Research ArticleOrgan Transplants

Inducing CTLA-4–Dependent Immune Regulation by Selective CD28 Blockade Promotes Regulatory T Cells in Organ Transplantation

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Science Translational Medicine  03 Feb 2010:
Vol. 2, Issue 17, pp. 17ra10
DOI: 10.1126/scitranslmed.3000116

Abstract

Transplantation is the treatment of choice for patients with end-stage organ failure. Its success is limited by side effects of immunosuppressive drugs, such as inhibitors of the calcineurin pathway that prevent rejection by reducing synthesis of interleukin-2 by T cells. Moreover, none of the existing drugs efficiently prevent the eventual rejection of the organ. Blocking the CD28-mediated T cell costimulation pathway is a nontoxic alternative immunosuppression strategy that is now achieved by blockade of CD80/86, the receptor for CD28 on antigen-presenting cells. However, interaction of CD80/86 with cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) is required for immune regulation. Therefore, CD28 blockade, instead of CD80/86 blockade, might preserve regulatory signals mediated by CTLA-4 and preserve immune regulation. By using monovalent antibodies, we identified true CD28 antagonists that induced CTLA-4–dependent decreased T cell function compatible with regulatory T (Treg) cell suppression. In transplantation experiments in primates, blocking CD28 augmented intragraft and peripheral blood Treg cells, induced molecular signatures of immune regulation, and prevented graft rejection and vasculopathy in synergy with calcineurin inhibition. These findings suggest that targeting costimulation blockade at CD28 preserves CTLA-4–dependent immune regulation and promotes allograft survival.

Introduction

T cells were identified as major players in immune responses after allotransplantation and in autoimmunity. T cell activation is induced by specific antigen recognition and reinforced by engagement of costimulatory molecules that regulate their differentiation into either pathogenic effector cells or anti-inflammatory regulatory cells. Costimulation by CD28 and cytotoxic T lymphocyte–associated antigen 4 (CTLA-4) contributes to determining this balance after initial antigen exposure. The current paradigm holds that constitutively expressed CD28 binds CD80/86 to provide a costimulatory signal important for sustaining T cell proliferation and proinflammatory responses (1). Furthermore, although CD28 signals are critical for regulatory T (Treg) cell homeostasis (2), CD28 engagement by CD80/86 molecules can inhibit Treg activity (3). CTLA-4, the other CD80/86 ligand, delivers antiproliferative signals to T cells (4), triggers indoleamine 2,3-dioxygenase (IDO) (5) production in antigen-presenting cells (APCs), and is essential for the suppressive function of Treg cells (6) and the induction of tolerance to allografts (7, 8). Targeting the CD28-CD80/86 pathway in patients with CTLA-4–Ig (immunoglobulin) reagents (belatacept, abatacept, and CD80/86 antagonists) is a promising alternative to current immunosuppressive treatments in autoimmunity (9, 10) and renal transplantation (11). However, CD80/86-specific blocking strategies inhibit CTLA-4 signals crucial to the function of Treg cells and do not reproducibly induce transplant tolerance (12, 13). We thus hypothesized that blocking CD28 without affecting CTLA-4 could be an effective strategy for modulating immune responses by preventing the maturation of pathogenic effectors while preserving the function of Treg cells. Here, we used non–cross-linking selective CD28 antagonists and showed that this treatment decreased the allogeneic immune response against kidney or heart transplant and prolonged allograft survival in two primate models.

Results

CTLA-4–dependent and CTLA-4–independent components of CD28 blockade

sc28AT, a monovalent fusion antibody, competes with CD80/86 for binding to CD28 (14). Because the binding epitope is different from the epitope of superagonistic CD28 antibodies (15) (fig. S1A), sc28AT did not induce T cell receptor (TCR)–independent activation and proliferation of human T cells (fig. S1B) or human Treg cells (fig. S1C) in vitro. We investigated in vitro the consequences of selective CD28 blockade with sc28AT on cognate human T cell interactions with B cells used as APCs by live cell dynamic microscopy. sc28AT prevented the formation of stable T cell–APC conjugates, increased T cell motility, and reduced T cell activation as measured by calcium flux (Fig. 1, A to C, and movies S1 to S3). The effect of sc28AT on conjugate formation and T cell motility was abolished by the simultaneous blockade of CTLA-4 (Fig. 1, A and B), suggesting that the interaction of CTLA-4 with CD80/86 was essential for sc28AT-mediated activity. Although a minority of T cells still established prolonged contacts with APCs in the presence of sc28AT, these contacts resulted in greatly reduced calcium influx relative to T cells interacting with APCs in the absence of CD28 blockade, and this reduction was not reversed by CTLA-4 blockade (Fig. 1C and fig. S2). The importance of CTLA-4–CD80/86 interaction for T cell motility was confirmed after CD80/86 targeting by either CD80/86 antibodies or CTLA-4–Ig (Fig. 1B), which did not modify T cell–APC contacts but prevented T cell activation (Fig. 1, A and C). These data reveal CTLA-4–dependent (promotion of T cell motility and inhibition of T cell–APC contacts) and CTLA-4–independent (inhibition of T cell activation) components in the action of monovalent antibodies against CD28. Furthermore, sc28AT reduced the proliferation of human and primate T cells in mixed lymphocyte reactions (MLRs; Fig. 2A) and decreased cytokine release after stimulation of Jurkat T cells with superantigens (Fig. 2B), showing that inhibition of long-term T cell–APC contacts by specifically antagonizing CD28-CD80/86 interaction suppresses effector T (Teff) cell activation.

Fig. 1.

CTLA-4–dependent impairment of T cell–APC contact after selective CD28 blockade. (A) Time-lapse microscopy of cognate contacts between EBV-specific T cells and B cell–EBV APCs in the presence of the indicated antibody. Results are shown as percentage of cells establishing short (≤5 min; black bars), medium (between 5 and 15 min; hatched bars), and long (≥15 min; white bars) contacts over 20 min. (B) T cells were cultured as in (A) and the cumulative distance moved of individual cells was recorded to assess T cell motility. (C) T cells were cultured as in (A) and calcium peaks were recorded. *P < 0.05, ***P < 0.001.

Fig. 2.

