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. Author manuscript; available in PMC: 2015 Dec 1.
Published in final edited form as: Am J Transplant. 2014 Nov 13;14(12):2704–2712. doi: 10.1111/ajt.12936

Use of CTLA4Ig for Induction of Mixed Chimerism and Renal Allograft Tolerance in Nonhuman Primates

Yohei Yamada 1,*, Takanori Ochiai 1,*, Svjetlan Boskovic 1, Ognjenka Nadazdin 1, Tetsu Oura 1, David Schoenfeld 2, Kate Cappetta 1, Rex-Neal Smith 3, Robert B Colvin 3, Joren C Madsen 1, David H Sachs 4, Gilles Benichou 1, A Benedict Cosimi 1, Tatsuo Kawai 1,5
PMCID: PMC4236265  NIHMSID: NIHMS615761  PMID: 25394378

Abstract

We have previously reported successful induction of renal allograft tolerance via a mixed chimerism approach in nonhuman primates (NHP). 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 CTLA4-Igs, Abatacept and Belatacept, were substituted for anti-CD154 mAb in the conditioning regimen (low dose total body irradiation, thymic irradiation, ATG and a one month post-transplant 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.

Introduction

We have previously reported long-term immunosuppression free renal allograft survival after induction of transient hematopoietic chimerism in nonhuman primates (NHP) (1–4). In the previous studies, we found that costimulatory blockade with anti-CD154 mAb significantly enhances chimerism induction and renal allograft tolerance (3). However, anti-CD154 mAb is not currently clinically available due to its thrombogenic side effects (5, 6), making that conditioning regimen inapplicable to clinical transplantation. In our initial clinical trial of tolerance induction for HLA-mismatched kidney allografts, we used the anti-CD2 mAb, MEDI507, chosen because of its unique properties of both T cell depletion and co-stimulatory blockade (7). Although this agent was effective (8, 9), its clinical availability is currently uncertain. Thus we have sought alternative approaches for adding costimulatory blockade to T cell depletion with ATG. We have tested two CTLA4Igs, Abatacept and Belatacept, approved for administration to patients with rheumatoid arthritis and kidney transplantation, respectively. These CTLA4Igs are fusion proteins composed of the Fc region of the immunoglobulin IgG1 fused to the extracellular domain of CTLA4. Abatacept and Belatacept differ by only 2 amino acids in the CTLA4 domain. In this NHP study, we found that Belatacept but not Abatacept, can be effectively substituted for anti-CD154 mAb in our previous successful regimen, thus potentially providing a clinically applicable alternative approach to costimulatory blockade in our nonmyeloablative conditioning regimen to promote chimerism and long-term renal allograft survival without maintenance immunosuppression.

Materials and methods

Animals

A total of 15 Cynomolgus monkeys (Groups A–C, including donor animals) that weighed 3 to 7 kg were used (Charles River Primates, Wilmington, MA). All cynomolgus monkey recipients received the same conditioning regimen with either Abatacept or Belatacept. All surgical procedures and postoperative care of animals were performed in accordance with National Institute of Health guidelines for the care and use of primates and were approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee.

Conditioning Regimens

All recipients underwent conditioning followed by MHC mismatched KTx and DBMT from the same donor. MHC characterization was performed as previously described (7,8). The conditioning regimen consisted of low-dose total body irradiation (TBI, 1.5 GyX2) on days −6 and −5(relative to KTx/DBMT), thymic irradiation (TI, 7 Gy) on day−1, equine ATG (Atgam, Pharmacia and Upjohn, Kalamazoo, MI, 50 mg/kg/day on days −2, −1 and 0) and Abatacept (Group A) or Belatacept (Group B) (CTLA-4 Ig provided by Bristol Meyer Squibb, MA), 20 mg/kg on Days 0 and +2, and 10 mg/kg on days +5 and +15) (Fig. 1a). In Groups A, B and D, a one month course of cyclosporine (CyA) was administered between days 1–28 to maintain therapeutic trough levels of CyA (250–350 ng/ml). In the attempt to reduce potential risks of over-immunosuppression, two additional monkeys (Group C) were treated with low-dose cyclosporine, which was not started until day 3 with target therapeutic levels 150–200 ng/ml during Belatacept treatment (Fig. 1B). Results of Groups A–C were compared with previously reported observations in recipients treated with anti-CD154 mAb (Group D).

