (2018) found a correlation between increased IL-15 expression and subsequent excessive NKG2D expression, and the manifestation of a number of autoimmune disorders, such as, type I diabetes, multiple sclerosis and inflammatory bowel diseases (e.g. Crohn's disease and coeliac disease).
- In addition, TLR2 expression was elevated in joints, cartilage and bones affected by rheumatoid arthritis, and its ligand, peptidoglycan, was identified in their synovial fluid.
b. B-Cells
- Known as B lymphocytes, B cells serve a role in the humoral immunity aspect of the adaptive immune system. They generate molecules called antibodies that are either secreted or inserted into the plasma membrane where they function as a component of B-cell receptors.
How do B cells develop?
- B cells first develop from haematopoietic stem cells (HSCs) that originate from bone marrow.
- HSCs initially differentiate into multipotent progrenitor (MPP) cells, then differentiaite into lymphoid progenitor (CLP) cells.
- From this stage, their development into B cells occurs in a number of steps, each indicated by various gene expression patterns and immunoglobulin H chain and L chain gene loci arrangements, with the latter caused by B cells undergoing V(D)J recombination as they develop.
- Like T cells, B cells experience 2 types of selection during their development in the bone marrow, both involving B cell receptors (BCR) on the cell surface.
- Positive selection: If both the pre-BCR and the BCR don't bind to antigen, they don't receive the signals to proceed to the next phase, thus its development will terminate.
- Negative selection: If BCR binds to self-antigen too strongly, then that B cell experiences 1 of 4 fates: clonal deletion, receptor editing, anergy, or ignorance (i.e. ignores signal to cease development and continues developing). This phase results in central tolerance, which only allows the survival of mature B cells that don't bind self-antigens present in the bone marrow.
- Immature cells migrate from the bone marrow into the spleen as transitional B cells, which occurs in 2 transitional stages: T1 and T2.
- B-cells migrating to and after entering the spleen are labelled T1. Within the spleen, T1 B-Cells transition to T2 B-Cells.
- T2 B cells differentiate into either follicular (FO) B cells or marginal zone (MZ) B cells depending on the signals they receive via BCR and other receptors.
- When its differentiation is complete, they become mature B Cells, or naive B cells.
How are B-cells activated?
- After B cells mature in the bone marrow, they migrate to the secondary lymphoid organs (SLOs), such as the spleen and lymph nodes via the bloodstream, which receive a constant input of antigen through circulating lymph.
- B cell initiate its activation at the SLO by binding to an antigen via its BCR.
- It is suggested BCRs diffuse through the membrane to interact with Lck and CD45 equally in frequency, which yield a net equilibrium of phosphorylation and non-phosphorylation.
- Nutt et al. (2015) outlined FO B cells preferentially experience T cell-dependent activation, whereas MZ and B1 B cells preferentially experience T cell-independent activation.
- Asokan et al. (2013) found B cell activation is augmented by CD21, which is a surface receptor as part of a complex of surface proteins CD19 and CD81 (all 3 are collectively known as the B cell coreceptor complex).
- Zabel & Weis (2001) found BCR initially binds to an antigen tagged with a fragment of the C3 complement protein, which triggers CD21 to bind that C3 fragment and co-ligate with the bound BCR. Subsequently, this results in signal transduction by CD19 and CD81 that decreases the activation threshold of the B cell.
i. T cell-dependent activation
- T cell-dependent (TD) antigens activate B cells with the aid of T Cells because they aren't capable to stimulate a humoral response in organisms lacking T cells.
- B cell take several days to respond to these antigens, even though the antibodies have higher affinity and a higher functional versatility than T cell-independent activation.
- When a BCR binds a TD antigen, it is absorbed by B cells through receptor-mediated endocytosis, then degraded, before being presented to T cells as peptide fragments in complex with MHC-II molecules on the cell membrane.
- T helper (Th) cells, usually follicular Th (TFH) cells, recognise and bind these MHC-II-peptide complexes via their T cell receptor (TCR).
- T cells subsequently express CD40L surface protein, as well as release cytokines such as IL-4 and IL-21.
- CD40L is a co-stimulatory factor that binds the B cell surface receptor CD40, which promotes B cell proliferation, immunoglobulin class switching, and somatic hypermutation as well as sustains T cell growth and differentiation, hence activates B cells.
- When T cell-derived cytokines bind to B cell cytokine receptors, it triggers B cell proliferation, immunoglobulin class switching, and somatic hypermutation as well as guide differentiation. When B cells receive the necessary signals, they are now activated.
- Activated B cells engage in a two-step differentiation process that results in both temporary plasmablasts for immediate protection and lasting plasma cells and memory B cells for persistent protection.
- The first step is the extrafollicular response, which occurs outside lymphoid follicles but in the SLO nonetheless. This involves activated B cells proliferating, experiencing immunoglobulin class switching, and differentiating into plasmablasts that generate early, weak antibodies that are primarily IgM.
- The second step involves activated B cells moving to a lymphoid follicle where it creates a germinal centre (GC), a specialised microenvironment where B cells experience considerable proliferation, immunoglobulin class switching, and affinity maturation governed by somatic hypermutation.
- These steps produce both high-affinity memory B cells and lasting plasma cells. The plasma cells produce an abundant amount of antibody and either remain within the SLO, or migrate to bone marrow.
ii. T cell-independent activation
- T cell-independent (TI) antigens activate B cells without T cell assistance, which includes foreign polysaccharides and unmethylated CpG DNA. They can trigger a humoral response in organisms lacking T cells.
- B cells respond rapidly to these antigens, but its generated antibodies are lower affinity and has less functional versatility than those generated from T cell-dependent activation.
- B cells activated by TI antigens require additional signals to complete its activation process. They are provided either by recognition and interaction with a common microbial constituent to toll-like receptors (TLRs) or by vast crosslinking of BCRs to repeated epitopes on a bacterial cell.
- B cells activated by TI antigens proceed to proliferate outside lymphoid follicles but still in SLOs, probably experience immunoglobulin class switching, and differentiate into short-lived plasmablasts that generate early, weak (primarily) IgM antibodies, as well as some populations of long-lived plasma cells.
iii. Memory B cell activation
- Memory B cells activate when they identify and bind their target antigen, which is shared by their parent B cell. Memory B cells can either be activated independently of T cells, such as those specific to certain viruses, or with T cell assistance.
- When memory B cells bind to antigen, it engulfs the antigen via receptor-mediated endocytosis, which degrades it. Then it presents an antigen fragment to T cells as small peptides in complex with MHC-II molecules on the cell membrane.
- Memory follicular T helper (TFH) cells subsequently bind these MHC-II-peptide complexes through their TCR.
- After TCR-MHC-II-peptide binding and the communication of other signals from the memory TFH cell, the memory B cell then activates and differentiates either into plasmablasts and plasma cells via an extrafollicular response or transitions to a germinal centre where they produce plasma cells and more memory B cells.
- However, more research is required to understand whether the memory B cells experience affinity maturation within these secondary GCs.
Describe the B-Cell Receptor
- The B cell receptor (BCR) is a transmembrane protein expressed on the surface of a B cell. It consists of a membrane-bound immunoglobulin molecule and a signal transduction moiety.
- The immunoglobulin molecule comprises of a type 1 transmembrane receptor protein, usually situated on the outer surface of these lymphocyte cells.
- BCR regulate B cell activation through biochemical signalling and physical acquisition of antigens from the immune synapses. Their main function is to collect and capture antigens by their biochemical modules for receptor clustering, cell spreading, creation of pulling forces, and receptor transport. This subsequently leads to endocytosis and antigen presentation.
- Merlo & Mandik-Nayak (2013) observed the mechanical activity of B cells follow a pattern of negative and positive feedback that regulate the amount of eliminated antigen by directly influencing the dynamic of BCR-antigen bonds.
- Dal Porto et al. (2014) found grouping and spreading increase the interaction between BCR and antigen, which increased sensitivity and amplification. On the other hand, pulling forces uncouple the antigen from the BCR, which indicates the quality of antigen binding being tested.
How is the structure of BCR developed?
 |
| B cell receptor general structure includes a membrane-bound immunoglobulin molecule and a signal transduction region. Disulfide bridges link the immunoglobulin isotype and the signal transduction region. |
- The first checkpoint in B cell development is the generation of a functional pre-BCR, consisting of 2 surrogate light chains and 2 immunoglobulin heavy chains, which are typically associated with Ig-α (or CD79A) and Ig-β (or CD79B) signalling molecules.
The BCR consists of 2 components:
(1) A membrane-bound immunoglobulin molecule of 1 isotype (IgD, IgM, IgA, IgG, or IgE). These are identical to a monomeric version of their secreted forms, except in the presence of integral membrane domain.
(2) The signal transduction moiety is a heterodimer called Ig-α/Ig-β (CD79), which is bound together by disulfide bridges. Each component of the dimer spans the plasma membrane and a cytoplasmic tail that exhibits an immunoreceptor tyrosine-based activation motif (ITAM).
- The BCR complex contains an antigen-binding subunit called the membrane immunogloblulin (mlg), which consists of 2 immunoglobulin light chains (IgLs) and 2 immunoglobulin heavy chains (IgHs) as well as 2 heterodimer subunits known as Ig-α and Ig-β.
- A combination of mIgM molecules, Ig-α and Ig-β is required for transport of mIgM molecules to the cell surface.
- Pier et al. (2005) found Pre-B cells that fail to generate Ig molecules usually carry both Ig-α and Ig-β to the cell surface.
- Studies identified a component within the BCR that recognises antigens consists of 3 distinct genetic regions, known as V, D, and J. These regions undergo recombination and splicing at the genetic level in a combinatorial process that is unique to the immune system.
- Studies reported several genes encode of these regions in the genome and combine in numerous ways to generate a variety of receptor molecules. It allows the body to prepare for the immense possibility of antigens that may invade the body.
- During the first stages of B cell development, heavy chain rearrangement of the BCR occurs. Subsequently this is followed by recombination of short JH (joining) and DH (diversity) regions in early pro-B cells that is supported by the enzymes RAG2 and RAG1.
- Hoehn et al. (2016) labelled the cell after D and J regions recombination as a "late pro-B" cell and stated the short DJ region can then be recombined with a longer segment of the VH gene.
- BCRs contain unique binding sites that depend on the complementarity of the epitope surface and the receptor surface, which usually occurs by non-covalent forces. If mature B cells contain a BCR that doesn't have that specific antigen present, then it only survives in the peripheral circulation for a limited time. Janeway et al. (2015) stated B cells will undergo apoptosis if they don't interact with any antigen within that timeframe. Alberts (2014) explained this allows optimal circulate of B-lymphocytes within the peripheral circulation.
- Brenzski & Monroe (2010) found the BCR structure specific to antigens are virtually similar to the generated antibodies. Nevertheless, there is structural dissimilarity in the C-terminal area of the heavy chains, which contains a short hydrophobic section that spans across the lipid bilayer of the membrane.
Describe the signalling pathways of BCR
- When the mIg subunits of the BCR binds a specific antigen, it triggers the BCR signalling pathway, similar for all receptors of the non-catalytic tyrosine-phosphorylated receptor family.
- This results in phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) in the associated Igα/Igβ heterodimer subunits by the tyrosine kinases of the Src family, including Blk, Lyn, and Fyn.
- A number of models have been hypothesised regarding the mechanisms behind phosphorylation induced by BCR-antigen binding, including conformational change of the receptor and aggregation of multiple receptors upon antigen binding.
- It is thought tyrosine kinase Syk binds to and is activated by phosphorylated ITAMs, which subsequent;y phosphorylates scaffold protein BLNK on multiple sites.
- A 2011 Janeway's immunobiology journal article suggested downstream signalling molecules are recruited to BLNK after phosphorylation, which activates them and signal transduction to the interior.
(a) IKK/NF-κB Transcription Factor Pathway
- After BCR recognises an antigen, CD79 and other proteins, and microsignalosomes proceed to activate PLC-γ before it integrates into the c-SMAC.
- c-SMAC cleaves PIP2 into IP3 and DAG (diacylglycerol).
- IP3 functions as a second messenger to significantly increase ionic calcium inside the cytosol (via secretion from the endoplasmic reticulum or influx from the extracellular environment via ion channels). This activates PKCβ from the calcium and DAG.
- PKCβ phosphorylates (either directly or indirectly) the NF-κB signalling complex protein CARMA1 (the complex itself comprising CARMA1, BCL10, and MALT1).
- This recruits and assembles IKK (IkB kinase) and TAK1 by a number of ubiquitylation enzymes also associated with the CARMA1/BCL10/MALT1 complex.
- MALT1 is a caspase-like protein that cleaves A20, an inhibitory protein of NF-κB signaling (that functions by ubiquitylating NF-κB's ubiquitylation substrates).
- TAK1 phosphorylates IKK trimer after its recruitment to the signalling complex by its associated ubiquitylation enzymes.
- IKK phosphorylates an inhibitor of and bound to NF-κB called IkB, which marks it for proteolytic degradation (hence destruction), ultimately releases cytosolic NF-κB.
- NF-κB migrates to the nucleus to bind to DNA at particular response elements, which recruits transcription molecules to initiate the transcription process.
(b) Ligand binding to the BCR resulting in phosphorylation of BCAP protein
- This results in binding and activation of a number of proteins with phosphotyrosine-binding SH2 domains, such as PI3K. Activated PI3K results in PIP2 phosphorylation, which creates PIP3.
- Proteins with PH (Pleckstrin homology) domains bind to PIP3 to be activated, which include proteins of the FoxO family. This triggers stimulate cell cycle progression, and protein kinase D, which enhances glucose metabolism.
- Another activated protein is Bam32, which recruits and activates small GTPases such as Rac1 and Cdc42. This subsequently results in cytoskeletal changes associated with BCR activation by modifying actin polymerisation.
What are the different types of B-cells?
a. Pro-B Cells
- When lymphoid progenitor cells receive the appropriate signals from bone marrow stromal cells, it triggers the development of B cells. Cytokines trigger TdT and recombinase (RAG-1 and RAG-2) synthesis in CD34+ lymphoid progenitors.
- These cells experience D-J combination on the H chain chromosome to become early pro B-cells, which subsequently start to express CD45 (B220) and Class II MHC. The late pro-B cell stage is concluded when a V segment combines with the D-JH segments.
b. Pre-B Cell
- When Pro-B cells express membrane m-chains with surrogate light chains in the pre-B receptor, they transition into pre-B cells. Pre-B cell receptors consist of surrogate L chains and signal transduction molecules Iga-Igb.
- Since the cytoplasmic tails of Ig heavy chains are too short to enter the cytoplasm and send out an antigen-binding signal, Iga-Igb signal transduction molecules contain ITAMs (Immunoreceptor Tyrosine Activation Motifs) that phosphorylate in response to antigen-BCR binding.
- Phosphorylated ITAMs trigger a cytoplasmic signalling cascade that stimulates the pre-B cell to stop recombination of H chain and start proliferation into a clone of B cells that generate the same m-chain.
- This results in the production of large pre-B cells.
c. Naive B Cell
- After proliferation, small pre-B cells experience V-J combining on 1 L-chain chromosome. When a L-chain is fully synthesised, it is expressed with m-chain on the membrane of an immature B cell.
- Since immature B cells are highly sensitive to antigen binding, they undergo apoptosis if they bind self-antigen in the bone marrow.
- B cells that don't bind self-antigen will express d-chain and both IgD and IgM on the membrane upon departure from the marrow. At this point, they become mature naive B cells.
d. B1 Cells
- B1 cells are a sub-class of B cell lymphocytes that play an important role in the humoral immune response. They tend to be found in peripheral sites, but less commonly found in the blood. They are initially produced in the foetus before self-renew itself in the periphery.
- They exhibit the same functions as other B cells such as generating antibodies antigen antigens and serving as antigen-presenting cells, but they don't have any memory capabilities.
What are the different types of B1 cells?
- Griffin et al. (2011) found human B1 cells exhibit a marker profile of CD20+CD27+CD43+CD70- as well as CD5+/-.
- Cunningham et al. (2014) identified the main difference between B1 cells and other B cells include the variable expression of CD5, CD86, IgD and IgM.
- In mice, B1 cells are further subdivided into B1a (CD5+) and B1b (CD5−) subtypes. B1b cells are produced by precursors in the adult bone marrow, whereas B1a cells aren't. Tung et al. (2006) found B1a and B1b express different levels of CD138.
- B1b cells recognise more types of antigens (e.g. intracellular antigens) than B1a cells, particularly a range of protective antigens called 'conserved factors'.
- Researchers suggested an additional role of B1a cells being the production of natural serum antibody. In contrast, B1b cells was identified to generate dynamic T cell independent (TI) antibodies and provide long-term protection after bacterial infection such as Borrelia hermsii and Streptococcus pneumoniae.
- B1a derived cells have a subset called innate response activator (IRA) B cells, which release GM-CSF and IL-3. Chousterman & Swirski (2015) found IRA B cells accumulate in the spleen in atherosclerosis, which results in extramedullary haematopoiesis and activating dendritic cell.
What is the role of B1 cells in the immune response?
- B1b cells tend to be involved in the antibody response during an infection or vaccination because they don't require an activation signal from a T helper cell to respond.
- B1 cells was found to express more IgM than IgG and its receptor have low affinities for numerous different antigens, a phenomenon known as polyspecificity. Polyspecific immunoglobulins tend to prefer other immunoglobulins, self antigens and common bacterial polysaccharides.
- There are higher populations of B1 cells in the peritoneal and pleural cavities than in the lymph nodes and spleen.
- Ghosn et al. (2008) found B1 cells express high levels of surface IgM (sIgM), CD11b, and low levels of surface IgD (sIgD), CD21, CD23, and the B cell isoform of CD45R (B220).
- Hayakawa et al. (1985) investigated B1 cells in adult mice models and discovered low levels of them in the spleen and secondary lymphoid tissues but are abundant in the pleural and peritoneal cavities.
- B1 cells are found to derived from precursors in the foetal liver and neonatal (but not adult) bone marrow, which comprises the earliest wave of mature peripheral B cells.
- Hayakawa et al. (1986) used sequence analysis to find antibodies secreted by B1 cells contain limited sets of V region genes and an elevated usage of λ light chains.
- However there is no evidence that B1 cell sequences undergo somatic hypermutation (SHM), nor contain many non-templated nucleotide (N) sequence insertions.