Monovalent CD28 antagonists block allorecognition but do not impede the function of Treg cells in vitro. (A) Mixed lymphocyte reaction using human (n = 10), baboon (n = 15), or macaque (n = 9) PBMCs. Black bars, mean ± SD in control conditions (mouse irrelevant IgG1); white bars, mean ± SD with sc28AT (10 μg/ml). (B) IL-2 secretion by Jurkat T cells stimulated with bacterial superantigen (staphylococcal enterotoxin E) and Raji B cells in the presence of sc28AT (n = 8) or CTLA-4–Ig (n = 3). Results are expressed as percentage of IL-2 secretion observed in the absence of antibody (100%). (C) Suppressive activity of human Treg is not impeded by CD28 blockade. Treg cells were added to CD4+CD25 T cells stimulated with allogeneic irradiated PBMCs at the indicated ratio in the presence of CD28 or CTLA-4 (10 μg/ml) blocking antibodies. Results are mean counts per minute (cpm) ± SD of one of three representative assays. (D) Suppressive activity of human Treg pretreated with CD28 or CTLA-4 blocking antibody. Treg cells were first cultured with allogeneic mDCs in the presence of sc28AT or antibody against CTLA-4 Fab fragments (10 μg/ml) for 18 hours, washed, and assessed in a suppression assay. Results are mean cpm ± SD of one of three representative assays. *P < 0.05, **P < 0.01, ***P < 0.001.

Compatibility of CD28 blockade with Treg function

Immunosuppressive drugs might either block or synergize with the suppressive activity of Treg cells. It was therefore important to analyze the consequences of CD28 blockade on the function of Treg cells. The suppression by Treg cells of the proliferation of naïve CD4+ T cells in response to allogeneic irradiated peripheral blood mononuclear cells (PBMCs) synergized with sc28AT, whereas addition of CTLA-4 antagonists blocked the suppression (Fig. 2C). To examine the effect of sc28AT specifically on Treg cells, we activated Treg cells with allogeneic mature dendritic cells (mDCs) in the presence of sc28AT and secondarily tested for their ability to suppress the proliferation of naïve T cells stimulated with allogeneic mDCs. Treg suppression was significantly increased when Treg cells were primed in the presence of sc28AT, whereas it was not modified by CTLA-4 blockade (Fig. 2D). Together, these data suggest that signals through CTLA-4 and CD28 regulate positively and negatively, respectively, the regulatory activity of Treg. In particular, the enhanced Treg immunosuppressive capacity observed after CD28 blockade with sc28AT in vitro prompted us to examine the potential effect of this CD28 antagonist in vivo.

Selective CD28 blockade in transplantation

To investigate the action of CD28 blockade in vivo, we used a life-sustaining kidney transplant model in baboons. The animals were divided into four groups (Fig. 3A). (i) Without immunosuppressive treatment, kidney transplant was rejected within a week (median graft survival of 6 days) with acute biopsy-proven cellular rejection (n = 3). (ii) With monotherapy with the calcineurin inhibitor tacrolimus for 90 days, 50% of recipients developed a renal graft failure with a biopsy-proven cellular acute rejection during the first week after transplantation. The other 50% had stable kidney function over the 3-month experiment but developed cellular acute rejection within a week after withdrawal of the drug. The median survival time (MST) in this group was 47 days (n = 4; rejection at 6, 7, 94, and 97 days). (iii) sc28AT induction monotherapy for 25 days (see pharmacokinetic profiles in fig. S3A and description of target cells in fig. S3C) modestly, but significantly, prolonged graft survival (n = 4; MST, 11 days). (iv) Combined administration of tacrolimus (0 to 90 days) and sc28AT (0 to 25 days) resulted in a significant increase in graft survival (n = 5; MST, 103 days: 23, 36, 103, 130, and 269 days). No rejection episode (clinical or biopsy-proven) developed in any of these bitherapy recipients even after complete withdrawal of immunosuppression at day 90. However, three animals in this group were euthanized at days 23, 36, and 130 after developing pyelonephritis or acute tubular necrosis and one animal was lost at day 103 from an anesthetic accident. Anatomopathological analyses excluded rejection in these animals.

Fig. 3.

Administration of sc28AT and tacrolimus prevents kidney allograft rejection in baboons. (A) Rejection-free survival after renal allotransplantation for baboons without therapy (n = 3) or treated with a 25-day induction therapy with sc28AT alone (n = 4), tacrolimus for 90 days (n = 4), and sc28AT plus tacrolimus (25 and 90 days, respectively; n = 5). Vertical hash marks, death of recipients without graft rejection as assessed by histology. White square, remaining living baboon. **P = 0.008 versus tacrolimus monotherapy; #P = 0.01 versus untreated controls. (B) Total lymphocyte counts (means ± SD, top panel) and CD3+ T cell counts (bottom panel) in untreated recipients (n = 3) and recipients treated with sc28AT monotherapy (n = 4). (C) Phenotype of blood T cells during the first week after transplantation in controls (n = 3; left) and sc28AT monotherapy recipients (n = 4; right). T cells expressing the specified marker on days 0, 1, 2, 4, and 6 from left (black bars) to right (white bars). *P < 0.05. (D) Donor-specific hyporesponsiveness. PBMCs were harvested in the sc28AT plus tacrolimus group and tested in MLR against donor cells before transplantation (black bars) and at day 90 (white bars) and against third-party cells at day 90 (gray bars). Results are mean stimulatory index ± SD of triplicate wells. Two experiments are shown. **P = 0.0014.

No differences were observed between groups with regard to CD3+, CD4+, or CD8+ T cell infiltration into the graft. In contrast, CD20+-infiltrating B cells were barely detectable after sc28AT treatment alone or in combination with tacrolimus, whereas they were abundant in kidney graft biopsies from untreated animals or animals treated with tacrolimus alone (fig. S4). Furthermore, mRNA concentrations of inflammatory cytokines interleukin-6 (IL-6) and interferon-γ (IFN-γ) were reduced in kidney graft biopsies 1 week after transplantation in animals treated with sc28AT relative to control untreated animals (fig. S5). In contrast, mRNA concentrations of transforming growth factor–β (TGF-β), as well as CD25, CTLA-4, Foxp3, and heme oxygenase-1 (HO-1), were increased in sc28AT-treated animals (fig. S5).

Absolute numbers of total lymphocytes and CD3+ T cells varied slightly within the normal range after sc28AT treatment (Fig. 3B) (16), indicating that sc28AT did not induce T cell depletion. In addition, the expression of activation markers on T cells was not markedly modified by CD28 blockade (Fig. 3C and fig. S6A), although we observed an increase in the percentage of CD25+ T cells 1 week after transplantation in the sc28AT group (Fig. 3C, right panel). Serum levels of IFN-γ, tumor necrosis factor–α, IL-2, IL-4, IL-5, or IL-6 cytokines were low and similar in animals receiving sc28AT and controls (fig. S6B). Thus, interaction of sc28AT with CD28 on T cells did not result in polyclonal T cell activation.

