Regulatory dendritic cell infusion prolongs kidney allograft survival in nonhuman primates

Abstract

We examined the influence of regulatory dendritic cells (DCreg), generated from cytokine-mobilized donor blood monocytes in vitamin D3 and IL-10, on renal allograft survival in a clinically relevant rhesus macaque model. DCreg expressed low MHC class II and costimulatory molecules, but comparatively high levels of programmed death ligand-1 (B7-H1), and were resistant to pro-inflammatory cytokine-induced maturation. They were infused intravenously (3.5-10 × 10(6) /kg), together with the B7-CD28 costimulation blocking agent CTLA4Ig, 7 days before renal transplantation. CTLA4Ig was given for up to 8 weeks and rapamycin, started on Day -2, was maintained with tapering of blood levels until full withdrawal at 6 months. Median graft survival time was 39.5 days in control monkeys (no DC infusion; n = 6) and 113.5 days (p < 0.05) in DCreg-treated animals (n = 6). No adverse events were associated with DCreg infusion, and there was no evidence of induction of host sensitization based on circulating donor-specific alloantibody levels. Immunologic monitoring also revealed regulation of donor-reactive memory CD95(+) T cells and reduced memory/regulatory T cell ratios in DCreg-treated monkeys compared with controls. Termination allograft histology showed moderate combined T cell- and Ab-mediated rejection in both groups. These findings justify further preclinical evaluation of DCreg therapy and their therapeutic potential in organ transplantation.

Keywords: Costimulation blockade; dendritic cells; memory T cells; rapamycin; renal transplant; rhesus macaques.

Figure 1. Phenotypic and functional characteristics of control DC and DCreg propagated from CD14⁺ blood monocytes mobilized in rhesus monkey leukapheresis products

(A), DC were propagated in GM-CSF + IL-4 from CD14⁺ monocytes, in the absence (control DC) or presence of VitD3/IL-10 (DCreg), as described in the Materials and Methods. Phenotypic data obtained using flow cytometry are shown for unstimulated control DC and DCreg, and also for both populations stimulated for 24 h with a potent, pro-inflammatory cytokine cocktail. (B), DCreg induce minimal allogeneic CD4⁺ and CD8⁺ T cell proliferation compared with control DC. Left: Proliferative responses of normal allogeneic CD4⁺ and CD8⁺ T cells in response to control DC or DCreg were evaluated. CFSE-labeled rhesus PBMC (0.2×10⁶) were cocultured with either control DC or DCreg (0.01×10⁶) at 20:1 ratio for 5 days. After gating on CD4⁺ and CD8⁺ T cells, proliferation was evaluated by CFSE dilution analysis. Right: Mean CFSE proliferation of allogeneic CD4⁺ and CD8⁺ T cells was significantly less in response to DCreg compared to control DC. Data are representative of 5 independent experiments using different monkeys.

Figure 2. Rhesus DCreg enhance apoptosis of alloreactive T memory cells

As in Figure 1, CFSE-labeled rhesus PBMC were cocultured with control DC or DCreg at 20:1 ratio for 5 days, followed by evaluation of T cell proliferation by CFSE dilution analysis. (A) Left: DCreg elicit less CD4⁺/CD8⁺ CD95⁺ T memory cell proliferation. Right: Mean of 5 experiments showing significantly less proliferation of CD95⁺ T cells in response to DCreg. (B) Left: DCreg elicit less granzyme B⁺ CD95⁺ effector memory T cells in comparison to control DC in 5-day MLR. Right: Mean of 3 independent experiments using different donor:responder pairs, showing less induction of granzyme B⁺ T cells after coculture with DCreg. Gating for CD4⁺ T cells was based on isotype controls. As the vast majority of CD8⁺ T cells were Granzyme B⁺ under all culture conditions, gating was based on granzyme B^hi populations. (C) Rhesus DCreg induce less allogeneic CD4⁺ and CD8⁺ T cell proliferation in 5-day CFSE-MLR, but enhance apoptosis of non-proliferating T cells compared with control DC. (D), DCreg induce higher incidences of CD4⁺/CD8⁺ CD95⁺ Annexin⁺ memory T cells than control DC after 5-day MLR culture. Data in C and D are representative of 2 independent experiments using different stimulator-responder pairs.

