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? But, our immune system generates a random set of antibodies by shuffling a relatively small number of genes. - waste of resources - would need a large genome - do not provide the immune system with the flexibility of fighting new infections in a specific way - generation of antibodies against self-antigens Disadvantages/problems:

3 B cell development Ig heavy chain VpreB plus  5 Ig light chain proB preB IImmature B Heavy chain gene rearrangement preB II Light chain gene rearrangement mature B

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

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

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

7 Proteins: VpreB +  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, Ig , Ig = containing variable regions for recognition of antigens Ig , Ig  = (1) for cell surface expression of immunoglobulin; (2) for signaling

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

9 5 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 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 Function of 5 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 V-AV-BV-CV-AV-BV-C Stochastic events Antigen-dependent selection Testing dependence on antigen selection during maturation of B cells

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

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

14 -p p p Y Y proB preB I Immature & mature B preB II Normal mouse  MT mouse CD22- CD43+ CD22+ CD43- MHC II+ CD23+ CD22- CD43+ XX CD22+ CD43- MHC II+ CD23+ CD22- CD43+

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

16 proB preB I Immature B preB II mature B 5  MT Syk BLNK Btk Vav CD45 Lyn Regulation of positive selection by signaling pathways

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

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

19 Receptor editing by secondary rearrangement V  segmentsJ  segments Recombination of V  and J  segments Constant region Secondary Recombination (1) (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 -p p p Y Y p p Y Y p p Y Y Strong signal leads to apoptosis Weak signal leads to anergy Deletion and anergy as mechanisms of immune tolerance - IgHEL/HEL mouse model HEL -p p p Y Y IgHEL

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

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

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

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

27 Figure 1

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31 Figure 5

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33 Figure 7