To investigate the alloreactivity of peripheral T cells in kidney recipients that received sc28AT and tacrolimus bitherapy and failed to reject the allograft, we performed ex vivo MLR. The proliferative response against donor cells was reduced after combination therapy, whereas the alloreactive response against cells from a third-party animal was preserved, suggesting the acquisition of donor-specific hyporesponsiveness in animals treated with sc28AT and tacrolimus (Fig. 3D).

Increase in Treg cells after transplantation and specific CD28 blockade

We determined levels of Treg cells with multiparameter flow cytometry. CD4+CD25+CD127lo Treg cells also expressed Foxp3, CTLA-4, and CD28 (Fig. 4A). After administration of sc28AT, alone or in combination with tacrolimus, the percentage of Treg cells increased by a factor of >2 within a week to reach 5 to 6% of CD4+ T cells relative to 3% in untreated recipients and 2% in recipients treated with tacrolimus alone (Fig. 4B). Absolute Treg counts similarly increased by factors of 2 to 3 in recipients treated with sc28AT alone or together with tacrolimus relative to their pretreatment levels and with untreated or tacrolimus-treated animals (Fig. 4C). Animal receiving tacrolimus alone did not display any change in levels of Treg during the 3-month treatment (Fig. 4, B and C). We investigated whether these phenotypically defined Treg cells were functional. CD25+ PBMCs harvested from control tacrolimus-treated ungrafted baboons were unable to suppress Teff cell proliferation (Fig. 4D). This is in agreement with a previous study showing that CD4+CD25+ T cells are barely detectable in peripheral blood of naïve baboons and this cell population does not exhibit suppressive activity (17). In contrast, CD25+ PBMCs obtained 90 days after transplant from kidney allograft recipients treated with sc28AT in combination with tacrolimus were very effective at suppressing Teff proliferation even at low Treg-to-Teff ratios (Fig. 4D).

Fig. 4.

Treg cell enrichment in the peripheral blood of kidney allograft baboon recipients treated with sc28AT. (A) CD25+CD127lo Treg cells analyzed in blood by flow cytometry after gating on CD3+CD4+ cells. These cells also expressed CD28 and intracellular CTLA-4 and Foxp3. (B and C) Kinetics of CD4+CD25+Foxp3+CD127lo Treg levels in blood, percentage of CD4+ T cells (B) or absolute number (C), in control untreated animals (n = 3), sc28AT monotherapy (n = 4), tacrolimus monotherapy (n = 4 until week 1 and then n = 2), and sc28AT plus tacrolimus (n = 5 up to 2 weeks, n = 4 at 1 month, and then n = 3). D, days; W, weeks; M, months. #P = 0.02, ##P = 0.0011 versus untreated group; *P < 0.05, **P < 0.01, ***P < 0.001 versus tacrolimus monotherapy group. (D) Suppressive activity of CD25+ PBMCs from sc28AT plus tacrolimus recipients at day 90 after transplantation (black bars; n = 3) or from control ungrafted baboons treated with tacrolimus (white bars; n = 4).

The increase in Treg cells detected after transplantation was not due to nonspecific expansion of existing CD4+CD25+ Treg cells by sc28AT, because infusion of sc28AT at the same dose in nontransplanted baboons during a week did not elicit changes to the frequency of Treg cells (fig. S7).

T cells expressing Foxp3 and CTLA-4 accumulate in the kidney allograft after CD28 inhibition

To better characterize the localization of Treg cells in kidney graft recipients that received a bitherapy, we examined kidney biopsies for the presence of cells expressing the Treg markers Foxp3 and CTLA-4. We observed increased infiltration with T cells expressing Foxp3, CTLA-4, or both molecules 1 week after transplantation in animals treated with sc28AT monotherapy relative to untreated controls (Fig. 5, A and B). In recipients that received sc28AT plus tacrolimus, the number of infiltrating Treg cells expressing Foxp3 and CTLA-4 was initially low and similar to levels observed in recipients treated with tacrolimus alone (Fig. 5C). However, numbers of infiltrating Treg cells increased by factors of 2 to 3 within 1 to 3 months after transplantation in recipients treated with sc28AT and tacrolimus, and Treg infiltration in kidney grafts was markedly higher in bitherapy recipients relative to recipients treated with tacrolimus alone (Fig. 5C).

Fig. 5.

Increased Treg cell infiltration in kidney allografts after selective CD28 blockade with sc28AT. (A) Confocal-like microscopy analysis of a kidney graft biopsy from a sc28AT plus tacrolimus–treated recipient 1 month after transplantation. Blue, CD3 staining; green, Foxp3 staining; red, CTLA-4 staining. Scale bars, 10 μm. (B) Quantitative evaluation of graft infiltration by T cells expressing Foxp3, CTLA-4, or both in control and sc28AT monotherapy recipients 1 week after transplant (expressed as percentage of CD3+ T cells). Data from individual animals are represented with a specific color and the four individual data points represent quadruplicate evaluations performed on two different tissue sections of the same animal. (C) Same as (B) in recipients treated with tacrolimus alone or sc28AT plus tacrolimus at 1 week, 1 month, and 3 months after transplant. *P < 0.05, **P < 0.01.

IDO expression in kidney transplant after CD28 inhibition

IDO is an enzyme that is up-regulated after engagement of CD80/86 on APCs by CTLA-4 on Teff or Treg cells (18). IDO limits T cell responses by tryptophan deprivation and by the proapoptotic action of tryptophan catabolites (19). Comparison of IDO mRNA levels in groups treated with or without sc28AT (alone or in combination with tacrolimus) showed that selective CD28 blockade was accompanied by a notable increase in intragraft IDO mRNA expression (Fig. 6A). IDO expression was detectable by fluorescence microscopy in graft biopsies from sc28AT-treated recipients (Fig. 6B) in association with CD31+ endothelial cells (Fig. 6C), DC-SIGN+–infiltrating cells (representing a subset of immature myeloid cells; Fig. 6D), and actin+ smooth muscle cells (Fig. 6E). IDO expression could not be detected in graft biopsies from untreated recipients or from recipients treated with tacrolimus alone (Fig. 6B).

Fig. 6.

Expression of IDO in allograft of sc28AT-treated recipients. (A) Quantitative PCR measurement of IDO mRNA transcripts 1 week after transplantation in kidney graft biopsies from control untreated (♦, n = 2) or tacrolimus alone–treated recipients (□, n = 2) and from sc28AT alone (▴, n = 4) or sc28AT plus tacrolimus recipients (○, n = 4). Each point represents the mean of duplicate measurements. *P = 0.04. (B) Immunohistology of biopsies from a control and a sc28AT-treated recipient labeled with an antibody against IDO (green) 1 week after transplantation. Scale bar, 100 μm. (C) Costaining of IDO (green) and CD31, an endothelial cell marker (red), showing a small blood vessel. (D) Costaining of IDO (green) and DC-SIGN, a C-type lectin expressed by immature myeloid cells (red), in the renal parenchyma. (E) Costaining of IDO (green) and smooth muscle actin (red), showing colocalization in an arteriole. Nuclei are in blue. Scale bars, 10 μm [(C) to (E)].