- Martin & Kearney (2001) observed B1 cell development depends on positive regulators of BCR signalling and the loss of negative regulators results in accumulation of B1 cells.
- Bendelac et al. (2001) suggested the self or foreign antigen plays a role in influencing the repertoire of the B1 cell compartment.
- Kantor & Herzenberg (1993) found B1 cells self-renew and spontaneously secrete IgM and IgG3 serum antibodies, which are polyreactive, self-reactive and able to bind to numerous common pathogen-associated carbohydrates.
- These natural serum antibodies are involved in the early stages of the immune response against bacteria and viruses but this requires complement fixating on them in order to effectively eliminate the antigen.
e. B2 Cell
(i) Follicular (FO) B-Cell
- Follicular (FO) B cells situate in primary and secondary lymphoid follicles (containing germinal centres) of secondary and tertiary lymphoid organs, including spleen and lymph nodes.
- FO B cell pathways in secondary lymphoid organs is thought to play an important role in antibody responses against proteins.
- A majority of mature B cells exiting the spleen become FO B cells, which express high levels of IgD, and CD23, but low levels of CD21 and IgM, and no CD1 or CD5.
- They assemble into the primary follicles of B cell zones concentrated around follicular dendritic cells in the white pulp of the spleen and the cortical areas of peripheral lymph nodes.
- Miller et al. (2002) used multiphoton-basede live imaging on lymph nodes to observe continuous movement of FO B cells within these follicular areas at velocities of ~6 µm per min.
- Bajenoff et al. (2006) suggested this movement along the processes of FDC serve as a guidance system for mature resting B cells in peripheral lymph nodes.
- More than 95% of the B cells in peripheral lymph nodes are FO B cells, which freely recirculate.
- Although the BCR repertoire of the FO B cell compartment appears under positive selection pressures during final maturation in the spleen, its diversity is significantly broader than B1 and MZ B cell compartments.
- McHeyzer-Williams LJ & McHeyzer-Williams MG (2005) stated FO B cells require CD40-CD40L-dependent TFH cell help in order to stimulate effective primary immune responses and antibody isotype switching, as well as generate high-affinity B cell memory.
(ii) Marginal Zone (MZ) B-Cell
https://onlinelibrary.wiley.com/doi/full/10.1111/sji.12920
- Marginal zone B cells (MZ B cells) are non-circulating mature B cells that separate anatomically into the marginal zone (MZ) of the spleen and other types of lymphoid tissue.
- MZ B cells situated in this region usually express low-affinity polyreactive B-cell receptors (BCR), high levels of IgM, Toll-like receptors (TLRs), CD21, CD1, CD9, CD27 with low to negligible levels of secreted-IgD, CD23, CD5, and CD11b, which are phenotypically distinct from follicular (FO) B cells and B1 cells.
- They are innate-like B cells specialised to assemble rapid T-independent, as well as T-dependent response against blood-borne pathogens. Appelgren et al. (2018) described MZ B cells as the main generators of IgM antibodies in humans.
How do MZ B cells develop and differentiate?
- The marginal zone of the spleen comprises of numerous subtypes of macrophages and dendritic cells intertwined with MZ B cells.
- MacLennan et al. (1985) stated MZ B cells don't fully develop until 2-3 weeks after birth in rodents and 1-2 years in humans.
- In humans, MZ B cells are found in the inner wall of the subcapsular sinus of lymph nodes, the epithelium of tonsillar crypts, and the sub-epithelial area of mucosa-associated lymphoid tissues including the sub-epithelial dome of intestinal Peyer's patches, as well as in peripheral blood.
- On the other hand, mice MZ B cells don't circulate in the peripheral blood and only limited to follicular shuttling.
Mice MZ B cells are identified as having:
-- High IgM levels
-- Low IgD levels
-- High CD21 levels
-- Low CD23 levels
-- High CD1d levels (MHC Class I-like molecule involved in the presentation of lipid molecules to NKT cells)
- In comparison, human MZ B cells express CD9 and CD27.
- Typical MZ B cells express polyreactive BCRs that bind to numerous microbial molecular patterns, as well as high levels of TLRs.
- Hardy (2008) identified MZ B cell development is dependent on the tyrosine kinase for Pyk-2 and NOTCH2 signalling, which result in proliferation.
How do MZ B cells activate and function?
- Richard Hardy (2008) found MZ B-cells are swiftly recruited early into the adaptive immune response in a T cell-independent manner.
- Balazs et al. (2002) stated MZ B-cells are ready in position for the first line of defence against systemic blood-borne antigens that infiltrate the circulation and become confined in the spleen.
- Turner & Mabbott (2017) discovered MZ B cells bind smaller blood-borne antigens, whereas dendritic cells, circulating granulocytes or MZ macrophages bind larger blood-borne antigens.
- It's known MZ B cells travel between the blood-filled marginal zone for antigen collection and the follicle for antigen delivery to follicular dendritic cells.
- Mice models demonstrated MZ B-cells shear flow via the LFA-1 integrin ligand ICAM-1 and interact or migrate down the flow via the VLA-4 integrin ligand VCAM-1.
- Tedford et al. (2017) found CXCR5/CXCL13 signalling is important for MZ B cells to enter the follicle, and sphingosine-1-phosphate signalling is essential for MZ B cells to exit from the follicle.
- It is hypothesised MZ B-cells respond to both T-dependent and T-independent antigens as well as microbial polysaccharide antigens of encapsulated bacteria such as Streptococcus pneumoniae, Haemophilus influenzae and Neisseria meningitidis.
- Palm et al. (2016) reported TLRs activate MZ B-cells after recognition of microbial molecular structures with the assistance of BCR.
- MZ B-cells offer a fast first line of defence against blood-borne pathogens and generate low-affinity antibodies of broad specificity prior to the induction of T-cell-dependent high-affinity antibody responses. Thus, Hendricks et al. (2018) suggested MZ B cells are essential for preventing sepsis.
- Lopes-Carvalho et al. (2005) found the activation threshold of MZ B cells is lower than the FO B cells, with an elevated tendency for plasma cell differentiation that contributes to the acceleration of the primary antibody response.
- Appelgren et al. (2018) recognised MZ B cells are the main generators of IgM antibodies in the human body.
- MZ B cells play an essential role in eliminating foreign pathogens and maintaining homeostasis via opsonisation of dead cells and cellular debris. Furthermore, MZ B cells play a role as antigen-presenting cells by activating CD4+ T cells more effectively than FO B cells because of their increased expression of MHC class II, CD80 and CD86 molecules.
- Turner et al. (2017) discovered deficiencies in MZ B cells increased the risk of pneumococcal infection, meningitis and insufficient antibody response to capsular polysaccharides.
How do MZ B cells become memory B cells?
- The immunoglobulin genes in Human MZ B cells in the spleen undergo somatic hypermutation, which implies these cells are produced through a germinal centre reaction to become memory B cells.
- Naive MZ B cells secrete low-affinity IgM antibodies, whereas memory MZ B cells express high-affinity Ig molecules.
- Aside from unswitched cells (IgM+), class-switched B cells are located in the human and rodent marginal zone (IgG+ and IgA+).
- Hendricks et al. (2018) found human MZ B-cells express CD27, which is a member of the TNF-receptor family.
Describe the role of MZ B cells in autoimmune diseases and tumours
- A majority of MZ BCRs are self-reactive, which may contribute to their expansion in some autoimmune diseases, such as arthritis, diabetes, and lupus.
- Nevertheless, eliminating self-antigens are an essential process that helps prevent the development of autoimmune diseases.
- Bron & Meuleman (2019) found MZ B cells are malignant in marginal zone lymphomas, which is a heterogeneous group of indolent lymphomas.
f. Plasmablast
Plasmablasts are a temporary, proliferating antibody-secreting cell that derive from B cell differentiation, as well as T cell-independent activation of B cells or the extrafollicular response from T cell-dependent activation of B cells. Nutt et al. (2015) found they are produced in the early stages of infection and its secreted antibodies have a weaker affinity towards their target antigen compared to plasma cells.
g. Plasma Cell (Effector B Cells)
- Also known as plasma B cells or effector B cells, plasma cells are white blood cells that originate in the lymphoid organs as B lymphocytes. Their main function is to release an abundant quantity of proteins called antibodies, as part of the immune response against specific antigens.
- The secreted antibodies migrate from the plasma cells to the site of the target antigen via the blood plasma and the lymphatic system, where they begin to neutralise or destroy the antigens.
Describe the structure of plasma cells
 |
| This is a micrograph of a plasmacytoma under H&E stain. It shows abundant (malignant) plasma cells with the occasional Mott cell, a plasma cell with intracytoplasmic Russell bodies (an eosinophilic uniformly staining membrane bound body which contains immunoglobulin). Other features of plasmacytomas (not apparent on the image) are: a prominent perinuclear hof (large Golgi bodies), and Dutcher bodies. Multiple myeloma (which is diagnosed using several clinical criteria) is, histologically, a plasmacytoma. |
- Under a light microscope, plasma cells are large lymphocytes with substantial basophilic cytoplasm, an abnormal nucleus with heterochromatin in a distinct cartwheel or clock face arrangement.
- Under electron microscopy, their cytoplasm contains a pale zone, as well as an extensive Golgi apparatus and centrioles. Plasma cells contain numerous rough endoplasmic reticulum and a Golgi apparatus that allows the production of immunoglobulins.
- Plasma cells also contain ribosomes, lysosomes, mitochondria, and the plasma membrane.
Surface antigens:
- Plasma cells express relatively few surface antigens such as CD138, CD78, and the Interleukin-6 receptor, as well as CD27 (a reliable biomarker).
- High levels of surface antigen CD138 (syndecan-1).
- High levels of CD319 (SLAMF7) expressed on regular human plasma cells as well as malignant plasma cells in multiple myeloma.
How do plasma cells develop?
- When B cells depart the bone marrow, they serve as antigen-presenting cell (APC) and absorb foreign antigens via receptor-mediated endocytosis.
- Antigenic peptides (fragments) are placed onto MHC-II molecules, and presented on its extracellular surface to CD4+ T cells (or Helper T cells). Th cells subsequently bind to the MHC II-antigen complex to activate the B cell.
- Activated B cells then differentiate into specialised B cells, which typically occurs in germinal centres of secondary lymphoid organs such as the spleen and lymph nodes.
- In the germinal centre, B cells may differentiate into memory B cells or plasmablasts (or immature plasma cells). Plasmablasts subsequently mature into plasma cells, which initiates the production of antibodies.
- Plasmablasts are considered the most immature plasma cells that release more antibodies than B cells, but less than plasma cells. Walport et al. (2010) found plasmablasts divide quite rapidly and can internalise antigens and present them to T cells. A plasmablast's fate can be either to undergo apoptosis or to irreversibly differentiate into a mature, fully differentiated plasma cell.
- Mature B cells require the transcription factors Blimp-1/PRDM1 and IRF4 to differentiate into plasma cells.
What are the functions of plasma cells?
- Walport et al. (2008) found plasma cells can't perform several functions of its precursors such as switching antibody classes, presenting antigens via MHC-II, and taking up antigen via surface immunoglobulin.
- Caligaris-Cappio & Ferrarini (1997) stated signals received from the T cell during differentiation influences the lifespan, production of class of antibodies, and the location the plasma cell migrates to.
- Since B cell differentiation via T cell-independent antigen stimulation can occur anywhere in the body, this yields temporary cells that release IgM antibodies.
The T cell-dependent processes are subdivided into two responses:
-- Primary response = T cells are present during the initial interaction by the B cell with the antigen. This yields temporary cells that situate in the extramedullary regions of lymph nodes.
-- Secondary response = It yields long-lasting plasma cells that secrete IgG and IgA, as well as travel to the bone marrow frequently.
e.g. If plasma cells matured where cytokine interferon-γ is present, it tends to produce IgG3 antibodies.
- Kierszenbaum (2002) found that although every B cell is specific to a single antigen, each B cell secretes numerous matching antibodies per second. In comparison, plasma cells only secretes a single of antibody in a single class of immunoglobulin.
-- Long-lived plasma cells (LLPC)
- After plasma cells undergo affinity maturation in germinal centres, they mature into 1 of 2 types of cells: short-lived plasma cells (SLPC) or long-lived plasma cells (LLPC).
- Radbruch et al. (2006) found LLPC typically situate in the bone marrow for an extended period of time and release antibodies that provides long-term protection. Another function of LLPC is maintenance of antibody production for a person's lifetime, which doesn't require antigen restimulation to generate antibodies.
- Halliley et al. (2015) identified the human LLPC population as CD19–CD38hiCD138+ cells.
- Manz & Radbruch (2002) stated the long-term survival of LLPC depends on a particular environment in the bone marrow, or a plasma cell survival niche.
- If LLPC leaves this survival niche, it leads to apoptosis. Nguyen et al. (2019) hypothesised the survival niche only supports a restricted number of LLPC, therefore the niche's environment aims to protect its LLPC cells as well as adapt to novel cells.
- Cassese et al. (2003) identified several molecules that promote the survival of LLPC, which include IL-5, IL-6, TNF-α, stromal cell-derived factor-1α, and signalling via CD44.
- A minority of LLPC situate in the gut-associated lymphoid tissue (GALT), where they secrete IgA antibodies to support mucosal immunity. Lemke et al. (2016) discovered plasma cells in the gut aren't guaranteed to be produced de novo from active B cells but LLPC do exist there nonetheless. This indicates a similar survival niche like in the bone marrow.
- Several studies reported a number of tissue specific niches in nasal-associated lymphoid tissues (NALT), human tonsillar lymphoid tissues and human mucosa or mucosa-associated lymphoid tissues (MALT) that promote the survival of LLPC.
- A number of experimental studies demonstrated the lack of antigen and B cells didn't influence the production of high-affinity antibodies by the LLPC. Furthermore, prolonged exhaustion of B cells (with anti-CD20 monoclonal antibody treatment that affects B cells but not PC) didn't influence antibody titres. These findings indicated the longevity of some plasma cells.
- Longmire et al. (1973) found LLPC in bone marrow are the principal source of circulating IgG in humans, which releases them at elevated levels independently of B cells.
- Studies by Mei et al. (2009) and Bohannon et al. (2016) found a number of plasma cells located in bone marrow produce IgG as well as IgM.
h. Memory B Cell
- A memory B cell (MBC) is a type of B lymphocyte that develops within germinal centres of secondary lymphoid organs, and plays an important role in the adaptive immune response.
What are the different subsets of memory B cells?
i. Germinal centre independent memory B cells
- This involves activated B cells differentiating into memory B cells prior to entry into the germinal centre. B cells with elevated interaction with T(FH) within the B cell follicle have an increased tendency to enter the germinal centre.
- Kurosaki et al. (2015) found B cells fated to develop into memory B cells independently from germinal centres undergo CD40 and cytokine signalling from T cells.
- Class switching can still occur before the memory B cell's interaction with the germinal centre, whereas somatic hypermutation only occurs after the memory B cell's interaction with the germinal centre.
- Researchers in 2017 implicated a positive benefit for the lack of somatic hypermutation. Decreased affinity maturation results in memory B cells that are less specialsied to a particular antigen, thus they have the capacity to recognise a diverse range of antigens.
ii. T-independent memory B cells
- They are a subset of B1 cells that typically reside in the peritoneal cavity.
- Kurosaki et al. (2015) found some B1 cells differentiate into memory B cells without communicating with a T cell upon reintroduction to antigen. They subsequently produce IgM antibodies in response to the infection.
iii. T-bet memory B cells
- They express a transcription factor called T-bet, which plays a role in class switching. Knox et al. (2019) suggested T-bet cells play an essential role in immune responses against intracellular bacterial and viral infections.
Describe the lifespan of memory B cells
- Seifert & Küppers (2016) estimated memory B cells can last for a few decades, which provides them the capacity to respond to numerous interactions with the same antigen.
- Suan et al. (2017) suggested a theory behind the memory B cell's longevity is the high expression of anti-apoptosis genes compared to other subsets of B cells.
- Moreover, it isn't necessary for memory B cells to continually interact with the antigen or T cells in order to achieve long-term survival.
- A experimental study by Anderson et al. (2006) estimated the half-life of memory B cells to be between 8 and 10 weeks. Furthermore, it was demonstrated the concentration of memory B cells remained constant for a period of approximately 8-20 weeks after the immunisation.
- Jones et al. (2015) used a mouse model to estimate the lifespan of memory B cells is at least 9 times greater than the lifespan of a follicular naïve B cell.
Describe the development and activation mechanisms of Breg cells
i. T cell dependent mechanisms
- Seifert & Küppers (2016) stated the T-cell dependent development pathway involves naïve follicular B cells being activated by antigen presenting follicular B helper T cells (TFH) during the initial infection, or primary immune response.
- Garside et al. (1998) found naïve B cells circulate through follicles in secondary lymphoid organs, such as spleen and lymph nodes, where they can be activated by a drifting foreign peptide entering through the lymph or by antigen presented by antigen presenting cells (APCs) such as dendritic cells (DCs).
- Moreover, B cells can be activated by binding foreign antigen in the periphery where they subsequently migrate into the secondary lymphoid organs. When the B cell binds to the peptide, it transducts a signal that triggers migration of the cells to the follicle's edge that borders the T cell area.
- When foreign peptides are consumed by the B cells, they disintegrate, and its fragment becomes expressed on MHC-II.
- A majority of B cells within the secondary lymphoid organs then enter B-cell follicles where a germinal centre develops. Suan, Sundling & Brink (2017) found a majority of those B cells will eventually differentiate into plasma cells or memory B cells within the germinal centre.
- The TFHs expressing the T cell receptors (TCRs) associated with the peptide at the border of the B cell follicle and T-cell zone tend to interact with the MHC-II ligand.
- Taylor et al. (2012) explained T cells subsequently express the CD40 ligand and initiate the release of cytokines. This stimulates the B cell proliferation and class switch recombination of the B cell's genes that alters its immunoglobulin type. This allows memory B cells to release a variety of antibodies in future immune responses.
- Depending on the expressed transcription factors, B cells subsequently either differentiate into plasma cells, germinal centre B cells, or memory B cells.
- Taylor et al. (2012) stated the activated B cells expressing the transcription factor Bcl-6 tend to enter B-cell follicles and experience germinal centre reactions.
- Seifert & Küppers (2016) stated B cells proliferate inside the germinal centre, which is followed by mutation of the BCR's genetic coding region, a process known as somatic hypermutation.
- These mutations either increase or decrease the affinity of the surface receptor for a specific antigen, a process known as 'affinity maturation'.
