Project 10 Mixed Bone Marrow Chimerism as a Treatment for Diabetes

Tolerance to Carbohydrate Antigens and Xenotransplants Megan Sykes, M.D. HCT for Tolerance Induction: Requirements • Conditioning must be minimally toxic (nonmyeloablative) • Conditioning must overcome HvG barrier to reliably allow mixed chimerism induction (confidence to remove non-specific immunosuppression). • GVHD unacceptable- must be reliably avoided. • Ability to cross full (two haplotype) HLA barriers desirable. Significance of Antibodies in Transplantation • a) Pre-formed antibodies (present before transplant) • Induced antibodies (post-transplant) Reflect sensitization to donor T cell-dependent Mostly IgG Natural antibodies (no specific sensitization; IgM predominant) i. ABO mismatched tx ii. Xenotransplantation b) Due to sensitization (mainly IgG) i. Pregnancy ii. Transfusion iii. Previous Transplant B Cell Tolerance • Could overcome natural antibody-mediated resistance • Could overcome resistance by pre-sensitized B cells • Could prevent sensitization post-transplant • Not necessary to prevent sensitization posttransplant if T cell tolerance is achieved Natural Antibodies in Xenograft Rejection • Humans and old world monkeys, unlike most species, lack a functional a1,3Gal transferase, the enzyme needed to produce a1,3Galβ1-4GlcNAc glycoproteins and glycolipids • Natural antibodies against the ubiquitously expressed a1,3Gal specificities cause hyperacute and delayed vascular rejection, and present a major obstacle to pig to human xenotransplantation • While aGal knockout pigs have overcome this obstacle, antibodies against other specificities are likely to be important as well Human xenoreactive NAbs predominantly recognize Gala1,3Gal carbohydrate epitopes on porcine cells GalT+/+ Anti-Gal NAb Experimental model GalT+/+ GalT-/- Anti-Gal NAb Low Levels of Anti-Gal Natural Antibodies Induce HAR/AHR of Grafts Lacking a Complement Regulatory Protein Survival DAF -/- and DAF +/+ heart grafts in Gal T -/- recipients Group Donor Recipient Treatmenta Rejected/ Total Presence of anti-aGal Antibodiesb IgM IgG Rejection time (day) Median survival (day) A DAF-/- Gal T -/- 7/7 + - 1, 1, 3, 4, 8, 9, 9 4.0 B DAF +/+ Gal T -/- 2/4 + - 13, 25, >92, >92, >58.5 C DAF -/- Gal T -/- RRBC i.p. 6/6 + + 1, 1, 5, 6, 7, 10 5.5 D DAF +/+ Gal T -/- RRBC i.p. 5/5 + + 2, 3, 3, 5, 13 3 E DAF -/- B6 8/8 - - 8, 11, 13, 13, 14, 14, 15, 15 13.5 -/- Low Levels of Anti-Gal Natural Antibodies Induce AHR In Mice Lacking a Complement Regulatory Protein (Minor Histocompatibility Antigen Disparity) DAF KOnaïve GalT KO 24 hr AHR WTnaïve GalT KO ACR DAF KOWT ACR GKO +WT GKO mixed chimeras Tolerization of anti-Gala1,3Gal natural antibody-forming B cells by induction of mixed chimerism Yang et al, J. Exp. Med. 187(8) 1335-42, 1998 Mixed chimerism induced with non-myeloablative conditioning prevents T cell-and anti-Gala1,3Gal-mediated graft rejection Ohdan et al, J. Clin.Invest. 