Unit 4: Major histocompatibility complex; T and B lymphocytes - Subjective Questions

The Major Histocompatibility Complex (MHC) is a large genomic region containing a cluster of closely linked genes that code for cell-surface glycoproteins essential for antigen presentation to T lymphocytes.

Key Points:

• In humans, MHC is called the HLA (Human Leukocyte Antigen) complex, located on the short arm of chromosome 6.
• In mice, it is called the H-2 complex, located on chromosome 17.

Significance:

• Antigen presentation: MHC molecules bind peptide fragments of antigens and display them to T cells.
• Self vs. non-self discrimination: Enables the immune system to distinguish self from foreign antigens.
• Graft rejection: MHC differences between donor and recipient are the primary cause of transplant rejection.
• T-cell activation: T cells recognize antigen only when presented on MHC (MHC restriction).
• Immune regulation: MHC polymorphism ensures a population can respond to a wide range of pathogens.

MHC Class I molecules are membrane-bound glycoproteins present on all nucleated cells.

Structural Components:

• Heavy (α) chain: A transmembrane polypeptide (~45 kDa) with three extracellular domains — α1, α2, and α3.
• β2-microglobulin: A small (~12 kDa) non-covalently associated polypeptide encoded outside the MHC region (chromosome 15 in humans). It does not span the membrane.

Peptide-binding groove:

• Formed by the α1 and α2 domains.
• Closed at both ends, accommodating short peptides of 8–10 amino acids.

Other features:

• The α3 domain is immunoglobulin-like and interacts with the CD8 co-receptor on cytotoxic T cells.
• A transmembrane region anchors the molecule to the cell surface.

Function: Presents endogenous (intracellular) antigens to CD8+ cytotoxic T lymphocytes.

Summary: Class I presents intracellular antigens for cytotoxic responses, while Class II presents extracellular antigens for helper responses.

MHC Class I Distribution:

• Expressed on all nucleated cells and platelets.
• Absent on mature red blood cells (RBCs).
• Highest expression on lymphocytes; lower on liver, muscle, and neural cells.

MHC Class II Distribution:

• Restricted to professional antigen-presenting cells (APCs):
  • Dendritic cells
  • Macrophages
  • B lymphocytes
• Also on thymic epithelial cells and (when induced by IFN-γ) endothelial cells.

Functional Importance:

• Class I on all cells: Allows any infected cell (e.g., virus-infected) to display endogenous antigens and be recognized/killed by CD8+ cytotoxic T cells — a universal surveillance system.
• Class II on APCs only: Restricts activation of CD4+ helper T cells to specialized cells that sample extracellular pathogens, ensuring controlled and appropriate initiation of adaptive responses.

This division of labor ensures efficient immune surveillance against both intracellular and extracellular threats.

The endogenous pathway processes antigens synthesized within the cell (e.g., viral or tumor proteins) for presentation on MHC Class I to CD8+ T cells.

Steps:

1. Protein degradation:

   • Cytosolic proteins are tagged with ubiquitin and degraded by the proteasome (specifically the immunoproteasome) into short peptides (8–10 aa).

2. Peptide transport:

   • Peptides are transported from the cytosol into the endoplasmic reticulum (ER) by the TAP (Transporter associated with Antigen Processing) protein, which is ATP-dependent.

3. Peptide loading:

   • In the ER, newly synthesized MHC Class I molecules are stabilized by chaperones (calnexin, calreticulin, tapasin, ERp57).
   • The peptide-loading complex loads the peptide onto the MHC Class I groove.

4. Transport to surface:

   • The stable peptide–MHC Class I complex travels via the Golgi apparatus to the cell surface.

5. Presentation:

   • The complex is displayed to CD8+ cytotoxic T lymphocytes.

Outcome: Recognition triggers killing of infected or abnormal cells.

The exogenous pathway processes antigens taken up from outside the cell for presentation on MHC Class II to CD4+ helper T cells.

Steps:

1. Antigen uptake:

   • Extracellular antigens are internalized by APCs via phagocytosis, endocytosis, or receptor-mediated endocytosis.

2. Endosomal degradation:

   • Antigens pass through increasingly acidic vesicles (early endosome → late endosome → lysosome).
   • Acidic proteases (cathepsins) degrade proteins into peptides (13–25 aa).

3. MHC Class II synthesis and protection:

   • MHC Class II is synthesized in the ER and associated with the invariant chain (Ii), which blocks the peptide-binding groove to prevent premature loading.

4. CLIP and HLA-DM:

   • The invariant chain is degraded, leaving a fragment called CLIP in the groove.
   • HLA-DM catalyzes removal of CLIP and exchange with the antigenic peptide.

