Addition of Anti-CD40 Monoclonal Antibody to Nonmyeloablative Conditioning with Belatacept Abrogated Allograft Tolerance Despite Induction of Mixed Chimerism

Abstract

BACKGROUND:

We recently reported anti-CD40 monoclonal antibody and rapamycin (aCD40/rapa) to be a reliable, nontoxic, immunosuppressive regimen for combined islet and kidney transplantation (CIKTx) in nonhuman primates (NHPs). In the current study, we attempted to induce allograft tolerance through the mixed chimerism approach using a conditioning regimen with aCD40 and belatacept (Bela).

METHODS:

Five CIKTx or kidney transplant (KTx) recipients were treated with aCD40/rapa for 4 months. All recipients then received a conditioning regimen including horse anti-thymocyte globulin (hATG) and aCD40/Bela. The results were compared with previous reports of recipients treated with Bela-based regimens.

RESULTS:

All 3 CIKTx recipients developed mixed chimerism, which was significantly superior to that observed in the previous Bela-based studies. Nevertheless, all CIKTx recipients in this study lost their islet and renal allografts as a result of cellular and humoral rejection on days 140, 89, and 84. The 2 KTx-alone recipients were treated with the same conditioning regimen and suffered rejection on days 127 and 116, despite the development of excellent chimerism. B lymphocyte reconstitution dominated by memory phenotypes was associated with early development of donor-specific antibodies in 4/5 recipients. In vitro assays showed no donor-specific regulatory T cell (Treg) expansion, which has been consistently observed in tolerant recipients with our mixed chimerism approach.

CONCLUSION:

Despite displaying excellent immunosuppressive efficacy, costimulatory blockade with anti-CD40 mAb (2C10R4) may inhibit the induction of renal or islet allograft tolerance via a mixed chimerism approach.

INTRODUCTION

We previously developed a clinically applicable conditioning regimen for simultaneous kidney and bone marrow transplantation (SKBMT) in nonhuman primates (NHPs) using horse anti-thymocyte globulin (hATG) and belatacept (Bela/hATG)¹. This protocol was subsequently modified for deceased donor transplantation by delaying donor bone marrow transplantation (DBMT) until 4 months after kidney transplantation (delayed DBMT)². In the delayed tolerance protocol, more profound T cell depletion with rabbit ATG (rATG) in place of hATG was required to suppress memory T cell (T_MEM) responses presumably induced by the renal allograft despite conventional immunosuppression administered until DBMT. However, when this delayed DBMT was applied to combined islet and kidney transplantation (CIKTx), a prominent inflammatory cytokine syndrome elicited by rATG³ resulted in loss of islet function. Therefore, we further revised the delayed DBMT regimen for CIKTx recipients by restoring hATG, expecting only moderate T cell depletion but less inflammatory responses. To suppress T_MEM responses after DBMT, we instead added anti-CD40 monoclonal antibody (mAb), which has been demonstrated to have inhibitory effects on T_MEM in NHP transplant models.^3,4 Furthermore, the synergistic effect of anti-CD40 mAb and CTLA4Ig has been suggested in murine allograft models⁵ and in an NHP bone marrow transplant model⁶.

In the current study, we evaluated the efficacy of dual costimulatory blockade (CB) with anti-CD40 mAb and belatacept for induction of allograft tolerance via the mixed chimerism approach.

MATERIALS AND METHODS

Animals and pair selections

A total of 10 cynomolgus monkeys, including donor animals, weighing 3–8 kg were used for this study (Charles River Primates, Wilmington, MA). Donors and recipients were paired based on ABO compatibility and major histocompatibility complex (MHC) mismatching. MHC characterization was performed as previously described^7,8. All surgical procedures as well as postoperative care of animals was performed in accordance with National Institutes of Health guidelines for the care and use of primates and approved by the Massachusetts General Hospital Institutional Animal Care and Use Committee.

Experimental design

The results from aCD40/Bela/hATG-treated animals were compared with results from previously reported recipients who received either Bela/hATG¹ or Bela/rATG².