104(3) 281-90, 1999 Ohdan et al, Tx 71:1532,2001 Rapid Disappearance of Anti-Gal Antibodies in RatMouse Mixed Chimeras, and Evidence for Tolerance of Anti-GalProducing Cells 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1 0.0 2 weeks 6 weeks OD 492 SERUM ANTI-GAL LEVELS * * 1:250 * * * 1:250 * 1:1250 1:6250 Serum dilution SPL 1:1250 1:6250 Conditioned GalT -/Mixed chimeras GalT +/+ Anti-Gal Ab-producing cells (spots per well) 40 30 20 10 0 ELISPOT ASSAY (13 wk) * * * 800 160 32 6.4 Cell dilution (x10 cells per well) Ohdan et al, Tx 71:1532,2001 Mixed Chimerism Prevents HAR, DXR, and Cellular Rejection of Cardiac Xenografts Ohdan et al, Tx 71:1532,2001 Percentage of heart graft survival 100 80 60 40 Rat GalT+/+ mixed chimeras Rat GalT-/- mixed chimeras Conditioned GalT +/+ Conditioned GalT -/- 20 0 0 5 10 85 90 95 100 Days after heart transplantation Mixed Chimerism Prevents HAR, DXR, and Cellular Rejection of Cardiac Xenografts Conditioned GalT-/-1 hour (HAR) 4 days (DVR) Conditioned GalT+/+ RatGalT-/- Chimera 117 days (no rejection) 6 days (cellular rejection) Summary: Rat to GalT-/- Mouse Mixed Chimeras • Mixed chimerism can be induced in GalT-/- mice using non-myeloablative conditioning and high doses of rat marrow. • Mixed chimerism in this model leads to rapid tolerization of anti-Gal-secreting B cells. • Mixed xenogeneic chimerism in this model leads to T and B cell tolerance, thereby preventing HAR, DXR, cellular and chronic rejection of primarily vascularized cardiac xenografts. • Our studies in ratmouse mixed chimeras prepared with non-myeloablative conditioning indicate that tolerance is also induced for IgM NAb of other (non-Gal) specificities (Aksentijevich et al, Transplantation 1992;53:1108) • Thus, even with the use of Gal knockout pigs, induction of mixed chimerism could avoid both T cell- and non-Gal antibody-mediated rejection. Addtionally, NK cells are also rendered tolerant to the xenogeneic donor in mixed chimeras. B cells with anti-Gal receptors are located in the spleen and peritoneal cavity of GalT-/- mice, but only splenic B cells produce anti-Gal Ab Unsorted Sorted SPL Unsorted 200 40 8 1.6 SPL 0.3% IgM 42.7% Sorted (Gal+/IgM+) 40 8 1.6 0.32 Per C Unsorted Per C 1.5% 200 40 8 1.6 71.0% Sorted (Gal+/IgM+) 40 8 1.6 0.32 Gal-BSA Cell dilution (x103 cells per well) Ohdan et al, JI 165:5518 ,2000 “B1-b-like” Phenotype of Anti-Gal and Most IgM-Producing Cells Ohdan et al, JI 165:5518 ,2000 Cell size sIgM CD21 CD23 CD5 Mac-1 CD43 B-2 cells B-1a cell B-1b cells Newly formed B cells Marginal zone B cells small large large small large + ++ ++ ++ ++ ++ ++ ++ + ++ + + + + + + and + + + + 493 + - B cells with anti-Gal receptors (PerC) large B cells with anti-Gal receptors (SPL) large Anti-Gal IgM-producing cells (SPL) large BMT in Presensitized Gal KO Mice B6 GalT (H-2b) -/- 7Gy Thymic irradiation 3Gy Whole body irradiation Anti-CD4 Anti-CD8 mAbs High serum anti-Gal IgG and IgM Day -5 levels Week -4 Day 0 Bone marrow transplantation Gal+ Rabbit RBC i.p. Ohdan et al, Xenotx.8:227, 2002 BALB/c GalT BMC(160x106) +/+ Tolerance of Anti-Gal-Producing Cells by 2 Weeks PostBMT in Presensitized Mice Receiving Non-Myeloablative BMT 20 Anti-Gal Ab -producing cells (spots per well) 15 SPLEEN 20 BONE MARROW 15 10 Conditioned GalT-/Mixed chimeras Conditioned GalT+/+ 10 5 * * 5 * 32 6.4 0 * 0 800 160 * 32 6.4 800 160 Cell dilution (x10 3 cells per well) Ohdan et al, Xenotx.8:227, 2002 Mechanisms of B Cell Tolerance 1. Deletion- Developing and mature B cells 2. Receptor Editing- Secondary light chain rearrangements in developing and ?GC B cells 3. Anergy- Newly formed B cells and ?B-1 cells 4. Follicular exclusion 5. Sequestration (e.g. in peritoneal cavity) Presence of Gal-binding splenic B cells 2 weeks post-BMT in mixed allogeneic chimeras prepared in GalT-/- mice