B Cell repertoire - role of B cell antigen receptors (BCR)

1. B Cell repertoire - role of B cell antigen receptors (BCR)

2. To generate enough antibodies for a large number of pathogens, adaptive immunity could have evolved by producing one gene for each antibody. Disadvantages/problems: - how does the body know of all the pathogens that they would encounter? - would need a large genome - do not provide the immune system with the flexibility of fighting new infections in a specific way But, our immune system generates a random set of antibodies by shuffling a relatively small number of genes. Disadvantages/problems: - waste of resources - generation of antibodies against self-antigens

3. B cell development preB I preB II Immature B mature B proB Heavy chain gene rearrangement Light chain gene rearrangement Ig heavy chain Ig light chain VpreB plus l 5

4. Generation of a diverse repertoire of immunoglobulins (Ig) (1) - heavy chain gene D segments Constant region VH segments JH segments Recombination of D and JH segments Recombination of V and DJH segments

5. Generation of a diverse repertoire of immunoglobulins (Ig) (2) - light chain gene Constant region V segments J segments Recombination of V and J segments

6. Generation of a diverse repertoire of antibodies (1) Random rearrangement of variable gene segments (2) Different combinations of heavy (IgH) and light chains (Igk or Igl) (3) Addition or deletion of nucleotides at the junction where the variable gene segments are joined

7. Proteins: VpreB + l 5 = surrogate light chain - part of pre-BCR RAG-1 + RAG-2 = recombinase nucleases = nick and digest hairpin loops of intermediate TdT (terminal deoxynucleotide transferase) = for N-addition IgH, Igk, Igl = containing variable regions for recognition of antigens Iga, Igb = (1) for cell surface expression of immunoglobulin; (2) for signaling

8. positive selection of B cells - BCR signals promote proliferation and differentiation mature B Immature B preB I proB preB II

9. Role of preB receptor: - suppress RAG genes expression, such that allelic exclusion of IgH is achieved - promote proliferation of preB cells to finish rearrangements of IgL l5 and VpreB KO mice - Delayed B cell development, but not abolished - normal numbers of peripheral B cells 6 months later - No apparent defect in allelic exclusion Function of l5 and VpreB - may select for heavy chain variable regions - shaping the primary B cell repertoire

10. Antigen-dependent maturation of B cells: - Fix IgH chain expression in transgenic mice - These mice utilize endogenous light chains - Isolated immature and mature B cells separately -> measure the usage of light chain variable gene segments Hypothesis: If maturation of immature B cells (to mature B cells) is random, antibody repertoire remains unchanged Result: Repertoire significantly changed Conclusion: Maturation of immature B cells is not random, suggesting antigen dependent

11. Testing dependence on antigen selection during maturation of B cells V-A V-B V-C V-A V-B V-C Stochastic events Antigen-dependent selection

12. Y Y -p Y Y -p Activation of B cell antigen receptor (BCR) signaling antigen BCR -p Y Lyn/ Fyn/ Blk -p Y p p p p Syk SLP65 Bruton tyrosine kinase PLC-g Proliferation

13. Ligand-independent signaling through (pre-) B cell BCR

14. Immature & mature B Y -p Y -p p p preB I proB preB II Normal mouse CD22- CD43+ CD22+ CD43- MHC II+ CD23+ mMT mouse CD22- CD43+ X X CD22- CD43+ CD22+ CD43- MHC II+ CD23+

15. Y -p Y -p Y Y Y -p -p -p Y Y Y -p -p -p p p p p p p p p Antigen-dependent and independent (pre-) BCR signals Strong signal Weak maintenance signal

16. Regulation of positive selection by signaling pathways BLNK Btk Vav CD45 Lyn l5 mMT Syk mature B Immature B preB I proB preB II

17. negative selection of B cells - BCR signals induces death or anergy mature B Immature B preB I proB preB II

18. deletion Rescued deletion Self-reactive Immature B Activated B B cell tolerance

19. Receptor editing by secondary rearrangement (1) Constant region Vk segments Jk segments Recombination of Vk and Jk segments Secondary Recombination (2) Rearrangement of the germline allele

20. Receptor editing: (1) Abnormally high or low levels of receptor signals stimulate receptor editing (2) Interleukin-4, in combination with LPS or anti-CD40, stimulate expression of RAG-1 and -2 genes (3) Interleukin-7, in combination with anti-CD40, stimulate RAG-1 and -2 genes expression

21. Receptor editing (continued): (4) c-myb and Pax-5 proteins activate RAG-2 gene expression (5) E2A activate RAG-1 and RAG-2 genes expression (6) OcaB, a transcription factor, promotes Ig gene recombination through the activation of germline gene transcription

22. Y Y Y Y -p -p -p -p Y Y Y Y -p -p -p -p p p p p p p p p Deletion and anergy as mechanisms of immune tolerance - IgHEL/HEL mouse model HEL IgHEL Weak signal leads to anergy Strong signal leads to apoptosis

23. Self-reactivity Shapes the B cell repertoire Strength of BCR signal Failure to enter the periphery Mature B cell Anergy Deletion B cell fate

24. Expression of Ig isotypes in normal B cells IgM IgG mature B Immature B preB I preB II proB Antigen-induced activation mMT

25. Discussion of Seagal et al, 2003, J. Exp. Med. Previous observation: Hypothesis/Proposal: Conclusion: Results:

26. PE PE FITC Staining for cell surface maker B CELL FITC

27. Figure 1

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33. Figure 7