Belatacept, a second generation CD28 antagonist, is the first biologic to receive clinical approval for use in long-term transplant immunosuppression. This new drug class prolongs graft survival through costimulatory blockade (CoB), a novel immunosuppression strategy that disrupts the vital costimulatory signals (such as CD28-B7 and CD40-CD154 interactions) required for full activation of alloreactive T cells (1–3). Conventional immunosuppression regimens typically rely on calcineurin inhibitors, all of which suffer from serious metabolic side effects such as hypertriglyceridemia, hypertension, and hyperglycemia (4). Calcineurin inhibitors are also nephrotoxic, contributing to chronic renal graft failure. Importantly, the phase III BENEFIT trial of belatacept demonstrated dramatically improved long-term renal function in belatacept-treated transplant recipients compared with conventional regimens (5, 6). However, the BENEFIT trial also paradoxically revealed that patients treated with belatacept suffered a higher incidence and severity of acute rejection.
Accumulating evidence suggests that alloreactive memory T cells may play a critical role in mediating this CoB-resistant transplant rejection. Memory T cells possess a lower costimulatory threshold than naïve T cells, and in experimental transplant systems, alloreactive memory T cells have proven resistant to CoB (7–11). In addition to their contribution to CoB-resistant rejection, these donor-specific memory T cells are of broader interest to the transplant community, as pretransplant levels of donor-reactive memory T cells are associated with acute rejection and worsened long-term graft function, even in patients treated with calcineurin inhibitors (7, 12–14). Thus, understanding the origins of alloreactive memory T cells and the mechanisms by which they contribute to transplant rejection is essential for improving the clinical outcomes of organ transplants, especially considering the increasing prominence of CoB as an immunosuppression strategy.
Alloreactive memory T cells can arise from prior exposure to donor major histocompatibility complex (MHC), whether through a failed prior transplant, blood transfusion, or pregnancy. More recently, several groups have described how alloreactive memory T cells can arise in transplant recipients without prior exposure to donor MHC through the process of heterologous immunity. Heterologous immunity is a by-product of infection, whereby a subset of pathogen-specific memory T cells can crossreact with donor antigens, enabling their recruitment into a rejection response (15). Recently published findings have highlighted the significant contribution of heterologous immunity to alloreactive memory responses in humans, finding that more than 40% of T cells raised against common viruses possess alloreactive potential (16).
To improve the clinical efficacy of costimulation blockade against an alloreactive memory response, several groups have attempted to couple CoB with adjunct immunosuppressive agents that target the memory T cells. Integrins such as leukocyte functional antigen-1 (LFA-1) and very late antigen-4 (VLA-4) are attractive targets for these adjunct therapies, as integrin expression is markedly up-regulated on the surface of memory alloreactive T cells (17). Integrins are heterodimeric cell surface receptors that play a vital role in T-cell adhesion, trafficking, and activation (18–23). Importantly, integrin antagonists are clinically relevant adjunct immunosuppressants, as anti-VLA-4 (natalizumab) and anti-LFA-1 (efalizumab) are clinically approved for treating autoimmune diseases such as multiple sclerosis, psoriasis, and Crohn’s disease (24, 25).
In experimental transplant systems using immunologically naïve recipients, integrin antagonists can dramatically prolong graft survival, either as monotherapy (26–35) or when coupled with CoB (36–41). In addition to suppressing naïve alloresponses, we have previously demonstrated that combined costimulatory and integrin blockade can prolong graft survival against memory alloresponses (17). However, the transplant system used in this earlier work did not address the ability of LFA-1 antagonism to synergize with costimulation blockade in inhibiting polyclonal allocrossreactive heterologous T-cell responses, potentially limiting its relevance to the clinically important phenomenon of heterologous immunity.
In this report, we address these critical concerns about the clinical relevance of combined costimulatory and integrin blockade, demonstrating that a regimen of CoB + anti-LFA-1 can inhibit transplant rejection by alloreactive memory T cells in a fully allogeneic transplant system that models heterologous immunity. This regimen effectively suppressed the ability of alloreactive memory T cells to proliferate, attenuated memory T-cell effector functions as measured by cytokine release, and promoted a selective retention of allo-specific FoxP3+ Tregs in the draining lymph nodes (dLNs). Given that an LFA-1 antagonist has already been clinically developed, these findings may offer a clinically translatable strategy to improve the efficacy of biologics such as belatacept in prolonging transplant survival.
