CUT-AND-PASTE GENES

1. CUT-AND-PASTE GENES Immunogenetics

2. Latin immunis, means, "exempt" in English. Immunogenetics

3. The immune response begins when a white blood cell called a macrophage encounters a virus and consumes it. Meanwhile, other viruses look for nearby cells to infect. Immunogenetics

4. Next, the macrophage digests the virus and displays pieces of the virus called antigens on its surface. Nearby cells have now become infected by the attacking viruses. Immunogenetics

5. Unique among the many different helper T cells (another class of white blood cells) in the body, one particular helper T cell now recognizes the antigen displayed and binds to the macrophage. Immunogenetics

6. This union stimulates the production of chemical signals-- such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) by the macrophage, and interleukin-2 (IL-2) and gamma interferon (IFN-y) by the T cell -- that allow intercellular communication. Immunogenetics

7. As part of the continuing process, IL-2 instructs other helper T's and a different class of T cells, the killer T's, to multiply. The proliferating helper T's in turn release substances that cause B cells to multiply and produce antibodies. Immunogenetics

8. The killer T cells now begin shooting holes in host cells that have been infected by viruses. Immunogenetics

9. The antibodies released by the B cells bind to antigens on the surfaces of free-floating viruses. Besides making it easier for macrophages to destroy viruses, this binding signals blood components called complement to puncture holes in the viruses. Immunogenetics

10. Finally, as the infection is brought under control, the activated T and B cells are turned off by suppressor T cells. However, a few "memory cells" remain behind to respond quickly if the same virus attacks again. Immunogenetics

11. Somatic recombination of antibody genes and the generation of antibody diversity There are a million to a billion different B and T cells, each of which corresponds to a different antibody or T cell receptor protein. The human genome is thought to contain less than 105 genes. How can a human make more antibodies than there are genes in its genome? Immunogenetics

12. Review • Immunoglobulins [antibodies] Antibody molecules have 4 polypeptide chains [2 heavy & 2 light chains]. Each polypeptide chain has constant and variable regions. An antibody molecule has two sites to bind to antigen. Secreted antibodies are grouped into 5 major classes: [IgG, IgA, IgM, IgD, IgE] Immunogenetics

13. Light and heavy chains have variable regions and constant regions (relative to amino acid sequence). Immunogenetics

14. The 110 amino acid variable regions are located at the amino-terminal ends and form the antigen binding site. • Within the variable regions, there are three hypervariable regions (5 to 10 amino acids). Immunogenetics

15. Constant and Variable Regions Immunogenetics

16. Antibody Domains • Both chains are composed of 110 amino acid repeating segments. • The repeating segments fold independently to form functional domains. • Each domains is held together by one intrachain disulfide bond. • The domains evolved by gene duplications. Immunogenetics

17. Three-Dimensional Structure • Each domain folds into a very similar three-dimensional structure. • In the variable domain are three hypervariable loops that form the antigen binding site. • Changing the length and amino acid sequence of the hypervariable loops does not disturb the three-dimensional structure. Immunogenetics

18. Immunogenetics

19. Immunogenetics

20. Immunogenetics

21. Antibody Diversity • During B cell development, separate gene segments are assembled in a genetic recombination event before the gene is transcribed. • Experiments done in 1976 showed that the C-coding and V-coding regions on different parts of the chromosome were spliced together. Immunogenetics

22. V-D-J-C Immunogenetics

23. Light chain rearrangements only involve VJ and C rearrangements. Immunogenetics

24. Immunogenetics

25. In the progenitor B cell, antibody gene rearrangement is initiated by a DH/JH rearrangement of one of the two H chain gene alleles. As soon as one functional H chain gene and one functional L gene are created, the rearrangement process terminates. However, if temporal cascade of rearrangements ultimately fails to create either a functional H chain or L chain gene, the B cell is eliminated. Immunogenetics

26. Rearrangement of antigen receptors is an irreversible event and absolutely required for lymphoid development. Immunogenetics

27. Development of B-cells 1) Gene rearrangements to produce highly variable antibody genes 2) Expression of surface immunoglobulin 3) Deletion or silencing of autoreactive B-cells Immunogenetics

28. Stem Cell Factor (SCF), Interleukin-7 Immunogenetics

29. allelic exclusion Allelic exclusion is the process by which lymphocytes express antigen receptors from only one of two possible alleles. Immunogenetics

30. Membrane bound antigens can crosslink the surface IgM on the surface of immature B cells. Examples of such antigens are MHC class I and class II molecules. Immature B cells that recognize such self antigens undergo apoptosis and are therefore removed from the repertoire. Immunogenetics

31. Stem cell Germline DNA Early pro-B cell Late pro-B cell Pre-B cell IgM expressed on cell surface Immature B cell Mature B cell Secreted Ig Plasma cell Immunogenetics

32. Immunogenetics

33. Four sources of antibody diversity: 1) Combinatorial diversity 2) Diversity due to heavy and light chain joining 3) Junctional diversity 4) Diversity due to somatic mutations Immunogenetics

34. Combinatorial Diversification • In the mouse variable light chain, about 300 different V-regions can be joined to 4 different J segments which are then joined to one C-region. • In the mouse variable heavy chain, 500 different V-regions can be joined to 4 J segments and 12 D segments. • What is the total number of possible combinations? Immunogenetics

35. Combinatorial Diversification • Light Variable Chain: • 300 x 4 = 1200 • Heavy Variable Chain: • 500 x 4 x 12 = 24,000 • Total possible combinations: • 1200 x 24,000 = 28,800,000 Immunogenetics

36. Junctional Diversification • Joining of the V segment to a J or D segment is not always precise in that random nucleotides can be inserted or lost at the junction. • It is estimated that junctional diversification increases the number of possible combinations by at least 108. Immunogenetics

37. How does this occur? Immunogenetics

38. VDJ recombination involves several steps, beginning with a cut to the DNA. These site-specific double- stranded breaks are initiated by two genes-called RAG-1 and RAG-2. Immunogenetics

39. Mechanisms of variable region DNA rearrangements • Recombination signal sequences (RSS's) • Recombination activating genes (RAG1 and RAG2) Immunogenetics

40. A protein, called Ku80, appears to do double duty in the processes of DNA repair and a kind of DNA recombination known as "VDJ joining." Immunogenetics

41. VDJ recombination also takes place in the T cells. Immunogenetics

42. Class Switch In immature (and naïve) B cells, the m (and d) chains are located adjacent to the VDJ region. At this point, IgM (and IgD) are expressed. Constant region gene segments located further downstream can be brought into proximity to the VDJ region by looping out and deleting intervening sequences. This process is irreversible and requires T cell signals. Immunogenetics

43. Alternative polyadenylation sites Immunogenetics

44. Alternative mRNA splice sites Immunogenetics

45. B cell derived tumors Chronic lymphocytic leukemia Blood Malignant Plasma Cells Multiple myeloma Plasma cell (secreting Ig of various isotypes, IgG, IgA, IgE...) Immunogenetics

46. Immunogenetics

47. Monoclonal Antibodies A hybridoma is a fusion of a cancer cell with a functional, normal cell - a hybrid of a lymphoma) which results in the generation of an immortal cell-line. Immunogenetics

48. Immunogenetics