Figure 2. Rhesus DCreg enhance apoptosis of alloreactive T memory cells

As in Figure 1, CFSE-labeled rhesus PBMC were cocultured with control DC or DCreg at 20:1 ratio for 5 days, followed by evaluation of T cell proliferation by CFSE dilution analysis. (A) Left: DCreg elicit less CD4⁺/CD8⁺ CD95⁺ T memory cell proliferation. Right: Mean of 5 experiments showing significantly less proliferation of CD95⁺ T cells in response to DCreg. (B) Left: DCreg elicit less granzyme B⁺ CD95⁺ effector memory T cells in comparison to control DC in 5-day MLR. Right: Mean of 3 independent experiments using different donor:responder pairs, showing less induction of granzyme B⁺ T cells after coculture with DCreg. Gating for CD4⁺ T cells was based on isotype controls. As the vast majority of CD8⁺ T cells were Granzyme B⁺ under all culture conditions, gating was based on granzyme B^hi populations. (C) Rhesus DCreg induce less allogeneic CD4⁺ and CD8⁺ T cell proliferation in 5-day CFSE-MLR, but enhance apoptosis of non-proliferating T cells compared with control DC. (D), DCreg induce higher incidences of CD4⁺/CD8⁺ CD95⁺ Annexin⁺ memory T cells than control DC after 5-day MLR culture. Data in C and D are representative of 2 independent experiments using different stimulator-responder pairs.

Figure 3. DCreg infusion prolongs MHC-mismatched renal allograft survival in rhesus monkeys

(A), Costimulation blockade/rapamycin-based immunosuppression protocol. Monkeys received DCreg (3–10×10⁶/kg i.v.) (n=6) or no cells (n=6) on day −7 relative to kidney transplantation on day 0. Each group of monkeys was given CTLA4Ig (25 mg/kg i.v.), either from day −7 to day 10 post-transplant (n=4 per group) or from day −7 to day 56 (n=2 per group). Intramuscular rapamycin was commenced on day −2 and whole blood trough levels maintained at 10–15 ng/ml (day 0-day 28); 5–10 ng/ml (day 29-day 150) and 1–5 ng/ml (day 151-day 180). Immunosuppressive treatment was stopped at day 180. (B), serum creatinine levels in control (n=6) and DCreg-treated animals (n=6) at various times post transplant; (C), body weight loss in each group at various times post-transplant (D), urinary protein/creatinine ratios in the same groups of monkeys, 6–8 weeks post-transplant and (E), actuarial graft survival data.

Figure 3. DCreg infusion prolongs MHC-mismatched renal allograft survival in rhesus monkeys

(A), Costimulation blockade/rapamycin-based immunosuppression protocol. Monkeys received DCreg (3–10×10⁶/kg i.v.) (n=6) or no cells (n=6) on day −7 relative to kidney transplantation on day 0. Each group of monkeys was given CTLA4Ig (25 mg/kg i.v.), either from day −7 to day 10 post-transplant (n=4 per group) or from day −7 to day 56 (n=2 per group). Intramuscular rapamycin was commenced on day −2 and whole blood trough levels maintained at 10–15 ng/ml (day 0-day 28); 5–10 ng/ml (day 29-day 150) and 1–5 ng/ml (day 151-day 180). Immunosuppressive treatment was stopped at day 180. (B), serum creatinine levels in control (n=6) and DCreg-treated animals (n=6) at various times post transplant; (C), body weight loss in each group at various times post-transplant (D), urinary protein/creatinine ratios in the same groups of monkeys, 6–8 weeks post-transplant and (E), actuarial graft survival data.

Figure 3. DCreg infusion prolongs MHC-mismatched renal allograft survival in rhesus monkeys

(A), Costimulation blockade/rapamycin-based immunosuppression protocol. Monkeys received DCreg (3–10×10⁶/kg i.v.) (n=6) or no cells (n=6) on day −7 relative to kidney transplantation on day 0. Each group of monkeys was given CTLA4Ig (25 mg/kg i.v.), either from day −7 to day 10 post-transplant (n=4 per group) or from day −7 to day 56 (n=2 per group). Intramuscular rapamycin was commenced on day −2 and whole blood trough levels maintained at 10–15 ng/ml (day 0-day 28); 5–10 ng/ml (day 29-day 150) and 1–5 ng/ml (day 151-day 180). Immunosuppressive treatment was stopped at day 180. (B), serum creatinine levels in control (n=6) and DCreg-treated animals (n=6) at various times post transplant; (C), body weight loss in each group at various times post-transplant (D), urinary protein/creatinine ratios in the same groups of monkeys, 6–8 weeks post-transplant and (E), actuarial graft survival data.

