Immunoglobulin Superfamily of Genes and Diversity of Antigen Recognition

1. Immunoglobulin Superfamily of Genes and Diversity of Antigen Recognition

2. 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

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

4. 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!!

5. 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

6. 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

7. 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

8. The BIG PICTURE B-cell Development Light Chain Generated

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

10. 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

11. Path of the Lymphocyte

12. When and Where does Somatic Recombination Occur? Isotype Switching VDJ

13. 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

14. 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

15. 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

16. 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

17. 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

18. 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

19. Mechanisms of Somatic Recombination • Mechanism: • 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

20. 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

21. 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.

22. 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

23. 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)

24. 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

25. 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

26. 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.

27. 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

28. 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

29. The BIG PICTURE B-cell Development Light Chain Generated

30. 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

31. 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

32. 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

33. 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

34. 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.

35. 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

36. 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

37. Questions? Comments?