5. Vesicle fusion and loading:

   • The MHC Class II vesicle fuses with the peptide-containing endosome (MIIC compartment), where peptide loading occurs.

6. Presentation:

   • The peptide–MHC II complex is displayed to CD4+ helper T cells.

Outcome: Activation of helper T cells that coordinate humoral and cellular immunity.

Cross-presentation is the process by which certain antigen-presenting cells (mainly dendritic cells) present exogenous antigens on MHC Class I molecules to CD8+ cytotoxic T cells, instead of the conventional MHC Class II route.

Mechanism:

• Exogenous antigens are internalized into endosomes/phagosomes.
• Instead of full lysosomal degradation, antigens are exported into the cytosol.
• They are then processed by the proteasome and loaded onto MHC Class I via the TAP-dependent pathway (or via a vacuolar TAP-independent route).

Immunological Importance:

• Priming CD8+ T cells: Allows activation of cytotoxic T cells against pathogens that do not directly infect dendritic cells (e.g., viruses infecting other cell types).
• Anti-tumor immunity: DCs can present tumor antigens acquired from dying tumor cells.
• Vaccine design: Critical for developing vaccines that stimulate CTL responses.
• Cross-tolerance: Can also induce tolerance to self-antigens when presented without co-stimulation.

The B-cell receptor (BCR) is a membrane-bound complex responsible for recognizing specific antigens.

Components:

1. Membrane-bound immunoglobulin (mIg):

   • A surface antibody (usually IgM and IgD on naïve B cells).
   • Composed of two heavy chains and two light chains.
   • Contains variable (V) regions that form the antigen-binding site.
   • The mIg recognizes antigen but has a short cytoplasmic tail and cannot signal alone.

2. Ig-α (CD79a) and Ig-β (CD79b) heterodimer:

   • Disulfide-linked signaling molecules associated with mIg.
   • Contain ITAMs (Immunoreceptor Tyrosine-based Activation Motifs) in their cytoplasmic tails.
   • Transduce the signal after antigen binding.

Function:

• The mIg provides antigen specificity.
• The Ig-α/Ig-β complex transmits activation signals into the cell.

Unlike the TCR, the BCR can recognize native, unprocessed antigens directly.

B-cell activation requires antigen recognition through the BCR and, usually, additional signals.

T-Dependent (TD) Activation:

• Occurs with protein antigens.

Steps:

1. Signal 1: BCR binds antigen; the antigen is internalized, processed, and presented on MHC Class II.
2. Signal 2: A CD4+ helper T cell recognizes the peptide–MHC II complex. The interaction of CD40 (B cell) with CD40L (T cell) provides co-stimulation.
3. Cytokine help: Helper T cells secrete cytokines (IL-4, IL-21) that drive proliferation and differentiation.

Outcomes: Isotype (class) switching, affinity maturation, memory B cell formation, and germinal center reactions.

T-Independent (TI) Activation:

• Occurs with non-protein antigens (e.g., polysaccharides, LPS).

Types:

• TI-1 antigens: Polyclonal activators (e.g., LPS) that stimulate B cells regardless of specificity.
• TI-2 antigens: Highly repetitive epitopes that crosslink many BCRs.

Outcomes: Mainly IgM production, little class switching, weak memory, and no germinal center.

Key difference: TD responses are stronger, produce memory and high-affinity antibodies, while TI responses are rapid but limited.

T-Cell Receptor (TCR) Structure:

• A heterodimer consisting of two polypeptide chains linked by a disulfide bond.
• Most T cells express the αβ TCR (~95%); a minority express the γδ TCR.
• Each chain has:
  • A variable (V) region — forms the antigen-binding site.
  • A constant (C) region.
  • A transmembrane region and a short cytoplasmic tail.

Associated Signaling Complex:

• The TCR is non-covalently associated with the CD3 complex (γ, δ, ε chains) and ζ (zeta) chains, which contain ITAMs for signal transduction.

Antigen Recognition:

• TCR recognizes processed peptide antigens presented by MHC molecules (MHC restriction) — it does not bind free antigen.

Comparison with BCR:

MHC restriction refers to the phenomenon whereby a T cell recognizes an antigenic peptide only when it is presented in association with a specific MHC molecule on the surface of another cell.

Key Points:

• T cells cannot recognize free/soluble antigen; they require peptide–MHC complexes.
• CD8+ T cells are MHC Class I restricted — they recognize peptides presented on MHC I.
• CD4+ T cells are MHC Class II restricted — they recognize peptides presented on MHC II.

Experimental Basis:

• Demonstrated by Zinkernagel and Doherty (1974), who showed that virus-specific cytotoxic T cells could only kill infected cells sharing the same MHC haplotype. They were awarded the Nobel Prize in 1996.

