Long-term Nonhuman Primate Renal Allograft Survival Without Ongoing Immunosuppression in Recipients of Delayed Donor Bone Marrow Transplantation

Abstract

Background: We have previously reported successful induction of renal allograft tolerance in nonhuman primates (NHP) after an initial posttransplant period of conventional immunosuppression (delayed tolerance) using a nonmyeloablative conditioning regimen consisting of anti-CD154 and anti-CD8 mAbs plus equine antithymocyte globulin (Atgam) and donor bone marrow transplantation (DBMT). Because these reagents are not currently clinically available, the protocol was revised to be applicable to human recipients of deceased donor allografts.

Method: Four cynomolgus monkeys received major histocompatibility complex-mismatched kidney allografts with conventional immunosuppression for 4 months. The recipients were then treated with a nonmyeloablative conditioning regimen consisting of thymoglobulin, belatacept, and DBMT. The results were compared with recipients treated with conditioning regimen consisting of Atgam and anti-CD154 mAb, with and without anti-CD8 mAb.

Results: In 4 consecutive NHP recipients treated with the modified conditioning regimen, homeostatic recovery of CD8 TEM was delayed until after day 20 and multilineage chimerism was successfully induced. Three of the 4 recipients achieved long-term allograft survival (>728, >540, >449 days) without ongoing maintenance immunosuppression. Posttransplant MLR showed loss of antidonor CD8 T cell and CD4 IFNγ responses with expansion of CD4FOXP3 regulatory T cells. However, the late development of donor-specific antibody in NHP recipients confirms the need for additional anti-B-cell depletion with agents, such as rituximab, as has been shown in our clinical trials.

Conclusions: This study provides proof of principle that induction of mixed chimerism and long-term renal allograft survival without immunosuppression after delayed DBMT is possible with clinically available reagents.

Figure 1. Conditioning regimen for delayed tolerance

All recipients initially underwent KTx alone and received conventional triple drug immunosuppression including tacrolimus, mycophenolate mofetil, and prednisone. Three to 4 months later, the recipients underwent conditioning and DBMT. In the Atgam/aCD154 regimen (Groups #1 and #2), low-dose total body irradiation (1.5 Gy × 2) on days -6 and -5 (relative to DBMT), thymic irradiation (7 Gy) on day -1, and Atgam (50mg/kg X3) were administered before DBMT. After DBMT, a short course of anti-CD154 mAb and a 1-month course of cyclosporine A (CyA) were administered, after which no further immunosuppression was given. In the thymo/bela regimen (Group #3), anti-CD8 mAb, Atgam, and anti-CD154 mAb were replaced with thymoglobulin (10 mg/kg on days -2 and -1) and belatacept.

Figure 2. Lymphocyte subsets after DBMT

With the Atgam/aCD154 regimen, rapid recovery of CD8⁺ effector memory T cells (CD8⁺ T_EM) and central memory T cells (CD8⁺ T_CM) was observed by day 12 (triangle). With the addition of anti-CD8 mAb to the Atgam/aCD154 regimen, prolonged deletion of CD8⁺T_EM and T_CM was observed for more than 30 days (open circle). With the thymo/bela regimen, deletion of CD8⁺T_EM and T_CM was less intense than that observed with anti-CD8 mAb, but more prominent than Atgam alone (red circle). There was no difference observed in other T cell subsets, such as NK cells and B cells (B cell counts were not monitored in Group A).

Figure 3. Chimerism in the myeloid and lymphoid lineages after DBMT

With the Atgam/aCD154 regimen (triangle), the recipients failed to develop chimerism (Figure 3, triangle). By adding anti-CD8 mAb to the Atgam/aCD154 regimen, 10/12 successfully developed mixed chimerism (Figure 3, open circle). With the thymo/bela regimen, all 4 recipients successfully developed chimerism in both lymphoid and myeloid lineages (red circle).