- Allman et al. (2019) reported BCRs expressed on the surface of B cells after somatic hypermutation are tested within the germinal centre for their affinity to the current antigen.
- Victora & Nussenzweig (2010) discovered BCRs with increased affinity after somatic hypermutation tend to receive survival signals by interacting with their cognate TFH cells.
- On the other hand, Suan et al. (2017) found BCRs with decreased affinity for antigen or B cells with increased auto-reactivity tend to be negatively selected and ultimately die through apoptosis.
- Weisel & Shlomchik (2017) found newly differentiated memory B cells migrate to the periphery where they will likely interact with antigen in the event of a future infection, i.e. Peyer's patch.
- Kurosaki et al. (2015) suggested there is a degree of randomness in the differentiation process into memory B cells.
- Shinnakasu & Kurosaki (2017) hypothesised the involvement of the transcription factor NF-κB and the cytokine IL-24 in the process of differentiation into memory B cells.
- Another theory suggested B cells with relatively lower affinity for antigen will differentiate into memory B cells, and B cells with relatively higher affinity for antigen will differentiate into plasma cells.
ii. T cell independent mechanisms
Some IgM+ memory B cells can be produced independently of the germinal centres, thus they don't undergo somatic hypermutations nor class switch recombination. However, not much is known about the mechanisms of such pathways.
Describe the primary and secondary responses of memory B cells
i. Primary response
- Suan et al. (2017) stated B cells differentiate into plasma cells upon infection with a pathogen, which result in the release of the first wave of antibodies in order to eliminate the infection.
- Gatto & Brink (2010) estimated a small proportion of B cells expressing BCRs cognate to the antigen tend to differentiate into memory B cells that survive for longer period of time in the body.
- In addition, memory B cells maintain their BCR expression, meaning they are capable of responding rapidly upon secondary exposure.
ii. Secondary response and memory
- Memory B cells specific to the antigen, as well as similar antigens respond in a secondary response. When memory B cells re-interact with the same specific antigen, they would proliferate and differentiate into plasma cells in order to respond to and eliminate the antigen.
- Memory B cells that don't differentiate into plasma cells may re-enter the germinal centres to experience additional class switching or somatic hypermutation for additional affinity maturation.
- Kurosaki et al. (2015) discovered differentiation of memory B cells into plasma cells took significantly less time to complete than differentiation by naïve B cells, which meant a more efficient secondary immune response by memory B cells.
- Seifert & Küppers (2016) asserted the efficiency and generation of the memory B cell response is the foundation for vaccines and booster shots.
- Zuccarino-Catania et al. (2013) discovered several surface proteins, such as CD80, PD-L2 and CD73, expressed only on the memory B cells, which play a role in the proliferation of these cells in multiple phenotypic subsets.
- Furthermore, memory B cells expressing CD80, PD-L2 and CD73 tend to transform into plasma cells. Conversely, memory B cells lacking those markers tend to form germinal centre cells.
- IgM+ memory B cells lack the expression of CD80 or CD73, whereas IgG+ express them. Chong et al. (2018) found IgG+ memory B cells tend to differentiate into antibody-secreting cells.
What are the markers of memory B cells?
- Memory B cells usually express a distinct surface marker CD27, however a few subsets don't express CD27. Weisel & Shlomchik (2017) suggested memory B cells lacking CD27 associate with exhausted B cells or specific autoimmune conditions such as HIV, lupus, or rheumatoid arthritis.
-- IgD = Memory B cells expressing only IgD are rare and tend to situate in the tonsils.
-- IgE = Memory B cells expressing IgE are scarce in healthy individuals. B cells often differentiate into plasma cells rather than memory B cells.
-- IgG = Memory B cells expressing IgG typically differentiate into plasma cells.
-- IgM = Memory B cells expressing IgM tend to situate in the tonsils, Peyer's patch, and lymph nodes. This subset has a tendency to proliferate and re-enter the germinal centre during a secondary immune response.
- The signals produced by TLR2 and Myd88 play a role in stimulating the production of specific-IgG1, anaphylactic-IgG1 and total-IgE antibodies. Komegae et al. (2013) reported the signal produced by TLR4 (stimulated by natterins) promotes the synthesis of the IgE antibody, which acts as an adjuvant.
- Another marker of B cells differentiating into memory B cells is the CCR6 receptor, which detects chemokines that supports the movement of B cells within the body.
- Suan et al. (2017) hypothesised memory B cells express CCR6 to be able to migrate out of the germinal centre and into the tissues to interact with the antigen.
- Memory B cells were found to express high levels of CCR6 receptor as well as demonstrate an elevated chemotactic response to the CCR6 ligand (CCL20) compared to naïve B cells.
- In mice models, double-knockout of CCR6 gene didn't have much effect on the primary humoral response and the maintenance of the memory B cells. However, memory B cells without CCR6 lack an effective secondary response upon re-exposure of the antigen.
- Elgueta et al. (2015) concluded CCR6 plays a crucial role in the recall of memory B cells to their cognate antigen as well as the appropriate anatomical positioning of memory B cells.
i. Regulatory B (Breg) Cell
- Regulatory B cells (Breg cells) are a small population of B cells that serve in immunomodulations and in suppression of immune responses.
- They were first discovered by Katz et al. in 1974 and found they suppress immune reaction independently of antibody production.
- Wolf et al. (1996) discovered an immunomodulation of experimental autoimmune encephalomyelitis (EAE) by B cells, which were verified by a Mizoguchi's et al. model of chronic colitis in 1997.
- A number of mouse models discovered Bregs in autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus (SLE).
How do Breg cells develop?
- Although Bregs can develop from different subsets of B cells, it is uncertain whether they derive from a specific progenitor or originate within conventional B cell subsets.
- Matsushita et al. (2008) found the markers of mouse Bregs were primarily CD5 and CD1d positive in a model of EAE or after exposition of Leishmania major.
- Evans et al. (2007) found mouse Bregs in a model of collagen-induced arthritis (CIA) were primarily CD21 and CD23 positive. Blair et al. (2010) found the markers of peripheral blood Breg cells are CD24 and CD38.
- Iwata et al. (2011) discovered peripheral blood Brefs were predominantly CD24 and CD27 positive after associating with anti-CD40 antibody and CpG bacterial DNA.
- van de Veen et al. (2013) discovered Breg cells were additionally CD25, CD71 and PD-L1-positive after interacting with CpG bacterial DNA and TLR9.
Describe the Breg cell's mechanisms of action
- Breg cells produces IL-10, a cytokine that inhibits inflammatory reactions moderated by T cells, particularly Th1 type immune reactions, which was demonstrated in models of EAE or contact hypersensitivity.
- Schaut et al. (2016) observed Breg cell subsets suppress Th1 action via IL-10 production during chronic infectious diseases such as visceral leishmaniasis.
- Researchers discovered Breg produces another anti-inflammatory cytokine called transforming growth factor (TGF-β) in mouse models of SLE and diabetes.
- Moreover, Breg was observed to express surface molecules, such as FasL, or PD-L1, that triggers apoptosis of target cells.
How are Breg cells activated?
- 2 signals are required to activate Breg cells, which are produced by (1) external pathogens and (2) endogenous signals generated by the action of body cells.
- Molecular structures attributable to pathogenic microorganisms recognise TLR receptors, which subsequently triggers a signal cascade at the latter stages of effector cytokine production.
- Rosser & Mauri (2015) reported the main endogenous signal is provided by the surface molecule CD40.
j. Transitional B Cell
- Transitional B cells exist at an the intermediate stage of B cell development between bone marrow immature cells and mature B cells in the spleen.
- They situate in the bone marrow, peripheral blood, and spleen, and a small proportion of immature B cells that survive the transitional phase become mature B cells in secondary lymphoid organs such as the spleen.
- The label "transitional B Cell" was first described in a 1995 mouse model involving developing B cells that are in the intermediate stage between immature bone marrow B lineage cells and fully mature naïve B cells in the peripheral blood and secondary lymphoid tissues.
- Wardman et al. (2003) hypothesised novel transitional cells that left the bone marrow undergo peripheral checks to suppress the generation of autoantibodies.
- Suryani et al. (2010) stated the transitional B cells surviving negative selection against autoreactivity will develop into naive B cells.
- Loder et al. (1999) suggested the transitional B cell compartment serves as an important negative selection checkpoint for autoreactive B cells, which results in a small proportion of immature B cells surviving the transition to the mature naive stage.
- Allman et al. (1999) found all transitional B cells demonstrated high levels of heat-stable antigen (HSA) relative to their mature counterparts and expressed the phenotypic surface markers AA4.
-- T1 and T2 Cells
In mice, there are 2 transitional B cell stages: T1 and T2.
- T1: This stage occurs during the B cell's migration from the bone marrow to its entry into the spleen.
- T2: This stage occurs within the spleen where immature B cells develop into mature B cells.
- In contrast, human transitional B cells are more homogenenous and express high levels of CD10, CD24, and CD38. They can also be located in the bone marrow, peripheral blood, and spleen.
- Cuss et al. (2008) found T1 B cells express the following surface marker profile IgMhiIgD−CD21−CD23−, whereas T2 B cells retain high levels of surface IgM but are IgD+CD21+ and CD23+ as well.
- More research is required to understand the differences in functions between T1 B cells and T2 B cells.
j. Lymphoplasmacytoid cell
This cell is a combination of B lymphocyte and plasma cell morphological features that is hypothesised to be associated with a subtype of plasma cells. Ribourtout & Zandecki (2015) discovered the lymphoplasmacytoid cell in pre-malignant and malignant plasma cell dyscrasias related to the production of IgM monoclonal proteins. Examples of dyscrasias include IgM monoclonal gammopathy of undetermined significance and Waldenström's macroglobulinemia.
What is a germinal centre?
- Germinal centres (GCs) are transiently developed structured within B cell zones (follicles) in secondary lymphoid organs such as lymph nodes, ileal Peyer's patches, and the spleen.
- It is the site of activation, proliferation and differentiation of mature B cells, as well as somatic hypermutation of the antibody genes during a typical immune response.
- B cells experience rapid and mutative cellular division in the germinal centre's dark zone, where they are centroblasts.
- After proliferation, B cells then migrate to the light zone to become centrocytes, where they undergo the selection process by follicular helper T (TFH) cells in the presence of follicular dendritic cells (FDCs).
- Germinal centres serve as the main generators of affinity matured B cells specialised in releasing antibodies that effectively recognize antigen (e.g. infectious agents), as wells as the produces of long-lived plasma cells and durable memory B cells.
Describe the process within GCs
 |
| Histologic comparison of cell types in a germinal centre, H&E stain: - Centrocytes = Small to medium size with angulated, elongated, cleaved, or twisted nuclei. - Centroblasts = Larger cells containing vesicular nuclei with 1-3 basophilic nucleoli apposing the nuclear membrane. - Follicular dendritic cells = Round nuclei, centrally located nucleoli, bland and dispersed chromatin, and flattening of adjacent nuclear membrane. |
- In the lymph nodes, mature peripheral B cells known as follicular B cells receive antigen from follicular dendritic cells (FDCs).
- They subsequently present it to cognate CD4+ TFH cells at the border separating the interfollicular T cell area and B cell zone (also known as lymphoid follicles).
- After a few cycles of cellular division, B cells then undergo somatic hypermutation that mutates the antibody-encoding DNA, which generates a diverse range of clones in the germinal centre.
- Somatic hypermutation involves pseudo-random substitutions biased towards genetic regions encoding the antigen recognition surface of the antibodies. This step highlights the importance of affinity maturation, whereby greater affinity antibodies are generated and selected for after antigen recognition.
- When centroblasts (maturing B cells) receive an unidentified stimulus, they migrate from the dark zone to the light zone. At that stage, they begin to express their modified BCRs on the surface to become centrocytes.
- At this stage, the centrocytes are in a state of activated apoptosis and compete for survival signals provided by FDCs and TFH cells. This process is known as germinal centre selection, which depends on the affinity of their surface antibody to the antigen.
- B cells with the most successful mutations yielding a higher affinity surface antibody towards antigen will gain a survival advantage over lower affinity B cell clones and those that have acquired deleterious mutations.
- Centroblasts then re-enter the dark zone to provide an opportunity for otherwise non-selected B cell mutants to acquite more mutations in order to improve affinity towards antigen. When they interact with T cells, this helps prevent the production of autoreactive germinal centre B cells.
- During the centroblast-centrocyte cycling stage, mature B cells acquire the last differentiation signal to leave the germinal centre as an antibody-producing plasma cell or a memory B cell ready for reactivation.
- Selected B cells may restart the entire cycle of mutative centroblast division and centrocyte selection in order to continuously improve its recognition of antigens over time.
c. Natural Killer Cells
- NK cells play a major role in the innate immune response by protecting the host from tumours and virally infected cells.
- They regulate the functions of other immune cells, such as macrophages and T cells, and distinguish infected cells and tumours from uninfected and regular cells by perceiving the changes of a surface molecule called MHC (major histocompatibility complex) class I.
- When NK cells interact with a family of cytokines known as interferons, they activate to release cytotoxic granules in order to disintegrate the infected cells.
- They are labelled natural killer cells because they don't require prior activation in order to eliminate cells lacking MHC class I.
For more detail on NK Cells, see the first part of "Are we immune to everything?"
d. X cell (Dual expressor lymphocyte)
- Ahmed et al. (2019) hypothesised an X lymphocyte expressing both a B-cell receptor and T-cell receptor associates with type 1 diabetes.
- There is a debate whether such a lymphocyte actually exists. Thus, more research is required to understand the nature and properties of X cells (i.e. dual expressers).
ii. Antigen Presentation
- Antigen presentation is a crucial immune process to trigger T cell immune response. Since T cell receptors (TCRs) only recognise fragmented antigens displayed on cell surfaces, antigen processing has to occur prior to the antigen fragment (bound to the major histocompatibility complex (MHC)) being transported to the cell surface.
- In the event of a viral or bacterial infection, the APC presents an endogenous or exogenous peptide fragment originated from the antigen by MHC molecules.
There are 2 types of MHC molecules:
-- MHC Class 1 (MHC-I) = Binds peptides from the cell cytosol
-- MHC Class 2 (MHC-II) = Binds peptides produced in the endocytic vesicles after internalisation
(A) Class I: Presentation of intracellular antigens
- When cytotoxic (CD8+) T cells encounter a foreign pathogen such as a virus, intracellular bacteria or a transformed tumour cell, they trigger mechanisms to disintegrate the harmful agent.
- Antigens generated endogenously within nucleated APCs bind to MHC-I molecules and are presented on the cell surface.
- This pathway allows the immune system to identify transformed or infected cells that express peptides from mutated or foreign proteins.
- In the presentation stage, cytosolic proteases in the proteasome fragment the antigenic proteins into small peptides.
- Those peptides are transported to the endoplasmic reticulum (ER) via heat shock proteins and the transporter associated with antigen processing (TAP) responsible for translocating the cytosolic peptides into the ER lumen in an ATP-dependent transport mechanism.
- A number of ER chaperones are involved in the assembly of MHC-I, which include calnexin, calreticulin, Erp57, protein disulfide isomerase (PDI), and tapasin.
- TAP, tapasin, MHS Class 1, ERp57 and calreticulin are components of the peptide-loading complex (PLC).
- Peptides are subsequently loaded onto MHC-I peptide binding groove between 2 α-helices at the bottom of α1 and α2 domains of the MHC class I molecule.
- Peptide-MHC-I complexes (pMHC-I) subsequently release from tapasin before they depart the ER to be transported to the cell surface by exocytic vesicles.
- pMHC-I complexes of APCs activate naïve CD8+ T cells in order for them to directly eliminate infected or transformed cells.
- At this stage, antigen can be presented directly or indirectly from virus-infected and non-infected cells.
- In the case of cross-presentation, MHC-I molecules expressed by a number of APCs such as plasmacytoid dendritic cells can present extracellular antigens, usually presented by MHC-II molecules.
- Since APCs are uninfected, this process stimulates local antiviral and anti-tumour immune responses immediately without trafficking the APCs in the local lymph nodes.
(B) Class II: Presentation of extracellular antigens
- Antigens originating from the extracellular space are enclosed into endocytic vesicles and then presented my MHC-II molecules on the surface of APC (e.g. dendritic cells, B cells or macrophages) to T-helper cells expressing CD4 molecule.
- Stern & Santambrogio (2016) found APCs typically internalise exogenous antigens by endocytosis, as well as chaperone-mediated autophagy, endosomal microautophagy, macroautophagy, or pinocytosis.
- In endocytosis, internalised antigens are enclosed in vesicles called endosomes. This antigen presentation pathway follows this order: (1) early endosomes (2) late endosomes (endolysosomes), and (3) lysosomes.
- Inside the lysosome, the antigen is hydrolysed by lysosome-associated enzymes e.g. acid-dependent hydrolases, glycosidases, proteases, lipases.
- MHC-II molecules are transported from the ER to the MHC-II loading compartment along with the protein invariant chain (Ii, CD74).
- A non-classical MHC-II molecule (HLA-DO and HLA-DM) then catalyses the exchange of a segment of CD74 (CLIP peptide) with the peptide antigen.
- Sinha & Bhattacharya stated peptide-MHC-II complexes (pMHC-II) are transported to the plasma membrane and the processed antigen is presented to the helper T cells in the lymph nodes.
- Flores-Romo (2011) found APCs mature via chemotactic signals during its migration to lymphoid tissues, where they lose the capacity to phagocytise but increase its capacity to communicate with T-cells by antigen-presentation.
- APCs require pMHC-II and costimulatory signals to fully activate naïve T helper cells.
- Another pathway of endogenous antigen processing and presentation over MHC-II molecules occurs in medullary thymic epithelial cells (mTEC) via autophagy.
- The action of AIRE and self-digestion of the expressed molecules presented on both MHC-I and MHC-II molecules results in random gene expression of the whole genome.
(C) Presentation of native intact antigens to B cells
- This process involves BCRs binding to intact native and undigested antigens, instead of to a peptide's linear sequence already digested into fragments and presented by MHC molecules.
- In lymph nodes, follicular dendritic cells present larger complexes of intact antigens to B cells in the form of immune complexes.
- Harwood & Batista (2010) suggested a number of APCs expressing reduced lvels of lysosomal enzymes are less likely to digest the antigen they have captured prior to its presentation to B cells.
What is major histocompatibility complex (MHC)?