Combined LFA-1 and CoB Prolongs Skin Graft Survival Against a Heterologous Immune Alloresponse
To study the impact of combined LFA-1 and CoB on transplant rejection mediated by an alloreactive memory response, we used a well-defined experimental model of heterologous immunity (15). In this system, naïve C57BL/6 mice are infected with lymphocytic choriomeningitis virus, followed by an infection with vaccinia virus 6 weeks later. These sequential infections generate pathogen-specific memory T cells that are crossreactive with BALB/c alloantigens (∼104 allocrossreactive memory CD4+ and CD8+ T cells per 108 splenocytes) (15). Six weeks after the final infection, the mice receive a simultaneous skin graft and bone marrow transplant from a fully allogeneic BALB/c donor (Fig. 1A). Although uninfected transplant recipients treated with CoB alone demonstrated indefinite graft survival, sequentially infected recipients treated with CoB alone promptly rejected their skin grafts with the same kinetics as untreated controls (Fig. 1B). Treatment with anti-LFA-1 alone also led to prompt rejection, but treatment with a combined regimen of CoB and anti-LFA-1 enabled prolonged skin graft survival, with a median survival time of more than 100 days (Fig. 1B). A donor bone marrow transplant was important for prolonged graft survival in this stringent transplant system, as even uninfected recipients achieved only a 22-day median skin graft survival time when treated with CoB alone in the absence of donor bone marrow (Fig. 1B). Similarly, maintenance anti-LFA-1 was required for the duration of transplant, as administration of anti-LFA-1 only during the first 6 days after transplant failed to prolong graft survival (see Figure S1, SDC, http://links.lww.com/TP/A655).
Although grafts explanted from untreated recipients showed a prominent cellular infiltrate, explanted grafts taken either early (day 11) or late (>100 days) posttransplant from recipients treated with combined costimulatory/LFA-1 blockade had no infiltration, closely resembling isografts or grafts explanted from uninfected recipients treated with CoB alone (Fig. 2A–D). Further immunohistochemistry with anti-CD3 revealed a lack of T cells in the grafts treated with the combined immunosuppression regimen (Fig. 2E–H).
Combined Blockade Surmounts Barrier Posed by Heterologous Immunity to Allogeneic Bone Marrow Engraftment
We also examined BALB/c bone marrow engraftment 8 weeks after transplant by assessing for hematopoietic chimerism in the peripheral blood of graft recipients. Using flow cytometry to determine the expression of donor MHC (H-2Kd), we found that sequentially infected recipients treated with either anti-LFA-1 or CoB alone failed to develop either lymphoid (CD3+) or myeloid (CD11b+) chimerism (Fig. 3A, B). In contrast, recipients treated with combined costimulatory and LFA-1 blockade demonstrated durable low-level (1%–6%) lymphoid and myeloid chimerism.
Intriguingly, although our earlier published work found coupling CoB to either anti-LFA-1 or anti-VLA-4 could markedly prolong graft survival, treatment with CoB + anti-VLA-4 was ineffective in this model of heterologous immunity (see Figure S2, Part A, SDC,http://links.lww.com/TP/A655). Treatment with CoB and anti-VLA-4 also failed to permit bone marrow engraftment and chimerism (see Figure S2, Part B, SDC,http://links.lww.com/TP/A655), consistent with previous evidence that VLA-4 is critical for homing of lymphocytes to the bone marrow (42–44).
Combined Blockade Inhibits Alloreactive T-Cell Proliferation and Effector Responses
Further ex vivo studies were performed to assess the mechanism by which combined costimulatory and LFA-1 blockade prolongs graft survival. First, we used an in vivo mixed lymphocyte reaction to assess the ability of these different regimens to suppress the proliferation of alloreactive T cells after induction of heterologous immunity. Anti-LFA-1 alone failed to suppress the proliferation of alloreactive splenocytes, whereas CoB alone had a modest effect (Fig. 4A, B). In contrast, combined CoB and anti-LFA-1 demonstrated the most pronounced inhibition of alloreactive recall proliferation, demonstrating the synergy of these regimens (Fig. 4A, B). Next, we evaluated how these different regimens impacted alloreactive T-cell effector mechanisms. Consistent with our previously published work (17), intracellular cytokine staining for interferon-gamma (IFN-γ) and tumor necrosis factor (TNF) revealed that after induction of heterologous immunity, transplant recipients treated with either CoB or anti-LFA-1 alone had a significant reduction in the percentage of splenocytes that were activated double producers of IFN-γ and TNF compared with untreated recipients (Fig. 4C, D). Combined CoB and anti-LFA-1 demonstrated an even more prominent inhibitory effect (Fig. 4C, D).