Maintenance immunosuppression before DBMT:

Three cynomolgus monkeys received CIKTx and 2 received KTx alone from MHC-mismatched donors. All monkeys were treated with anti-CD40 mAb⁹ (NIH Nonhuman Primate Reagent Resource, Boston, MA, Cat# PR-4047, RRID:AB_2716325) (2C10R4: 20 mg/kg i.v. on days 0, 2, 5, 7, 9, and weekly at 10 mg/kg) plus daily rapamycin (LC laboratories, Woburn, MA) i.m. from day 0 to maintain trough levels at 10–15 ng/ml. Anti-inflammatory therapy, including tocilizumab (Chugai Pharm, Tokyo, Japan) (anti-IL-6R mAb: 10 mg/kg i.v. on days 0 and 5) and etanercept (Immunex, Seattle, WA) (TNF-alpha receptor fusion protein: 25 mg i.v. on days 0, 3, 7, and 10), was administered to all recipients (Fig. 1A)³.

Figure 1: The DBMT conditioning regimens with or without anti-CD40 mAb.

aCD40/Bela/hATG: Two to 3 weeks before combined islet and kidney transplantation (CIKTx), cynomolgus monkey recipients received streptozotocin (STZ: 75 mg/kg) for induction of diabetes. After transplantation, the CIKTx or kidney transplant (KTx)-alone recipients received the same immunosuppressive therapy with anti-CD40 mAb and rapamycin for 4 months. Anti-inflammatory therapies included anti-IL-6R mAb (10 mg/kg) and TNF-alpha receptor fusion protein (Etanercept: 25 mg) up to day 10 (A).³ At four months after transplantation, all recipients were conditioned with total body irradiation (TBI: 1.5 Gy × 2 on days −6 and −5, relative to DBMT), local thymic irradiation (TI: 7 Gy on day −1, relative to DBMT), and horse anti-thymocyte globulin (hATG: 50 mg/kg ×c 3 on days −2, −1 and 0, relative to DBMT). Following donor bone marrow transplantation (DBMT), the recipients were treated with dual costimulatory blockade (CB) with anti-CD40 mAb (20 mg/kg, on post-DBMT days 0, 2, 5, 7, 9, and 12) and belatacept (20 mg/kg, on post-DBMT days 0, 2, 5, and 15). Cyclosporine A (CyA) was administered for 28 days following DBMT, after which no immunosuppression was administered (A).

Bela/hATG: In a previous study, the conditioning regimen consisted of low-dose TBI (1.5 Gy × 2) on days −6 and −5 relative to simultaneous kidney and bone marrow transplantation (SKBMT), TI on day-1, hATG (50 mg/kg × 3 on days −2, −1, and 0) and belatacept (20 mg/kg × 4 on days 0, 2, 5, and 15). A 1-month course of CyA was administered to maintain therapeutic trough levels of 250–350 ng/ml (B).¹

Bela/rATG: In the previous deceased donor study, all recipients initially underwent KTx alone with a conventional triple drug immunosuppressive regimen consisting of tacrolimus, mycophenolate mofetil, and prednisone (B). Four months after KTx alone, the recipients underwent conditioning and DBMT (delayed-DBMT). The conditioning regimen for DBMT is same as the Bela/hATG regimen, except the hATG was replaced with rATG (B).²

Conditioning regimen for DBMT used in this study:

aCD40/Bela/hATG (n=5):

Four months after CIKTx or KTx alone, all NHP recipients were conditioned with total body irradiation (TBI: 1.5 Gy on days −6 and −5, relative to the day of DBMT), local thymic irradiation (TI: 7 Gy on day −1), and hATG (Atgam: Pharmacia and Upjohn, Kalamazoo, MI: 50 mg/kg i.v. on days −2, −1 and 0). Following DBMT, the recipients were treated with anti-CD40 mAb (2C10R4: 20 mg/kg i.v. on days 0 and 2, 5, 7, 9 and 12 post-DBMT) and belatacept (Bela: provided by Bristol Meyer Squibb, New York, NY: 20 mg/kg i.v. on days 0, 2, 5, and 15 post-DBMT). Cyclosporine A (CyA: Novartis, Basel, Switzerland) was administered i.m. on days 1–28 post DBMT, after which no immunosuppression was administered (Fig. 1A).

