Immunoglobulin Superfamily of Genes and Diversity of Antigen Recognition

Outline Immunoglobulin Superfamily Antigen Recognition Members: Antibodies T-Cell Receptor Immense Diversity: The Problem: Package all that Information The Solution: Somatic Recombination Development of a Lymphocyte: A Time for Diversity Somatic Recombination VDJ: The Making of the Variable Region Isotype Switching

Antigen Recognition Members of the Ig Superfamily Cellular Adhesion Cell-Cell Interactions Antigen Recognition undergo DNA recombination

The Problem: All That Information Typically, most individuals recognize 107 – 109 different antigens If each antigen receptor was a gene, then genes dedicated to this purpose alone would take a LARGE portion of the genome!!

Discovery of Immunoglobulin Gene Organization Dreyer and Bennett Amino acid sequencing of Ig Same isotype of protein had highly variable V-region Must come from different genes that are joined at DNA/RNA level Tonegawa Ig genes are different in a cell committed to the synthesis of that protein (lymphoid-tumor vs. stem cell) Difference due to events that must occur during the development of that cell

The Solution: Packaging Germline DNA: NO functional Ig/TCR protein can be made without extensive processing Genes are packaged into loci each containing the pieces (gene segments) of a functional antibody or TCR The gene segments are put together in a step-wise fashion that results in a functional protein Recombination allows for the “piecing together” of genes Splicing finalizes the RNA transcripts END RESULT: Antibody or TCR

Immunoglobulin Gene Segments Antigen Recognition Receptor Gene Loci Gene Segments are located in different genes on different CHROMOSOMES! Each locus: Sets of gene segments Gene segments (exons) separated by non-coding regions (introns) Immunoglobulin Gene Segments TCR Gene Segments

The BIG PICTURE B-cell Development Light Chain Generated

Immense Diversity in Antigen Recognition Typically, most individuals recognize 107 – 109 different antigens

Outline Immunoglobulin Superfamily Antigen Recognition Members: Antibodies T-Cell Receptor Immense Diversity: The Problem: Package all that Information The Solution: Somatic Recombination Development of a Lymphocyte: A Time for Diversity Somatic Recombination VDJ: The Making of the Variable Region Isotype Switching

Path of the Lymphocyte

When and Where does Somatic Recombination Occur? Isotype Switching VDJ

Steps of Lymphocyte Development Development of Lymphocytes Steps of Lymphocyte Development Development (T or BM) Functional Receptor or DIE Positive Selection (T or BM) Interact w/ appropriate Receptor (MHC) or DIE Negative Selection (T or BM) React to self and DIE IV. Functional Competence (SLT) Somatic Recombination occurs during development- formation of functional receptor

B-Cell Development Occurs in the bone marrow Roughly 109 cell are produced per day Occurs in a highly controlled, stepwise fashion Mistakes are not tolerated

T-Cell Development Occurs mostly in the thymus – progenitors come from bone marrow Occurs in a highly controlled, stepwise fashion Mistakes are not tolerated 95% of the cells never make it to the state of naïve mature T-cell

Resident Cells of the Bone Marrow and Thymus Play An Important Role in Lymphocyte Development Stromal cells of both the bone marrow and thymus play an important role in development of lymphocytes Other cell types are also present (epithelial cells/dendritic cells/macrophage) Secrete cytokines to stimulate growth (IL-7 = lymphopoietic growth factor) Help with positive and negative selection

Outline Immunoglobulin Superfamily Antigen Recognition Members: Antibodies T-Cell Receptor Immense Diversity: The Problem: Package all that Information The Solution: Somatic Recombination Development of a Lymphocyte: A Time for Diversity Somatic Recombination VDJ: The Making of the Variable Region Isotype Switching

Somatic Recombination Somatic Recombination = process of DNA recombination by which functional genes encoding variable regions of Ag R are formed during lymphocyte development. Rearrangements occur in strict developmental sequences Two levels of diversity: Combinatorial Junctional

Mechanisms of Somatic Recombination Each gene segment (V, D, and J) has an adjacent Recombination Signal Sequence (RSS) at the 3' end of each V segment at both ends of each D segment at the 5' end of each J segment These are recognized by two proteins encoded by two Recombination Activating Genes RAG-1 and RAG-2 The RAG-1 and RAG-2 proteins cut through both strands of DNA at the RSS forming

Mechanisms of Somatic Recombination Combinatorial Diversity Structures to know: heptamer/nonamer: Conserved 12 or 23 nucleotide spacer: Not Conserved 12/23 Rule: Recombination will occur only with segment that have a 12 or 23 at the 5’ region

Mechanisms of Somatic Recombination Combinatorial Diversity: Brings regulatory elements (promoters/enhancers) closer together, thus strongly promoting expression of the gene elements in proximity of one another.

