Microbiology and Immunology Past Paper PDF 2024-2025

MICROBIOLOGY AND IMMUNOLOGY (PBBS505A&B) 2024-2025 Session -8 Generation of Diversity Rahul Vijay, DVM, Ph,D [email protected]  Lecture Objectives How diversity is generated in B and T cells  V(D)J r...

MICROBIOLOGY AND IMMUNOLOGY (PBBS505A&B) 2024-2025 Session -8 Generation of Diversity Rahul Vijay, DVM, Ph,D [email protected]  Lecture Objectives How diversity is generated in B and T cells  V(D)J recombination  Junctional diversity  Combinatorial diversity  Major cells of the Adaptive Immune Response B lymphocytes T lymphocytes B Cell Receptor T Cell Receptor (BCR) (TCR) BCRs from B cells detects a unique TCRs on each T cells detects a unique extracellular antigen. intracellular antigen in the context of Major Histocompatibility (MHC) molecules. Secreted form of BCR is called the antibody (immunoglobulin ) TCR is not secreted Antibody can bind to antigens and neutralize them Structural Comparison of BCR or Immunoglobulin and TCR B lymphocytes T lymphocytes TCR BCR  Antigen binding sites are formed by variable regions of The heavy and light chains in a BCR (antibody) The a and b chains in a TCR  The Hypervariability (HV) regions or Complementarity Determining Regions (CDR) of both the heavy chain and the light chain contribute to antigen binding of an antibody molecule. Each variable region has three HV or CDR domains. TCRs also have three HV or CDR in each of its chains. These HV/CDR regions together decides the specificity of the receptor for its antigen  The total number of unique antigens that an individual's adaptive immune system can recognize is called immune repertoire. Thus the immune repertoire will be comprised of all the BCR and TCR specificities : 108 -1013. However, the human genome contains ‘only’ around 2.5x104 protein coding genes. Key Question How can the immune system recognize so many different foreign antigens?  Hypotheses to explain this enormous diversity: i. Germline theory – there is a separate gene for each difference immunoglobulin chain and that the repertoire is largely inherited. ii. Somatic diversification theory - proposed that the observed repertoire is generated from a limited number of inherited V-region sequences that undergo alteration within cells during the individual’s lifetime. Some elements of both theories are correct - DNA sequence encoding each variable region is generated by rearrangements of a relatively small group of inherited gene segments. Molecular Mechanisms for the Generation of Diversity Takes place during B and T cell development Also after B cell activation – Somatic Hypermutation Rearrangement of distinct gene segments and combinations of different chains Variable regions in each heavy and light chain in BCR or TCR are different due to somatic recombination of: i. Multiple V gene segments in the germline ii. J and D gene segments in the germline iii. Imperfect joining of these segments  Human Immunoglobulin Genes (BCR genes) Three unlinked chromosomal regions: Light chains - kappa (k) or lambda (l) Heavy chains (variable and constant regions) (chromosome 22) (chromosome 2) (chromosome 14)  Generation of B cell diversity DNA rearrangements must take place at the correct locations relative to the V, D, or J gene segment coding regions- V(D)J Recombination  V(D)J Recombination these DNA rearrangements must be regulated such that a V gene segment is joined to a D or a J and not joined to another V gene segment. This is made possible by these conserved noncoding DNA sequences called Recombination Signal Sequences (RSS), found near to the points at which recombination should take place. 12/23 rule is always followed except in the rare instance of D-D joining in heavy chain. You do not have to remember the heptamer or nonamer sequences  Enzymes involved in V(D)J Recombination The complex of enzymes that act in concert to carryout Somatic V(D)J recombination - V(D)J recombinase V(D)J recombinase Lymphoid specific Non homologous end joining (NHEJ) Only in developing T and B cells pathway enzymes Ubiquitously expressed Recombination activating genes – Repairs double stranded DNA breaks (endonucleases) Intrinsically imprecise RAG-1 Creates Junctional diversity RAG-2 Ku70:Ku80 – binds DNA ends Terminal deoxynucleotidyl Artemis:DNA-PK complex – open the transferase (TdT) – adds nucleotide DNA hairpin loops randomly to the single strand DNA – DNA ligase IV – ligase enzyme creates junctional diversity XRCC4 – DNA repair protein  Deficiency of one of the RAG genes, DNA-PK, Ku or Artemis results in incomplete lymphocyte development and thus lack of B and T cells – Severe Combined Immune Deficiency (SCID). In humans mutations in RAG1 or RAG2 in an inherited disorder called Omenn Syndrome. Defects in Artemis in humans produce a combined immunodeficiency of B and T cells resulting in increased radiosensitivity; due to their inability to repair double stranded DNA breaks – radiation sensitive SCID (RS- SCID).  Diversity in Immunoglobulin repertoire contributed by 4 main events 1. Combinatorial diversity 2. Junctional diversity - caused by diversity of the V gene - caused by randomness of the V(D)J recombination segments in the light chain process (addition and subtraction of nucleotides at the -3x106 different antibody molecules joints between gene segments. 3. Combinatorial diversity from joining of 4. Somatic hypermutation light and heavy chains - happening in activated B cells upon an - due to possible different combinations of infection heavy and light chains V regions that pair to - point mutations into rearranged V regions form the antigen binding site in the genes immunoglobulin molecule - Enzymes involved Activation-Induced Cytidine Deaminase (AID) and Uracil-DNA Glycosylase (UNG) Combinatorial diversity + Junctional diversity = 1013 different antibody molecules  Antibody or Ig isotypes Do not cause diversity in the antigen binding sites. 5 main classes on Ig classes – IgD, IgM, IgG, IgE and IgA. IgM, IgG, IgE and IgA are almost mostly seen in only in activated B cells, while IgD is seen only in resting (naïve) B cells These classes are differentiated by the constant regions (encoded by C-regions genes) of their heavy chain. IgA and IgM can form multimers increasing the ‘avidity’ of the molecule Avidity: number of antigen binding sites. Affinity: the strength of antigen binding. Antibody or Ig isotypes and their subclasses The one that causes an allergic reaction Generation of T cell diversity The TCR Complex Occurs in the thymus V(D)J Recombination also happens in TCR gene loci (Chromosome 14) (Chromosome 7) Generation of Diversity in the TCR 12/23 rule is applicable in TCR rearrangement as well  Generation of Diversity Somatic hypermutation does not occur in T cells!! Quiz 1. Which among the following is incorrect? a. VDJ recombination occurs both chains of Ig and TCR a and b chains b. VDJ recombination occurs in Ig heavy chain and TCR b chains c. Only VJ recombination occurs in Ig light chains and TCR a chains d. Somatic hypermutation occurs before V(D)J recombination. Quiz 2. Which among the following is (are) correct? a. IgG causes the allergic episode b. IgD formed following somatic hypermutation. c. RAG gene mutations causes disruption of both B and T cells. d. Somatic hypermutation happens in the thymus in T cells.