Conditioning regiments from previous studies

Bela/hATG (n=5)¹:

The conditioning regimen consisted of low-dose TBI (1.5 Gy × 2) on days −6 and −5 relative to simultaneous kidney and bone marrow transplantation (SKBMT), TI on day-1, hATG (50 mg/kg × 3 on days −2, −1, and 0, relative to SKBMT), and belatacept (20 mg/kg × 4 on days 0, 2, 5, and 15, relative to SKBMT). A 1-month course of CyA was administered to maintain therapeutic trough levels of 250–350 ng/ml (Fig. 1B)¹.

Bela/rATG (n=4)²:

All recipients initially underwent KTx alone with a conventional triple drug immunosuppressive regimen consisting of tacrolimus (Astellas Pharma, Inc., Osaka, Japan) (starting with 0.1 mg/kg/day i.m. to maintain trough levels of 10–20 ng/dl), mycophenolate mofetil (Roche, Inc., Nutley, NJ) (200 mg/day), and prednisone (starting with 40 mg/day and tapering to a 1 mg/day maintenance dose in 2 weeks) (Fig. 1B). Four months after KTx alone, the recipients underwent conditioning and DBMT (delayed-DBMT). The conditioning regimen for DBMT was identical to the Bela/hATG regimen, with the exception of hATG, which was replaced by rATG (Thymoglobulin: Bridgewater, NJ: 10 mg/kg on day −2 and −1, relative to DBMT) (Fig. 1B)².

Diabetes induction and management

As previously reported³, CIKTx recipients received streptozotocin (STZ) i.v. at a dose of 75 mg/kg (Zanosar, Teva Parenteral Medicines, Irvine, CA) 2 – 3 weeks before planned CIKTx after which blood glucose (BG) levels were monitored twice daily and measured by Accu-Check active II (Roche, Indianapolis, IN). After administration of STZ, all animals developed diabetes, defined as 3 consecutive fasting blood glucose (FBG) readings >250 mg/dL with C-peptide levels <50 pg/mL (C-peptide, Luminex, Merck Millipore, Billerica, MA). Regular insulin (Neutral Protamine Hagedorn and Humulin R, Eli Lilly Co., Indianapolis, IN) and Lantus (Eli Lilly Co.) were administered via a sliding scale to maintain BG levels <200 mg/dL.

Islet isolation and CIKTx

As previously reported³, the protocol for islet isolation was based on a modified human islet isolation protocol¹⁰. Under general anesthesia, monkeys underwent heterotopic KTx and bilateral native nephrectomies as previously described¹¹, followed by islet allograft infusion into a branch of the inferior mesentery vein. The islet was gravity-infused slowly over a period of approximately 10–15 min.

Definition of islet rejection and BG control after rejection

The loss of islet function was diagnosed when the FBG level exceeded 250 mg/dL for 3 consecutive days. The date of graft rejection was defined as day 1 of the first 3 consecutive days of rejection. Subsequent to rejection, recipient animals were treated with exogenous insulin to maintain BG levels <200 mg/dL³.

Flow cytometric analyses

Peripheral blood mononuclear cells (PBMCs) were labeled with a combination of the following mAbs: CD3 (SP34–2), CD4 (L200), CD8 (SK1), CD21 (B-ly4), CD27 (M-T271), CD28 (CD28.2), CD95 (DX2), and IgM (G20–127) (BD Pharmingen, San Jose, CA), CD20 (2H7) (Biolegend, Inc., San Diego, CA) and FOXP3 (236A/E7) (eBioscience, Inc., San Diego, CA). Based on previous reports, which suggest that long-term allograft survival is associated with a predominance of immature B lymphocyte reconstitution,^12,13 CD3^-CD20⁺CD21⁺IgM⁺ or CD3^-CD20⁺CD21^-CD27⁺ B cells representing immature or mature B cell phenotypes, respectively, were monitored before and after conditioning. The fluorescence of the stained samples was analyzed using FACSverse (BD Biosciences, San Jose, CA) and Accuri flow cytometers (BD PharMingen), and FlowJo software (Tree Star, Inc., Ashland, OR).

