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bone marrow

B. Distinct mechanisms regulate central vs peripheral tolerance. self Ag. IgM. preB. imm. B. imm. B. receptor editing. RECEPTOR SELECTION. bone marrow. peripheral immune organs. tissue restricted self Ag. CLONAL SELECTION. IgM. IgD. die-- by BAFF starvation?.

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bone marrow

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  1. B Distinct mechanisms regulate central vs peripheral tolerance self Ag IgM preB imm. B imm. B receptor editing RECEPTOR SELECTION bone marrow peripheral immune organs tissue restricted self Ag CLONAL SELECTION IgM IgD die-- by BAFF starvation?

  2. A fact that you may find surprising • Most randomly generated antigen receptors are autoreactive • This is a consequence of the size/flexibility of antigen receptors that allow antibodies to cross react • Tolerance is a major problem for the immune system which is dealt with in a series of steps to reduce the frequency and affinity of self reactive cells

  3. * * * * * * * * * * non-self self Imagine a single cell with a receptor that sees 10 different epitopes, at random

  4. * * * * * * * * * non-self self As the reactivity is random, sometimes the cell will see self epitopes, leading to tolerance making the cell unusable or deleted, unavailable to respond to non-self

  5. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * This cell is more useful! non-self self Imagine a single cell with a receptor that sees 100 different epitopes, at random

  6. * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * non-self self A cell with a receptor that sees 100 different epitopes is more likely to be self reactive

  7. Because the ability to see foreign antigens increases linearly with antigen receptor cross reactivity, while the chance of a cell being eliminated by tolerance increases exponentially, there is an optimal extent of multireactivity that can be calculated. It turns out that at this optimum, which is independent of assumptions about the actual size of the self antigen repertoire, ~62% (1-e-1) of cells should be autoreactive. However, the ability of cells to edit may allow the extent of cross reactivity to be even higher. Nemazee 1996 Immunol.Today 17:25

  8. In the bone marrow Self antigen Receptor-less cell (pre B) Innocuous receptor Autoreactive receptor Recombinase ON Recombinase OFF Recombinase ON

  9. Receptor editing Self antigen Recombination STOP signal L-chain gene recombination continues

  10. 2nd chain gene recombination 1st chain gene recombination g/d edited TCRa a/b T cell lineage pre-TCR TCR b b proT a/b a/b positive selection Thymus small CD4+/CD8+ double-positive large preT cell CD4+ or CD8+ single-positive thymocyte edited L-chain editing H/L B cell lineage pre-BCR BCR mH mH H/L H/L proB CLP positive selection Bone marrow large preB cell small preB cell immature (editing competent) B cell mature B cell

  11. Vn V1 D1 ........... Dn J1 ......... Jn // Constant region exons // // 1st chain locus V(D)J recombination V DJ Constant region exons V DJ C 1st chain Lymphocyte 2nd chain V J C Ag receptor Constant region exon/s V J V(D)J recombination 2nd chain locus Vn V1 J1 ......... Jn // Constant region exon/s // Asymmetry in antigen receptor gene structure (and function)

  12. One consequence of the asymmetry of antigen receptor genes Antibody diversity is concentrated in H-chain CDR3 Davis MM, Semin Immunol. 2004

  13. But Vh replacement may overcome this restriction “The editing locus” Ig-L (k+l), TCR-a, TCR-g ? C V4 V3 V1 V2 J J 1o V4 V3 C V1 V2 J J 2o V1 V2 C J Compatible signal sequences facilitate receptor editing “The first chain”: Ig-H, TCR-d, TCR-b? C V4 V3 V1 V2 J J D D 1o V4 V3 V1 V2 C J J D Incompatible signal sequences

  14. Sometimes productive Never productive, permanently inactivate the locus The Ig k locus gene organization facilitates editing VJ V’s VJ0 iE Ck 3’E RS IRS1 J3 J4 J5 VJ-intron-RS VRS

  15. Most V-to-Jk5 Receptor editing occurs frequently in normal B cells: 27 out of 57 VkJk-intron-RS loci from normal l+ cells are in-frame and potentially functional Retter and Nemazee 1998J Exp Med 188:1231.