CD28 blockade inhibits acute and chronic rejection of heart allografts in macaques in synergy with calcineurin inhibition

We tested sc28AT in a more stringent model of heterotopic heart transplantation in the cynomolgus macaque (20) using sc28AT alone or in combination with a calcineurin inhibitor [cyclosporin A (CsA)]. The pharmacokinetic profiles are presented in fig. S3B. sc28AT bound to target T cells in the blood and showed tissue penetration in the spleen, lymph nodes, and, to some extent, thymus (fig. S3D). In macaques treated for 20 days with sc28AT monotherapy at 2 mg/kg (n = 3) or 10 mg/kg (n = 3), cardiac allografts had a median survival of 13 and 34 days, respectively (Fig. 7A). Thus, treatment with sc28AT alone resulted in a significant prolongation of graft survival relative to untreated monkeys (MST, 6 days; n = 5). Another group received CsA monotherapy that was dosed at 10 to 25 mg/kg intramuscularly daily to achieve therapeutic trough levels of >400 ng/ml (21). Four of eight treated animals exhibited symptomatic acute allograft rejection on days 7, 23, 44, and 71. One recipient died of an infection on day 26. The other three recipients exhibited graft survival >80 days without any evidence of clinical rejection, and transplanted hearts were electively explanted around day 90.

Fig. 7.

Selective CD28 inhibition prolongs cardiac allograft survival and prevents CAV in macaques. (A) Cardiac allograft survival for monkeys without therapy (n = 5) or treated with sc28AT monotherapy at 2 mg/kg per day (n = 3) or 10 mg/kg per day (n = 3), CsA monotherapy (n = 6), CsA plus sc28AT at 0.4 mg/kg bitherapy (n = 2), or CsA plus sc28AT at 2 mg/kg bitherapy (n = 3). *P < 0.05, ***P < 0.001 versus control untreated recipients. (B) A representative vessel from a cardiac allograft treated with CsA (day 72, left panel) shows grade 2 CAV with distinct neointimal thickening and 10 to 50% (estimated at 25% in this instance) luminal narrowing. In contrast, a representative graft artery from a recipient treated with sc28AT plus CsA shows an absence of neointimal proliferation (day 80, bottom panel). (C) CAV incidence and severity at days 70 to 90, graded as described in Materials and Methods, were significantly lower (P < 0.05) in CD28 blockade plus CsA combination therapy (n = 4) relative to CsA alone (n = 5).

When CsA was combined with sc28AT at 0.4 mg/kg daily, one of two animals developed symptomatic rejection at day 49. When sc28AT was dosed at 2 mg/kg daily and combined with CsA, all three recipients displayed prolonged graft survival of >80 days. One animal was killed with a beating graft at day 80 because of a lymphoma, and the two remaining functional heart grafts were electively explanted around day 90 in the absence of any detectable clinical rejection. The International Society for Heart and Lung Transplantation (ISHLT) rejection scores were consistently lower in protocol biopsies and explanted grafts from monkeys treated with sc28AT plus CsA relative to animals treated with CsA alone (fig. S8). Whereas all grafts treated with CsA monotherapy exhibited severe cardiac allograft vasculopathy (CAV) at the time of explant, CAV incidence and severity were significantly reduced when sc28AT was combined with CsA (Fig. 7, B and C).

In addition, long-term graft survival induced by CsA and CD28 blockade was associated with a control of alloantibody production that was otherwise induced in heart graft recipients under monotherapy during the first month after transplantation (fig. S9). We also analyzed levels of CD4+CD25+CD127lo Treg cells in the blood of two sc28AT monotherapy–treated recipients by flow cytometry. As expected, >80% of CD4+CD25+CD127lo Treg cells expressed Foxp3 and >50% expressed CTLA-4. Treg cells represented 3.5 ± 0.1% of CD4+ T cells before transplantation, and their level increased to reach 6.3 ± 0.4% of CD4+ T cells at 3 weeks after transplantation in sc28AT-treated recipients.

The synergy observed between sc28AT and CsA suggested that sc28AT might be used as calcineurin-sparing regimen to reduce allograft rejection while decreasing calcineurin toxicity. We therefore explored whether sc28AT combined with a subtherapeutic CsA regimen (CsA tapered to trough levels of 100 to 300 ng/ml within 2 weeks after transplant) modulates alloimmunity and CAV. Relative to monotherapy with sc28AT (MST, 22 days; range, 8 to 36; n = 6) or subtherapeutic CsA (MST, 38 days; range, 12 to 49; n = 7), addition of CD28 blockade prolonged graft survival (MST, 59 days; range, 21 to >88; n = 6; P < 0.05) (fig. S10A). Subtherapeutic CsA–treated grafts showed severe CAV as early as 2 weeks after transplantation (day 14 CAV score, 1.5 ± 0.8), whereas added CD28 blockade was associated with significantly attenuated CAV scores (day 14, 0 ± 0.01; P = 0.036) (fig. S10B).

Discussion

Our study tested the immunoregulatory effect of selective CD28 blockade on kidney and heart allografts in primates. It had been shown previously in rodents that antibody-mediated CD28 downmodulation delayed acute rejection (22) or inhibited chronic rejection (23, 24) after organ transplantation. The lack of a CD28-specific modulating or antagonist antibody for humans or nonhuman primates has prevented verification of this effect in primates or in humans, where the role of Treg cells in transplant outcome is still a matter of debate (25, 26). We found that sc28AT, a chimeric human/primate CD28 monovalent antagonist antibody (27), selectively blocked CD28 interactions with its ligands without sharing superagonistic activity because it could not stimulate human Teff or Treg cells in vitro, a feature characteristic of superagonistic antibodies. Also, sc28AT did not activate or deplete T cells in primates in vivo and did not increase Treg levels in nontransplanted primates. Thus, two major features differentiate sc28AT from a superagonistic antibody. First, sc28AT is monovalent and, as such, cannot induce CD28 receptor cross-linking (27). Second, it does not bind the target epitope on CD28 that is essential for inducing the TCR-independent superagonistic signal (15). Using sc28AT in vivo, we found that sc28AT synergized with calcineurin inhibitors to promote acceptance of both kidney and heart allografts in nonhuman primates. Furthermore, because an important goal of clinical transplantation is to eliminate calcineurin inhibitors or limit their use (because of their toxicity), induction treatment with sc28AT was also associated with a subtherapeutic regimen of CsA in the heart transplant model. This resulted in a significant delay in the occurrence of first rejection episodes and reduced the severity of CAV. These observations confirmed the synergy between calcineurin inhibition and CD28 blockade. However, because several grafts in this series were ultimately rejected, it also indicated the importance of a strong immunosuppression in the early post-operation period in this experimental mismatched transplantation setting. Finally, our data from the baboon model suggest that selective blockade of CD28 directly impaired Teff cells while promoting regulation provided by Treg cells, CTLA-4, and other immunomodulatory mediators such as IDO, HO-1, and TGF-β.