Detection of chimerism

After standard water shock treatment, peripheral blood cells were stained with fluorescein isothiothianate (FITC)-conjugated mouse anti-human HLA class I (Bw6) mAb (Miltenyi Biotec, San Diego, CA), which cross-reacts with cynomolgus monkeys. The cells were incubated for 30 min at 4°C, and then washed and analyzed on FACScan (Becton Dickinson, Mountain View, CA). In all experiments, the percentage of cells stained with antibody was determined using a one-color fluorescence histogram and compared with those obtained from donor and pretreatment frozen recipient cells, which were used as positive and negative controls. The percentage of cells considered positive was determined using the fluorescence level at the beginning of the positive peak for the positive control stain as the cutoff value and by subtracting the percentage of cells stained with an isotype control. By using forward and 90° light scatter (FSC and SSC, respectively) dot plots, the lymphocyte (FSC- and SSC-low), granulocyte (SSC-high), and monocyte (FSC-high but SSC-low) populations were gated, and chimerism was determined separately for each population. Nonviable cells were excluded by propidium iodide (Thermo Fischer Scientific, Grand Island, NY) staining.

Mixed lymphocyte culture

T cells were purified from PBMCs by negative selection with a pan T cell isolation kit (Miltenyi Biotec, San Diego, CA). The isolated T cells were labeled with carboxyfluorescein succinimidyl ester (CFSE) (Life Technologies, Carlsbad, CA) at a concentration of 3 μM per 10⁷ cells at 37°C for 5 min and cultured in 96-well V-bottom plates with irradiated PBMCs or T cell activation beads (αCD2/αCD3/αCD28 mAb) (Miltenyi Biotec, San Diego, CA). After 5 days, the cells were stained with antibodies and CFSE dilution was assessed by flow cytometry¹⁴. Results were compared with those of previously published tolerant recipients¹⁴.

Detection of donor specific antibody (DSA)

Anti-donor alloantibody was detected by flow cytometric analysis. Donor PBMCs were first incubated with recipient sera for 30 min at 4°C. After washing with FACS medium, FITCconjugated mouse anti-human IgG mAb was added and incubated for 30 min at 4°C and then washed. After washing, PBMCs were fixed with 2% paraformaldehyde. Cells were then acquired and analyzed with a cytometer. A positive reaction was defined as a shift greater than 10 channels in mean fluorescence intensity of donor lymphocytes when using test sera compared with a pretransplant serum control.

Statistical analyses

All measured outcomes are summarized as the mean ± standard deviation unless otherwise specified. Between-group pairwise comparison of means was performed by linear contrast in a random intercept longitudinal linear mixed effects model setting. Statistical analyses were performed with GraphPad PRISM 7.01 (GraphPad Software, Inc, San Diego, CA). We used Mantel-Cox Log-rank test to analyze the survival of different groups; P values lower than 0.05 have been considered statistically significant.

RESULTS

Nonmyeloablative conditioning with aCD40/Bela/hATG successfully induced multilineage mixed chimerism but failed to achieve tolerance in CIKTx.

Four months after CIKTx, all 3 recipients were treated with the conditioning regimen summarized in Fig. 1 followed by DBMT. After DBMT, recipients were treated with anti-CD40 mAb and belatacept and a 1-month course of CyA, before discontinuing all immunosuppression. All 3 CIKTx recipients successfully developed excellent multilineage mixed chimerism up to day 68 (Fig. 2). Nevertheless, all 3 CIKTx recipients rejected both islet and renal allografts by days 84–140 post-DBMT (Table 1). Kidney allografts at autopsy showed acute T cell mediated rejection (TCMR) as well as antibody mediated rejection (AMR) with positive C4d staining in all 3 CIKTx recipients (Figs. 3A, 3B, and 3C). Mononuclear cell infiltration was also observed in the transplanted islet (Fig. 3D).