  16. Evidence for the high likelihood of a cell to be autoreactive -Evidence of editing on the Igk locus in a large fraction of IglB cells (Retter and Nemazee 1998J Exp Med 188:1231; Brauninger 2001 EJI) -A dearth of arginines in antibody combining sites, except in disease- there appears to be ~40% loss in B cells because of this selection pressure alone (Louzoun et al. 2002 Semin.Immunol. 14:239) -A decreasing frequency of multireactive B cells with developmental progression, starting with 75% at the bone marrow stage to less than 30% at later stages (Wardemann et al 2003, Science 301:1374 -Random Ig H/L transgene pairs are often autoreactive and lead to editing in vivo (Novobrantseva et al. 2005. Int.Immunol. 17:343 )

  17. Knock-in version of 3-83 Ig mouse 3-83 VkJk2 Vk Jk RS Ck Ig k Eik E3’k 3-83 VHDHJH Cm Cd Ig H Eim Pelanda et al., Immunity (1997) 7:765

  18. Summary -Most generated B cells are autoreactive. -Most immature B cells are editing-competent. -Editing has a low threshold affinity in anti-MHC Tg (3-83). -Editing can occur often under normal conditions. -Tolerance is likely to be the major inducer of editing.

  19. Degeneracy and lymphocyte tolerance • The randomly generated antibody repertoire MUST (and does) generate a high frequency of autoreactive B cells. Selection for the optimal level of degeneracy regulates the size and flexibility of the antibody combining site. • Central tolerance by receptor editing avoids much of the cell loss that would occur if clonal deletion was the major tolerance mechanism. • There is a division of labor between antibody H-chain genes and L-chain genes: H genesgeneratediversity L genes facilitate editing. • Immune tolerance in peripheral B cells is highly context dependent, and is regulated by B cell competition for cytokines such as BAFF.

  20. Djemel Aït-Azzouzene VH VH VL VL DCH1 DCk + Linker CH2 CH3 Macroself Ag: IgCk-reactive custom superantigen as a probe for tolerance regulation. anti-k VH VL VL VH Fv Fab scFv CH1 Ck CH2 Fc CH3 H-2Kb Tm Cy rat IgG1 187.1

  21. L-chain specific tolerance in k-macroself transgenics lymphocyte gate B220 gate Ait-Azzouzene et al. 2005 J. Exp. Med. 201:817-828.

  22. Induced tolerance in Wild type → Macroself Tg radiation chimeras

  23. A B220intermed IgM+/B220intermed 90 90 80 80 70 70 60 60 % BrdU+ 50 50 40 40 30 30 20 20 10 10 0 0 0 0 20 20 40 40 60 60 80 80 100 100 hours hours Non-Tg Tg Non-Tg Tg 4 4 4 4 10 10 10 10 3 3 3 3 10 10 10 10 B 2 2 2 2 10 10 10 10 Bone marrow Spleen 1 1 1 1 10 10 10 10 Littermate Tg Littermate Tg 0 0 0 0 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 4 67 41 0.1 28 1 58 0.1 10 <0.1 4 4 4 4 10 10 10 10 3 3 3 3 10 10 10 10 2 2 2 2 10 10 10 10 13 <0.1 1 1 1 1 10 10 10 10 surface Igk B220 0 0 0 0 10 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 3 64 43 0.3 32 1 56 0.1 11 <0.1 13 11 Intracellular Igk Lymphocyte gate No increase in bone marrow B cell turnover in k-macroself Tg mice

  24. 100 80 60 40 20 0 0 1 2 3 4 10 10 10 10 10 100 100 80 80 60 60 40 40 20 20 0 0 4 4 0 1 2 3 0 1 2 3 10 10 10 10 10 10 10 10 10 10 100 80 60 40 20 0 0 1 2 3 4 10 10 10 10 10 Transgenic mice expressing high or low levels of macroself Ag pUlik Tg intron Ubiquitin C promoter VDJh CH2 Li VJ CH3 Tm Cy 3’UTR 1 kb Bone marrow Spleen pUliklow pUlikhigh Macroself antigen expression

