Unit 2: Working of Immune System: Comprehensive Study Notes

1. Major Histocompatibility Complex (MHC) and Antigen Processing

Structure and Functions of MHC

The Major Histocompatibility Complex (MHC) is a group of genomic genes encoding cell-surface proteins essential for the adaptive immune system to recognize foreign molecules.

MHC molecules display peptide fragments on the cell surface for recognition by T cells. T cells only recognize antigens when bound to MHC proteins (MHC restriction).

• MHC Class I: Consists of a heavy α chain linked non-covalently to β₂-microglobulin. Expressed on almost all nucleated cells. Presents antigens to CD8⁺ cytotoxic T lymphocytes (CTLs).
• MHC Class II: Consists of two transmembrane glycoprotein chains (α and β chains). Expressed primarily on professional antigen-presenting cells (APCs) such as dendritic cells, macrophages, and B cells. Presents antigens to CD4⁺ helper T cells.

Endogenous Pathway of Antigen Processing and Presentation

The endogenous pathway processes antigens originating within the cell (e.g., viral proteins or tumor antigens) for presentation on MHC Class I molecules.

1. Protein Degradation: Intracellular proteins are tagged with ubiquitin and degraded into peptide fragments by the proteasome.
2. Peptide Transport: Transporter associated with antigen processing (TAP) translocates the peptide fragments from the cytosol into the lumen of the Endoplasmic Reticulum (ER).
3. Loading onto MHC Class I: Newly synthesized MHC Class I α chain and β₂-microglobulin assemble in the ER with the help of chaperones (calnexin, calreticulin, tapasin). Peptides are loaded into the peptide-binding groove.
4. Surface Presentation: The MHC Class I-peptide complex is transported via the Golgi apparatus to the plasma membrane for CD8⁺ T cell surveillance.

Exogenous Pathway of Antigen Processing and Presentation

The exogenous pathway processes extracellular antigens ingested by antigen-presenting cells (APCs) for presentation on MHC Class II molecules.

1. Antigen Internalization: Extracellular antigens are internalized into the cell by endocytosis, phagocytosis, or receptor-mediated endocytosis into endosomes.
2. Proteolysis: Endosomes fuse with lysosomes, where acid hydrolases break down the internalized antigens into short peptides.
3. MHC Class II Synthesis and Transport: MHC Class II molecules are synthesized in the ER, bound to an Invariant chain (Ii) that blocks the peptide-binding groove and prevents premature binding of endogenous peptides.
4. CLIP Removal and Peptide Loading: The MHC Class II-Ii complex is routed to endosomal compartments. The invariant chain is degraded, leaving a small fragment called CLIP (Class II-associated invariant chain peptide) in the groove. HLA-DM facilitates the exchange of CLIP for an antigenic peptide.
5. Surface Presentation: The stable MHC Class II-peptide complex moves to the cell surface to be recognized by CD4⁺ helper T cells.

2. Components and Pathways of Complement System

The complement system consists of over 30 soluble plasma proteins and membrane-bound receptors that act in a enzymatic cascade to amplify immune responses, promote inflammation, and eliminate pathogens.

Pathways of Complement Activation

1. Classical Pathway: Initiated by antibody-antigen complexes. Immunoglobulin IgG or IgM bound to pathogen surface binds the C1 complex (C1q, C1r, C1s). C1 cleaves C4 and C2 to form C4b2a (C3 convertase).
2. Lectin Pathway: Initiated independent of antibodies. Mannose-binding lectin (MBL) or ficolins bind to specific carbohydrate motifs (like mannose) on microbial surfaces, activating MBL-associated serine proteases (MASPs), which cleave C4 and C2 to form C3 convertase (C4b2a).
3. Alternative Pathway: Acts as an amplification loop and provides antibody-independent innate defense. Spontaneous hydrolysis of C3 (C3 tick-over) produces C3(H₂O), which binds Factor B. Factor D cleaves Factor B to yield C3 convertase (C3bBb), stabilized by Properdin.