Figure 2: Development of peripheral blood mixed chimerism after DBMT.

Excellent hematopoietic chimerism was induced in both myeloid (A) and lymphoid (B) lineages in all recipients treated with the aCD40/Bela/hATG regimen (closed circle). The levels of chimerism were significantly superior to those observed in previous studies of belatacept-based regimens (Bela/hATG: open circle¹ or Bela/rATG: open square²). *: p<0.05, **: p<0.01.

Table 1:

Chimerism and allograft survival in belatacept-based regimens

Regimen	Allograft	N	aCD40	1Bela	Chimerism	Graft survival following DBMT (days)
aCD40/Bela/2hATG	Islet+Kidney	3	+	+	3/3	140, 89, 84
aCD40/Bela/2hATG	Kidney	2	+	+	2/2	127, 116
Bela/3rATG(A)	Kidney	4	−	+	4/4	820, >658, >449, 4108
Bela/2hATG*(B)	Kidney	5	−	+	4/5	>861, >796, >378, 5156, 125

^1:belatacept

^2:horse anti-thymocyte globulin

^3:rabbit anti-thymocyte globulin

^4:died from an anesthesia complication w/o rejection

^5:failed to develop mixed chimerism

^*:Donor bone marrow transplantation was performed simultaneously with kidney transplantation.

^(A):Reference²

^(B):Reference¹

Figure 3: Islet and kidney allograft histopathological findings.

The histopathologic findings of the renal and islet allografts in recipients of aCD40/Bela/hATG. The kidney allografts of all 3 CIKTx recipients showed mononuclear cell infiltration (PAS, 100x) (A), dense tubulointerstitial infiltrates (PAS, 200x) (B), and C4d deposition (IHC C4d, 200x) (C) at autopsy. Mononuclear cell infiltration was also observed in the transplanted islet (HE, 400x) (D). A protocol kidney biopsy from an monkey undergoing KTx alone before conditioning showed minimal mononuclear cell infiltration and no evidence of rejection (PAS, 200x) (E); however, at autopsy (day 116), it had histologic features of antibody-mediated rejection with dense tubulointerstitial infiltrates (PAS, 100x) as well as transmural vasculitis (F) with C4d deposition (not shown).

Conditioning with aCD40/Bela/hATG also failed to induce tolerance in KTx alone recipients despite successful induction of chimerism.

The failure to induce allograft tolerance despite successful induction of chimerism in 3 CIKTx recipients prompted us to examine the same conditioning regimen in KTx alone recipients. Two cynomolgus monkey recipients underwent KTx with maintenance immunosuppression using aCD40/rapa. Both recipients did well without rejection for 4 months (Fig. 3E). At that point they received the same conditioning regimen used for the CIKTx, and then underwent DBMT. Both developed excellent mixed chimerism, and in one, the chimerism was prolonged lasting up to 3 months post-DBMT (Fig. 2). The levels and duration of chimerism in all 5 recipients treated with the aCD40/Bela/hATG regimen were in fact significantly superior to those observed in the previously reported recipients that achieved long-term immunosuppression-free survival with either Bela/rATG in the delayed-DBMT² or Bela/hATG in the SKBMT protocol¹. However, both KTx alone recipients treated with the aCD40/Bela/hATG regimen rejected their renal allografts on days 116 and 127 (Table 1, Fig. 3F).

Addition of anti-CD40 mAb therapy resulted in early B cell sensitization

There was no significant difference in overall CD3^-CD20⁺ B cell reconstitution between the aCD40/Bela/hATG and Bela/rATG regimens (Fig. 4A). Subset analysis at 2 months after DBMT, however, revealed significantly more memory B cells (CD3^-CD20⁺CD21^-CD27⁺), but significantly fewer immature B cells (CD3^-CD20⁺CD21⁺IgM⁺) in the aCD40/Bela/hATG recipients, compared with the Bela/rATG group (Fig. 4B). The memory phenotype dominant B cell subsets observed in the anti-CD40 mAb-treated recipients were consistently associated with early development of DSA, while DSA was suppressed in recipients treated with the Bela/rATG regimen (Fig. 4C).