We showed that sc28AT antagonized T cell activation in vitro by preventing the formation of stable T cell–APC conjugates and by increasing T cell motility, which was otherwise reduced after cognate interactions with APCs through a TCR-induced stop signal (28). Antagonistic antibody against CTLA-4 reversed the effect of sc28AT treatment, in accordance with findings that CTLA-4 overrides the TCR-induced stop signal (28). Similar results were obtained when CTLA-4 engagement was prevented by CTLA-4–Ig or antibodies against CD80/86. These data suggest an intrinsic role for CTLA-4 in the T cell–inhibitory effect of sc28AT. However, another interpretation might be that in the context of deficient CTLA-4 signals, T cells can overcome CD28 blockade, possibly because of a decrease in the activation threshold, and manage enough signaling to lead to efficient interaction. In contrast, the reduction of calcium peaks in T cells that established a stable contact with APCs despite CD28 blockade could not be reversed by the simultaneous blockade of CTLA-4. Thus, the mechanism of action of selective CD28 blockade on T cells involves both CTLA-4–dependent and CTLA-4–independent components. Notably, sc28AT caused impaired alloreactive T cell proliferation in MLR in vitro, which could contribute to allograft survival in vivo by skewing the Teff-Treg balance toward regulation.

An emerging hypothesis presents CD28 and CTLA-4 molecules as a “rheostat” for targeting T cell responses toward immunity or regulation, respectively (29). If CD28 is blocked, then CTLA-4–CD80/86 interactions would be favored and T cell responses would be shifted toward regulation. In vitro, we could block the suppressive activity of human Treg cells with Fab antibodies against CTLA-4, as has been shown in mice (30). In contrast, sc28AT did not block but instead increased the suppressive activity of Treg cells in accordance with data showing that CD28 stimulation abolishes the suppressive function of Treg cells (1).

In kidney or heart allograft transplantation in primates, acute rejection was prevented, chronic rejection was attenuated, and functional Treg cells were increased after treatment with sc28AT combined with calcineurin inhibitors. Donor-specific hyporesponsiveness was demonstrated in the kidney transplant model. Early graft biopsies showed less infiltration by CD20+ B cells, which have been associated with severe acute rejection (31). The reduction in B cell infiltration might be attributed classically to reduced help by T cells or to a direct effect of Treg cells on B cells (32). Indeed, sc28AT treatment favored graft infiltration by T cells expressing Foxp3 and CTLA-4, which is associated with the suppressive function of Treg cells (6). Finally, examination of graft tissue revealed that selective CD28 blockade resulted in reduced levels of inflammatory cytokines but increased expression of immunoregulatory TGF-β, HO-1, and IDO. The effects on B and Treg cells might not be clinically relevant shortly after transplantation at a time when alloreactive T cells are strongly stimulated and Treg cells have not yet infiltrated the allograft. Indeed, the full control of acute rejection in the early post-transplant period could be achieved only in bitherapy with therapeutic levels of calcineurin inhibitors. However, Treg cells (and other induced immune regulation mechanisms) might become relevant to prevent late rejection events, because allograft survival persisted after withdrawal of the immunosuppressive treatment in several recipients. In addition, Treg cells could contribute to limit chronic rejection because CAV was consistently reduced in heart grafts in macaques. Furthermore, the exclusive availability of CD80/86 ligands for CTLA-4 binding and the role of CTLA-4 in immune regulation suggest that CTLA-4 could be important in this process.

What mechanisms underlie the induction of Treg cells in sc28AT-treated animals? The observation that the administration of sc28AT to nongrafted primates did not alter the frequency of Treg cells excluded a direct effect of the monovalent antibody. Rather, allogeneic T cells might have been driven to differentiate into Treg cells in vivo in response to the allograft because CD28 signaling was absent, whereas CTLA-4–CD80/86 interactions could take place. Indeed, although the survival of natural Treg cells is strictly CD28-dependent (2), the generation of adaptive Treg cells can occur in situations of suboptimal costimulation (33) or in the absence of CD28 signals (34). Additionally, selective CTLA-4 engagement can induce adaptive Treg cells with alloantigen specificity (35). The observation that PBMCs from kidney graft recipients treated with sc28AT and tacrolimus were hyporesponsive to donor APCs but not third-party cells is consistent with this possibility. In contrast to clinical immunosuppression achieved by a CD80/86 antagonist, which was not associated with induction of Treg cells and not relying on immune regulation (36, 37), our findings are consistent with previously reported downstream mechanisms of suppression attributed to Treg cells in transplantation, including release of TGF-β, expression of CTLA-4, and induction of IDO and HO-1 enzymes (38).

Our study shows that selective blockade of CD28 costimulation after transplantation reduced alloreactivity and increased the pool of peripheral Treg cells. In addition, Treg cells accumulated in the graft, where they likely modulated pathogenic T cells and promoted prolonged allograft survival. Whether selective CD28 blockade has significant practical advantages relative to CD80/86 blockade (11), as our model predicts, remains to be formally tested. However, the efficacy of CD28 antagonists in combination with conventional immunosuppression to inhibit acute and chronic allograft rejection in primates is promising. Although superagonistic activity has been excluded here, any translation of a CD28 antagonist into clinical application must first reevaluate that point.