Figure 4. DCreg infusion exerts no consistent effect on anti-donor T cell proliferative responses

(A), Ex vivo anti-donor and anti-third party proliferative responses of circulating CD4⁺ and CD8⁺ T cells from control or DCreg-treated rhesus renal allograft recipients before, and 4–6 and 8–12 weeks post-transplant. Each graph represents the response of one recipient from the DCreg group and its concurrent recipient from the control group against the same donor cells or 3^rd party cells. CFSE-MLRs were performed as described in the Materials and Methods. PBMC from each recipient were cocultured with T cell-depleted PBMC from either donor or third party at 1:1 ratio for 5 days. (B), proliferative responses of T cells from a DCreg-treated monkey (M113) whose transplant survived 300 days, showing initial depression (day 180) of anti-donor and anti-third party responses, but later recovery of anti-donor reactivity (day 240) after withdrawal of all immunosuppression, followed by enhanced anti-donor reactivity at the time of rejection (day 300).

Figure 4. DCreg infusion exerts no consistent effect on anti-donor T cell proliferative responses

(A), Ex vivo anti-donor and anti-third party proliferative responses of circulating CD4⁺ and CD8⁺ T cells from control or DCreg-treated rhesus renal allograft recipients before, and 4–6 and 8–12 weeks post-transplant. Each graph represents the response of one recipient from the DCreg group and its concurrent recipient from the control group against the same donor cells or 3^rd party cells. CFSE-MLRs were performed as described in the Materials and Methods. PBMC from each recipient were cocultured with T cell-depleted PBMC from either donor or third party at 1:1 ratio for 5 days. (B), proliferative responses of T cells from a DCreg-treated monkey (M113) whose transplant survived 300 days, showing initial depression (day 180) of anti-donor and anti-third party responses, but later recovery of anti-donor reactivity (day 240) after withdrawal of all immunosuppression, followed by enhanced anti-donor reactivity at the time of rejection (day 300).

Figure 5. DCreg infusion suppresses CD4⁺ and CD8⁺ Tmem in renal-allografted monkeys and enhances PD-1 and CTLA4 expression on donor-reactive Tmem

(A), Absolute numbers of circulating CD4⁺ and CD8⁺ CD95⁺ Tmem in control and DCreg-treated renal allograft recipients at various times pre- and post-transplant. (B, C) Incidences of PD1⁺CTLA4⁺ Tmem in ex-vivo-stimulated CD4⁺ and CD8⁺ T cell populations from (B) M112 and M143 (control) monkeys, and (C) M147 and M148 (DCreg-treated) monkeys. Recipient PBMC obtained 4 weeks (and 8 weeks in the DCreg group monkeys) post transplant, were cocultured with either donor or third party stimulators for 5 days as in Figure 4. CD4⁺ and CD8⁺ CD95⁺ T cells were then analyzed for cell surface PD-1 and CTLA4 expression. POD = post-operative day.

Figure 5. DCreg infusion suppresses CD4⁺ and CD8⁺ Tmem in renal-allografted monkeys and enhances PD-1 and CTLA4 expression on donor-reactive Tmem

(A), Absolute numbers of circulating CD4⁺ and CD8⁺ CD95⁺ Tmem in control and DCreg-treated renal allograft recipients at various times pre- and post-transplant. (B, C) Incidences of PD1⁺CTLA4⁺ Tmem in ex-vivo-stimulated CD4⁺ and CD8⁺ T cell populations from (B) M112 and M143 (control) monkeys, and (C) M147 and M148 (DCreg-treated) monkeys. Recipient PBMC obtained 4 weeks (and 8 weeks in the DCreg group monkeys) post transplant, were cocultured with either donor or third party stimulators for 5 days as in Figure 4. CD4⁺ and CD8⁺ CD95⁺ T cells were then analyzed for cell surface PD-1 and CTLA4 expression. POD = post-operative day.

Figure 5. DCreg infusion suppresses CD4⁺ and CD8⁺ Tmem in renal-allografted monkeys and enhances PD-1 and CTLA4 expression on donor-reactive Tmem

(A), Absolute numbers of circulating CD4⁺ and CD8⁺ CD95⁺ Tmem in control and DCreg-treated renal allograft recipients at various times pre- and post-transplant. (B, C) Incidences of PD1⁺CTLA4⁺ Tmem in ex-vivo-stimulated CD4⁺ and CD8⁺ T cell populations from (B) M112 and M143 (control) monkeys, and (C) M147 and M148 (DCreg-treated) monkeys. Recipient PBMC obtained 4 weeks (and 8 weeks in the DCreg group monkeys) post transplant, were cocultured with either donor or third party stimulators for 5 days as in Figure 4. CD4⁺ and CD8⁺ CD95⁺ T cells were then analyzed for cell surface PD-1 and CTLA4 expression. POD = post-operative day.