Figure 4: Dual CB therapy induced early B cell sensitization.

B cell (CD3^-CD20⁺ cells) reconstitution, including immature (CD21⁺IgM⁺) and memory (CD21^-CD27⁺) phenotypes, relative to those before DBMT conditioning were closely monitored. These values were compared with 3 previously reported “delayed-DBMT” KTx recipients who were conditioned with rabbit anti-thymocyte globulin and belatacept (rATG/Bela).² (A) Peripheral blood B cell counts rapidly declined after the initiation of DBMT conditioning and then recovered by day 70 in both aCD40/Bela/hATG and Bela/rATG. There was no significant difference in B cell reconstitution between aCD40/Bela/hATG (n=5: closed circle with S.D.) and rATG/Bela (n=3: open circle with S.D.). (B) In rATG/Bela recipients (n=3), immature B cells (CD3^-CD20⁺CD21⁺IgM⁺) were dominant at 2 months after DBMT (left panel, open circle). In contrast, B cells with memory phenotype (CD3^-CD20⁺CD21^-CD27⁺) were dominant at 2 months after DBMT in the aCD40/Bela/hATG recipients (n=5) (right panel, closed circle). *: p<0.05, N.S.: not significant. (C) Rapid development of DSA was observed in 4/5 aCD40/Bela/hATG treated recipients (left panel), while DSA development was suppressed in the Bela/rATG recipients (right panel).

Positive anti-donor CD8⁺ T cell responses and absence of donor-antigen-specific Treg expansion in vitro in recipients of the aCD40/Bela/hATG protocol.

A consistent immunologic feature observed in our previous studies included loss of anti-donor CD8⁺ T cell responses (Figs. 5A and 5B), but sustained anti-donor CD4⁺ T cell responses (Figs. 5C and 5D) with significant donor specific expansion of CD4⁺FOXP3⁺ regulatory T cells (Treg) in tolerant recipients (Figs. 5E and 5F) (data from previously published studies in 8 recipients that achieved renal allograft tolerance)¹⁴. In cynomolgus monkeys, peripheral blood Tregs comprised 1–2% of the CD4⁺ T cells, but expanded to 8 ± 5.4% after donor antigen stimulation in tolerant recipients. In contrast, sustained anti-donor responses were observed in both CD8⁺ (Figs. 5A and 5B) and CD4+ T cells (Figs. 5C and 5D), and no donor specific Treg expansion (Figs. 5E and 5F) was detected in any of the 5 CIKTx or KTx-alone recipients treated with the aCD40/Bela/hATG regimen.

Figure 5: Neither loss of anti-donor CD8⁺ responses nor donor-antigen-specific Treg expansion was observed in aCD40/Bela/hATG treated animals in vitro.

CD3⁺ T cells isolated from aCD40/Bela/hATG treated recipients were labeled CFSE and were cultured with irradiated self or donor peripheral mononuclear cells (PBMCs) for 5 days. Cultured cells were then stained for CD4, CD8, and FOXP3. Representative flow cytometric data (A, CD8; C, CD4; E, CD4 gated FOXP3) and mean % proliferation relative to the response to self antigens (B, CD8; D, CD4; F, CD4 gated FOXP3) are shown compared with those of previously published results in 8 tolerant recipients.¹⁴ The loss of anti-donor CD8⁺ T cell response was not observed in the aCD40/Bela/hATG treated recipients, which was significantly higher than those observed in the tolerant recipients (A and B). Substantial anti-donor CD4⁺ T cell proliferation was observed in the aCD40/Bela/hATG treated animals, comparable to that observed in tolerant recipients (C and D). Among these proliferated CD4⁺ cells after donor stimulation, a significant proportion of proliferating CD4⁺ cells were found in FOXP3 intolerant recipients, while such FOXP3⁺ cell proliferation was minimal in the aCD40/Bela/hATG treated animals (E and F). Proliferated cells (%) = Proliferated cells (%) with donor antigens - Proliferated cells (%) with self antigens. Data are presented as the mean ± SD. *P<0.05.

