Curative islet and hematopoietic cell transplantation in diabetic mice without toxic bone marrow conditioning

Abstract

Mixed hematopoietic chimerism can promote immune tolerance of donor-matched transplanted tissues, like pancreatic islets. However, adoption of this strategy is limited by the toxicity of standard treatments that enable donor hematopoietic cell engraftment. Here, we address these concerns with a non-myeloablative conditioning regimen that enables hematopoietic chimerism and allograft tolerance across fully mismatched major histocompatibility complex (MHC) barriers. Treatment with an αCD117 antibody, targeting c-Kit, administered with T cell-depleting antibodies and low-dose radiation permits durable multi-lineage chimerism in immunocompetent mice following hematopoietic cell transplant. In diabetic mice, co-transplantation of donor-matched islets and hematopoietic cells durably corrects diabetes without chronic immunosuppression and no appreciable evidence of graft-versus-host disease (GVHD). Donor-derived thymic antigen-presenting cells and host-derived peripheral regulatory T cells are likely mediators of allotolerance. These findings provide the foundation for safer bone marrow conditioning and cell transplantation regimens to establish hematopoietic chimerism and islet allograft tolerance.

Keywords: CP: Cell biology; CP: Immunology; allogeneic transplantation; bone marrow; diabetes mellitus; hematopoietic cell transplantation; immunologic tolerance; islet transplantation; mixed chimerism.

Figure 1.. Non-myeloablative conditioning promotes durable donor chimerism across fully mismatched MHC barriers

(A) Transplantation model and strains used. (B) Reduced intensity conditioning regimen. 500 ug αCD117 is administered on day −6, then 200 cGy XRT on day −3, then 300 ug of αCD4/CD8 each on days −2 through 0. On day 0, mice are transplanted with 1.5E6 Lin⁻cKit⁺ HSPCs after the final TCD treatment. (C) Chimerism analysis of peripheral blood 4 weeks after conditioning and HCT including overall chimerism, CD3⁺ T cells, CD11b⁺ myeloid cells, and CD19⁺ B cells. (D and E) Longitudinal chimerism analysis of peripheral blood in 2 separate experimental cohorts. (D: cohort 1; E: cohort 2; n = 5 per cohort) (F) Chimerism analysis of recipient bone marrow at 15 or 18 weeks post-HCT including CD49b⁺ NK cells and Lin⁻Sca1⁺cKit⁺ (LSK) cells. (G) Chimerism analysis of recipient spleen at 15 or 18 weeks post-HCT (n = 10, sum of 2 independent experiments). HSPCs, hematopoietic stem and progenitor cells; XRT, X-ray therapy; TCD, T cell depletion; HCT, hematopoietic cell transplant. X-ray therapy; TCD, T cell depletion; HCT, hematopoietic cell transplant. (C–G) Data show mean ± SEM.

Figure 2.. Histology of short- and long-term islet transplants in mixed chimeras

(A) Experimental transplantation schematic. Mixed chimeras received B6 islets in the left kidney and BALB/c (n = 7) or FVB (n = 3) islets in the opposite kidney. Conditioned non-chimeras received B6 islets in the left kidney and BALB/c islets in the opposite kidney (n = 3). (B) Experimental timeline for islet transplants. Some islet grafts transplanted in mice 13 weeks post-HCT were recovered after 2 weeks (short-term). Other islet grafts (long-term) transplanted in mice 5 weeks post-HCT were recovered after 14 weeks. Chimerism was verified at 4 weeks post-HCT. (C) Representative BALB/c and B6 islet grafts 2 weeks after islet transplantation in mixed chimeras stained for insulin and CD3 or CD45 (n = 2). (D) Representative FVB and B6 islet grafts 2 weeks after islet transplantation in mixed chimeras stained for insulin and CD3 or CD45 (n = 3). (E) Representative BALB/c and B6 islet grafts 2 weeks after islet transplantation in conditioned non-chimeras stained for insulin and CD3 or CD45 (n = 3). (F) Representative BALB/c and B6 islet grafts 13 weeks after islet transplantation in mixed chimeras stained for insulin (n = 5). Scale bars: 100 μm for (C)–(E) and 200 μm for (F).

Figure 3.. Mixed chimerism in diabetic B6 RIPDTR mice results in long-term, donor-matched islet graft tolerance and diabetes reversal

(A) Experimental conditioning and transplantation outline. Conditioning was initiated on day −6 with αCD117, and diabetes was induced on day −4.5 by DT injection. 300 cGy XRT was given on day −3. On day 0 after the final TCD, B6 RIP-DTR mice received islet grafts from B6, BALB/c, or FVB donors. Following islet transplantation, mice receive 1.5E6 Lin⁻cKit⁺ HSPCs from BALB/c donors. (B and C) Longitudinal chimerism analysis demonstrating robust multi-lineage chimerism up to 20 weeks after HCT (B: n = 7 males) or up to 16 weeks after HCT (C: n = 4 females). (D) Chimerism of different cell subtypes in mixed chimera bone marrow at 16 or 20 weeks after HCT. (E) Non-fasting blood glucose of mice that received B6 islets. One mouse transiently required insulin after islet transplantation but eventually became insulinin dependent (black line). Vertical dotted line denotes islet transplant and HCT. Arrow indicates nephrectomy in 2 of 3 mice. (F) Non-fasting blood glucose of mice that received BALB/c islets. Arrow indicates nephrectomy in 3 of 4 mice. (G) Non-fasting blood glucose of mice that received FVB islets (n = 4). Data shown here do not include glycemia measurements after reversion to hyperglycemia. (H) Representative B6 islet graft stained for insulin and CD45 at 20 weeks after transplantation in a mixed chimeric recipient (n = 3). (I) Representative BALB/c islet graft 20 weeks after transplantation in a recipient that developed mixed chimerism (n = 4). (J) Representative FVB islet graft 20 weeks after transplantation in a recipient that developed mixed chimerism (n = 4). Scale bars in (H–J): 200 μm. DT, diphtheria toxin; XRT, X-ray therapy; TCD, T cell depletion; HCT, hematopoietic cell transplant. (B–D) Data show mean ± SEM.

Figure 4.. Analysis of cell subsets that modulate central and peripheral tolerance

(A) Chimerism analysis of CD11c⁺ dendritic cell subsets in the thymus of B6 RIP-DTR mixed chimeras at 16 or 20 weeks post-HCT. pDCs = B220⁺PDCA1⁺, mDC = B220⁻SIRPα⁺CD8⁻, tDC = B220⁻SIRPα⁻CD8⁺. (B) Frequency of thymic CD25^hi tTregs among host CD4⁺ T cells in chimeras versus non-chimeras. (C) Frequency of splenic CD25^hi Tregs among host CD4⁺ T cells in the spleen of chimeras compared with non-chimeras. (D) Frequency of splenic pDCs among CD11c⁺ cells in chimeras versus non-chimeras (n = 11 for chimeras and n = 5 for conditioned non-chimeras, sum of 2 experiments). Data show mean ± SEM. *p < 0.05, **p < 0.01.