- The major histocompatibility complex (MHC) is a locus on vertebrate DNA that contains a group of closely linked polymorphic genes coding for cell surface proteins associated with the adaptive immune system, known as MHC molecules.
- MHC was first described by British immunologist Peter Gorer in 1936.
- In 1941, Clarence Little first recognised MHC genes in inbred mice strains by transplanting tumours across different strains and observed their rejection according to strains of host vs. donor.
- In 1951, Snell & Higgins selectively bred 2 mouse strains, discovered a new strain nearly identical to one of the progenitor strains, but contrasting in tissue compatibility upon transplantation. This lead to the discovery of the MHC locus.
- In 1963, Jean Dausset demonstrated the existence of MHC genes in humans and discovered the first human leukocyte antigen, now labelled the HLA-A2 protein.
- In the late 1970s, Baruj Benacerraf demonstrated polymorphic mHC genes not only determine an individual's distinctive constitution of antigens but also regulate the interaction among the various cells of the immune system.
- Benacerraf, Snell & Dausset were awarded the 1980 Nobel Prize in Physiology or Medicine for their discoveries concerning “genetically determined structures on the cell surface that regulate immunological reactions”.
- In 1999, a consortium of sequencing centres from the UK, USA and Japan in Nature published the first fully sequenced and annotated MHC for humans.
- Belov et al. (2006) discovered the grey short-tailed opossum (Monodelphis domestica) contained MHC that spans 3.95 Mb, yielding 114 genes, 87 of which were shared with humans.
- As of 2019, the IPD-MHC database contains 77 species for sequences of MHC.
Describe the genetics of MHC
- In spite of the differences in the amount of genes in MHC genome across different species, the overall assembly of the locus is quite consistent.
- In human MHC, it exists on chromosome 6, between the flanking genetic markers MOG and COL11A2 (from 6p22.1 to 6p21.3 about 29Mb to 33Mb on the hg38 assembly). It contains 224 genes spanning 3.6 megabase pairs (3 600 000 bases).
- The human MHC is also labelled the HLA (human leukocyte antigen) complex, which is similar to SLA (Swine leukocyte antigens), BoLA (Bovine leukocyte antigens), DLA for dogs, Histocompatibility system 2 (H-2) in mice, RT1 in rats, and B-locus in chickens.
- There are 2 subgroups in the MHC gene family: MHC class I, MHC class II, and MHC class III. There are 2 types of genes coding for the proteins MHC-I molecules and MHC-II molecules directly involved in antigen presentation.
- The IMGT database outlined these genes are highly polymorphic, 19031 alleles of class I HLA, and 7183 of class II HLA are deposited for human.
What are the different types of MHC?
a. Class I
- MHC-I molecules are located on the cell surface of all nucleated cells, as well as platelets in vertebrates.
- Kulski et al. (2002) discovered the MHC-I genes in the most recent common ancestor of all living jawed vertebrates. It is hypothesised this gene family experienced numerous divergent evolutionary paths after its emergence in jawed vertebrates.
- Azevedo et al. (2015) reported cases of trans-species polymorphisms in MHC-I genes, where a specific allele in an evolutionary related MHC-I gene exists in 2 species. It is thought to be caused by strong pathogen-mediated balancing selection by pathogens that infected both species.
- The birth-and-death evolution of MHC-I genes theory claims gene duplication events cause the genome to contain numerous copies of a gene that can subsequently undergo different evolutionary processes.
- Nei & Rooney (2005) stated these processes may lead to the death of one copy of the gene, or produce 2 new genes with divergent function.
- Hughes (1995) suggested human MHC-Ib loci (HLA-E, -F, and -G) and MHC-I pseudogenes may emerge from MHC-Ia loci (HLA-A, -B, and -C) in this birth-and-death process.
Describe the structure of MHC-I
- MHC-I molecules are heterodimers that contains 2 polypeptide chains, α and β2-microglobulin (B2M). The 2 chains are linked non-covalently with B2M and the α3 domain.
- Only the α chain is polymorphic and encoded by a HLA gene, whereas the B2M subunit is not polymorphic and encoding by the β2 microglobulin gene.
- The α3 domain spans the plasma membrane and interacts with the CD8 co-receptor of T-cells, which fixes the MHC-I molecule in one spot.
- The T cell receptor (TCR) on the surface of the cytotoxic T cell subsequently binds its α1-α2 heterodimer ligand, and checks the coupled peptide for antigenicity.
- Both the α1 and α2 domains then fold to create a groove for peptides to bind to. A majority of peptides MHC-I molecules bind to are about 8-10 amino acids long.
What are the genes and isotypes of MHC-I?
- Very polymorphic: HLA-A, HLA-B, HLA-C
- Less polymorphic: HLA-E, HLA-F, HLA-G, HLA-K (pseudogene), HLA-L (pseudogene)
What are the functions of MHC-I?
- MHC-I molecules bind peptides produced from degradation of cytosolic proteins by the proteasome. The MHC-I:peptide complex is subsequently inserted via endoplasmic reticulum into the external plasma membrane of the cell.
- Since the epitope peptide is bound on extracellular segments of MHC-I molecule, its main function is to express intracellular proteins to cytotoxic T cells (CTLs).
- Nevertheless, MHC-I may present peptides produced by exogenous proteins, in a process called cross-presentation.
- A typical cell expresses peptides from typical cellular protein turnover on its MHC-I, and central and peripheral tolerance mechanisms prevent CTLs from being activated in response to them.
- When a cell expresses foreign proteins, such as after being infected by a virus, a small proportion of MHC-I then presents these peptides on the cell surface. Subsequently, CTLs specific for the MHC:peptide complex would recognise and kill the presenting cells.
- MHC-I molecule can also function as an inhibitory ligand for natural killer cells (NKs). When levels of surface MHC-I decrease, a number of viruses and tumours employ a pathway to evade CTL responses, which activates NK cells killing.
PirB and visual plasticity
- Syken et al. (2006) discovered a MHC-I-binding receptor called paired-immunoglobulin-like receptor B (PirB) being involved in the regulation of visual plasticity.
- PirB is expressed in the central nervous system (CNS) and reduces ocular dominance plasticity during the developmental critical period and adulthood.
- Double knockout of PirB genes in mice models lead to increased ocular dominance plasticity in all mice regardless of age, particularly during the critical period. This finding indicates PirB is associated with modulation of synaptic plasticity in the visual cortex.
How are MHC-I molecules synthesised?
- In the cytosol, the proteasome is a macromolecule that contains 28 subunits, half of which influence proteolytic activity. It degrades intracellular proteins into peptide fragments, which are subsequently released into the cytosol.
- Other functions of proteasomes include ligation of spliced peptides, production of non-contiguous sequences that aren't linearly templated in the genome.
- Faridi et al. (2018) suggested spliced peptide segments may have originated from the same protein (cis-splicing) or different proteins (trans-splicing).
- Peptides are translocated from the cytosol into the endoplasmic reticulum (ER) before they interact with MHC-I molecule, whose peptide-binding site is in the ER lumen.
Describe the role of MHC-I in peptide translocation and peptide loading
 |
| This is a simplified diagram of cytoplasmic protein degradation by the proteasome, transport into endoplasmic reticulum by TAP complex, loading on MHC class I, and transport to the surface for presentation. |
- Peptide translocation from the cytosol into the ER lumen is triggered by the transporter associated with antigen processing (TAP). TAP is a type of ABC transporter with the structure of a heterodimeric multimembrane-spanning polypeptide containing TAP1 and TAP2.
- Those 2 subunits together form a peptide binding site and 2 ATP binding sites facing the cytosol. TAP binds peptides on the cytoplasmic side and translocates them under ATP consumption into the ER lumen. The MHC-I molecule is subsequently loaded with peptides in the ER lumen.
- Blees et al. (2017) stated the peptide-loading process involves a number of other molecules that form a multimeric complex called the peptide loading complex, which contains TAP, tapasin, calreticulin, calnexin, and Erp57 (PDIA3).
- Before its dissociation, calnexin stabilises MHC-I α chains prior to β2m binding in order to the completee assembly of the MHC molecule.
- If a MHC molecule doesn't bind a peptide, it becomes inherently unstably, thus it requires binding of the chaperones calreticulin and Erp57 to achieve stability.
- Studies found tapasin binds to the MHC molecule and connects it to the TAP proteins, then facilitates peptide selection in a process called peptide editing. This triggers facilitation of enhanced peptide loading and co-localisation.
- When the peptide is loaded onto MHC-I, the entire complex dissociates and departs the ER through the secretory pathway to approach the cell surface, which involves a number of posttranslational modifications of the MHC molecule.
- A few of the posttranslational modifications occur in the ER that involve altering the N-glycan regions of the protein, followed by modifications to the N-glycans in the Golgi apparatus. N-glycans achieve full maturation prior to approaching the cell surface.
How is peptide removed?
- If peptides don't bind to MHC-I molecules in the ER lumen, they are transported from the ER via the sec61 channel into the cytosol.
- When they reach the cytosol, they may truncate in size, and translocate by TAP back into the ER to bind to MHC-I molecule for a second attempt.
How does MHC-I affect viruses?
- As viruses trigger cellular expression of viral proteins, some of them are marked for degradation, with the emerging peptide fragments entering the ER and binding to MHC-I molecules.
- During the MHC-I-dependent pathway of antigen presentation, virus-infected cells signal T-cells to produce abnormal proteins as a result of infection.
- A majority of virus-infected cells would be triggered to undergo apoptosis via cell-mediated immunity, which decreases the risk of adjacent cells being infected.
- Nevertheless, numerous viruses evolved to down-regulate or block the presentation of MHC-I molecules on the cell surface. This results in the activation of NK cells, which then recognise the cell as aberrant, indicating it could be infected by viruses attempting to avoid immune destruction.
- Wang et al. (2008) discovered a number of human cancers down-regulated MHC-I, which provided transformed cells the ability to evade normal immune surveillance.
b. Class II
- MHC-II molecules are usually found only on professional antigen-presenting cells (APCs) such as dendritic cells, mononuclear phagocytes, some endothelial cells, thymic epithelial cells, and B cells.
- MHC-II molecules are heterodimers containing 2 homogenous peptides, α and β chains, both of which are encoded in the MHC.
- Each domain within the HLA gene is encoded by a different exon within the gene, and a few genes have further domains encoding leader sequence, transmembrane sequences, etc. Those molecules contain both extracellular regions, as well as a transmembrane sequence and a cytoplasmic tail.
- The α1 and β1 regions combine to create a membrane-distal peptide-binding domain, while the α2 and β2 regions combine to create a membrane-proximal immunoglobulin-like domain.
- The antigen binding groove is composed of 2 α-helixes walls and a β-sheet. Since the antigen-binding groove of MHC-II is open at both ends, the presented antigens are longer, about 15-24 amino acid residues in length.
What genes associate with MHC-II?
Where is MHC-II expressed?
- Ting & Trowsdale (2002) found MHC-II are constitutively expressed in professional, antigen-presenting cells, as well as other cells stimulated by interferon-γ.
- MHC-II is also expressed on the epithelial cells in the thymus and on APCs in the periphery, as well as group 3 innate lymphoid cells.
- Its expression is regulated in APCs by a MHC-II transactivator called CIITA, as well as non-professional APCs.
- CIITA is only expressed on professional APCs, but its activity is regulated by non-professional APCs.
- Roche & Furuta (2015) found IFN-γ stimulates CIITA expression and converts MHC-II-negative monocytes into functional APCs that express MHC-II on their surfaces.
How is MHC-II synthesised?
- In the ER, the α and β chains of MHC-II are created first and then combined wth a polypeptide known as the invariant chain.
- The peptide-binding cleft on nascent MHC-II protein in the rough ER is occluded by the invariant chain (li; a trimer) in order to cover it from binding cellular peptides or peptides from the endogenous pathway.
- The invariant chain also enables the export of MHC-II from the ER to the Golgi apparatus, which subsequently fuses with the late endosome containing endocytosed, degraded proteins.
- The invariant chain then disintegrates in stages by proteases called cathepsins, with only a small fragment remaining known as CLIP. The role of CLIP is to maintain blockage of the peptide binding cleft on MHC-II.
- A MHC-II-like structure called HLA-DM promotes removal CLIP for MHC-II to bind peptides with higher affinities. Finally, the stable MHC-II is presented on the cell surface.
What are the functions of MHC-II?
- When MHC-II is packed with extracellular proteins, it presents the extracellular pathogens to immune cells such as CD4+ T helper cells. Owen et al. (2013) stated that when peptide binding is stable, it avoids detachment and degradation of the peptide, which can be achieved without secure attachment to MHC-II.
- This avoids T cell recognition of the antigen, T cell recruitment, and yields an appropriate immune response. A proper immune response includes localised inflammation and swelling due to recruited phagocytes or an antibody immune response due to activated B cells.
-- Recycling of MHC-II complexes
- After MHC-II are synthesised and presented on APCs, they aren't expressed on the cell surface indefinitely because of the internalisation of the plasma membrane by APCs.
- In some cells, antigens bind to recycled MHC-II molecules whilst in the early endosome stage, while other cells such as dendritic cells internalise antigens via receptor-mediated endocytosis and produce MHC-II as well as peptide in the endosomal-lysosomal antigen processing compartment.
- Roche & Furuta (2015) hypothesised the existent MHC-II complexes are mature dendritic cells can be recycled and developed into novel MHC-II molecules as well as peptide after antigen internalisation.
Describe the pathways of MHC-II antigen presentation
- 2 kinases called PIK3R2 and PIP5K1A phosphorylate Phosphatidylinositol (PIP), which create substrates for PSD4 to load GTP on.
- PSD4 (Pleckstrin and Sec7 Domain containing 4) is a GEF (Guanine nucleotide Exchange Factor) that loads ARL14/ARF7 with GTP.
- ARL14/ARF7 is a small GTPase protein selectively expressed on immune cells, which is localised within MHC-II compartments in immature dendritic cells.
- ARF7EP is an effector of ARL14/ARF7 that interacts with MYO1E, which binds itself to actin myofibres. The protein MYO1E controls MHC-II compartments with an actin-based process.
- This pathway leads to the maintenance of MHC-II loaded vesicles within the immature dendritic cell, which prevents its translocation to the cell membrane.
Describe the MHC-II's role in disease
-- Bare lymphocyte syndrome
- Steimle et al. (2015) found mutations in MHC-II genes that code from transcription factors regulating MHC-II gene expression result in bare lymphocyte syndrome. This diminishes CD4 T-cells and a number of immunoglobulin isotypes, despite normal levels of both CD8 Cells and B cells.
- Deficient MHC-II molecules can't present antigens to T cells and properly activate T cells. This means T cells can't proliferate, and release cytokines to activate neighbouring immune cells.
- This compromises the entire immune response cascade, which includes B cells. This means a reduction in T cell levels, and T cells are unable to interact and activate B cells. Furthermore, B cells aren't activated and are unable to differentiate into plasma cells, which leads to defective antibodies.
- Serrano-Martín et al. (2017) stated the only treatment for bare lymphocyte syndrome is a bone-marrow transport, however this fails to cure the disease and most patients die by the age of 10 years.
-- Type I diabetes
- HLA-II genes are known to be associated with the risk of inheriting Type I diabetes, accounting for 40-50% of heritability. Type I diabetes risk is higher for alleles of these genes linked to peptide binding to MHC-II.
- A number of allele polymorphisms were identified by Xie et al. (2014) to increase the risk of Type I diabetes are DRB1 and DQB1.
c. Class III
- MHC-III is a group of proteins in the major histocompatibility complex (MHC) family, but not much is well known about its structure and function.
- It's known they don't take part in antigen presentation and a majority of MHC-III molecules take part in cellular signalling.
- Their gene cluster, located between those of MHC-I and MHC-II, was discovered when genes (related to complement components C2, C4, and factor B) were discovered in between class I and class I genes on the short (p) arm of human chromosome 6.
- This cluster contains genes coding for different signalling molecules such as tumour necrosis factors (TNFs) and heat shock proteins.
- A 1999 study identified more than 60 MHC-III genes, which is roughly 28% of the total MHC genes (224).
- Deakin et al. (2006) discovered the region within MHC-III gene cluster comprising of genes for TNF is also known as MHC-IV.
- MHC-III proteins are created by liver cells (hepatocytes) and special white blood cells (macrophages), among others.
Describe the gene structure of MHC-III
- MHC-III genes are located on human chromosome 6, specifically 6p21.3, about 700 kb long and comprises of 61 genes.
- It includes numerous retroelements such as human endogenous retrovirus (HERV) and Alu elements, as well as G11/C4/Z/CYP21/X/Y genes that are between 142 and 214 kb long.
- Human MHC-III genes share genetic similarity with other animals such as mouse, frog (Xenopus tropicalis), and gray short-tailed opossum.
- However, mice MHC-III lack NCR3, MIC and MCCD1, and opossum MHC-III lack NCR3 and LST1.
- Furthermore, Shiina et al. (2008) found MHC-III in birds (chicken and quail) contain only a single gene that codes for a complement component gene (C4).
- Sambrook et al. (2008) found fish MHC-III genes were distributed across different chromosomes.
Describe the functions of MHC
- MHC is the tissue-antigen that allows T cells to bind to, recognise, and tolerate itself. It serves as the chaperone for intracellular peptides that binds with MHCs to become a molecular complex and then present to TCRs as potential foreign antigens.
- MHC subsequently communicates with TCR and its co-receptors to optimise binding conditions for the TCR-antigen interaction (i.e. maximise antigen binding affinity and specificity) and signal transduction effectiveness.
- The MHC-peptide complex is a complex of auto-antigen / allo-antigen. The auto-antigen component being tolerated by the T cells, but the allo-antigen component activating the T cells.
- Kindt et al. (2007) stated the antigen presentation process involves MHC molecules binding to both TCR and CD4/CD8 co-receptors on T lymphocytes, and the antigen epitope situated in the peptide-binding groove of MHC interacting with the variable Ig-Like domain of the TCR to activate T-cells.
- A MHC molecule called HLA-B27 is known to increase the risk of an autoimmune condition called 'ankylosing spondylitis' and other associated inflammatory diseases, but the mechanisms involving aberrant antigen presentation or T cell activation are poorly understood.
- When MHC molecules bind to peptide epitopes to form a complex, they serve as ligands for TCRs to bind to. T cells activate after binding to the peptide-binding grooves of any MHC molecule they aren't trained to recognise during positive selection in the thymus, a process known as 'tissue allorecognition'.