Materials and Methods

Reagents

A nonactivating human CD28-specific single-chain Fv antibody fragment was developed from the CD28-specific CD28.3 clone (14) and linked to α1-antitrypsin (sc28AT) to prolong its half-life in the serum in vivo (27). sc28AT was produced by TcL Pharma and by a National Institutes of Health (NIH) production platform (K. Reimann, Beth Israel Deaconess Medical Center, Boston, MA) from transformed Chinese hamster ovary cells and purified from supernatant by ion exchange chromatography. sc28AT was quantified by two complementary specific sandwich enzyme-linked immunosorbent assays (ELISAs) (TcL Pharma). sc28AT cross-reacts with CD28 from cynomolgus monkey, baboon, and marmoset but not from dog, rabbit, rat, and mouse and has the same binding affinity as the parental murine Fab fragment (27). Antibody against human CTLA-4 (clone 147.1) was provided by Medarex. Fab monovalent fragments were prepared with Immunopure IgG1 Fab Preparation kit (Pierce). Antibodies against human CD80 (clone M24) and CD86 (clone 1G10) were provided by Innogenetics. CTLA-4–Ig (LEA29Y) was prepared from transfected Cos cells in our laboratory. Fluorescent monoclonal antibodies (mAbs) against human CD3 (SP34-2), CD4 (L200), CD8 (RPA-T8), CD28 (28.6), CD25 (M-A251), CD45RO (UCHL1), CD62L (FMC46), CD127 (hIL-7R-M21), CTLA-4 (BNI3), and HLA-DR (G46-6) were from BD Biosciences. Biotinylated antibody against human α1-antitrypsin (Abcam) was used with streptavidin-phycoerythrin (Beckman Coulter) for sc28AT staining on T cells. The APC-conjugated antibody against human Foxp3 staining kit (PCH101 and 236A/E7) was purchased from eBioscience and used according to the manufacturer’s instructions. Primary antibodies used for immunohistochemical staining were rabbit antibodies against human CD3 (DAKO) and human IDO-1 [provided by I. Anegon, Nantes, France (39)] and mouse antibodies against human CD11b (BEAR1; Beckman Coulter), human CD20 (L26; DAKO), human CD31 (LCI4; Serotec), human CTLA-4 (BNI3; BD Biosciences), human Foxp3 (236A/E7; eBioscience), human DC-SIGN (DCN46; BD Biosciences), and human α–smooth muscle actin (O.N.5; Abcam).

Epitope analysis

The determination of the epitope recognized by the CD28.3 antibody was performed by Agrobio. Briefly, CD28.3 Fab fragments were added to human CD28 (R&D Systems) immobilized on Sepharose. Immune complexes were reduced and alkylated with iodoacetamide (55 mM, 1 hour) before chymotrypsin (1 mg/50 mg of bound antibody) was added for 4 hours at 18° to 25°C. The Sepharose was then washed with 25 mM ammonium carbonate followed by 50 mM glycine (pH 2.5). Eluted peptides were then concentrated on a C18 matrix and analyzed by matrix-assisted laser desorption/ionization–time of flight (TOF)-TOF. The epitope was determined by peptide mass fingerprinting.

Live cell dynamic microscopy

A human Epstein-Barr virus (EBV)–specific CD4+CD28+ T cell clone (2 × 105 cells) (40) was stained with the Fura-2 acetoxymethyl ester probe (0.5 μM for 30 min; Interchim), washed, and added to 4 × 105 human EBV-transformed B lymphoblastoid cell lines [pool of cells from three donors, obtained as described (41)] on a coverslip coated with poly-l-lysine (0.001%; Sigma). Using the MetaFluor image analysis software (Molecular Devices), we acquired bright-field and fluorescent images at 15-s intervals on a Leica microscope (Leica Microsystems). Individual T cell–APC interactions and individual T cell calcium peaks were recorded manually over a 20-min incubation period with MetaFluor (version 7.1.7) and MetaMorph (version 7.5.6; Roper Scientific) software. A calcium peak was recorded when fluorescence levels reached twice the baseline level. T cells were tracked with the ImageJ free software (version 1.41). The total number of cells analyzed was 60 cells for control condition (from 11 experiments), 31 cells for sc28AT condition (from 7 experiments), and 29 cells for sc28AT plus antibody against CTLA-4 (from 4 experiments), 13 cells for antibody against CD80/86 (from 3 experiments), and 26 cells for CTLA-4–Ig condition (from 5 experiments). Antibodies were all used at 10 μg/ml. Data are presented as mean ± SD for each condition.

Mixed lymphocyte reactions

PBMCs were isolated from whole blood by density centrifugation over Ficoll-Paque (Eurobio). Freshly isolated PBMCs were incubated with allogeneic irradiated PBMCs (105 cells per well of each cell type) for 5 days at 37°C and 5% CO2 in complete medium (RPMI 1640, 10% heat-inactivated allogeneic pooled sera, 2 mM l-glutamine, penicillin (100 U/ml), streptomycin (0.1 mg/ml), 1% nonessential amino acids, 1 mM sodium pyruvate, and 5 mM Hepes, all from Sigma). Cells were pulsed with 1 μCi of [3H]thymidine during the final 8 hours of culture and then harvested and counted in a scintillation counter. In other experiments, PBMCs were maintained in culture without allogeneic cells and assayed similarly as above.

Cytokine secretion assays

To assess IL-2 production by T cells, we stimulated 105 Jurkat T cells with 2 × 104 Raji B cells in microtiter plates in the presence of staphylococcus enterotoxin E (5 ng/ml) for 48 hours at 37°C and 5% CO2 in complete medium. IL-2 secretion was evaluated in the supernatant with the Max Set Deluxe Human IL-2 ELISA kit (BioLegend). To analyze the synthesis of multiple cytokines by human PBMCs, we cultured 105 PBMCs from healthy humans in triplicate for 5 days in complete medium with control or antibodies to CD28. After 48 hours, 30 μl of supernatant was collected from each triplicate, pooled, and analyzed for cytokine concentration with a Human Th1/Th2 Cytokine kit (BD Biosciences).

Suppression assays

All experiments were performed with PBMCs obtained from healthy donors. CD4+ T cells were enriched from PBMCs by negative selection with CD4+ T Cell Isolation Kit II (Miltenyi) and an autoMACS separator (Miltenyi). Enriched CD4+ cells were then stained with mAbs against human CD4, CD25, and CD127 at 4°C for 30 min. CD4+CD25hiCD127lo Treg cells and CD4+CD25 naïve T cells were then sorted (purity routinely above 95%) with a high-speed cell sorter (FACSAria, BD Biosciences) and FACSDiva software (BD Biosciences). CD4+CD25 cells (2 × 104) were cocultured with 105 allogeneic irradiated PBMCs and autologous CD4+CD25hiCD127lo Treg cells at a 1:1 or 1:0.25 ratio for 5 days at 37°C and 5% CO2 in human complete medium. Blocking antibodies were added at the start of the culture at 10 μg/ml. Proliferation was assessed by [3H]thymidine incorporation during the last 8 hours of culture.

Allogeneic mDCs were generated from monocytes as described (42). Briefly, monocytes were enriched by elutriation (>85% CD14+) and cultured for 6 days in medium supplemented with IL-4 (40 ng/ml; R&D Systems) and granulocyte-macrophage colony-stimulating factor (1000 IU/ml; Gentaur). Cells were harvested on day 5 and cultured for 24 hours with lipopolysaccharide for maturation (1 μg/ml; Escherichia coli 0111:B4, Sigma). Allogeneic mDCs (5 × 103) were then cocultured with 5 × 104 Treg cells in complete medium with the indicated blocking antibodies (10 μg/ml) for 18 hours. mDCs or Treg cells were washed and added to 5 × 104 CD4+CD25 cells (same donor as Treg cells) stimulated with 5 × 103 allogeneic mDCs (same donor as mDCs used for Treg activation). Cells were then cultured for 5 additional days, and proliferative responses were assessed by [3H]thymidine incorporation.

