Use of CTLA4Ig for induction of mixed chimerism and renal allograft tolerance in nonhuman primates

Abstract

We have previously reported successful induction of renal allograft tolerance via a mixed chimerism approach in nonhuman primates. In those studies, we found that costimulatory blockade with anti-CD154 mAb was an effective adjunctive therapy for induction of renal allograft tolerance. However, since anti-CD154 mAb is not clinically available, we have evaluated CTLA4Ig as an alternative agent for effecting costimulation blockade in this treatment protocol. Two CTLA4Igs, abatacept and belatacept, were substituted for anti-CD154 mAb in the conditioning regimen (low dose total body irradiation, thymic irradiation, anti-thymocyte globulin and a 1-month posttransplant course of cyclosporine [CyA]). Three recipients treated with the abatacept regimen failed to develop comparable lymphoid chimerism to that achieved with anti-CD154 mAb treatment and these recipients rejected their kidney allografts early. With the belatacept regimen, four of five recipients developed chimerism and three of these achieved long-term renal allograft survival (>861, >796 and >378 days) without maintenance immunosuppression. Neither chimerism nor long-term allograft survival were achieved in two recipients treated with the belatacept regimen but with a lower, subtherapeutic dose of CyA. This study indicates that CD28/B7 blockade with belatacept can provide a clinically applicable alternative to anti-CD154 mAb for promoting chimerism and renal allograft tolerance.

Keywords: Basic (laboratory) research/science; bone marrow/hematopoietic stem cell transplantation; kidney transplantation/nephrology; tolerance: chimerism; tolerance: costimulation blockade; tolerance: experimental.

Fig. 1. Conditioning regimens and cyclosporine levels

(A) All recipients received a nonmyeloablative conditioning regimen which consisted of low dose total body irradiation (TBI 1,5 Gy X2 on days −6 and −5), thymic irraditation (TI; 7 Gy on day-1) and a three day pre-transplant course of horse anti-thymocyte globulin (Atgam on days −2, −1 and 0). Recipients received simultaneous kidney and bone marrow transplantation on day 0, followed by costimulatory blockade (Abatacept in Group A, Belatacept in Groups B and C and anti-CD154 mAb in Group D) for 2 weeks and a one-month course of cyclosporine. In Group C, lower cyclosporine dosages was administered. (B) In Groups A, B and D, cyclosporine was started on day 0 with target therapeutic levels 250–350 ng/ml for the first month(the graph shows only Group B). In Group C, cyclosporine was not started until day 3 with target therapeutic levels 150–200 ng/ml for the first 2 weeks. CyA injection was discontinued on day 28. Because intramuclularly injected oral-prep CyA is slowly absobed, CyA levels did not become nondetectable until day 70–80.

Fig. 2. Peripheral chimerism post DBMT

(A) Although statisitcally not significant, Group B recipients developed chimerism longer than Group D. Lymphoid chimerism observed in Groups A and C was limited (<0.5%). Statistical analysis: Group A vs. B (p=0.07), A vs. C (p=0.98), A vs. D (p=0.18), B vs. C (p=0.24), B vs. D (p=0.79), C vs. D (p=0.5) (B) Groups A and B recipients developed excellent myeloid chimerism comparable to that observed in Group D. Group C recipients failed to develop any myeloid chimerism. Statistical analysis: Group A vs. B (p=0.2), A vs. C (p=0.85), A vs. D (p=0.93), B vs. C (p=0.08), B vs. D (p=0.28), C vs. D (p=0.5)

Fig. 3. Histopathology findings of renal allografts of long-term survivors

(A)The renal allograft in M5710 examined on day 861at autopsy showed minor transplant glomerulopathy without cellular infiltration or interstitial fibrosis. (B, C) Autopsy samples taken from M8010 (B) and from M2611(C) on days 796 and 378 showed no diagnostic abnormality.

Fig. 4. Renal allograft survival

There were statistically significant differences between Groups B vs. A (p<0.005) or B vs. C (p<0.01), but no difference was observed between Groups B vs. D.

Fig. 5. Lymphocyte subsets after transplantation in recipients in Group B (Belatacept) and Group D (anti-CD154 mAb)

(A, B) There was no significant difference in the numbers of memory CD4 or CD8 T cells between Groups B and D (TEM=T effector memory, TCM=T central Memory). (C) In Group D, significant enrichment of activated Tregs (A-Tregs: CD45RA⁻Foxp3^high) was observed among CD4 T cells but no such enrichment of Tregs was observed in Group B. On the other hand, there was no significant difference observed in resting Tregs (R-Treg, CD45RA⁺Foxp3⁺). (D) There was no statistically significant difference between the two groups in memory (CD27⁺) or naïve (CD27⁻) B cells.

Fig. 6. MLR in Group B recipients

Anti-donor and third party responses were monitored by measuring the frequency of IFNγ prodcing cells using the ELISPOT assay in three long-term animals. In M5710, following global hyporesponsiveness of memory T cells on day 42, anti-donor response became detectable on day 100 as his immune function returned. Specific loss of anti-donor response was then observed on 643 days after Tx. M8010 (Fig. 6B) and M2611(Fig. 6C), anti-donor and third party CD4 and CD8 Tmem responses were evaluated separately. In both animals, while CD8 Tmem responses show non-specific suppressive pattern even at 300 days after Tx, CD4 Tmem responses show donor specific hyporesponsiveness. Anti-donor response in M3809, that eventually rejected the renal allograft on day 125, was weaker on day 90 than those against third party cells. M812, another monkey that rejected renal allograft on day 156, showed global hyporensponse on day 95..

Fig. 6. MLR in Group B recipients

Anti-donor and third party responses were monitored by measuring the frequency of IFNγ prodcing cells using the ELISPOT assay in three long-term animals. In M5710, following global hyporesponsiveness of memory T cells on day 42, anti-donor response became detectable on day 100 as his immune function returned. Specific loss of anti-donor response was then observed on 643 days after Tx. M8010 (Fig. 6B) and M2611(Fig. 6C), anti-donor and third party CD4 and CD8 Tmem responses were evaluated separately. In both animals, while CD8 Tmem responses show non-specific suppressive pattern even at 300 days after Tx, CD4 Tmem responses show donor specific hyporesponsiveness. Anti-donor response in M3809, that eventually rejected the renal allograft on day 125, was weaker on day 90 than those against third party cells. M812, another monkey that rejected renal allograft on day 156, showed global hyporensponse on day 95..