Significance:

• Ensures T cells respond to antigens only in the context of self-cells, linking antigen recognition to cell-to-cell contact.
• Establishes the dual specificity of the TCR — for both peptide and self-MHC.

T-cell maturation occurs in the thymus, where progenitor cells from the bone marrow develop into mature T cells.

Stages (based on CD4/CD8 expression):

1. Double-Negative (DN) Stage:

   • Thymocytes lack both CD4 and CD8 (CD4⁻CD8⁻).
   • Located in the subcapsular region of the cortex.
   • TCR β-chain gene rearrangement occurs; the pre-TCR is formed.
   • Progresses through DN1 → DN4 substages.

2. Double-Positive (DP) Stage:

   • Thymocytes express both CD4 and CD8 (CD4⁺CD8⁺).
   • TCR α-chain rearrangement completes the αβ TCR.
   • Cells undergo positive and negative selection in the cortex and medulla.

3. Single-Positive (SP) Stage:

   • Cells express either CD4 or CD8 (CD4⁺ or CD8⁺).
   • Mature naïve T cells that exit the thymus to peripheral lymphoid organs.

Key Events:

• Gene rearrangement by RAG-1/RAG-2 generates TCR diversity.
• Thymic selection ensures self-tolerance and MHC restriction.

Only about 1–5% of thymocytes survive selection and mature.

During T-cell development in the thymus, immature thymocytes undergo two critical selection processes to ensure a functional and self-tolerant T-cell repertoire.

Positive Selection:

• Occurs in the thymic cortex.
• Involves interaction of double-positive (CD4⁺CD8⁺) thymocytes with cortical thymic epithelial cells (cTECs) expressing self-MHC + self-peptides.
• Purpose: To select T cells whose TCRs can recognize self-MHC (i.e., are MHC-restricted).
• Thymocytes that bind self-MHC with moderate affinity survive; those that cannot bind die by neglect (apoptosis).
• Determines CD4 vs CD8 lineage commitment (MHC II → CD4; MHC I → CD8).

Negative Selection:

• Occurs mainly in the thymic medulla (and cortico-medullary junction).
• Involves dendritic cells and medullary thymic epithelial cells (mTECs) presenting self-antigens (aided by the AIRE gene).
• Purpose: To eliminate T cells that bind self-antigens too strongly, preventing autoimmunity (central tolerance).
• Strongly self-reactive thymocytes undergo apoptosis (clonal deletion).

Summary:

• Positive selection ensures usefulness (MHC recognition).
• Negative selection ensures safety (self-tolerance).

B-cell maturation is antigen-independent and occurs in the bone marrow, progressing through defined stages marked by immunoglobulin gene rearrangement.

Stages:

1. Pro-B cell:

   • Earliest committed stage.
   • Heavy-chain D-J and V-DJ rearrangement begins.
   • Expresses markers like CD19, CD45R.

2. Pre-B cell:

   • Complete μ heavy chain is produced and expressed with the surrogate light chain as the pre-BCR.
   • Signals successful heavy-chain rearrangement and triggers proliferation.
   • Light-chain (κ or λ) rearrangement begins.

3. Immature B cell:

   • Expresses complete membrane IgM (mIgM) on the surface.
   • Undergoes negative selection — strongly self-reactive cells are deleted, anergized, or undergo receptor editing.

4. Mature (naïve) B cell:

   • Expresses both IgM and IgD.
   • Exits bone marrow and migrates to peripheral lymphoid organs, ready to respond to antigen.

Key Points:

• Gene rearrangement by RAG-1/RAG-2 generates antibody diversity.
• Central tolerance ensures removal of self-reactive B cells.

The invariant chain (Ii) and CLIP play crucial regulatory roles in ensuring correct peptide loading onto MHC Class II molecules.

Invariant Chain (Ii):

• A non-polymorphic protein that associates with newly synthesized MHC Class II in the endoplasmic reticulum.
• Functions:
  • Blocks the peptide-binding groove, preventing premature binding of endogenous peptides in the ER.
  • Guides transport of MHC Class II to the endosomal/lysosomal compartment (MIIC) through targeting signals.
  • Promotes proper folding of the MHC II molecule.

CLIP (Class II-associated Invariant chain Peptide):

• As the invariant chain is degraded by proteases in the endosome, a small residual fragment called CLIP remains lodged in the peptide-binding groove.
• CLIP acts as a placeholder until an antigenic peptide is ready.

Role of HLA-DM:

• The non-classical MHC molecule HLA-DM catalyzes the removal of CLIP and facilitates loading of high-affinity antigenic peptides.