Baboon Treg suppression assays

CD25+ and CD25 cells were prepared from PBMCs during the third month after transplantation with fluorescein isothiocyanate (FITC) mAb against human CD25 and specific anti-FITC microbeads (Miltenyi) and positive or negative selection, respectively. To determine suppression, we mixed 105 carboxyfluorescein succinimidyl ester (CFSE)–labeled CD25 PBMCs with different numbers of unlabeled CD25+ PBMCs (at ratios of 1:0.5 to 1:0.0078) in duplicate wells of a plate previously coated with antibodies against human CD3 (10 μg/ml; 2 hours at 37°C). The proliferative response was evaluated after 3 days by measuring the percentage of CFSE-diluted cells by flow cytometry. The percentage of suppression was calculated by comparing the percentage of proliferated cells (CFSE dilution) in the presence of indicated numbers of CD25+ PBMCs to the percentage of proliferated cells in the absence of CD25+ cells (= maximal proliferation).

Animals

Cynomolgus monkeys (Macaca fascicularis) (2 to 3 kg) were obtained from Covance Research Products and Three Springs Scientific Inc. Baboons (Papio anubis) (6 to 15 kg) were obtained from the Centre National de la Recherche Scientifique Primatology Center. Experiments performed on macaques were in accordance with the guidelines of the Institutional Animal Care and Use Committee of the University of Maryland Medical School and carried out in compliance with the Department of Health and Human Services–NIH Guide for the Care and Use of Laboratory Animals. Experiments performed in baboons complied with recommendations of the Institutional Ethical Guidelines of the Institut National de la Santé et de la Recherche Médicale (France). The donor-recipient combinations were chosen according to blood group compatibility, major histocompatibility complex (MHC) mismatching by DRB MHC class II locus typing, and verification of MHC incompatibility by MLR (stimulatory index > 5).

Renal transplantation in baboons

Renal allotransplantation was performed in binephrectomized recipients, as described (43). Twenty-four–hour diuresis was monitored daily, as well as blood urea nitrogen. Transplantectomies were performed when plasma creatinine levels rose up to 400 μM, and surgical biopsies were performed for histological examination before euthanasia. One biopsy fragment was collected for hematoxylin and eosin (H&E)–blinded histological examination by a pathologist. A second fragment was snap-frozen in liquid nitrogen for mRNA extraction, and a third fragment was placed in Tissue-Tek (Sakura Finetek) for immunohistochemical staining. Protocol biopsies were performed at 1 week, 1 month, and 3 months after transplantation. Control groups either were untreated (n = 3) or received tacrolimus alone (Prograf, provided by Astellas) (n = 4). Tacrolimus was given once daily from day 0 to day 90 after transplantation (0.05 to 0.15 mg/kg intramuscularly) to achieve target therapeutic trough levels (10 to 20 ng/ml). sc28AT was administered once daily (4 mg/kg intravenously) during the first 25 days after transplantation. Blood samples were collected for fluorescence-activated cell sorting analysis and assessment of serum levels of sc28AT and cytokines (Non-Human Primate Th1/Th2 Cytokine kit, BD Biosciences) before the daily administration of sc28AT.

Cardiac transplantation in cynomolgus monkeys

All recipient animals underwent heterotopic intra-abdominal cardiac allograft transplantation, as described (44). Surgical cardiac biopsies were performed on postoperative days 7, 14, and 28 and monthly thereafter until graft explant. Graft function was monitored daily by palpation and implanted telemetry (Data Sciences International). Clinical acute graft rejection was detected as consistent high body temperature (>38.5°C) coupled with either a decrease in graft heart rate (to <120 beats per minute or a drop of >40 beats per minute from a stable baseline) or an increase in graft diastolic pressure of >10 mmHg. Graft rejection was defined as loss of contraction by telemetry and confirmed at explant and was always preceded by signs of acute rejection. Control groups either were left untreated (n = 5) or received CsA (Neoral, Novartis) (n = 6). CsA was given once daily (5 to 25 mg/kg intramuscularly) to achieve target therapeutic (>400 ng/ml) or subtherapeutic (100 to 300 ng/ml) trough levels. sc28AT was given as indicated in Fig. 7. In three therapeutic CsA-treated animals, a first episode of symptomatic acute rejection was treated with three daily steroid boluses (10 mg/kg; Solu-Medrol, Pharmacia). Rejection was reversed in two of the three treated animals. In one therapeutic CsA-treated animal, suspected rejection based on histological analysis of the biopsy tissue sample was also treated with a 3-day course of steroids. One recipient died of infection different from the ones that rejected the heart at day 26 and was excluded from statistical analysis. Cellular infiltrates were analyzed on H&E-stained paraffin sections and graded for acute rejection by ISHLT criteria (45). CAV incidence in beating hearts explanted after day 70 (therapeutic CsA group) or in weekly sample biopsies (subtherapeutic CsA group) was recorded as percent of arteries and arteriolar vessels involved (CAV score ≥ 1) at each time point. CAV severity was scored in these explanted hearts as follows: grade 0, normal arterial morphology; grade 1, activated endothelial cells with enlarged nuclei and/or adherent leukocytes, without luminal narrowing (<10%); grade 2, distinct neointimal thickening, luminal narrowing <50%; grade 3, extensive neointimal proliferation with >50% luminal occlusion. Scoring was independently performed for each explanted heart by three evaluators (T.Z., R.N.P., and B.N.) blinded with respect to the treatment group. The mean CAV score for each biopsy or explant was calculated with the following equation: (#grade 0 vessels x0 + #grade 1 vessels x1 + #grade 2 vessels x2 + #grade 3 vessels x3)/total number of arterial vessels scored. Individual means were averaged to calculate the group mean ± SD for each treatment group.