presensitized with rabbit RBC 4 weeks prior to BMT. Mixed chimera Percentage of Gal-binding B cells 0.3 0.25 P<0.05 0.2 0.15 0.1 0.05 0 Conditioned GalT -/Mixed chimeras CD19 BSA Gal-BSA Kawahara et al, Am J Transplant 5:2821, 2005 Absence of B cells with receptors for Gal in the spleens and peritoneal cavity of GalT+/+GalT-/- pre-sensitized mixed chimeras (20 weeks after BMT) SPL Conditioned GalT-/- Per C 0.06% 1.3% GalT-/Mixed chimeras CD19 0.01% CD19 0.2% Conditioned GalT+/+ BSA 0.01% 0.2% Gal-BSA BSA Gal-BSA Ohdan et al, Xenotx.8:227, 2002 Adoptive transplantation experimental design 10 x 10 Ly5.1 B6 BMCs 3Gy WBI Without donor cell depletion 6 With donor cell depletion 2 or 12 weeks 1 x 107 spleen cells 3Gy WBI B6 GalT-/-,Igh-/(both Gal and B cell deficient mice) B6 GalT-/-(Ly5.2) (Ly5.2) With CD4 depletion Kawahara et al, AJT 5:2821, 2005 Tolerance of Anti-Gal-Producing Cells is Broken by Parking in the Absence of Gal Antigen at 2 Weeks, but not 12 Weeks Post-Gal+GalT/- BMT: Evidence for Early Anergy and Late Deletion of Anti-GalProducing Cells Time of adoptive transfer: Mean (OD492) 2 weeks * * * *P < 0.01 0.7 0.6 12 weeks * * Serum Anti-Gal at 2 Weeks 0.6 0.5 0.4 0.3 0.2 0.1 0 Depleted chimera 0.5 0.4 0.3 0.2 0.1 0 Untreated Control Undepleted Depleted chimera chimera GalT-/-,Igh-/-mice Control Undepleted Transfer: Untreated chimera GalT-/-,Igh-/-mice Kawahara et al, 5:2821, 2005 Conclusions • B cell tolerance to Gal develops rapidly, via a nondeletional mechanism, in naïve and pre-sensitized Gal KO mice receiving Gal+ BMT with nonmyelaoblative conditioning • Early tolerance is dependent on the persistence of donor antigen and probably involves anergy • Long-term tolerance may reflect deletion/receptor editing of newly developing B cells • CR1/2, which is expressed on B cells and FDC, plays a role in self-tolerance; – CD21/35 and C4 help to prevent autoimmune disease in mouse models. • Prodeus AP et al., Immunity 1998 – Humans lacking C1q or C4 frequently develop SLE. • Walport et al., Immunol. Today 1991 → These results suggest that B cell tolerance may be dependent on CD21/CD35. • We hypothesized that CR1/2 might play a role in the tolerization of anti-Gal-producing cells via mixed chimerism Experimental design Anti-CD4/8 mAbs 3Gy TBI 7Gy TBI RRBC immunization GalT KO (B6) GalT CR2 DKO (B6) BALB/c B6 WT BMT analysis Chimerism Anti-Gal Ab Anti-Gal Ab-producing cells Summary • CR1/2 expression on stromal cells restores baseline anti-Gal NAb levels. • Stromal CR1/2 expression allows induction of B cell tolerance via mixed chimerism. • CR1/2 expression is not required for induced anti-Gal IgM responses. • CR1/2 expression on stromal cells plays an important role in the tolerance of anti-Gal Abproducing B cells via mixed chimerism. Conclusions • CR1/2 expression on non-hematopoietic cells, presumably follicular dendritic cells, is essential for the maintenance of anti-Gal Nab production. • Stromal (presumably FDC) CR1/2 is required for induction of B cell tolerance via mixed chimerism. • These observations suggest the existence of previously-undefined interactions between B-1 cells and FDC. Contributors BMT Section, Transplantation Biology Research Center • Hideki Ohdan • Toshiyasu Kawahara • Ichiro Shimizu • Philip Bardwell • Fabienne Haspot Collaborators •Mike Carroll, Center for Blood Research •Ed Medof, Case Western/Cleveland Clinic