Mechanisms of Somatic Recombination Junctional Diversity Addition or removal of nucleotides between V/D, D/J or V/J segments at the time of joining

Mechanisms of Somatic Recombination Junctional Diversity RAG mediated cleavage Endonuclease may remove Nu P-nucleotides = make blunt ends from “over hangs” made by RAG enzymes N-nucleotides = new nucleotides randomly added by TdT (Terminal Deoxyribonucleotidyl Transferase)

Outline Immunoglobulin Superfamily Antigen Recognition Members: Antibodies T-Cell Receptor Immense Diversity: The Problem: Package all that Information The Solution: Somatic Recombination Development of a Lymphocyte: A Time for Diversity Somatic Recombination VDJ: The Making of the Variable Region: B-Cells/T-Cells Isotype Switching

B-Cell Development PRE-B CELL The first rearrangement is DH and JH. Then, the DJ joins with the V gene segment of chromosome 14. The heavy chain is coupled with a surrogate light chain. Expressed on cell surface Called pre-B-Cell Receptor (pre-BCR) Pre-BCR important for survival signals and allelic exclusion

B-Cell Development IMMATURE B-CELL The V joins with the J gene segment of chromosome 2 for k or 22 for l. Expressed on cell surface as a complete IgM IgD/IgM is the final set of markers present on the surface of a mature B-cell.

Heavy and Light Chain Gene Recombination Heavy chain 1st D to J V to DJ Light chain 2nd V to J Primary RNA transcript is spliced to remove introns Final heavy and light chains are assembled into functional protein

Coexpression of IgM and IgD Does Not Require Isotype Switching IgM and IgD C gene segments are part of the same open reading frame (ORF) Variations in splicing of primary RNA transcript will result in generation of IgM or IgD Other isotypes available only by isotype switching

The BIG PICTURE B-cell Development Light Chain Generated

T-Cell Development PRE-T CELL The first rearrangement is D and J. Then, the DJ joins with the V gene segment of chromosome 7. This makes the beta chain of the TCR. The beta chain is coupled with an invariant protein. Expressed on cell surface Called pre-T-Cell Receptor (pre-TCR) Pre-TCR important for survival signals and allelic exclusion

T-Cell Development IMMATURE T-CELL The V joins with the J gene segment of chromosome 14 (alpha or 7 for lambda). This makes the alpha chain of the TCR. The beta chain assembled with alpha chain to complete TCR Expressed on cell surface

TCR a and b Chain Gene Recombination and Expression Beta chain 1st D to J V to DJ Alpha chain 2nd V to J Primary RNA transcript is spliced to remove introns Final alpha and beta chains are assembled into functional protein

Outline Immunoglobulin Superfamily Antigen Recognition Members: Antibodies T-Cell Receptor Immense Diversity: The Problem: Package all that Information The Solution: Somatic Recombination Development of a Lymphocyte: A Time for Diversity Somatic Recombination VDJ: The Making of the Variable Region: B-Cells/T-Cells Isotype Switching

Isotype Switching During an antibody response to a T-dependent antigen a switch occurs in the class of Ig produced from IgM to some other class (except IgD). During class switching another DNA rearrangement occurs between a switch site (Sµ) in the intron between the rearranged VDJ regions and the Cµ gene and another switch site before one of the other heavy chain constant region genes. This recombination event brings the VDJ region close to one of the other constant region genes and allows expression of a new class of heavy chain.

Isotype Switching Switch Recombination rearranged VDJ gene segment in a B-cell recombines with a downstream C region gene and the intervening DNA is deleted Involves nucleotide sequences known as switch regions that are in the 5’ of each CH locus Cytokines and CD40 stimulate the read through of these regions and I exons which contain nonsense and many stop codons, these stimulate recombination downstream C regions IL-4

Outline/Summary Immunoglobulin Superfamily Antigen Recognition Members: Antibodies T-Cell Receptor Immense Diversity: The Problem: Package all that Information The Solution: Somatic Recombination Development of a Lymphocyte: A Time for Diversity Somatic Recombination VDJ: The Making of the Variable Region T-cell/B-cell Isotype Switching

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