Immunohistochemical staining

Serial frozen sections (10 μm) were prepared from surgical or protocol renal biopsies. Slides were air-dried at room temperature for 1 hour before acetone fixation for 10 min at room temperature. Sections were saturated with phosphate-buffered saline containing 10% baboon serum, 2% normal goat serum, and 4% bovine serum albumin. Sections were incubated overnight with primary antibodies at 4°C, followed by fluorescent secondary antibodies and nuclear staining (4′,6-diamidino-2-phenylindole; Invitrogen). For intracellular immunostaining (CTLA-4, Foxp3, and IDO), sections were permeabilized with 0.5% saponin (Sigma) in the saturating solution. Treg infiltration was determined by a triple staining with antibodies against human CD3 (followed by Alexa Fluor 350–conjugated antibody against rabbit IgG; Invitrogen), human CTLA-4 (followed by Alexa Fluor 594–conjugated antibody against mouse IgG2a; Invitrogen), and human Foxp3 (followed by Alexa Fluor 488–conjugated antibody against mouse IgG1; Invitrogen). The percentage of CD3 cells expressing Foxp3, CTLA-4, or both was quantified manually (by an investigator blinded to the experimental conditions) on four different pictures from two different areas of each tissue section separated by at least 100 μm. IDO localization was determined by double staining with antibodies against human IDO-1 (followed by FITC-conjugated antibody against rabbit IgG; Jackson ImmunoResearch) and human CD31, human DC-SIGN, or human α–smooth muscle actin (followed by Alexa Fluor 568–conjugated antibody against mouse IgG; Invitrogen). The specificity of IDO-1 staining was confirmed by competition experiments with the IDO-1 peptide that had been used to raise the antibody. Slides were analyzed with standard fluorescence microscopy or confocal-like microscopy (ApoTome, Carl Zeiss) and the AxioVision imaging software (Carl Zeiss).

Messenger RNA analysis

Messenger RNA was extracted from snap-frozen renal biopsies with RNA microextraction kit (Qiagen) according to the manufacturer’s instructions. The quality and quantity of mRNA were controlled by infrared spectrometry (NanoDrop, Thermo Scientific). Messenger RNA was amplified and retrotranscribed with the Omniscript RT kit (Qiagen), and real-time quantitative polymerase chain reaction (PCR) was then performed, as previously described (43), with an ABI Prism 7700 Sequence Detection System (Perkin-Elmer). Amplifications were performed for hypoxanthine phosphoribosyltransferase (probe Hs99999909_m1; Applied Biosystems), IDO (forward, 5′-ACGGTCTGGTGTATGAAGGGT-3′; reverse, 5′-CACGGACTGAGGGATTTGACT-3′), IL-2 receptor (probe Hs00907778_m1; Applied Biosystems), CTLA-4 (forward, 5′-TCTTCATCCCTGTCTTCTCCAA-3′; reverse, 5′-GGTCAACTCATTCCCCATCA-3′), Foxp3 (forward, 5′-CCCTGCCCTTCTCATCCA-3′; reverse, 5′-GTGGCCCGGATGTGAAAA-3′), HO-1 (probe Hs00157965_m1; Applied Biosystems), IFN-γ (forward, 5′-TGGGTTCTCTTGGCTGTTACTG-3′; reverse, 5′-TTAATGTCTTCCTTGATGGTCTCC-3′), and IL-6 (probe Hs00174131_m1; Applied Biosystems).

Statistical analyses

Graft survival times were plotted with the Kaplan-Meier representation, and survival time between different groups was evaluated with a log-rank test. Continuous variables were expressed as the mean ± SD, unless otherwise indicated, and compared with the Mann-Whitney nonparametric test. MLR data were analyzed with unpaired t tests. Discrete variables (that is, incidence of early rejection) were compared with a contingency table and the χ2 test. P values of <0.05 were considered statistically significant. All statistical analyses were performed on a personal computer with the statistical package SPSS for Windows XP (version 11.0, SPSS) or GraphPad InStat (version 5.1, GraphPad Software).

Supplementary Material

www.sciencetranslationalmedicine.org/cgi/content/full/2/17/17ra10/DC1

Fig. S1. Nonagonistic properties of monovalent fragments from the CD28.3 antibody.

Fig. S2. Calcium flux profiles in vitro.

Fig. S3. Pharmacokinetic and pharmacodynamic aspects of sc28AT.

Fig. S4. Immunohistological analyses of kidney graft biopsies.

Fig. S5. qPCR measurement of mRNA transcripts in kidney graft biopsies.

Fig. S6. Nonagonistic properties of sc28AT in vivo.

Fig. S7. Sc28AT daily injection in naïve baboons does not enhance peripheral regulatory T cells.

Fig. S8. Representative histological analysis of cynomolgus monkey heart allografts at 3 months after transplantation (H&E staining).

Fig. S9. Long-term graft survival induced by CsA and CD28 blockade is associated with control of alloantibody production.

Fig. S10. Selective CD28 blockade synergized with low dose of cyclosporin A to delay cardiac allograft rejection and cardiac allograft vasculopathy in macaques.

Movies S1 to S3. Time-lapse microscopy analyses of T cell motility in vitro.

Footnotes

  • * These authors contributed equally to this work.

  • These authors contributed equally to this work.

References and Notes

  1. Acknowledgments: We thank A. Laaris, C. Avon, X. Cheng, N. Sangrampurkar, E. Welty, D. Minault, C. Lefeuvre, S. Lebas-Bernardet, P. Hulin (IFR26, Nantes, France), and T. Haudebourg for technical assistance. Funding: Roche Organ Transplantation Research Foundation grant 466230972 (B.V.); Progreffe Foundation (Nantes, France); TcL Pharma (Nantes, France); NIH grant UO1 AI 066719, American Society of Transplant Surgeons Mid-Career Award, Department of Defense Operational Requirements Document contract N00014-04-1-0821, and American Heart Association Grant-in-Aid (R.N.P.); and Maryland Restitution Fund Program Other Tobacco Related Diseases research grant (A.M.A. and R.N.P.). Author contributions: N.P. performed baboon experiment and in vitro experiments, interpreted the data, and prepared the manuscript. A.M.A. initiated and designed the M. fascicularis study, performed experiments, interpreted the data, and prepared the manuscript. T.Z. conducted heart transplantations and histology analysis in M. fascicularis. N.D. and C.M. performed experiments. B.N. conducted heart transplantations and histology analysis in M. fascicularis. X.T. conducted kidney transplantations in baboon. G.W. assisted with heart transplantations in M. fascicularis. K.R. analyzed the kidney biopsies. J.H. assisted with kidney transplantations in baboon. B.M. prepared and purified recombinant molecules. G.K. conducted kidney transplantations in baboons. F.C. prepared antibodies and antibody fragments and performed ELISA. E.A.-L. assisted with immunohistological analyses. J.-P.S. analyzed the data and edited the manuscript. R.N.P. founded the research, designed M. fascicularis experiments, analyzed the data, and edited the manuscript. G.B. organized and designed the baboon experiments, supervised the analyses, and edited the manuscript. B.V. founded the research, designed experiments, performed experiments, analyzed the data, and prepared the manuscript. Competing interests: B.V. and J.-P.S. are shareholders in TcL Pharma, a company that is developing CD28 antagonists.
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