Describe the roles of MHC in:
i. Antigen processing
ii. MHC-restricted antigen recognition
- MHC-restricted antigen recognition, or MHC restriction, describes the interaction between a T cell and a self-major histocompatibility complex molecule and a foreign peptide bound to it, but the T cell only responds to the antigen when it binds to a particular MHC molecule.
- When TCRs recognise only some MHC molecules but not others after the selection process, this becomes instrumental to MHC restriction.
- The purpose of MHC restriction is to prevent an excess number of wandering lymphocytes, thus conserve energy of cell-building resources.
- MHC restriction in T cells occurs during heir development in the thymus, specifically positive selection. This means only the thymocytes capable of binding, with the optimal affinity, with the MHC molecules to receive a survival signal and process to the next phase of selection.
- MHC restriction plays a critical role in allowing T cells to function appropriately after departing the thymus. It permits certain TCRs to bind to MHC and identify cells infected by intracellular pathogens, viral proteins and bearing genetic defects.
- The germline model hypothesises that MHC restriction developed from evolutionary pressure supporting T cell receptors that are capable of binding to MHC.
- The selection model hypothesises not all TCRs demonstrate MHC restriction, however only the TCRs with MHC restriction are expressed after being positively selected in the thymus. It suggests T cells can recognise a variety of peptide epitopes independent of MHC molecules before undergoing thymic selection. Therefore, only T cells with affinity to MHC are signalled to survive after CD4 or CD8 co-receptors also bind to the MHC molecule.
iii. Sexual mate selection
- The major histocompatibility complex in sexual selection discusses how MHC molecules provide immune system surveillance of the population of protein molecules in a host's cells.MHC complex genes have been demonstrated to maintain a significantly high level of allelic diversity across time and across various populations.
2 non-mutually exclusive hypotheses were proposed to explain how the co-evolutionary arms race between hosts and parasites yielded an extensive source of genetic variation impacting an organism's fitness.
1. MHC-heterozygote advantage hypothesis
- If individuals heterozygous at the MHC are unaffected by parasites than those that are homozygous, then there are advantages for females to select mates with MHC genes different from their own. This would lead to MHC-heterozygous offspring, referred to as disassortative mating.
- The hypothesis states that individuals with a htereozygous MHC can identify a wider range of pathogens and therefore induce a specific immune response against a higher number of pathogens, thus gaining an immunity advantage.
- O'Dwyer & Nevitt (2009) expressed concern the MHC-heterozygote advantage hypothesis is yet to be thoroughly tested. Nevertheless, numerous studies concluded non-MHC immune genes across species demonstrate heterozygote disadvantage, or the absence of any advantage.
- A mice model by Ilmonen et al. (2007) challenged this hypothesis by asserting increased MHC-heterozygous females demonstrated decreased fitness compared to homozygotes. Other animal studies conducted by McClelland et al. (2003) and Takahata et al. (1994) reported similar findings that excess heterozygosity reduces fitness.
2. Optimality Hypothesis
- This hypothesis suggests excess variability in MHC genes may lead to failure of T-cells to distinguish themselves from non-selves, therefore increase the risk of autoimmune disease. Antonides et al. (2019) stated this provides higher fitness to individuals without significant MHC diversity.
- Woelfing et al. (2009) argued it is optimal for an animal species to exhibit intermediate levels of MHC heterozygosity.
3. The Red Queen Hypothesis
- This hypothesis suggests MHC diversity is maintained by parasites. If an individual's MHC alleles provide different resistances to a certain parasite, therefore the allele with the highest resistance is favoured, selected for, and as a result spread throughout the population.
- Recombination and mutation generates new variants among offspring, which promotes a rapid response to evolving parasites or pathogens with significantly reduced generation periods.
- Nevertheless, if a particular allele becomes more common, selection pressure on parasites to avoid being recognised by this common allele increases. This distributes the parasite's advantage of escaping recognition, and facilitate selection against formerly resistant alleles.
- This allows the parasite to evade the cycle of frequency-dependent selection, which would avoid the co-evolutionary arms race that augments the maintenance of MHC diversity.
4. Inbreeding avoidance hypothesis
- This hypothesis suggests inbreeding increases the amount of overall homozygosity, which may be accompanied by the expression of recessive diseases and mutations, as well as the loss of any potential heterozygote advantage.
- Bernatchez & Landry (2003) dismissed this hypothesis because they argued relatedness has no association with choice of mate.
Describe the relationship between the olfaction and MHC
- Milinski et al. (2005) found MHC-based sexual selection associated with olfactory pathways in a number of vertebrate animals such as fish, mice, humans, primates, birds, and reptiles. Yamazaki et al. (1999) predicted olfaction acts to personally identify individuals based upon the MHC genes.
- Chemosensation allows humans to perceive, as well as assess, and respond to environmental olfactory cues known as pheromones.
- Phermones serve to convey one's species, sex, and genetic identity. Yamazaki et al. (1999) stated the MHC genes provide the instructions for the development of a set of unique olfactory codes.
- Despite the mechanism behind the recognition of MHC-specific odours being unknown, it is hypothesised that proteins bound to the peptide-binding groove of the MHC may create the odourant.
- Studies demonstrated receptors in the vomeronasal organ of mice are activated by peptides sharing several characteristics to MHC proteins. It is suggested commensal microflora or microorganisms lining the epithelial surfaces open to the external environment, such as the GIT and vagina, degrade the MHC-peptide complex fragments that were shed from the cell surface.
iv. Evolutionary diversity
- Sznarkowska et al. (2020) found most mammals share similar MHC variants to humans, who carry higher allelic diversity, which may be due to gene duplication of MHC regions.
- A majority of HLA alleles are shared with chimpanzee MHC alleles than other human alleles of the same gene.
- Balancing selection = Any natural selection process by which no single allele is absolutely most fit, such as frequency-dependent selection and heterozygote advantage.
- Pathogenic coevolution = Common alleles are under substantial pathogenic pressure, which prompts positive selection of uncommon alleles i.e. moving targets for pathogens. As pathogenic pressure on the previously common alleles decreases, their prevalence in the population equilibrates, and continue to circulate in a large population.
- Zeisset & Beebee (2014) suggested genetic drift as a major driving force in a number of species.
- Sommer (2005) hypothesised MHC diverisity may be a possible indicator for conservation, as larger, stable populations tend to exhibit considerable MHC diversity compared to smaller, isolated populations.
- Animal species known to have relatively low MHC diversity include the cheetah (Acinonyx jubatus), Eurasian beaver (Castor fiber), and giant panda (Ailuropoda melanoleuca).
- A 2007 study by Siddle et al. stated the low MHC diversity in the Tasmanian devil (Sarcophilus harrisii) may be associated with disease susceptibility. This is due to an antigen of a transmissible tumour, involved in devil facial tumour disease, being recognised as a self antigen.
- Shum et al. (2001) found the MHC-II allelic polymorphisms in ray-finned fish (such as rainbow trout) is similar to that in mammals and predominantly outlines to the peptide binding groove.
- Aoyagi et al. (2002) found significant amount of allelic polymorphisms in MHC-I of numerous teleost fishes compared to mammals. In that regard, the sequence identity levels between alleles can be extremely scarce and the variation extends beyond the peptide binding groove.
- Yamaguchi & Dijkstra (2013) theorised this type of MHC-I allelic variation may be a factor in allograft rejection, which helps fish prevent grafting of cancer cells through their mucosal skin.
- Abi Rached et al. (1999) suggested the MHC loci emerged from the two-round duplications in vertebrates of a single ProtoMHC locus, and the new domain organizations of the MHC genes were produced by subsequent cis-duplication and exon shuffling in a mechanism labelled "the MHC Big Bang.
- Suurväli et al. (2014) identified an association between the genes in the MHC locus and intracellular intrinsic immunity in the basal Metazoan Trichoplax adhaerens.
- In a transplant procedure (e.g. an organ or stem cell transplant), MHC molecules serve as antigens and can induce an immune response in the recipient, thereby triggering transplant rejection.
- Abbas & Lichtman (2009) identified and described MHC molecules in transplant rejection between mice of different strains and verified their role in presenting peptide antigens to cytotoxic T lymphocytes.
What is human leukocyte antigen (HLA)?
The human leukocyte antigen (HLA) system or complex is a complex of genes on chromosome 6 in humans that encode cell-surface proteins involved in the regulation of the immune system. A 2020 study described the HLA system as the human version of the major histocompatibility complex (MHC) found in many animals.
How is HLA classified?
MHC-I proteins create a functional receptor on a majority nucleated cells of the body.
There are 3 major and 3 minor MHC-I genes in HLA. β2-microglobulin binds with major and minor gene subunits to create a heterodimer.
There are 3 major and 2 minor MHC-II proteins encoded by the HLA. The MHC-II genes coalesce to produce heterodimeric (αβ) protein receptors usually expressed on the surface of antigen-presenting cells.
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| Codominant expression of HLA genes |
Nomenclature:
- HLA alleles typically start with HLA- and the locus name, then " * " and a/an (even) number of digits that specifies the allele.
- The first 2 digits define a group of alleles, known as supertypes.
- The 3rd through 4th digits define a non-synonymous allele.
- The 5th through 6th digits define any synonymous mutations within the coding frame of the gene.
- The 7th and 8th digits differentiate mutations outside the coding region.
- Letters including L, N, Q, or S defines an allele's expression level or other non-genomic data known about it.
- Therefore, a HLA allele with a complete description may be up to 9 digits long, not including the HLA-prefix and locus notation.
MHC Class I
MHC Class II
Describe the functions of HLA in:
i. Infectious disease
- Proteins from the pathogen are fragmented into peptides during phagocytosis and loaded onto HLA antigens (i.e. MHC-II). They are subsequently exhibited by the antigen-presenting cells to CD4+ helper T cells, which later induce a broad range of chemical effects and intercellular interactions to destroy the pathogen.
- Proteins (both native and foreign, such as viral proteins) created inside a majority cells are displayed on HLAs (MHC-I) on the cell surface. CD8+ T cells recognise these infected cells and subsequently proceed to destroy them.
- Each person contains 3 HLA types and 4 isoforms of DP, 4 isoforms of DQ and 4 Isoforms of DR (2 of DRB1, and 2 of DRB3, DRB4, or DRB5) for a total of 12 isoforms, which can bind a wide range of peptides.
ii. Graft rejection
- Any cell expressing another type of HLA is considered "non-self" and is identified as an invader by the host's immune system, which leads to the rejection of the tissue bearing those cells . In the case of transplanted tissue or organs, this results in transplant rejection.
- Since HLA plays an essential role in transplantation, HLA loci are often typed by serology and PCR.
- Agarwal et al. (2017) demonstrated relevance of high resolution HLA typing (HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DQB1 and HLA-DPB1) in transplantation when identifiying a full match, even in the scenario of a donor being related.
iii. Autoimmunity
- Studies stated people having particular HLA antigens have a higher likelihood of developing particular autoimmune diseases, such as type I diabetes, ankylosing spondylitis, rheumatoid arthritis, celiac disease, SLE (systemic lupus erythematosus), myasthenia gravis, inclusion body myositis, Sjögren syndrome, and narcolepsy.
- Although HLA typing resulted in some improvement and earlier diagnosis of celiac disease and type 1 diabetes, DQ2 typing requires either high-resolution B1*typing (resolving *02:01 from *02:02), DQA1*typing, or DR serotyping.
- HLA serotyping is increasing in use as a tool in diagnosing autoimmune conditions such as celiac disease. So far, it is the only effective means of discriminating between first-degree relatives that are at risk from those that are not as risk, prior to the emergence of occasionally irreversible symptoms such as allergies and secondary autoimmune disease.
iv. Cancer
- It's known DR3-DQ2 homozygotes are within the highest risk group, with roughly 80% of gluten-sensitive enteropathy cases related to associated T-cell lymphoma.
- Nevertheless, HLA molecules offer protection in identifying increases in antigens that haven't been tolerated due to its scarcity in the normal state.
Describe the role of HLA in allelic variation
- Apanius et al. (1997) suggested a heterozygous selection mechanism process acting on these loci as an attempt to explain for the variability.
- Wedekind et al. (1995) proposed a sexual selection theory that females can detect males with different HLA relative to their own type.
- Although the DQ and DP encoding loci have less alleles, several combinations of A1:B1 can yield a theoretical potential of 7,755 DQ and 5,270 DP αβ heterodimers, respectively.
- Each person can carry 4 variable DQ and DP isoforms, which increases the potential number of antigens that these receptors can present to the immune system.
- The variable positions of DP, DR, and DQ demonstrated that peptide antigen contact residues on MHC-II molecules are often the location of variation in the protein primary structure.
- Combining intense allelic variation and/or subunit pairing would allow MHC-II peptide receptors to bind a virtually limitless variation of peptides of 9 amino acids or longer in length. This protects interbreeding subpopulations from nascent or epidemic diseases.
- Individuals in a population often have different haplotypes, which associates with greater diversity. This diversity boosts the survival of such groups, and impedes evolution of epitopes in pathogens, which would otherwise be blocked from the immune system.
What is an antibody?
- Known as an immunoglobulin (Ig), an antibody is a T-shaped protein that identifies and neutralises foreign pathogens such as bacteria and viruses. The main function of an antibody is to detect a unique molecule of the pathogen called the antigen.
How were antibodies discovered?
- In October 1891, Paul Ehrlich first coined the term Antikörper (the German word for antibody) in the conclusion of his publication "Experimental Studies on Immunity".
- However, the term wasn't accepted immediatly and numerous alterative terms for antibody were suggested such as Immunkörper, Amboceptor, Zwischenkörper, substance sensibilisatrice, copula, Desmon, philocytase, fixateur, and Immunisin.
- The first study of antibodies was conducted in 1890 by Emil von Behring and Kitasato Shibasaburō, which reported antibody activity against diphtheria and tetanus toxins.They promoted the theory of humoral immunity and suggested a mediator in the serum may react with a foreign antigen.
- This concept motivated Ehrlich to suggest the side-chain theory for antibody and antigen interaction in 1897. This theory conjectured receptors (side-chains) on the cell surface could bind specifically to toxins in a "lock-and-key" interaction, which stimulates the production of antibodies.
- In 1904, Almroth Wright proposed soluble antibodies covered bacteria to tag them for phagocytosis and neutralisation, a process he named 'opsonisation'.
- In the 1920s, Michael Heidelberger and Oswald Avery observed antigens are composed of protein and can be precipitated by antibodies.
- In the late 1930s, John Marrack first investigated the biochemical characteristics of the interactions between antigen and antibody.
- In the 1940s, Linus Pauling experimentally confirmed the lock-and-key theory hypothesised by Ehrlich by demonstrating the interactions between antibodies and antigens that depends more on their shape than their chemical composition.
- In 1948, Astrid Fagraeus discovered antibodies were generated by B cells, in the form of plasma cells.
- In the early 1960s, Gerald Edelman and Joseph Gally discovered the structure of the antibody light chain, which is the same protein as the Bence-Jones protein described by Henry Bence Jones in 1845.
- In the 1970s, Edelman then discovered the heavy and light chains of antibodies were connected by disulfide bonds. Furthermore, Rodney Porter discovered the antibody-binding (Fab) and antibody tail (Fc) regions of IgG.
- Edelman & Porter extrapolated the structure and complete amino acid sequence of IgG, a discovery which they were jointly awarded the 1972 Nobel Prize in Physiology or Medicine.
- In 1973, David Givol prepared and characterised the Fv fragment of the antibody.
- In the 1960s, a majority of researchers investigated the isotypes IgM and IgG, but other isotypes were also researched.
- In 1963, Thomas Tomasi discovered the secretory antibody IgA.
- In 1964, David S. Rowe and John L. Fahey discovered IgD.
- In 1966, Kimishige Ishizaka and Teruko Ishizaka discovered IgE and its involvement in allergic reactions.
- In 1976, Susumu Tonegawa demonstrated the self-rearrangment of antibody genetic material to produce a diverse range of antibodies.
Describe the structure of the antibody
- Antibodies are proteins weighing about 150 kDa and about 10nm in length, whose Y shape is formed by 3 globular regions.
- In humans and a majority of mammals, an antibody unit comprises of 4 polypeptide chains; 2 identical heavy chains and 2 identical light chains linked by disulfide bonds. Each chain consists of a series of domains, with each around 110 amino acids long.
- Light chains comprise of 1 variable domain (VL) and 1 constant domain (CL), while heavy chains comprise of 1 variable domain (VH) and 3-4 constant domains CH1, CH2, ...
- An antibody is structurally divided into 2 antigen-binding fragments (Fab), which consist of 1 VL, VH, CL and CH1 domain each, as well as the crystallisable fragment (Fc) to create the trunk of the Y shape.
- Delves et al. (2017) described a hinge region between the heavy chains, whose flexibility allows antibodies to bind to epitope pairs at a range of distances, create complexes (dimers, trimers, etc.), and bind effector molecules more efficiently.
a. Antigen binding site
- The variable domain is described as the FV region, with the Fab subregion binding to an antigen.
- Each variable domain consists of 3 hypervariable regions, meaning the amino acid sequences there vary considerably between each antibody.
- After the protein folds, these hypervariable regions produce 3 loops of β-strands that are localised near one another on the surface of the antibody. These loops are called the complementarity-determining regions (CDRs) because their shape complements that of the antigen.
- 3 CDRs from each of the heavy and light chains combine to create an antibody-binding site whose shape can range from a small pocket to a large protrusion into a groove in an antigen.
- Janeway (2001) stated that having 2 identical antibody-binding sides allows stronger binding of antibody molecules to multivalent antigen (i.e. repeating sites such as polysaccharides in bacterial cell walls), as well as producing antibody complexes and larger antigen-antibody complexes.
b. Fragment crystallisable (Fc) region
- The fragment crystallisable region (Fc region) is located on the tail section of the antibody, which interacts with cell surface receptors called Fc receptors and a number of proteins of the complement system.
- In IgG, IgA and IgD isotypes, the Fc region contains 2 identical protein fragments that originated from the 2nd and 3rd constant domains of the antibody's 2 heavy chains.
- In IgM and IgE isotypes, their Fc regions contain 3 heavy chain constant chains (CH domains 2-4) in each polypeptide chain.
- Stadlmann et al. (2009) found the Fc regions of IgGs carry a highly conserved N-glycosylation site, which plays an important role in Fc receptor-mediated activity. The N-glycans bound to this site are primarily core-fucosylated diantennary structures of the complex type.