Fig. 1. Conditioning regimens and cyclosporine levels.

Fig. 1

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(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.

Renal and bone marrow transplantation

Kidney transplantation (KTx) was performed as reported previously (10). The recipients also underwent unilateral native nephrectomy and ligation of the contralateral ureter on day 0. The remaining native (hydronephrotic) kidney was removed 60–80 days after transplantation. Bone marrow was harvested from the surviving original kidney donor’s iliac bones by multiple percutaneous aspirations. If the animal was sacrificed at the time of donor nephrectomy, bone marrow cells were also harvested from the vertebral bones after euthanasia and kept frozen until DBMT. The unprocessed bone marrow cells (1.0 – 3.0 X 108 mononuclear cells/kg) were infused intravenously.

Flow cytometric analyses, detection of chimerism, cell sorting and invitro MLR

PBMCs, peripheral lymph nodes, spleen and bone marrow cells were labeled with a combination of the following mAbs: CD3 PerCP (SP 34–2), CD4 PerCP (L-200), CD8 PerCP (SK1), CD8 APC (SK1), CD95 FITC (DX2), CD95 APC (DX2), and CD28PE (CD28.2) (BD Pharmingen, San Jose, CA). For chimerism analyses, we used an anti-MHC class I HLA mAb (H38, One Lambda, Inc, CA) reacting specifically with certain MHC class I antigens. The recipient and donor pairs were chosen based on their differential reactivity to this mAb. The fluorescence of the stained samples was analyzed using FACS Calibur and FACS Scan flow cytometers and Cell Quest Software (BD), or FlowJo software. For assessing the effect of CTLA4Ig on memory T cell functions, the monoclonal antibodies anti-CD16, anti-CD95, anti-CD4 and anti-CD8 were used to purify CD4 Tmems (CD16−CD4+CD95+) and CD8 Tmems(CD16−CD8+CD95+) using a FACS Vantage cell sorter (BD Immunocytometry System), then those purified populations were tested for their IFNγ production in ELISPOT assay as previously described (11, 12). The purity of sorted cells was consistently > 95% as previously described.

Measurement of T cell-mediated alloresponses by ELISPOT

ELISPOT plates (Millipore, Bedford, MA) were pre-coated with 5 μg/ml of capture antibodies against γIFN (Mabtech, Sweden) in PBS and stored overnight at 4°C. The responding cells, CD4 Tmems and CD8 Tmems, separated from fresh PBMCs were co-cultured with an equal number of irradiated donor PBMCs as stimulating cells (1.5 × 105 cells/well), or unstimulated in medium alone, or with PHA at 1 μg/ml (Sigma). After 44 hours incubation at 37°C, the plates were washed and biotinylated detection antibodies (Mabtech, Sweden) were added (4°C OVN). After 5 washes with PBS, streptavidin-horseradish-peroxidase conjugate in PBS BSA 0.5% (Dako, Glostrup, Denmark) was added for 2 hrs at room temperature, followed by 5 washes. Finally, 50 μl/well of tetramethylbenzidine (TMB) liquid substrate (Sigma-Aldrich) was added and incubated for 30 min in the dark. The resulting spots were counted with an ELISPOT image analyzer (CTL Inc., Cleveland, OH), as described elsewhere (13).

Histological analyses

Protocol renal biopsies were obtained every 2–4 months in recipients with stable function as well as whenever a rise in serum creatinine occurred. Tissue was processed for routine microscopy and a portion frozen for immunofluorescence staining. Other organs obtained surgically (lymph nodes, native kidney and spleen) were similarly processed. Following euthanasia of any monkey, complete autopsies were performed for histopathologic examination of the renal allograft, lymph nodes, heart, lung, liver, pancreas, thymus and skin.

