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  1. Origin of diversityBibliography Antibody diversity: one enzyme to rule them all Michel C Nussenzweig & Frederick W Alt Nature Medicine 10;1304-5;2004 Immunobiology Janeway et al 6th ed 2005 (or 7th ed. 2008)

  2. Clonal selection theory יצירת מאגר של לימפוציטים בשלים תא אב יחיד מתמיין למספר גדול של לימפוציטים שלכל אחד מהם ספציפיות שונה חלוקה והתמיינות של לימפוציטים ספציפיים ליצירת קלון של תאים אפקטוריים סילוק של לימפוציטים לא בשלים המגיבים עם אנטיגנים עצמיים Memory cells B-cells: plasma cells Ab secretion T cells: T-helper T-killer

  3. Figure 3-1 part 3 of 3

  4. Figure 3-6 CDR1 CDR2 CDR3 CDR1 CDR2 CDR3

  5. Figure 4-2 The chance for successful rearrangement is 33%

  6. Ontogeny of the B Lymphocyte • Early differentiation • Many stages defined by Ig gene rearrangements • Progenitor cells • Hematopoietic stem cells • Pro-B • Pre-B • Immature B

  7. Pathway of B cell Differentiation Antigen Independent Antigen Dependent IgG “Memory” cell Cytoplasmic m chain Heavy chain + surrogate light chain “Heavy” plus “light” chain ((חליפית) Mature IgM + IgD Pre-Pro B Pro B Pre-B1 Pre-B2 B “Plasmablast” Immature IgM D--->JH VH-->DJH VL-->JL

  8. Ontogeny of the B Lymphocyte • Process of differentiation • Signals that promote survival and proliferation of early cells • Adhesive interactions with stroma (non-lymphoid cells that make up marrow matrix) • Secretion of IL7

  9. Ontogeny of the B Lymphocyte • Process of differentiation (continued) • Early cells • Pro-B – heavy chain D gene segment rearranges to J segment • Pre-B – Heavy chain V gene segment rearranges to join DJ region • Rearranged VDJ put close to heavy chain constant m gene • Synthesizes a m heavy chain

  10. Figure 7-4 part 1 of 3 V-DJ rearrangement D-J rearrangement

  11. Figure 4-2

  12. Figure 7-6 part 1 of 2

  13. DJ rearrangement on both chromosomes

  14. Ontogeny of the B Lymphocyte • Pre-B cell – expresses mchain as a trans-membrane molecule at cell surface • In conjunction with products of two non-rearranged genes • Lambda 5 and VpreB • Function as surrogate light chains • Also in conjunction with additional trans-membrane molecules linked by disulfides • Ig alpha (CD79a) • Ig beta (CD79b) • Combination of tran-smembrane molecules (pre-B cell receptor; pre-BCR)

  15. mheavy chain with surrogate light chains IL-7R (IL-7Ra and IL-2 gc chain) Iga and Igb proteins IL-7 and adhesive interactions between B cells and stromal cells important for proliferation Signal Transduction; 1. clonal expansion 2. allelic exclusion 3. light chain rearrangement l5 KO blocks L chain rearrangement and B cell differentiation It does not block second H-chain rearrangement

  16. Figure 7-18

  17. Sensitive to self antigens

  18. Figure 7-6 part 2 of 2 Sensitive to self antigens

  19. Allelic exclusion

  20. Ontogeny of the B Lymphocyte • Immature B cells • L chains pair with mH chains to form monomeric IgM then inserted into membrane • Recognize Ag and respond to it • Long-lasting inactivation instead of expansion and differentiation • Interact with self-Ag in bone marrow – inactivate cell • Called negative selection • Important for development of self-tolerance in B-lineage • Cells with potential reactivity to self prevented from responding (central tolerance)

  21. Ontogeny of the B Lymphocyte • Development of self-tolerance • Immature B cell exposed in bone marrow to: • Self-molecule on surface cells • Apoptosis (deletion) • Non-cell surface molecule (soluble Ag) • Cell is inactivated but not deleted (anergized) • Reactivation of VDJ recombinase (receptor editing) • Ig L chain genes undergo secondary rearrangement • Use unrearranged V or J segments • Generates specificity for non-self Ag – rescued from inactivation

  22. After gene rearrangements and production of a functional molecule, the cell tests whether its specificity is anti-self Maintenance of tolerance requires the persistence of antigen because self-antigens are always present but foreign antigens are transient

  23. Before clonal deletion of an anti-self B cell, the cell can attempt receptor editingof the light chain Fig 7.26 RAGs expression is still on So, light chain can use repeated rearrangements and can receptor edit. Repeated rearrangements are to make a functional molecule whereas receptor editing is to avoid clonal deletion of anti-self specific B cells. Immature B cell “edits” light chain if it binds antigen (gets negative selection signal). This could rescue the cell from negative selection (i.e., death). (needs signal to edit) Immature T cells continues to rearrange a chain until the cell gets positive selection signal. (will eventually die if it does not receive positive selection in a few days). (needs signal to stop “editing”)

  24. Figure 7-18

  25. Allelic exclusion

  26. Figure 3-11

  27. Figure 4-12

  28. Figure 4-15 Va are mixed with Vd

  29. In a TCR a locus there are about 70 V gene segments and 60 J segments. This provides for many attempts at a productive rearrangement. Rearrangements stop when there is positive selection.

  30. Figure 7-12

  31. Figure 4-2

  32. Mechanisms contributing to generation of primary antibody diversity in humans VxJx(D) 200 120 6000 Hxk H-L chain associations 1,200,000 Hxl 720,000

  33. Levels of Regulation of Ig Gene Expression & GOD • V-(D)-J rearrangement • Class switch recombination (CSR) • Somatic hypermutation (SH) • Receptor editing\revision

  34. V(D)J Recombination : RAG RAG 1 & 2 required • RAG 1 or RAG 2 K.O. mice • Also SCID in Man • No VDJ recombination (B or T-cells) • No dsDNA breaks

  35. RAG function • RAG 1 & 2 transfected into fibroblasts + synthetic V-D-J substrate Recombines exons Transcription control In synthetic substrate transfected into cell lines and mice, deletion of any of promoters or enhancers blocks rearrangement