- Stadlmann et al. (2008) found trace amounts of these N-glycan carry bisecting GlcNAc and α-2,6 linked sialic acid residues.
- Fc binds to a diverse range of cell receptors and complement proteins, which regulates various physiological effects of antibodies such as detection of opsonized particles, cell lysis, degranulation of mast cells, basophils, and eosinophils; and other processes.
c. Immunoglobulin light chain
The immunoglobulin light chain is the small polypeptide subunit of an antibody (immunoglobulin). A human antibody molecule contains 2 types of light chains:
-- Kappa (κ) chain = Encoded by the immunoglobulin kappa locus (IGK@) on chromosome 2
-- Lambda (λ) chain = Encoded by the immunoglobulin lambda locus (IGL@) on chromosome 22
Each light chain is composed of 2 tandem immunoglobulin domains:
-- 1 constant (CL) domain
-- 1 variable domain (VL) crucial for binding antigen
- A light chain protein is roughly 211-217 amino acids in length.
- The constant region determines the class of light chain (κ or λ) will be.
- There are 4 subtypes of λ chains: λ1, λ2, λ3, λ7.
d. Immunoglobulin heavy chain
- The immunoglobulin heavy chain (IgH) is the large polypeptide subunit of an antibody, with its gene loci located on chromosome 14 in human genome.
- There are 5 types of mammalian immunoglobulin heavy chain: γ, δ, α, μ and ε.
- Each of which define classes of immunoglobulins: IgG, IgD, IgA, IgM and IgE, respectively.
- Heavy chains α and γ are roughly 450 amino acids in length, whereas heavy chains μ and ε are roughly 550 amino acids in length.
Each heavy chain contains 2 regions:
- A constant region = Identical for all immunoglobulins of same class but differs between classes
-- Heavy chains μ and ε contain a constant region consisting of 4 domains.
- A variable region = This varies between different B cells, but is identical for immunoglobulins produced by the same B cell clone or B cell. It consists of a single immunoglobulin domain, which is approximately 110 amino acids long.
Describe the different classes of antibody
i. IgA
Immunoglobulin A (IgA) is an antibody that is is highly abundant in mucosal membranes as part of its immune function. It makes up up to 15% of total immunoglobulins released throughout the body.
What are different forms of IgA?
-- IgA1 vs. IgA2
There are 2 isotypes of IgA: IgA1 and IgA2. Both are heavily glycosylated proteins.
-- Serum vs. secretory IgA
Describe the physiology of IgA
-- Serum IgA
- Snoeck et al. (2006) found IgA binds an Fc receptor called FcαRI (or CD89) expressed on immune effector cells to induce inflammatory responses in the blood.
- FcαRI ligation via IgA containing immune complexes induces antibody-dependent cell-mediated cytotoxicity (ADCC), degranulation of eosinophils and basophils, phagocytosis by monocytes, macrophages, and neutrophils, and stimulation of respiratory burst activity by polymorphonuclear leukocytes.
-- Secretory IgA
- Snoeck et al. (2006) suggested higher levels of IgA in mucosal areas associated with the interaction between plasma cells that produce polymeric IgA (pIgA), and mucosal epithelial cells that express polymeric immunoglobulin receptor (pIgR).
- Plasma cells produce polymeric IgA in the lamina propria adjacent to mucosal surfaces. It then binds to the pIgR on the basolateral surface of epithelial cells, and is internalised by the cell via endocytosis.
- The receptor-IgA complex travels through the cellular compartments before being secreted on the luminal surface of the epithelial cells, whilst still bound to the receptor.
- Kaetzel et al. (1991) found this is followed by receptor proteolysis, and dimeric IgA plus the secretory component (sIgA) are to diffuse throughout the lumen.
- In the gut, IgA binds to the mucus layer covering the epithelial cells in order to create a barrier that can neutralising infectious pathogens before they approach the epithelial cells.
- Mantis et al. (2011) stated the generation of sIgA against specific antigens depends on the sampling of M cells and underlying dendritic cells, T cell activation, and B cell class switching in GALT, mesenteric lymph nodes, and isolated lymphoid follicles in the small intestine.
- sIgA mainly blockades epithelial receptors (e.g. by binding their ligands on pathogens), sterically inhibits binding to epithelial cells and acts by immune exclusion.
- Immune exclusion involves crosslinking agglutinating polyvalent antigens or pathogens with antibody, which traps them in the mucus layer, and/or eliminating them peristaltically.
- The oligosaccharide chains of the IgA component interacts with the mucus layer that situates above epithelial cells. Because sIgA is a poor opsonin and activator of complement, binding a pathogen is inadequate to contain it, since specific epitopes may to be bound to sterically block access to the epithelium.
- Maverakis (2015) found IgA clearance is regulated partially by asialoglycoprotein receptors, which recognises galactose-terminating IgA N-glycans.
ii. IgD
- Immunoglobulin D (IgD) isotype makes up roughly 1% of proteins in the plasma membranes of immature B-lymphocytes where it is typically co-expressed with another cell surface antibody called IgM.
- It is produced in a secreted form at small levels in the blood serum, making up 0.25% of immunoglobulins in serum.
- Rogentine et al. (1966) estimated the relative molecular mass and half-life of secreted IgD is 185 kDa and 2.8 days, respectively.
- Secreted IgD exists as a monomeric antibody containing 2 delta (δ) heavy chains of the, and 2 Ig light chains.
Describe the method of co-expression in IgD
- Murphy & Weaver (2016) suggested zinc finger protein 318 (ZNF318) is associated with the facilitation of IgD expression and control of the alternative splicing of the long pre-mRNA.
- In addition, immature B cells expressing the μ transcript lack ZFP318 expression, whereas mature B cells express both IgM and IgD because both δ and μ transcripts have been created and ZFP318 is expressed.
- In the human Heavy-Chain Locus, 3' of the V-D-J casette is a series of C (constant) genes), each correlating with an Ig isotype. The Cμ (IgM) gene is 3' and closest to the V-D-J cassette, whereas the Cδ gene situates 3' to Cμ.
- Alternative slicing selects either Cμ or Cδ to appear on the functional mRNA (μ mRNA and δ mRNA respectively).
- Researchers suggest alternative splicing occurs due to 2 polyadenylation sites, with 1 located between the Cμ and Cδ, and the other 3' of Cδ (polyadenylation in the latter site would result in Cμ being spliced away along with the intron). However, the exact mechanism of how the polyadenylation site is unknown.
- This yields a functional mRNA containing the V-D-J and C regions contiguous, and its translation produces either a μ heavy chain or δ heavy chain. The heavy chains subsequent;y link with either κ or λ light chains to produce the final IgM or IgD antibody.
Describe the structural diversity of IgD
- IgD has structural diversity throughout the evolution of vertebrates due to its structurally flexible locus complementing IgM function. Bengtén et al. (2002) suggested IgD plays an important role in substituting IgM function in case of IgM deficiencies.
- Ohta & Flajnik (2006) found alternative splicing is elevated across all jawed vertebrates but class switch recombination is only observed in higher vertebrates, which increases IgD diversification.
- Preud'homme et al. (2000) found jawed fishes contained IgD with a highly diverse constant region that contains amplifications of Cδ exons.
- In humans and primates, IgD contains 3 Cδ domains and a long H region with an amino-terminal region rich in alanine and threonine residues.
- Iwase et al. (1996) found C-terminal regions contain an abundance of lysine, glutamate and arginin residues altered with O-glycosylation for binding a putative IgD receptor on the surface of activated T cells.
- Swenson et al. (1998) found the H region of human IgD interacts with heparin and heparan sulphate proteglycans expressed in the basophiles and mast cells.
- In comparison, mouse IgD has a shorter H region and different amino acid composition modified with N-glycosylation.
What are the functions of IgD?
- IgD provides signals to B cells for its activation in order to prepare them for immune response. When B cells leave the bone marrow to populate peripheral lymphoid tissues, they begin express IgD, along with IgM.
- Übelhart et al. (2016) discovered IgD signalling is induced solely by repetitive multivalent immunogens, whereas IgM signalling is induced either by soluble monomeric or by multivalent immunogens.
- Edholm et al. (2011) found Cδ knockout mice unable to produce IgD didn't show any major B cell intrinsic defects.
- Chen et al. (2009) discovered IgD activates basophils and mast cells to release antimicrobial factors important for respiratory immune defense in humans, as well as B cell homeostatic factors.
How does IgD activate the immune system?
- Researchers discovered both innate and adaptive immune responses are stimulated by membrane-anchored IgD that serve as a component of B-cell receptor (BCR) complexes or secreted IgD form that binds to monocytes, mast cells and basophils respectively.
- Nguyen et al. (2010) demonstrated the first treatments with a monoclonal anti-IgD antibody that successfully attenuated disease severity in an animal model of collagen-induced arthritis.
- Studies by Kulkarni et al. (2019) and Tue Nguyen (2019) confirmed this novel therapeutic effect by anti-IgD antibody treatment in mouse models of epidermolysis bullosa acquisita and in chronic contact hypersensitivity respectively.
- Yujing Wu (2016) demonstrated secreted IgD levels are typically higher in patients with an autoimmune disease, as well as associated with increased activation of peripheral blood mononuclear cells in Rheumatoid Arthritis (RA) patients. This lead to a hypothesis that IgD may be an immunotherapeutic target for the management of RA.
- Tue Nguyen (2022) stated activated immune responses via IgD-BCR and secreted IgD elicit suppressive effects on autoimmune diseases and allergic inflammations, which indicated a potential immune regulatory function of IgD.
iii. IgE
- Immunoglobulin E (IgE) antibody isotype is found only in mammals that is produced by plasma cells.
- Its monomer structure contains 2 heavy chains (ε chain) and 2 light chains, with the ε chain consisting of 4 Ig-like constant domains (Cε1–Cε4).
- In the mid-1960s, Kimishige and Teruko Ishizaka (1966), and Gunnar Johansson and Hans Bennich (1967) discovered IgE antibody isotype .
Describe the IgE receptors
IgE primes the IgE-mediated allergic response by binding to Fc receptors expressed on the surface of mast cells and basophils. Fc receptors are also located on eosinophils, monocytes, macrophages and platelets in humans.
There are 2 types of Fcε receptors:
-- FcεRI (type I Fcε receptor)= High-affinity IgE receptor
-- FcεRII (type II Fcε receptor) or CD23 = Low-affinity IgE receptor
- IgE upregulates expression of both types of Fcε receptors on mast cells, basophils, and the antigen-presenting dendritic cells.
- When antigens bind to IgE-FcεRI complex on mast cells, bound IgE crosslinks with aggregated underlying FcεRI, which results in degranulation (i.e. the release of mediators) and the secretion of type 2 cytokines such as interleukin (IL)-3 and stem cell factor (SCF).
- This promotes survival and accumulation of mast cells, as well as IL-4, IL-5, IL-13, and IL-33, which in turn activates group 2-innate lymphoid cells (ILC2 or natural helper cells).
- Since basophils share a common haemopoietic progenitor with mast cells, they also release type 2 cytokines, including IL-4 and IL-13, and other inflammatory mediators upon the cross-linking of their surface bound IgE by antigens.
- Ewart et al. (2002) found the FcεRII is always expressed on B cells, but IL-4 can trigger its expression on the surfaces of macrophages, eosinophils, platelets, and some T cells nonetheless.
Describe the functions of IgE
-- Parasite hypothesis
- Epidemiological studies demonstrated IgE levels increases upon infection by parasites such as Schistosoma mansoni, Necator americanus and nematodes in humans.
-- Toxic hypothesis of allergic disease
- In 1981, Margie Profet hypothesised allergic reactions evolved over time as a last line of defence to protect against venoms or noxious toxins.
- Researchers in the 2010s discovered IgE-antibodies were involve in the acquisition of resistance against honey bee and Russell's viper venoms
- They concluded a small dose of bee venom provided immunity to a significantly larger, fatal dose, which indicated this IgE-associated, adaptive immune response evolved to protect the host against potentially toxic amounts of venom, such as bee stings and snakebites.
- Tsai et al. (2015) found phospholipase A2, a major allergen of bee venom, triggers a Th2 immune response, which stimulates the production of IgE antibodies.
-- Cancer
- Karagiannis (2003) suggested IgE may be involved in the stimulation of cytotoxic responses against cells expressing trace amounts of early cancer markers. However, more research is required to understood this phenomenon.
- Busse et al. (2012) implicated the unlikelihood of a causal relationship between anti-IgE treatments such as omalizumab and cancer malignancy.
What are the roles of IgE in disease?
- Atopic individuals exhibit 10 times the usual IgE levels in their blood, which may not necessarily lead to symptoms as observed in asthmatics with typical IgE levels in their blood. Takhar et al. (2005) discovered IgE production occurs locally in the nasal mucosa.
- When IgE specifically recognises an allergen (e.g. dust mite, grass or pollen), it creates a unique interaction with its high-affinity receptor FcεRI in order to prime basophils and mast cells to release inflammatory molecules such as histamine, leukotrienes, and certain interleukins.
- The effects of these chemicals triggers numerous symptoms associated with allergic reactions, which includes airway constriction in asthma, local inflammation in eczema, increased mucus secretion in allergic rhinitis, and increased vascular permeability.
- Elkayam et al. (1995) found elevated IgE levels in many autoimmune disorders such as SLE, rheumatoid arthritis (RA), and psoriasis, which indicates a hypersensitivity reaction in such diseases.
- Conrad et al. (2007) suggested regulation of IgE levels via control of B cell differentiation to antibody-secreting plasma cells associated with the "low-affinity" receptor FcεRII (CD23).
- Holm et al. (2011) stated FcεRII may facilitate antigen presentation, which is an IgE-dependent mechanism through which B cells expressing FcεRII may present allergen to and trigger specific T helper cells. This maintains Th2 response, which is one of the hallmarks of increased antibody production.
- Cox et al. (2008) stated the diagnosis of an allergy requires a review of the patient's medical history and detection of an allergen-specific IgE when conducting a skin or blood test.
- Although specific IgE testing is a reliable test for detecting allergies, it doesn't show that indiscriminate IgE testing or testing for immunoglobulin G (IgG) can support allergy diagnosis.
iv. IgG
Immunoglobulin G (IgG) is the most common type of antibody circulating in the blood, which represents about 75% of serum antibodies in humans.
Describe the structure of IgG
- IgG antibodies are large globular proteins composed of 4 peptide chains, which weighs about 150 kDa. It contains 2 identical γ heavy chains weighing about 50 kDa and 2 identical light chains weighing about 25 kDa, forming a tetrameric quaternary structure.
- Disulfide bonds link the 2 heavy chains and to a light chain each. The resulting tetramer contains 2 identical halves, which combined create the Y-like shape. In addition, each end of the fork contains an identical antigen binding site.
- Brian Cobb (2019) found the Fc regions of IgGs carry a highly conserved N-glycosylation site at asparagine 297 in the constant region of the heavy chain.
- Parekh et al. (1985) found the N-glycans connected to this site are predominantly core-fucosylated biantennary structures of the complex type. Furthermore, Stadlmann et al. (2008) discovered trace amounts of these N-glycans containing bisecting GlcNAc and α-2,6-linked sialic acid residues.
- de Haan et al. (2019) reported an association between the N-glycan composition in IgG and a number of autoimmune, infectious and metabolic diseases.
What are the subclasses of IgG?
Note: IgG affinity to Fc receptors on phagocytic cells is specific to individual species from which the antibody originates as well as the class. The structure of the hinge regions influences the biological characteristics of each of the 4 IgG classes.
- A 2013 study proposed the Temporal Model of human IgE and IgE function that suggests IgG3 (and IgE) respond the earliest in an immune response, which facilitates IgG-mediated defences to combine with IgM-mediated defences in eliminating foreign antigens. This subsequently stimulates the production of higher affinity subtypes IgG1 and IgG2.
- In any immune complexes formed, the relative balance of these subclasses may determine the strength of the inflammatory processes that arise.
- If antigen levels persist in the body, high affinity IgG4 production occurs, which reduces inflammation by diminishing FcR-mediated processes.
- Gao et al. (2014) asserted the relative ability of different IgG subclasses to fix complement may explain the anti-donor antibody responses harming a graft after organ transplantation.
- A mouse model of autoantibody mediated anaemia using IgG isotype switch variants of an anti erythrocytes autoantibody demonstrated IgG2a activated complement more effectively than IgG1.
- Furthermore, IgG2a isotype can interact with Fc-γ-R effectively, which suggests higher levels of IgG1 (in regards to IgG2a autoantibodies) is necessary to trigger autoantibody mediated pathology.
v. IgM
- Immunoglobulin M (IgM) is the largest antibody in vertebrates, as well as the first antibody to respond to initial exposure to an antigen.
- In 1937, Heidelberger & Pedersen observed antibodies in horses immunised with pneumococcus polysaccharide that were significantly larger than the typical rabbit γ-globulin, and were estimated to weigh about 990,000 daltons.
- Due to its large size, this antibody was originally labelled as γ-macroglobulin, before subsequent terminology changed it to IgM, with M standing for "macro".
- In 1943, Waldenström discovered homogeneous IgM in several multiple myeloma patients that were produced by tumour cells.
- In the 1960s, mice models of tumours that produce immunoglobulin were developed, which provided a source of homogeneous immunoglobulins of various isotypes, including IgM.
Describe the structure of IgM
- IgG contains µ heavy chain of about 576 amino acids, which includes a variable domain (VH ~110 amino acids), 4 distinct constant region domains (Cµ1, Cµ2, Cµ3, Cµ4, each about 110 amino acids), and a “tailpiece” of about 20 amino acids. The µ heavy chain carries oligosaccharides at 5 asparagine residues.
- Like typical antibodies, it also contains light chains (λ or κ) of about 220 amino acids, which consists of a variable domain (VL, about 110 amino acids), and constant domain (CL, about 110 amino acids long).
- IgM is a multimeric structure in the form of a pentamer that is illustrated in Figure 1.
Figure 1. Schematic model of IgM by Heyman & Shulman (2016)
- A) The µL heterodimer (halfmer) with variable (VH, VL) and constant region (Cµ1, Cµ2, Cµ3, Cµ4tp; CL) domains. The red arrowheads are cysteines that mediate disulfide bonds between µ chains, so that a cysteine disulfide bond appears as a red double arrowhead (red diamond).