Statistical analyses

We used an analysis of variance to compare the levels of chimerism in each group. For comparison of the T/B subsets, we used unpaired Student’s t -test. Renal allograft survival was analyzed using Kaplan-Meier analysis using GraphPad Prism Pro (GraphPad Software, Inc, San Diego, CA).

Results

Belatacept but not Abatacept promoted mixed chimerism and renal allograft tolerance

Recipients received the conditioning regimen with either Abatacept (Group A) or Belatacept (Group B) in place of anti-CD154 mAb (Fig. 1A). In the attempt to reduce potential risks of over-immunosuppression, two additional monkeys (Group C) were treated with a lower dose of cyclosporine, which was not started until day 3, with target therapeutic levels 150–200 ng/ml, instead of 250–350 ng/ml during Belatacept treatment (Fig. 1B). Results of Groups A–C were compared with previously reported observations in recipients treated with anti-CD154 mAb (Group D).

Although 2/3 recipients in Group A developed similar levels of myeloid chimerism comparable to Group D (Fig. 2), lymphoid chimerism was limited (<5%). All three rejected their renal allografts early (day 56, 89 and 121) after the immunosuppression was discontinued. In Group B, one recipient (M812) failed to develop chimerism and the kidney was rejected on day 156. However, 4/5 recipients developed significantly higher levels of chimerism (Fig. 2) than that previously achieved in Group D and three of them survived long-term without immunosuppression. The renal allograft of one long-term survivor (M5710) on day 861 showed minor transplant glomerulopathy without C4d staining or interstitial fibrosis (Fig. 3A). No diagnostic abnormality was found in M8010 and M2611 (Figs. 3B and 3C). Although two early allograft losses due to acute rejection were observed in Group B, long term allograft survival was comparable to the recipients treated with the anti-CD154 mAb (Group D)(Fig. 4). Two recipients who were treated with the Belatacept regimen but with lower, subtherapeutic doses of CyA (Group C) failed to develop chimerism (Fig. 2), and both rejected their allograft early (76 and 126 days).

Fig. 2. Peripheral chimerism post DBMT.

Fig. 2

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(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.

Fig. 3

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(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.

Fig. 4

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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.

Enrichment of Tregs was not observed after Belatacept treatment

We compared the effect of Belatacept and anti-CD154 mAb treatment on the recovery of lymphocyte subsets. Significantly higher levels of activated Tregs (A-Tregs: CD45RA−Foxp3high) among CD4 T cells were observed during the first 40 days after transplantation in Group D recipients than that observed in Group B. On the other hand, there was no significant difference observed in resting Tregs (R-Treg, CD45RA+Foxp3+) (Fig. 5C), CD4 or CD8 memory T cells (Fig. 5A, B), naive T cells (data not shown) NK/NKT cells (data not shown), nor of memory (CD27+) and naïve (CD27−) B cells. In both groups, while the number of peripheral naïve B cells started to recover around day 50 post DBMT, the count of peripheral memory B cells (B Mem) remained low until day 80 (Fig. 5D).

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

Fig. 5

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(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−Foxp3high) 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.

Donor specific hyporesponsiveness in the long-term survivors treated with Belatacept

In Group B long-term survivors (M5710, M8010 and M2611), anti-donor and third party responses were monitored by measuring the frequency of IFNγ producing cells using ELISPOT assay. In M5710 (Fig. 6A), following the detection of global hyporesponsiveness of memory T cells on day 42, anti-donor response returned on day 100 as his immune function returned. Specific loss of anti-donor response was then observed by day 643 post Tx. In M8010 (Fig. 6B) and M2611 (Fig. 6C), anti-donor and third party CD4 and CD8 memory T cells (Tmem) responses were evaluated separately. In both recipients, while global suppression of CD8 Tmem alloresponses was observed, CD4 Tmem ELISPOT showed donor specific hyporesponsiveness after transplantation. However, these MLR assays may not be useful as a tool to predict the eventual outcome of the transplanted kidney as MLR in M3809, that eventually rejected the renal allograft on day 125, also showed donor specific hyporesponsiveness on day 90. M812, another monkey that rejected renal allograft on day 156, showed global hyporensponsiveness on day 95.