  25. Transgenic mice expressing high or low levels of macroself Ag manifest distinct tolerance phenotypes peripheral deletion editing Aït-Azzouzene, D et al (2006) J Immunol. 176:939

  26. A C non Tg pUliklow non Tg pUliklow 69 30 1 <0.5 22 34 5 39 3 61 1 35 B220 HSA 37 58 3 2 IgM B220 CD23 CD21 AA4.1 IgD 4 4 10 10 Spleen, Igk gate Spleen, Igk gate 3 3 10 10 B D pUlikhigh non Tg Spleen, Igk gate 2 2 10 10 Igk Igl 80 64 5 1 1 10 10 0 0 10 10 7 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 7 16 pUliklow CD23 Igk Igl 40 50 4 4 10 10 3 3 10 10 39 19 2 2 10 10 CD21 2 23 1 1 10 10 0 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 pUliklow Non-Tg Remaining k+ B cells in low-expressing Tg spleen are immature

  27. k+ Peritoneal B-1 cells are spared in pUIiklow mice

  28. Peritoneal k+ cells of pUIiklowmice are not short-lived

  29. BAFF overexpression protects mature B cells from clonal deletion

  30. . . . B cell competition deletes mouse Ck+ spleen cells in macroself mice, regardless of BAFF overexpression k macroself/ BAFF Tg BAFF Tg BAFF Tg k-macroself Human Ck +/+ (not reactive) 28 6 28 6 51 <0.5 51 <0.5 33 33 33 33 49 <0.5 49 <0.5 x mouse Ck+/+ human Ck+/+ k k hC hC 21 4 21 4 35 <0.5 35 <0.5 55 20 55 20 65 <0.5 65 <0.5 k-macroself Ckh/m k k mC mC

  31. SUMMARY Excess BAFF can partly suppress peripheral deletion provided that competition from non-autoreactive B cells is minimal (i.e., 6%, not 50%)

  32. B-1 cells in the peritoneum are also eliminated by low level superantigen when competing B cells are present

  33. Tolerance steps in the preimmune repertoire Generated repertoire 20 20 16 16 13 13 14 14 15 15 17 17 19 19 18 18 4 4 5 5 3 3 9 9 6 6 8 8 7 7 1 1 2 2 11 11 10 10 12 12 Receptor editing in bone marrow Tolerance Edited repertoire 20 20 ’ ’ 13 13 15 15 19 19 16 16 ’ ’ 14 14 ’ ’ 18 18 17 17 ’ ’ 4 4 5 5 3 3 6 6 9 9 8 8 1 1 7 7 ’ ’ 2 2 10 10 12 12 Tolerance Peripherally tolerized repertoire Deletion in the peripheral immune tissue 13 13 15 15 16 16 ’ ’ 14 14 ’ ’ 17 17 ’ ’ 4 4 5 5 3 3 6 6 1 1 2 2 10 10 12 12

  34. In the peripheral lymphoid tissues Autoreactivity of increasing affinity 4 5 3 2 9 6 8 7 1 11 10 12 4 5 3 2 6 7 1

  35. The Scripps Research Institute Amanda Gavin Bao Duong José Luis Vela Christoph Huber Takayuki Ota Patrick Skog Min Lim David Russell Susan Tiegs Julie Lang Valérie Kouskoff Marc Retter Laurent Verkoczy Djemel Aït-Azzouzene Acknowledgments Kurt Bürki Roberta Pelanda/ K. Rajewsky Michel Nussenzweig Funding: NIAID, NIGMS • Discussion points: • Tolerance is a fact • Editing regulates antigen receptor gene structure • Size of the antibody combining site • Why antigen receptors aren't single chain • Retention of k/l • Role of competition in regulating tolerance

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