Combined Blockade Promotes Retention of Tregs in Draining LNs
Finally, we evaluated whether dominant tolerance mechanisms involving FoxP3+ Tregs could potentially contribute to the observed prolongation in graft survival in the recipients of combined blockade. Examining the dLNs of BALB/c graft recipients in which heterologous immunity had been induced, we found that the percentage of CD4+FoxP3+ Tregs was significantly higher in recipients treated with combined CoB and anti-LFA-1 at both early and late time points compared with untreated recipients or recipients treated with CoB alone (Fig. 4E, F). Importantly, although this accumulation of Tregs in the dLNs may contribute to graft survival, it is not sufficient by itself, as a similar accumulation was observed in the recipients treated with anti-LFA-1 alone, despite their early graft rejection (Fig. 4E, F).
Combined Blockade Suppresses Cytokine Responses of Human Memory CD8+ T Cells
We next extended our findings to human alloreactive memory T cells. Peripheral blood mononuclear cells (PBMCs) were obtained from human responder-stimulator pairs, none of which had prior history of transfusion, pregnancy, or solid organ transplant. These responder and stimulator PBMCs were cocultured along with different immunosuppressant reagents, after which IFN-γ and TNF cytokine production by CD8+CD45RA− memory T cells was determined through intracellular cytokine staining. Given the wide variation in alloreactive T-cell precursor frequency between different responder-stimulator pairings (45), the data were normalized against the peak cytokine response obtained with no treatment. Although treatment with belatacept alone failed to attenuate the percentage of cytokine producers among the alloreactive memory T-cell population compared with untreated controls, combined therapy with belatacept and anti-LFA-1 monoclonal antibody (mAb) led to a statistically significant reduction in IFN-γ production by the alloreactive memory T cells and a trend toward lower TNF production (Fig. 5A, B). Thus, combined integrin and CoB also seems to have efficacy against human heterologous alloreactive memory T-cell effector responses in vitro.
Compared with immunosuppression with calcineurin inhibitors, CoB offers improved long-term graft function. However, relatively higher rates of blockade-resistant transplant rejection pose a potential impediment to the widespread clinical adoption of immunosuppressants based on CoB, such as belatacept (5). Compelling evidence now suggests that alloreactive memory T cells may be prime mediators of this blockade-resistant rejection, as these memory T cells are known to possess diminished requirements for costimulation (7–11). Recipients possessing higher precursor frequencies of donor-reactive memory T cells may therefore be uniquely vulnerable to CoB-resistant rejection.
Importantly, the obstacle posed by alloreactive memory T cells may impact a large proportion of transplant patients, not just those previously sensitized to donor MHC by a failed prior transplant, blood transfusion, or pregnancy. Instead, recent evidence highlights heterologous immunity as a prominent source of alloreactive memory responses (15, 16). Thus, the clinical success of belatacept in this subset of patients may ultimately require adjunct immunosuppression to surmount the barrier posed by alloreactive memory T cells.
In the search for adjunct immunosuppressants that enhance the efficacy of CoB, we have focused on integrin antagonists. Earlier work by our group demonstrated that in a murine transplant system, donor-specific memory T-cell effector responses are dependent on LFA-1 engagement (17). Our previous work used an experimental transplant system in which ovalbumin-specific transgenic T cells were primed by infection with ovalbumin-expressing Listeria. After memory induction, these mice were challenged with a skin graft from a transgenic mouse that ubiquitously expresses membrane-bound ovalbumin. Several limitations with this system restricted the clinical relevance of our earlier findings. First, this system used a fully MHC-matched transplant pairing, with rejection targeted against only a nominal antigen (ovalbumin). Second, this earlier transplant system did not model crossreactive heterologous immunity, as the epitope used to prime the memory T cells was identical to the antigen recognized on the donor graft. In true heterologous immunity, pathogen-specific memory T cells likely recognize a crossreactive, nonidentical donor antigen, for which the T cells may possess altered affinity.