Fig. 6. MLR in Group B recipients.

Fig. 6

Fig. 6

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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..

Discussion

Promotion of mixed chimerism and allograft tolerance by costimulatory blockade has been regularly achieved in rodent studies (14–18). In one study, by combining CD154 blockade and high-dose bone marrow cell infusion, stable mixed chimerism and skin allograft tolerance has been achieved even without cytoreductive therapy (19). Different from rodent studies, where stable chimerism leads to allograft tolerance via thymic deletion mechanisms (20), the chimerism induced in our NHP and human recipients has always been transient (1, 3, 21) and peripheral mechanisms of tolerance induction are assumed to play a major role after disappearance of chimerism. Therefore, addition of costimulatory blockade may be even more important to promote renal allograft tolerance in our approach. We previously evaluated CD154 blockade in our NHP conditioning regimen and observed significant promotion of mixed chimerism and renal allograft tolerance following the addition of anti-CD154 mAb (3). Unfortunately, anti-CD154 mAb is not clinically available due to its thrombogenic side effects (5). Therefore, the major goal of this study was to identify clinically available agents that provide costimulatory blockade and can promote chimerism and renal allograft tolerance. For clinical use, the CTLA4Igs, Abatacept and Belatacept, are the only costimulatory blockers currently approved by the FDA. The first generation CTLA4Ig proved to be very effective to prolong allograft survival in rodent models (22–25) and the anti-human reagent, Abatacept, was found clinically effective in the treatment for rheumatoid arthritis (26). However, administration of Abatacept in NHP transplant models (27, 28) failed to prolong renal allograft survival, presumably due to its low affinity to B-7 molecules. In our current study, Abatacept also failed to induce lymphoid chimerism comparable to that observed with anti-CD154 mAb treatment and the Abatacept treated recipients rejected their allografts soon after discontinuation of immunosuppression. We subsequently evaluated the second generation CTLA4Ig, Belatacept, for which avidity to B7 ligands, especially CD86 is significantly improved (29). In contrast to Abatacept, Belatacept induced excellent chimerism and long-term results comparable to the anti-CD154 mAb regimen. The reason of the better outcomes by Belatacept is presumably due to superior affinity to B-7 molecule, but the differences in their pharmacodynamics or pharmacokinetics might also have resulted in different outcomes. Although the conclusion should be made cautiously due to the limited number of animals, the current study suggested that effective costimulatory blockade is indeed critical for induction of tolerance in our approach.

The group in Emory University has recently demonstrated successful induction of prolonged mixed chimerism using the conditioning regimen consisted of busulfan, basilliximab, anti-CD40 mAB, Belatacept and sirolimus. The chimerism induced with this conditioning regimen was myeloid dominant with very little T cell chimerism but lasted as long as 200 days. However chimerism eventually disappeared after dicontinuation of immunosuppression (30). In their subsequent study, they performed combined kidney and bone marrow transplantation using the same conditioning regimen (31). In contrast to our results, all five recipients rejected their kidney allografts despite successful chimerism induction. The discrepancy in the results could be attributed to differences in treatments of the conditioning regimen (e.g. lack of thymic irradiation in Emory’s protocol) but more importantly to their lack of T cell chimerism. In our study, the long-term renal allograft survival is related to the levels of lymphoid chimerism (including T cell chimerism) (32, 33) and myeloid chimerism has been irrelevant to induction of the renal allograft tolerance. A similar observation was also made in the current study and all recipients treated with Abatacept developed myeloid chimerism with very little lymphoid chimerism consistently rejected the kidney allograft. We speculate that interactions between some donor cells in the lymphoid lineage and recipient residual T cells are important for induction of renal allograft tolerance.