DISCUSSION

The CD40/CD154 pathway is pivotal to generating an effective immune response against foreign antigens.^15,16 Blockade of this pathway with anti-CD154 mAb has been demonstrated to promote allograft tolerance in various animal models,^17–19 including our NHP model using the mixed chimerism approach.²⁰ However, the clinical application of this approach has been delayed because of the adverse thrombophilic effects of anti-CD154 mAb observed in NHPs²¹ and humans.²² Blocking its counterpart, CD40, with anti-CD40 mAb has therefore been tested with encouraging observations in various preclinical^23,24 and clinical studies.²⁵ We also recently reported that the combination of anti-CD40 mAb and rapamycin provides effective immunosuppression without toxicity in combined islet and kidney transplantation (CIKTx).³ Since the immunosuppressive effects of anti-CD40 mAb on T_MEM have been demonstrated in previous studies,^3,4 we added anti-CD40 mAb to induce mixed chimerism in the delayed-DBMT protocol. As expected, all 3 CIKTx recipients developed excellent chimerism (Fig. 2), which was superior to that observed in recipients who achieved long-term immunosuppression-free renal allograft survival with the belatacept-based regimens.^1,2 Nevertheless, none of CIKTx recipients achieved long-term allograft survival and succumbed to acute antibody-mediated rejection 140 days after DBMT (Table 1). To exclude the possibility that the exceptionally high antigenic or inflammatory properties of the islet allografts induced strong alloimmune responses, which could have triggered rejection of both islet and renal allografts, we tested the same aCD40/Bela/hATG regimen in 2 KTx alone recipients. Both KTx recipients also failed to achieve long-term immunosuppression-free renal allograft survival despite successful induction of chimerism (Table 1). These findings rule out the notion that failure of tolerance induction with the aCD40/Bela/hATG regimen is due to the islet allograft. This was an unexpected outcome since renal allograft loss due to acute rejection is rare in recipients who successfully develop mixed chimerism with our conditioning regimen. As shown in Fig. 6 and Table 1, allograft survival was significantly shorter in the aCD40/Bela/hATG group compared to recipients in previous studies who successfully developed mixed chimerism following treatment with the Bela/hATG¹ or Bela/rATG² regimen.

Figure 6: Renal allograft survival curves in belatacept-based conditioning regimens.

Renal allograft survival in recipients treated with aCD40/Bela/hATG (n=5, red line) was significantly shorter than that reported for either simultaneous kidney and bone marrow transplantation (SKBMT) recipients treated with the Bela/hATG regimen (n=4, black dotted line),¹ or “delayed-DBMT” recipients treated with the Bela/rATG (n=3, black line)² regimen. Mantel-Cox Log-rank test was used. *: p<0.05.

These observations have caused us to conclude that anti-CD40 mAb may inhibit the induction of allograft tolerance, at least in our mixed chimerism approach. Ramakrishnan et al²⁶ also reported failure of renal allograft tolerance despite induction of chimerism in recipients treated with nonmyeloablative conditioning regimen with dual CD28/CD40 (3A8) costimulatory blockade. They attributed the failure of tolerance to the compartmentalized nature of the chimerism, which displayed poor lymphoid chimerism. Our studies have consistently shown that induction of donor chimerism, including the lymphoid lineage, is critical for induction of stable tolerance.^1,20,27 In the current study, donor chimerism was achieved; therefore, this explanation does not account for our observations.