Terminal Pathway and Membrane Attack Complex (MAC)

All three pathways converge at the formation of C3 convertase, which cleaves C3 into C3a and C3b.

C3b joins C3 convertase to form C5 convertase, which cleaves C5 into C5a and C5b. C5b sequentially recruits C6, C7, C8, and multiple C9 molecules to assemble the Membrane Attack Complex (MAC: C5b-C9), forming pores in pathogen membranes leading to osmotic lysis.

Functions of the Complement System

• Opsonization: C3b and C4b coat pathogens, enhancing their recognition and phagocytosis by macrophages and neutrophils expressing complement receptors.
• Inflammation (Anaphylatoxins): C3a, C4a, and C5a trigger degranulation of mast cells and basophils, increasing vascular permeability and attracting leukocytes (chemotaxis). C5a is the most potent anaphylatoxin.
• Lysis: Assembly of the Membrane Attack Complex (C5b-C9) directly lyses Gram-negative bacteria and enveloped viruses.
• Clearance of Immune Complexes: C3b binds immune complexes and facilitates their clearance by erythrocyte transport to the liver and spleen.

3. Properties, Types, and Functions of Cytokines

Cytokines are small, soluble proteins or glycoproteins secreted by various immune and non-immune cells that act as chemical messengers to mediate cell-to-cell communication.

Properties of Cytokines

• Pleiotropy: A single cytokine exerts different biological effects on different target cell types.
• Redundancy: Multiple distinct cytokines mediate the same or similar biological functions.
• Synergy: The combined effect of two cytokines is greater than the sum of their individual effects.
• Antagonism: One cytokine inhibits or blocks the effect of another cytokine.
• Cascade Induction: One cytokine acts on a target cell to induce the secretion of one or more additional cytokines.

Types and Classifications

4. Monoclonal Antibodies: Production and Applications

Monoclonal antibodies (mAbs) are identical antibodies produced by a single clone of B lymphocytes, targeting a single specific epitope on an antigen.

Production via Hybridoma Technology

Developed by Georges Köhler and César Milstein, hybridoma technology combines the specific antibody-producing capability of B cells with the indefinite lifespan of myeloma cells.

1. Immunization: A host animal (typically a mouse) is injected with the target antigen to stimulate specific antibody-producing plasma B cells in the spleen.
2. Isolation of B Cells: Plasma cells secreting desired antibodies are harvested from the mouse spleen.
3. Fusion: Splenic B cells are fused with HGPRT-deficient (Hypoxanthine-Guanine Phosphoribosyltransferase negative) immortal myeloma cells using Polyethylene Glycol (PEG) or electrofusion.
4. Selection in HAT Medium: The fused cell mixture is cultured in Hypoxanthine-Aminopterin-Thymidine (HAT) medium.
   • Aminopterin blocks the de novo nucleotide synthesis pathway.
   • Unfused myeloma cells lack HGPRT and cannot use the salvage pathway, so they die.
   • Unfused primary B cells have HGPRT but have a limited lifespan and die naturally.
   • Only hybridoma cells survive: they inherit immortality from myeloma cells and HGPRT enzyme from B cells to utilize the salvage pathway.
5. Screening and Cloning: Surviving hybridomas are screened (e.g., via ELISA) to identify clones producing the desired antibody, followed by single-cell cloning and expansion.

Applications of Monoclonal Antibodies

• Diagnostic Applications: Used in ELISA, Western blotting, flow cytometry, pregnancy test kits (detecting hCG), and blood typing.
• Therapeutic Applications: Used in targeted cancer immunotherapies (e.g., Trastuzumab for HER2⁺ breast cancer, Rituximab for CD20⁺ non-Hodgkin lymphoma) and autoimmune disease treatments (e.g., Infliximab blocking TNF-α in rheumatoid arthritis).
• Research Applications: Identification of cell-surface markers (CD molecules), protein purification via affinity chromatography.

5. General Introduction to Vaccines and Various Generations

A vaccine is a biological preparation that provides active acquired immunity against a specific infectious disease by simulating a primary pathogen exposure without causing disease.

Generations and Types of Vaccines