Since an increased incidence of post-transplant lymphoproliferative disease (PTLD) has been reported in some clinical trials with Belatacept, we additionally evaluated the Belatacept regimen targeting lower CyA levels. With our nonmyeloablative conditioning regimen, the NHP recipients typically develop pancytopenia with significant T cell depletion induced by the synergistic effects of TBI and Atgam during the first 2 post-transplant weeks. In addition, since Belatacept was administered intensively during the first two weeks and has a half-life of 8–10 days, therapeutic levels of CyA may not be necessary during this period. Thus, in two recipients, initiation of CyA administration was delayed until post-transplant day 3 and subsequent doses during the first two weeks were also reduced to maintain trough levels under 200 ng/ml. Unfortunately, neither of these two recipients developed chimerism and both rejected their allografts soon after discontinuation of CyA. Thus, although successful CNI sparing can be accomplished in kidney transplant patients treated with Belatacept (34–39), we could not achieve this in our NHP recipients in whom chimerism was not induced even with severe leukopenia resulting from the other conditioning regimen components (TBI, TI and Atgam). This observation may indicate that costimulatory blockade with Belatacept alone is not sufficient to achieve engraftment of donor hematopoietic stem cells.

In lymphocyte subset analyses, we found no enrichment of A-Tregs in the peripheral blood in recipients treated with Belatacept, while significant enrichment of A-Tregs was observed after anti-CD154 mAb treatment. This observation raises a concern that CTLA-4Ig might prevent expansion of Tregs, which has been reported to promote chimerism and allograft tolerance induction (40, 41). Such inhibition of Treg expansion after CTLA4Ig has also been reported in rodents and clinical studies (42, 43). Nevertheless, in the current study, the level of chimerism induced after Belatacept was significantly better than that observed with anti-CD154 treatment and Treg levels in the peripheral blood may not be relevant to induction of chimerism.

In conclusion, this is the first report of successful immunosuppression free, long-term survival using Belatacept in a preclinical primate kidney transplant model. We believe that the findings of this study may be directly applicable to clinical kidney transplantation.

Table 1.

Treatment groups and outcomes

Group N Costimulatory Blockade CyA Trough2 Monkey ID Chimerism Pathology Renal Alllograft Survival
100 200 300 400 500 days
A 3 Abatacept 250–350 M1605 − AR3 graphic file with name nihms615761t1.jpg
M905 + AR graphic file with name nihms615761t2.jpg
M1005 + AR graphic file with name nihms615761t3.jpg
B 5 Belatacept 250–350 M5710 + Minor glom.4 graphic file with name nihms615761t4.jpg
M8010 + NDAR5 graphic file with name nihms615761t5.jpg
M2611 + NDAR graphic file with name nihms615761t6.jpg
M812 − AR graphic file with name nihms615761t7.jpg
M3809 + AR graphic file with name nihms615761t8.jpg
C 2 Belatacept 150–200 M913 − AR graphic file with name nihms615761t9.jpg
M613 − AR graphic file with name nihms615761t10.jpg
D1 8 Anti-CD154 mAb 250–350 M5898 + NADR graphic file with name nihms615761t11.jpg
M2800 + NADR graphic file with name nihms615761t12.jpg
M1900 + CR7 graphic file with name nihms615761t13.jpg
M200 + NDAR graphic file with name nihms615761t14.jpg
M4498 + CR graphic file with name nihms615761t15.jpg
M5598 + CR graphic file with name nihms615761t16.jpg
M2198 + NDAR graphic file with name nihms615761t17.jpg
M300 − AR graphic file with name nihms615761t18.jpg
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1

historical data,

2

during days-0–28 after transplantation,

3

acute rejection,

4

minor glomeruopathy,

5

no diagnostic abnormality,

6

euethanized with normal kidney function due to self mutilation,

7

chronic rejection

Acknowledgments

This study was supported in part by NIH AI102405-01, PO-1 HL 18646-25 and a research fund provided by Bristol Myers Squib. We thank Ms. Susan Shea for technical assistance.

Funding:

Part of this study was conducted by research fund provided by Bristol Myers Squib.

List of abbreviations

NHP

nonhuman primates

CyA

cyclosporine

TMEM

memory T cells

TEM

effector memory T cells

TCM

central memory T cells

KTx

kidney transplantation

DBM

donor bone marrow

DBMT

donor bone marrow transplantation

Footnotes

Disclosure

The authors of this manuscript have no conflicts of interest to disclose as described by the American Journal of Transplantation.

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