Figure 4

A. Renal allograft survival after DBMT Renal allograft survival was significantly superior in recipients of the Thymo/bela regimen (red line), compared with recipients of the Atgam/aCD154 (p<0.03) regimen (dotted gray line). There was no statistically significant difference between thymo/bela and Atgam/aCD8/aCD154 (black line). B. Serum creatinine levels after DBMT in Group 3 recipients Serum creatinine was stable around 1.2 mg/dl during the entire observation period in Recipient #2, while Recipient #3 received his kidney from a relatively small recipient and his baseline creatinine levels were around 1.7 mg/dl. After the appearance of DSA, the baseline creatinine levels became slightly elevated (2.0 mg/dl). In Recipient #1, serum creatinine levels gradually increased from 1.0 to 2.1 mg/dl over 700 days. C. DSA titers after DBMT. Anti-T cell DSA has never been detected in any of the 3 recipients. However, anti-B cell DSA, presumably against MHC-class II, became positive after day 100 post-DBMT in Recipient #1. Recipient #2 transiently developed weak DSA after day 400. The cut-off median fluorescent intensity (MFI) for positive DSA (dotted line) was defined as 3 times higher than the pretransplant DSA titer.

Figure 4

A. Renal allograft survival after DBMT Renal allograft survival was significantly superior in recipients of the Thymo/bela regimen (red line), compared with recipients of the Atgam/aCD154 (p<0.03) regimen (dotted gray line). There was no statistically significant difference between thymo/bela and Atgam/aCD8/aCD154 (black line). B. Serum creatinine levels after DBMT in Group 3 recipients Serum creatinine was stable around 1.2 mg/dl during the entire observation period in Recipient #2, while Recipient #3 received his kidney from a relatively small recipient and his baseline creatinine levels were around 1.7 mg/dl. After the appearance of DSA, the baseline creatinine levels became slightly elevated (2.0 mg/dl). In Recipient #1, serum creatinine levels gradually increased from 1.0 to 2.1 mg/dl over 700 days. C. DSA titers after DBMT. Anti-T cell DSA has never been detected in any of the 3 recipients. However, anti-B cell DSA, presumably against MHC-class II, became positive after day 100 post-DBMT in Recipient #1. Recipient #2 transiently developed weak DSA after day 400. The cut-off median fluorescent intensity (MFI) for positive DSA (dotted line) was defined as 3 times higher than the pretransplant DSA titer.

Figure 5. Renal allograft biopsies in long-term survivors

The renal allograft biopsy acquired from Recipient #2 on day 449 post-DBMT showed no evidence of rejection (A). The biopsy from Recipient #3 on day 299 showed no rejection but a subsequent biopsy on day 411 showed the development of transplant glomerulopathy (C) with positive C4d (D). A pre-DBMT protocol biopsy from Recipient #1 performed on day 72 after KTx (15 days before DBMT) showed positive C4d staining (E). Although the biopsy from Recipient #1 on day 236 showed no rejection with focal nodular aggregates (F), a biopsy on day 399 and later biopsies showed features of chronic antibody-mediated rejection (G) with persistent diffuse C4d staining (H). A representative biopsy with Foxp3 staining (brown) from Recipient #2 showed Treg-rich organized structures (TOLS).

Figure 6. CFSE-MLR in 2 long-term survivors

A: Representative flow cytometry dot plots on T cell (CD8 and CD4) responses to donor antigens after DBMT. B: Recipient #2 T cell responses pre-and post DBMT: Recipient #2 exhibited a significant anti-donor CD8⁺ T cell response before DBMT. It was markedly decreased after conditioning and DBMT. Although a reduction of the anti-third-party CD8⁺ T cell response was also observed, it remained 2 times higher than that observed with donor antigen stimulation. In contrast, substantial anti-donor CD4⁺ T cell proliferation persisted even after DBMT (left panel). Among these proliferated CD4⁺ cells, a marked reduction of Th1 (CD4⁺INF-γ) responses was observed after DBMT (right panel), while there was an upward trend in donor reactive regulatory T cell (Treg) (CD4⁺FOXP3⁺) expansion after DBMT. Treg expansion was also observed by third-party stimulation, but it was lower than that achieved by donor stimulation (right panel). C: Recipient #3 T cell responses: T cell (CD8⁺, CD4⁺, CD4⁺INF-γ and CD4⁺FOXP3⁺) responses similar to Recipient #2 were observed in Recipient #3.

Figure 7. A hypothesis of renal allograft tolerance via transient mixed chimerism

Chimeric donor cells provide significant donor-antigen presentation under co-stimulatory blockade. This may result in the generation of donor-specific memory T cells that can produce cytokines (eg Th3 produces TGF-β) upon stimulation by donor antigens. Upon encountering donor antigens in the graft, these T cells produce local cytokines that favor the conversion of non-Treg cells to Treg in the renal allograft.