It is far more likely that the failure of tolerance induction in the aCD40/Bela/hATG recipients was due to an inadequate regulatory response. Since the major mechanism of renal allograft tolerance via transient mixed chimerism is considered regulatory rather than deletional,^14,28 systemic delivery of donor antigens by donor chimeric lymphoid cells may be necessary to induce antigen-specific unresponsiveness. Pilat et al²⁹ recently demonstrated that induction of mixed chimerism and skin allograft tolerance were not achieved by nonmyeloablative conditioning (low dose TBI, rapamycin with anti-CD4 or anti-CD8 mAb) with CTLA4Ig when a CD40−/− mouse is used either as a recipient or a donor. Since the CD40 signal is critical for activation and maturation of the antigen-presenting cells (APCs) required for full T cell activation,³⁰ it is plausible that absence of CD40 signaling in either donor (direct pathway) or recipient (indirect pathway) APCs may have interfered with maturation of APCs in our NHP recipient, resulting in insufficient donor antigen presentation to the recipient regulatory cells. We hypothesize that such donor antigen presentation by donor chimeric cells is needed to induce the immunologic features of peripheral tolerance (donor-specific Treg expansion associated with the loss of anti-donor CD8⁺ T cell responses) observed in our tolerant recipients.¹⁴ In contrast, such immunologic features, especially donor-specific Treg expansion, which can promote robust tolerance to the donor,³¹ were not observed in those recipients treated with the aCD40/Bela/hATG regimen (Fig. 5). Although blockade of the CD154 molecule (CD40 ligand) by anti-CD154 mAb with or without donor-specific transfusion has been shown to generate donor-specific Tregs in murine transplant models^32,33, blockade of its counter ligand, CD40, molecule may result in failure of Treg expansion and T cell tolerance. Pan et al have shown that CD40 is required for myeloid derived suppressor cell (MDSC) mediated T cell suppression and Treg expansion. In their murine tumor model, treatment with anti-CD40 reversed T cell tolerance and prevented tumor-associated expansion of Tregs³⁴. Similar to their observation, treatment with anti-CD40 mAb failed to induce Treg expansion and allograft tolerance in our NHP model.

In the current study, we observed early DSA development associated with B cell reconstitution predominantly with memory-phenotype B cells in recipients treated with aCD40/Bela/ATG regimens (Fig. 4). Since significant memory-type B cell predominance was not observed in the recipients treated with the Bela/ATG regimen, the enhanced B cell differentiation may have been caused by anti-CD40 mAb. Although enhancement of B cell differentiation by the anti-CD40 mAb clone, 2C10R4, was not observed in vitro (data not shown), the agonistic effect of 2C10R4 on B cells was observed when it was used in vivo. Conversely, effective suppression of B cell differentiation by anti-CD40 mAb has been reported in NHPs. Kim et al¹³ showed that additional treatment with anti-CD40 mAb (2C10R4) or belatacept for 2 months consistently suppressed the DSA development in KTx recipients treated with anti-CD3 immunotoxin, tacrolimus, and alefacept in NHPs. This DSA suppression was associated with predominance of immature B cells and depressed memory B cells. Furthermore, Aoyagi et al²⁴ reported that long-term (6 months) treatment with anti-CD40 mAb (ASKP1240) monotherapy effectively suppressed DSA development, while 2 weeks of treatment failed to suppress DSA in their NHP KTx model. These studies may indicate that B cell differentiation is effectively inhibited as long as anti-CD40 mAb is administered. Since anti-CD40 mAb was administered only up to day 12 post-DBMT in our conditioning regimen, the short duration of the antibody administration after DBMT may have resulted in homeostatic proliferation of memory B cells which led to early DSA development.

Although anti-CD40 mAb appears to be an excellent immunosuppressive agent, it may inhibit the induction of allograft tolerance by abrogating Treg expansion, in addition to its other agonistic effects on B cells. Although the outcome of the current study was negative, we think it is important to document these findings for future studies on tolerance induction involving costimulatory blockade.

ABBREVIATIONS:

AMR

antibody mediated rejection

ATG

antithymocyte globulin

CB

costimulatory blockade

CIKTx

combined islet and kidney transplantation

CyA

cyclosporine A

DBMT

donor bone marrow transplantation

KTx

kidney transplantation

mAb

monoclonal antibody

MHC

major histocompatibility complex

SKBMT

simultaneous kidney and bone marrow transplantation

TBI

total body irradiation

TCMR

T cell mediated rejection

TI

thymic irradiation