- B) The IgM “monomer” (µL)2, with the disulfide bonds between Cµ2 domains symbolised by a red double arrowhead.
- C,D) Both are models of the IgM pentamer containing J cain. As in (B), the red double arrowhead symbolises the disulfide bonds between Cµ2 domains and the disulfide bonds between Cµ4tp domains, and the long double-headed arrows symbolise the Cµ3 disulfide bonds.
- In Figure 1C, the Cµ3 disulfide bonds link µ chains in parallel with the Cµ4tp disulfide bonds, and these disulfide bonds link µ chains in series with the Cµ2 disulfide bonds.
- In Figure 1D, the Cµ2 and Cµ4tp disulfide bonds link µ chains in parallel and both types link µ chains in series with the Cµ3 disulfide bonds.
Figure 2. Alternative methods of linking µ chains
- A,B) They illustrate 2 of numerous possible models of inter-µ chain disulfide bonding in hexameric IgM. Like in Figure 1, a red double arrowhead symbolises the Cµ2 disulfide bonds and the Cµ4tp disulfide bonds, and the long double-headed arrows symbolise Cµ3 disulfide bonds. Each type of disulfide bond (Cµ2-Cµ2; Cµ3-Cµ3; Cµ4tp-Cµ4tp) links µ chains in series with each of the others.
- C) This schematic of pentameric IgM depicts how J chain might link µ chains that aren't connected via Cµ3 disulfide bonds.
What are the molecular requirements for producing polymeric IgM?
- Sørensen et al. (2000) stated the J chain would be essential for creating the polymeric immunoglobulins, and IgA polymerisation depends heavily on the J chain. However, Cattaneo & Neuberger (1987) discovered the formation of IgM was efficient without the J chain.
- The γ heavy chain contains inter-γ bonds that are formed by cysteines in the hinge, meaning each γ chain binds to only one other γ chain. In comparison, the Cµ2 and Cµ3 domains and the tailpiece each include a cysteine that create a disulfide bond with another µ chain. In addition, the cysteines in the Cµ2 domains modulate the creation of monomeric IgM (µL)2.
- Davis et al. (1989) stated the creation of polymeric IgM is hindered if the tailpiece from the µ heavy chain is removed.
- Smith et al. (1995) demonstrated that cells expressing a modified γ heavy chain that includes the tailpiece would result in the production of polymeric IgG.
- Several studies suggested models of pentameric IgM that contains between 1 and 3 J molecules per polymer, which may be due to incomplete radiolabeling or imprecisely quantitating an Ouchterlony line, or due to heterogeneity in the IgM preparations.
Describe the tertiary and quaternary structure of the µ constant region
- The domains of the µ heavy chain consist of overlying β-sheets containing 7 strands, which are stabilised by intra-domain disulfide bonds.
- Müller et al. (2013) described the IgM constant region as having a “mushroom-like” structure, where the Cµ2-Cµ3 domains forms a disk analohous to the head of a mushroom and the Cµ4tp domains protrude like a short stem of a mushroom.
Describe the functions of IgM
IgM interacts with a number of other physiological molecules:
- IgM binds complement component C1 and activates the classical pathway, which results in opsonisation of antigens and cytolysis.
- IgM binds to polyimmunoglobulin receptor (pIgR) in a mechanism that pulls IgM to mucosal surfaces, such as the gut lumen and into breast milk, which depends on J chain.
- 2 other Fc receptors, Fcα/µ-R and Fcµ-R, binds polymeric IgM and IgA, which mediates endocytosis. Its expression in the gut indicates an involvement in mucosal immunity. Shima et al. (2010) found Fcµ-R binds IgM exclusively and regulates cellular uptake of IgM-conjugated antigen.
- When endogenous antibodies are produced after a primary immunisation, it influences the antibody response to a booster immunisation. Depending on the type of antigen and the isotype of the antibody, the regulatory effects can suppress or enhance the antibody response.
- Urbaniak & Greiss (2000) administered IgG antibodies with xenogenic erythrocytes, which lead to virtual suppression of the erythrocyte-specific antibody response. It is used clinically to prevent Rh-negative mothers from becoming immunised against foetal Rh-positive erythrocytes, which significantly decreased the incidence of haemolytic disease in newborns.
- Sörman et al. (2014) found antigen-specific IgM substantially augments the antibody response, particularly in the case of large antigens.
- IgM-mediated enhancement doesn't occur in animals with depleted levels of complement component C3, nor in mutant animals lacking complement receptors 1 and 2. This suggests mutant IgM that doesn't activate complement wouldn't augment the immune response.
vi. IgY
- Immunoglobulin Y (IgY) is a major antibody in bird, reptile, and lungfish blood, as well as chicken egg yolk.
- Leslie & Clem first coined the term Immunoglobulin Y in 1969 when they demonstrated differences between the immunoglobulins found in chicken eggs, and immunoglobulin G.
- Studies found the structure and function of IgY is considerably different to mammalian IgG, and it doesn't cross-react with antibodies matured against mammalian IgG.
Describe the characteristics of IgY
- IgY is composed of 2 light and 2 heavy chains, with its heavy chains weighing roughly 65,100 atomic mass units (amu) and its light chains weighing roughly 18,700 amu. This sums up to a molar mass of around 167,000 amu.
- Compared to IgG, IgY doesn't bind to Protein A, Protein G, or cellular Fc receptors, nor activate the complement system.
What are the functions of antibodies?
 |
| 1. Antibodies (A) and pathogens (B) free roam in the blood. 2. The antibodies bind to pathogens, and can do so in different formations such as: (a) opsonisation, (b) neutralisation, and (c) agglutination. 3. A phagocyte (C) approaches the pathogen, and the Fc region (D) of the antibody binds to one of the Fc receptors (E) of the phagocyte. 4. Phagocytosis occurs as the pathogen is ingested. |
The main categories of antibody function include:
- Neutralisation = Neutralising antibodies block portions of the surface of a bacterial cell or virion to limit its pathogenicity.
- Agglutination = Antibodies combine foreign cells into large clumps that become attractive targets for phagocytosis.
- Precipitation = Antibodies combine serum-soluble antigens, which results in its precipitation out of solution in clumps that become attractive targets for phagocytosis.
- Complement activation (fixation) = Antibodies cling onto a foreign cell, which stimulate complement to attack it with a membrane attack complex. This results in:
- Lysis of the foreign cell
- Facilitation of inflammation by chemotactically attracting inflammatory cells
- Signalling immune cells to present present antibody fragments to T cells, or
- Downregulation of other immune cells to avoid autoimmunity.
- Borghesi & Milcarek (2006) found activated B cells differentiate into either antibody-producing cells called plasma cells or memory cells that last in the body for years afterward in order for the immune system to remember an antigen and respond quickly upon future exposures.
- Antibodies first appear during the prenatal and neonatal stages of life as part of passive immunisation from the mother.
- Early endogenous antibody production typically occurs during the first few years of life, which varies for different types of antibodies.
- Antibodies circulate freely in the bloodstream as part of the humoral immune system and specifically respond to only 1 antigen. They play 3 important roles in immunity:
- Binding to antigens to prevent them from infiltrating or harming cells
- Opsonises the pathogen to trigger removal of pathogens by immune cells such as macrophages
- Triggers other immune responses such as the complement pathway or vasoactive amine degranulation to stimulate destruction of pathogens.
i. Activation of complement
- When antiodies bind to surface antigens (on bacteria, for instance), it subsequently attracts the first component of the complement cascade with their Fc region and trigger activation of the "classical" complement system.
- This leads to the destruction of bacteria in 2 different ways.
- Antibody and complement molecules binds to microbe to mark it for ingestion by phagocytes in a process known as opsonisation.
- Several complement system components form a membrane attack complex to help antibodies directly destroy the bacterium in a process known as bacteriolysis.
ii. Activation of effector cells
- Antibodies bind to pathogens to agglutinate them in order to fight off pathogens replicating outside cells. Since an antibody has at least 2 paratopes, it can bind more than 1 antigen by interacting with identical epitopes expressed on the surfaces of the antigens.
- Pier et al. (2004) found antibodies coat the pathogen to trigger effector functions against the pathogen in cells that recognise their Fc region. When those effector cells recognise the coated pathogens with their Fc receptors, which interact with the Fc region of IgA, IgG and IgE antibodies.
- This triggers the immune cells's effector function that ultimately destroys the invading microbe:
- Phagocytes engulf the pathogen
- Mast cells and neutrophils degranulate
- Natural killer cells release cytokines and cytotoxic molecules
iii. Natural antibodies
- Humans and higher primates produce "natural antibodies" in serum without any previous infection, vaccination, or any exposure to a foreign antigen or passive immunisation.
- Natural antibodies activate the classical complement pathway, which result in lysis of enveloped virus particles prior to the activation of the adaptive immune response.
- In 2009, Vincent Racaniello discovered natural antibodies act against a terminal sugar on glycosylated cell surface proteins called the disaccharide galactose α(1,3)-galactose (α-Gal), which are usually produced by bacteria contained in the human gut.
- Milland & Sandrin (2006) suggested the rejection of xenotransplantated organs is due to natural antibodies circulating in the serum of the recipient binding to α-Gal antigens expressed on the donor tissue.
How is immunoglobulin diversity achieved?
- Mian et al. (1991) stated successful recognition and elimination of numerous types of microbes requires a diverse range of antibodies, which means their amino acid composition has to vary in order to interact with many different antigens.
- Fanning et al. (1996) estimated humans generate about 10 billion different antibodies, each having the capacity to bind a distinct epitope of an antigen. Nevertheless, the number of genes available to produce an enormous repertoire of different antibodies in a single individual is limited by the size of the human genome.
- Nemazee (2006) hypothesised a number of complex genetic mechanisms evolved over time to allow vertebrate B cells generate a diverse pool of antibodies from a relatively small number of antibody genes.
a. Domain variability
- The chromosome region containing heavy chain genes (IGH@) is located on chromosome 14, and the loci containing λ and κ light chain genes (IGL@ and IGK@) are located on chromsomes 22 and 2 respectively in humans.
- One of the domains is labelled the variable domain, which exists in each heavy and light chain of every antibody. However, they can vary in different antibodies produced by distinct B cells.
- The differences between the variable domains are situated on 3 loops called hypervariable regions (HV-1, HV-2 and HV-3) or complementarity-determining regions (CDR1, CDR2 and CDR3). The heavy chain locus contains about 65 different variable domain genes that all vary in their CDRs.
- When these genes are combined with an genes associated with other domains of the antibody, this generates a diverse range of an antibodies with a high degree of variability. This process is known V(D)J recombination, which will be discussed in detail below.
b. V(D)J recombination
https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/v-d-j-recombination
- V(D)J recombination is a process of somatic recombination that situates only in developing lymphocytes during the early stages of T and B cell maturation.
- This process yields a tremendously diverse repertoire of antibodies/immunoglobulins and T cell receptors (TCRs) expressed on B cells and T cells, respectively.
- In mammals, it usually occurs in the primary lymphoid organs; bone marrow for B cells and thymus for T cells. This process randomly rearranges variable (V), joining (J), and sometimes, diversity (D) gene segments, which produces novel amino acid sequences in the antigen-binding regions of immunoglobulins and TCRs.
- This gives antibodies and TCRs the capacity to recognise a wide range of antigens from virtually all pathogens including bacteria, viruses, parasites, and worms as well as "altered self cells" in cancer.
- However, some TCRs and antibodies may react to allergens in nature (such as pollen) or match host tissues and trigger autoimmunity.
- Susumu Tonegawa first discovered the genetic principle for generation of antibody diversity, which lead to him winning the 1987 Nobel Prize in Physiology or Medicine.
Describe the mechanism of V(D)J recombination
i. Key enzymes and components
- This process is moderated by a diverse family of enzymes called VDJ recombinase. The key enzymes involved in this process are:
- Recombination activating genes 1 and 2 (RAG)
- Terminal deoxynucleotidyl transferase (TdT)
- Artemis nuclease, a member of the ubiquitous non-homologous end joining (NHEJ) pathway for DNA repair.
- Other enzymes involved include:
- DNA-dependent protein kinase (DNA-PK)
- X-ray repair cross-complementing protein 4 (XRCC4)
- DNA ligase IV, non-homologous end-joining factor 1 (NHEJ1, also known as Cernunnos or XRCC4-like factor [XLF])
- Paralog of XRCC4 and XLF (PAXX)
- DNA polymerases λ and μ.
- A few enzymes such as RAG and TdT are specific to lymphocytes, whereas other enzymes such as NHEJ components are specific to other cell types and even ubiquitously.
- Maintenance of specificity of recombination requires V(D)J recombinase to recognise and bind to recombination signal sequences (RSSs) bordering the variable (V), diversity (D), and joining (J) genes segments.
- RSSs consist of 3 components: (1) a heptamer of 7 conserver nucleotides, (2) a spacer region 12 or 23 base pairs long, and (3) a nonamer of 9 conserved nucleotides.
- Although the majority of RSSs vary in sequence, the consensus heptamer and nonamer sequences are CACAGTG and ACAAAAACC, respectively.
- Ramsden et al. (1994) found the length of the spacer region is highly conserved, despite its sequence being poorly conserved. The spacer region's length corresponds to roughly one (12 base pairs) or two turns (23 base pairs) of the DNA helix.
- Based on the 12/23 Rule, van Gent et al. (1996) found gene segments primed for recombinaion are typically adjacent to RSSs of different spacer lengths (i.e., one has a "12RSS" and one has a "23RSS").
ii. Process
- V(D)J recombinase (via RAG1 activity) binds a RSS flanking a coding gene segment (V, D, or J) and produces a single-strand nick in the DNA between the first base of the RSS (just before the heptamer) and the coding segment.
- Since this step is energetically neutral (i.e. no requirement for ATP hydrolysis), it leads to the production of the free 3' hydroxyl group and a 5' phosphate group on the same strand.
- Schatz & Swanson (2011) found the reactive hydroxyl group is placed by the recombinase to target the phosphodiester bond of opposite strand, which creates 2 DNA ends: a hairpin (stem-loop) on the coding segment and a blunt end on the signal segment.
- When the formation of DNA nicks and hairpins occurs on both strands simultaneously in a complex, it is called a recombination centre.
- The blunt signal ends are ligated together to create a circular piece of DNA consisting of the intervening sequences between the coding segments known as a signal joint.
- Processing of coding ends starts when DNA-PK binds to each broken DNA end and recruits several other proteins including Artemis, XRCC4, DNA ligase IV, Cernunnos, and several DNA polymerases.
- DNA-PK forms a complex that results in its autophosphorylation, which activates Artemis. This, in turn, opens the coding end hairpins.
- If the coding end hairpins open at the centre, it results in a blunt DNA end. Nevertheless, in numerous cases, the opening occurs off-centre, which leads to additional bases remaining on 1 strand called an overhand. When DNA repair enzymes clear up the overhang, it results in palindromic (P) nucleotides.
- XRCC4, Cernunnos, and DNA-PK align the DNA ends and recruit terminal deoxynucleotidyl transferase (TdT) to add non-templated (N) nucleotides to the coding end.
- Although the addition of nucleotides is relatively random, TdT prefers G/C nucleotides to one strand in a 5' to 3' direction.
- Exonucleases excises bases from the coding ends (including any P or N nucleotides that could have formed). DNA polymerases λ and μ subsequently inserts additional nucleotides as requires to make the 2 ends compatible for joining. This stochastic process may result in any combination of the inclusion of P and N nucleotides, as well as exonucleolytic removal.
- The processed coding ends are ligated together by DNA ligase. This yields a highly variable paratope, even after recombination of the same gene segments.
- This process generates immunoglobulins and T cell receptors to antigens that neither the organism nor its ancestor(s) have previously encountered, which gives the adaptive immune response the capacity to respond to novel pathogens that develop or pathogens that frequently change.
c. Somatic hypermutation
Somatic hypermutation (SHM) is a cellular process that allows the immune system to adapt to novel pathogens it interacts with by diversifying B cell receptors. It involves a programmed mechanism of mutations that influence the variable regions of immunoglobulin genes. Unlike germline mutation, SHM affects only an animal's individual immune cells, and the mutations aren't passed on to the organism's offspring.
- AID requires a single strand to initiate the SHM process.
- Transcription by RNA polymerase II (RNA Pol II) exposes the single-stranded DNA template for AID.
- AID deaminates a cytosine to produce an uracil, which can subsequently be processed by different pathways.
- Replication over the uracil results in C to T or G to A transition mutations.
- Processing by uracil DNA glycosylase (UNG) generates an abasic site (Φ) that is cleaved by the apurinic/apyrimidinic endonuclease (APE1), which removes this site and subsequently Polβ resynthesises the DNA.
- Recognition of the U-G mismatch by MutSα (torus shape) is followed by the action of Exo1 and Polη spreads mutations (indicated as "N") to surrounding A-T nucleotides.
- UNG and Msh2/Msh6 can also get involved in high fidelity base excision repair (BER) and mismatch repair (MMR) pathways, which results in error-free repair.
- While DNA polymerases synthesise new DNA, it tends to introduce mutations at the position of the deaminated cytosine itself or neighbouring base pairs.
- Transcription and translation of the immunoglobulin variable region DNA occurs during B cell division.
- These mutations in the rapidly proliferating population of B cells results in the production of B cells with various receptors and varying specificity for the antigen, from which the B cells with highest affinities for the antigen are selected.
- Oprea (1999) stated the B cells with the highest affinity are selected to differentiate into antibody-producing plasma cells and long-lived memory B cells.
- The hypermutation process recruits cells that auto-select against the biosignature of an organism's own cells. Metzger (2011) suggested failure of this auto-selection process may result in auto-immune responses.
Describe the models of SHM
i. DNA deamination model
- The Neuberger "DNA deamination model" is based on the activation-induced cytidine deaminase (AID) and short-patch error-prone DNA repair by DNA polymerase-eta operating around AID C-to-U lesions.
- It only partially explains the origins of the entire spectrum of somatic mutations at A:T and G:C base pairs observed in SHM in B lymphocytes in vivo during an antigen-driven immune response. However, it fails to explain the mechanism behind the creation of strand biased mutations.
- Studies highlighted the key feature of this model is its critical dependence on the gap-filling, error-prone, DNA repair synthesis properties of DNA polymerase-eta targeting A:T base pairs at AID-mediated C-to-U lesions or ssDNA nicks.
- Delbos et al. (2007) identified DNA polymerase as the only known error-prone polymerase involved in SHM in vivo.