Significance: This system ensures MHC Class II presents exogenous peptides rather than endogenous ones, maintaining pathway specificity.

The MHC is characterized by two important genetic features: polygeny and polymorphism.

Polygeny:

• Refers to the presence of several different MHC genes within the complex.
• Class I: HLA-A, HLA-B, HLA-C.
• Class II: HLA-DP, HLA-DQ, HLA-DR.
• Each individual therefore expresses multiple different MHC molecules with distinct peptide-binding specificities.

Polymorphism:

• Refers to the existence of multiple alleles (variants) of each MHC gene within the population.
• MHC genes are the most polymorphic genes known, with thousands of alleles.
• The polymorphism is concentrated in the peptide-binding groove, altering which peptides can be bound.

Codominant Expression:

• Both maternal and paternal MHC alleles are expressed, increasing the variety of MHC molecules per individual.

Biological Significance:

• Broad antigen presentation: Enables recognition of a wide diversity of pathogen-derived peptides.
• Population-level survival: Ensures that at least some individuals can respond to any given pathogen, protecting the species.
• Transplantation: High polymorphism makes MHC matching between donors and recipients difficult.

Full activation of a naïve T cell requires two distinct signals provided by an antigen-presenting cell (APC).

Signal 1 — Antigen-specific signal:

• Interaction of the TCR with the peptide–MHC complex on the APC.
• Co-receptors CD4 (MHC II) or CD8 (MHC I) stabilize this interaction.
• Provides specificity.

Signal 2 — Co-stimulatory signal:

• Interaction between CD28 on the T cell and B7 molecules (CD80/CD86) on the APC.
• Confirms that the antigen is presented in a proper immunological context (e.g., during infection).

Signal 3 (often included) — Cytokine signal:

• Cytokines secreted by the APC direct the differentiation of the T cell into specific subsets.

Consequences of Only Signal 1 (no co-stimulation):

• The T cell becomes anergic (functionally unresponsive) or undergoes apoptosis.
• This mechanism promotes peripheral tolerance, preventing responses to self-antigens presented in the absence of danger signals.

Summary: Both antigen recognition and co-stimulation are essential; the two-signal model prevents inappropriate immune activation.

T cells are divided into two major populations based on their TCR chain composition.

Key Points:

• αβ T cells dominate conventional adaptive responses and require antigen processing.
• γδ T cells respond quickly to stress-induced molecules and microbial metabolites, functioning at epithelial barriers with limited diversity but rapid action.

During B-cell maturation in the bone marrow, immature B cells expressing self-reactive receptors must be controlled to prevent autoimmunity. Two key mechanisms operate:

1. Receptor Editing:

• When an immature B cell's mIgM strongly recognizes a self-antigen, the cell does not immediately die.
• Instead, RAG-1/RAG-2 genes are re-expressed, allowing a new light-chain gene rearrangement.
• This changes the specificity of the BCR — potentially making it non-self-reactive.
• If editing produces a harmless receptor, the cell survives.
• Receptor editing is the primary mechanism for dealing with self-reactivity.

2. Clonal Deletion:

• If receptor editing fails to eliminate self-reactivity, the strongly self-reactive B cell undergoes apoptosis (programmed cell death).
• This permanently removes dangerous clones from the repertoire.

3. Anergy (related mechanism):

• B cells that recognize soluble self-antigens with lower avidity may be rendered functionally unresponsive (anergic) rather than deleted.

Significance:

• These mechanisms collectively establish central B-cell tolerance, ensuring that mature B cells do not react against the body's own tissues.

The AIRE (AutoImmune REgulator) gene plays a central role in establishing central tolerance during T-cell development.

Role of AIRE:

• Expressed by medullary thymic epithelial cells (mTECs) in the thymus.
• AIRE is a transcriptional regulator that promotes the expression of a wide range of tissue-specific antigens (TSAs) — proteins normally found only in peripheral organs (e.g., insulin, thyroglobulin).
• This phenomenon is called promiscuous gene expression.

Function in Negative Selection:

• By displaying these tissue-restricted self-antigens on MHC molecules in the thymus, AIRE allows developing T cells to encounter self-antigens they would otherwise never see.
• Strongly self-reactive thymocytes are then eliminated via negative selection (clonal deletion) or diverted to become regulatory T cells (Tregs).

Consequences of AIRE Deficiency:

• Failure to express tissue-specific antigens in the thymus.
• Self-reactive T cells escape into the periphery.
• Leads to the multi-organ autoimmune disorder APECED / APS-1 (Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Dystrophy), characterized by autoimmune attack on multiple endocrine organs.

Significance: AIRE is essential for broadening the scope of central tolerance beyond thymus-expressed proteins.