- Franklin et al. (2004) stated studies tend to ignore the Y family DNA polymerase enzyme is also an efficient reverse transcriptase as demonstrated in in vitro assays.
ii. Reverse transcriptase model
- A more controversial hypothesis is an RNA/Reverse Transcriptase-based mechanism, which attempts to explain the creation of the entire spectrum of strand-biased mutations at A:T and G:C base pairs. Mutations of A are observed to exceed mutations of T (A>>>T) and mutations of G are observed to exceed mutations of C (G>>>C).
- This model suggests error-prone cDNA synthesis occurs via an RNA-dependent DNA polymerase that copies the base modified Ig pre-mRNA template and integrated the now error-filled cDNA copy back into the normal chromosomal site.
- Steele (2009) identified the errors in Ig pre-mRNA are due to a combination of adenosine-to-inosine (A-to-I) RNA editing, and RNA polymerase II transcription elongation complex copying uracil and abasic sites (arising as AID-mediated lesions) into the nascent pre-mRNA using the transcribed (TS) DNA as the copying template strand.
- Steele (2016) stated this mechanisms essentially depends on AID C-to-U DNA lesions and long tract error-prone cDNA synthesis of the transcribed strand by DNA polymerase-eta functions as a reverse transcriptase.
- Steele (2016) critically evaluarted the molecular evidence for and against each mechanism and demonstrated all the findings on SHM published since 1980 directly or indirectly this RNA/RT-based mechanism.
- Zheng et al. (2017) demonstratred Adenosine Deaminase enzymes acting on RNA (ADARs) can A-to-I edit both the RNA and DNA moieties of RNA:DNA hybrids in biochemical assays in vitro. It's found RNA:DNA hybrids of about 11 nucleotides in length are transient structures that materialise at transcription bubbles in vivo during RNA polymerase II elongation.
- Findings by Zheng et al. asserted that the RNA moiety would need to be initially A-to-I RNA edited before being reverse transcribed and integrated to produce the strong A>>>T strand biased mutation signatures at A:T base pairs observed in all SHM and cancer hypermutation data sets.
- Lindley (2013) discovered that the Ig-SHM-like strand-biased mutations in cancer genome protein-coding genes are additionally in "codon-context". She described this process 'targeted somatic mutation (TSM)' to emphasise somatic mutations are significantly more targeted than previously thought in somatic tissues associated with disease.
- Lindley et al. (2016) suggested the TSM process serves as an "in-frame DNA reader", by which DNA and RNA deaminases at transcribed regions are directed in their mutagenic action, by the codon reading frame of the DNA.
d. Affinity maturation
- Affinity maturation is a process that involves TFH cell-activated B cells create antibodies with increased affinity for antigen during the course of an immune response. If a host is repeated exposed to the same antigen, it creates antibodies of successively higher affinities.
- This process mainly occurs on membrane immunoglobulin of germinal centre B cells and as a direct result of somatic hypermutation (SHM) and selection by TFH cells.
Describe the affinity maturation process
i. In vivo
Affinity maturation is suggested to involve 2 interrelated processes, which occurs in the germinal centres of the secondary lymphoid organs.
1. Somatic hypermutation:
- This process involves mutations in the variable, antigen-binding coding sequences (i.e. complementarity-determining regions (CDR)) of the immunoglobulin genes. The mutation rate is up to 1 million times higher than in cell lines outside the lymphoid system.
- Teng & Papavasiliou (2007) estimated the increased mutation yields 1-2 mutations per CDR, and therefore, per cell generation. This influences the binding specificity and binding affinities of the resultant antibodies.
2. Clonal selection:
- This process involves B cells competing for limiting growth resources, such as the availability of antigen and paracrine signals from TFH cells.
- The follicular dendritic cells (FDCs) of the germinal centres present antigen to the B cells, and the B cell progeny with the highest affinities for antigen have a greater likelihood of positive selection leading to their survival.
- Positive selection process is determined by a cross-stimulation between TFH cells and their cognate antigen presenting GC B cells.
- Since a limited number of TFH cells situate in the germinal centre, only highly competitive B cells conjugate with TFH cells and therefore receive T cell-dependent survival signals. On the other hand, B cell progeny with minimal affinity with antigen won't be positively selected, hence eliminated.
- Roskos et al. (2007) stated several rounds of selection would result in antibodies with effectively increased affinities for antigen.
ii. In vitro
- The in vitro affinity maturation process shares similarities with in vivo affinity maturation, as it is also based on the principles of mutation and selection. It optimises antibodies, antibody fragments or other peptide molecules such as antibody mimetics.
- Along with chain shuffling, radiation, chemical mutagens or error-prone PCR is utilised to induce random mutations inside the CDRs, which increases the genetic diversity of the antibodies.
- Roskos et al. (2007) stated 2 or 3 rounds of mutation and selection utilising display methods such as phage display typically yields antibody fragments with affinities with low molar mass.
e. Clonal Selection
- This scientific theory attempts to describe the functions of cells of the immune system (lymphocytes) in response to specific antigens invading the body.
- In 1900, Paul Ehrlich proposed the "side chain theory" of antibody production that suggested particular cells express different "side chains" on their surface that interact with different antigens. He suggested a matching side chain would bind to an antigen, which subsequently halts the production of all other side chains in the cell and initiates the synthesis and release of antigen-binding side chain as a solubly antibody.
- In 1955, Danish immunologist Niels Jerne conjectured the existence of a diverse range of soluble antibodies in the serum prior to any infection. When an antigen infiltrates the body, it is virtually guaranteed only one type of antibody would be selected to match it. Burnet (1976) suggested this occurs by specific cells phagocytosing the immune complexes and subsequently reproducing the same antibody structure.
- In 1957, David W. Talmage conjectured that antigens bind to antibodies on the surface of antibody-producing cells, which are specifically selected for proliferation whose synthesised antibodies has affinity for the antigen.
- In 1957, Australian immunologist Frank Macfarlane Burney published a paper titled "A modification of Jerne's theory of antibody production using the concept of clonal selection" in the Australian Journal of Science. He elaborated Talmage's hypotheses and labelled the resulting theory the "clonal selection theory".
- In his 1959 book The Clonal Selection Theory of Acquired Immunity, Burnet expounded the clonal selection memory, where he defined immunological memory as the cloning of 2 types of lymphocyte.
- He suggested one lymphocyte clone serves to fight infection while the other clone survives and remains in the immune system for a long time to build immunity to that antigen.
- Burnet's hypothesis suggests a specific antigen activates (i.e. selects) only its counter-specific cell in a pre-existing group of lymphocytes (specifically B cells), which subsequently stimulates that particular cell to multiply, yielding identical clones for antibody production.
- In 1958, Gustav Nossal and Joshua Lederberg demonstrated only one B cell always produces only one antibody, which was the first piece of evidence to directly support the clonal selection theory.
Postulates:
- Each lymphocyte bears one type of receptor with a unique specificity that was produced by V(D)J recombination.
- Occupation of the receptor is required to activate the lymphocyte.
- The differentiated effector cells derived from an activated lymphocyte express receptors of identical specificity as the parent cell.
- Those lymphocytes expressing receptors for self molecules (i.e., endogenous antigens produced within the body) are signalled for destruction at an early stage.
f. Class switching
- Also known as isotype switching, isotypic commutation or class-switch recombination (CSR), immunoglublin class switching is a biological mechanism that alters a B cell's production of immunoglobulin from one type to another, e.g. IgM to IgG.
Describe the mechanism of class switching
- This process occurs after a mature B cell is activated via its membrane-bound antibody molecule (or B cell receptor) to produce different classes of antibody.
- The antibody isotypes share the same variable domains as the original antibody produced in the immature B cell during the process of V(D)J recombination, but the constant domains in their heavy chains vary.
- Naïve mature B cells initially generate both IgM and IgD, which are the first 2 heavy chain segments in the immunoglobulin locus. When these B cells are activated by antigen, they begin proliferating.
- If the activated B cells interacts with certain signaling molecules via their CD40 and cytokine receptors (both modulated by T helper cells), this triggers antibody class switching of the B-cells antibodies to produce IgG, IgA or IgE antibodies.
- It results in changes to the antibody heavy chain's constant region, but the variable region remains unchanged. Because the variable region remains the same, class switching doesn't influence antigen specificity. This means the antibody retains affinity for the same antigens, but it interacts with different effector molecules.
- This results in different daughter cells from the same activated B cell generating antibodies of different isotypes e.g. (IgG1, IgG2, etc.).
In humans, the order of the heavy chain exons is:
- μ - IgM
- δ - IgD
- γ3 - IgG3
- γ1 - IgG1
- α1 - IgA1
- γ2 - IgG2
- γ4 - IgG4
- ε - IgE
- α2 - IgA2
Class switch recombination (CSR):
- Sections of the antibody heavy chain locus are excised from the chromosome, and the gene segments adjacent to the removed section are rejoined to retain a functional antibody gene that produces antibody of a different isotype.
- Double-stranded breaks occur in DNA at conserved nucleotide motifs called switch (S) regions, which are upstream from gene segments that encode the constant regions of antibody heavy chains. They usually situate adjacent to all heavy chain constant region genes with the exception of the δ-chain.
- DNA is nicked and snipped at 2 selected S-regions by a series of enzymes, including activation-induced (cytidine) deaminase (AID), uracil DNA glycosylase and apyrimidic/apurinic (AP)-endonucleases.
- The intervening DNA between the S-regions is then removed from the chromsome, which eliminates unwanted μ or δ heavy chain constant region exons and facilitates substitution of a γ, α or ε constant region gene segment.
- The free ends of the DNA are rejoined by non-homologous end joining (NHEJ) that connects the variable domain exon to the appropriate downstream constant domain exon of the antibody heavy chain. Yan et al. (2007) found the free ends of DNA may be reconnected by an alternative pathway biased toward microhomology joins if NHEJ doesn't occur.
- Although CSR is typically a deletional process that rearranges a chromosome in "cis", it can also occur as an inter-chromosomal translocation that combines immunoglobulin heavy chain genes from both alleles (depending upon the Ig class).
What gene regulatory sequences are responsible for class switching?
- Class switching requires S regions to be initially transcribed and then spliced out of the immunoglobulin heavy chain transcripts (where they situate within introns).
- Pinaud et al. (2011) found chromatin remodelling, accessibility to transcription and to AID and synapsis of fragmented S regions are regulated by a super-enhancer, located downstream the most distal Calpha gene, the 3' regulatory region (3'RR).
- Péron et al. (2012) stated AID occasionally targets 3'RR super-enhancer, which stimulates DNA breaks and junction with Sμ. This subsequently removes the Ig heavy chain locus and defines locus suicide recombination (LSR).
Table 1. Class switching in mice
Table 2. Class switching in humans
g. Specificity designations
- A monospecific antibody has affinity for the same antigen or epitope, they are labelled monospecific, whereas a bispecific antibody has affinity for 2 different antigens or 2 different epitopes on the same antigen.
- If a group of antibodies have affinity for various antigens or microorganisms, they are known as polyvalent (or unspecific).
h. Asymmetry
- Asymmetrical (heterodimeric) antibodies provide increased flexibility and novel formats for binding a variety of drugs to the antibody arms.
- The "knobs-into-holes" format is specific to the heavy chain component of the constant region in heterodimeric antibodies.
- The "knobs" component is constructed by replacement of a small amino acid with a larger amino acid in order to fit into the "hole" component, which is constructed by replacement of a large amino acid with a smaller amino acid.
- Disulfide bonds between each chain links the "knobs" with the "holes" to form the "knobs-into-holes" shape, which facilitates antibody dependent cell mediated cytotoxicity.
- Single chain variable fragments (scFv) are linked to the variable domain of the heavy and light chain via a short linker peptide. This linker contains glycine for flexibility, and serine/threonine for specificity.
- Gunasekaran et al. (2010) found a hinge region links 2 different scFv fragments to the constant domain of the heavy chain or the constant domain of the light chain. This provides bispecificity to the antibody, meaning it has binding specificities of 2 different antigens.
- Gao et al. (1999) described artificial antibodies as diverse protein motifs that employ the same functions as a typical antibody molecule, but aren't limited by the loop and framework structural constraints of the natural antibody.
i. Junctional diversity
Junctional diversity is defined as the introduction of DNA sequence variations introduced by the improper joining of gene segments during the process of V(D)J recombination.
Describe the process of junctional diversity
- This phenomenon includes somatic recombination or V(D)J recombination, during which the different variable gene segments of TCRs and immunoglobulins are rearranged and unutilised segments are spliced out.
- The process introduces double-strand breaks between the required segments, and the ends subsequently create hairpin loops and rejoin together to create a single strand.
- Janeway et al. (2005) stated this joining process is prone to errors, which may lead to extra nucleotides being added or subtracted and, therefore, result in junctional diversity.
- Junctional diversity would result in frame-shift mutations and, therefore, creation of non-functional proteins.
- Recombination activating gene-1 and -2 (RAG1 and RAG2), along with DNA repair proteins, such as Artemis, are involved in single-stranded cleavage of the hairpin loops and addition of a series of palindromic, 'P' nucleotides.
- Terminal deoxynucleotidyl transferase (TdT) enzyme subsequently adds more random 'N' nucleotides.
- Wyman & Kanaar (2006) described exonucleases splice out unpaired nucleotides and the gaps are filled by DNA synthesis and repair machinery. However, the mechanism behind how exonucleases are responsible for this truncation of the junction is not well-known.
Describe the physiological regulation of the immune system
 |
| This diagram is a time-course of an immune response starting with the initial pathogen encounter, (or initial vaccination) and results in the formation and maintenance of active immunological memory. |
Studies reported close communication between the immune system and other bodily systems such as endocrine and nervous systems. Wilcox et al. (2017) stated the immune system plays an essential role in embryogenesis, as well as regeneration and tissue repair.
a. Hormones
- The sensitivity of the immune system can be modulated by a number of different hormones.
- A number of female sex hormones, such as oestrogen and progesterone, are known to stimulate both the innate and adaptive immune responses. On the other hand, Fimmel & Zouboulis (2005) found male sex hormones such as testosterone suppressed immune function.
- Nagpal et al. (2005) found other hormones such as prolactin, growth hormone and vitamin D play a role in regulating the immune system.
b. Vitamin D
- Despite a number of cellular studies suggesting vitamin D has receptors and possible functions in the immune system, a 2021 report by the US Institute of Health didn't find any clinical evidence that proves vitamin D deficiency increases the risk for immune diseases or vitamin D supplementation lowers immune disease risk.
- A 2011 United States Institute of Medicine report concluded no direct link between calcium or vitamin D intake and outcomes related to... immune functioning and autoimmune disorders, and infections.
c. Sleep and rest
- Bryant et al. (2004) asserted the immune system function is influenced by sleep deprivation.
- Krueger & Majde (2003) stated regulation of non-rapid eye movement (REM) sleep may be influenced by complex feedback loops featuring a number of cytokines produced in response to infection, such as interleukin-1 (IL-1) and tumor necrosis factor-α (TNF-α).
- Majde & Krueger (2005) suggested the immune response to infection lead to changes in the sleep cycle, including an increase in slow-wave sleep relative to REM sleep.
- Taylor et al. (2017) found sleep deprived people who received active immunisations decreased antibody production and diminished immune response, compared to adequately rested people.
- Furthermore, proteins such as NFIL3, were discovered to be closely associated with both T-cell differentiation and circadian rhythms, which may be impacted by the disruptions of natural light and dark cycles through instances of sleep deprivation.
- Krueger (2008) stated these disturbances can result in increased risk of chronic conditions such as chronic pain, asthma, and heart disease.
- During the early slow-wave-sleep stage, reductions in blood levels of cortisol, epinephrine, and norepinephrine increases the blood levels of the hormones leptin, pituitary growth hormone, and prolactin.
- These signals trigger a pro-inflammatory state by releasing the pro-inflammatory cytokines interleukin-1 (IL-1), interleukin-12 (IL-12), TNF-alpha (TNF-α) and IFN-gamma (IFN-γ). These cytokines subsequently induce a number of immune functions such as immune cell activation, proliferation, and differentiation.
- The levels of undifferentiated or immaturely differentiated cells such as naïve and central memory T cells peak during the evolving adaptive immune response.
- The group of hormones produced during this period (such as leptin, pituitary growth hormone, and prolactin) facilitates the interactions between APCs and T-cells, as well as skews the Th1/Th2 cytokine balance towards an environment that supports Th1. This increases the overall Th cell proliferation, and naïve T cell migration to lymph nodes.
- Besedovsky et al. (2012) suggested this process augments the production of long-lasting immune memory through the stimulation of Th1 immune responses.
- During periods of wakefulness, levels of differentiated effector cells, such as cytotoxic natural killer cells and cytotoxic T lymphocytes, peak to induce an effective response against any intruding pathogens. It's known levels of anti-inflammatory molecules, such as cortisol and catecholamines, peak during wakefulness periods too.
- Inflammation during wakefulness can manifest in cognitive and physical impairments, while inflammation during deep sleep may be caused by increased levels of melatonin.
- A 2014 study stated inflammation induces oxidative stress and melatonin during periods of sleep actively hinders the production of free radicals.
d. Repair and regeneration
- Innate immune cells such as macrophages and neutrophils, as well as other immune cells such as γδ T cells, innate lymphoid cells (ILCs), and regulatory T cells (Tregs), play a crucial role in tissue repair after an injury.
- Godwin et al. (2017) hypothesised organisms that can regenerate limbs (such as axolotls) may be less immunocompetent than organisms unable to regenerate.
If you have read everything up to this point, you can appreciate the sheer complexity of the body's immune system and its sophisticated capacity to develop mechanisms that not only attack pathogens that infiltrate the body but also remember it after eliminating it from the body, as well as prepare it for any pathogen the immune system it has not encountered before. So theoretically, our immune system can develop immunity to a majority of, if not, all pathogens that exist on Earth.
For a majority of your life, you wouldn't be aware your immune system is consistently active 24/7 to keep you alive and healthy unless you begin feeling ill or show symptoms of sickness. Thanks to the immune system, you and every other animal species are able to defend against the microscopic pathogens that threaten to kill us. It is one of the key bodily systems behind our longer life expectancies over the generations.
However, the immune system is still susceptible to being compromised if any of its mechanisms fail or evaded by stealthy means. The next blog part will discuss the diseases and medical conditions associated with failures of the immune system, the medical manipulations of the immune system, and how immunology came to be and evolved over time.
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