To address these limitations, we turned to a system previously used by our group to model heterologous immunity in a fully allogeneic transplant pairing (15). Consistent with our previous results, we find that although CoB alone could not prolong graft survival against a heterologous immunity memory response, an immunosuppression regimen using combined costimulatory and LFA-1 blockade did enable durable graft survival.
In contrast to our earlier results, however, the regimen of CoB + anti-VLA-4 failed to prolong graft survival. This difference may be explained by the impact of VLA-4 blockade on bone marrow engraftment. Several groups have demonstrated that VLA-4 is required for T-cell and hematopoietic stem-cell homing to the bone marrow (42–44, 46), and unlike recipients treated with CoB + anti-LFA-1, those treated with CoB + anti-VLA-4 failed to demonstrate successful engraftment of the BALB/c bone marrow transplant. Establishment of durable mixed chimerism may be required for long-term allogeneic skin graft survival in this stringent transplant system, explaining why CoB + anti-VLA-4 failed to prolong graft survival. Alternatively, this difference may reflect different integrin utilization between low-affinity memory T cells (e.g., crossreactive T cells generated by heterologous immunity) and high-affinity memory T cells (e.g., ovalbumin-specific transgenic T cells used in our previous transplant system) (47).
We explored several mechanisms by which combined costimulatory and LFA-1 blockade could prolong graft survival. Generally, the combined costimulatory/integrin blockade seemed to potently suppress recall proliferation of alloreactive T cells after induction of heterologous immunity. Anti-LFA-1 also attenuated allo-specific T-cell effector responses such as cytokine production when coupled with CoB. Finally, the combined blockade strategy promoted the potential for dominant immunosuppression through FoxP3+ Tregs, as the relative ratio of Tregs to CD4+FoxP3− effector cells in the dLNs was markedly increased, potentially enabling better immunoregulation and suppression of alloresponses. This effect seems mediated predominantly by anti-LFA-1, as we have previously reported (48).
The clinical potential of immunosuppression based on combined costimulatory and LFA-1 blockade is perhaps best reflected in its ability to inhibit human alloreactive memory T cells, as assessed by cytokine production. In this in vitro experiment, belatacept alone was relatively ineffective in suppressing these effector mechanisms. Although it is obviously impossible to identify the source of alloreactive memory in our human responder-stimulator pairs, these alloreactive memory T cells may have arisen through heterologous immunity, as none of our subjects had a previous history of solid organ transplant, blood transfusion, or pregnancy. Importantly, the frequency of alloreactive memory CD8+ T cells in our subjects was low [as is often the case (45)], and it remains possible that this combined blockade regimen may not be as effective at attenuating effector responses in individuals possessing a higher precursor frequency of alloreactive memory T cells (49).
In addition to our findings in the in vitro human allostimulation assay, the clinical potential of LFA-1 antagonists in transplantation is also enhanced by the development of a clinically-approved antagonist, efalizumab. However, there are several important limitations that might impact the clinical translation of combined costimulatory and integrin blockade. Belatacept therapy confers an increased risk of posttransplant lymphoproliferative disease (5), and it will be vital to define whether the addition of efalizumab would further increase these risks. An additional limitation of this regimen is that the CoB we used included anti-CD154, the clinical development of which has been complicated by its known prothrombotic effects (2). Earlier work has demonstrated that even in uninfected C57BL/6 recipients of BALB/c skin grafts, treatment with combined cytotoxic T-lymphocyte antigen 4 Ig and anti-LFA-1 (in the absence of anti-CD154) achieved a median survival time of only 45.5 days (39). Although anti-CD154 is critical for prolonged skin graft survival, it may not be required for the less stringent immune barrier posed by kidney, liver, or heart transplantation. Furthermore, the ongoing clinical development of both domain-specific antibodies against CD154 that lack thrombogenic side effects and CD40-specific monoclonals may also improve the translational potential of this regimen (50, 51).
Perhaps, the most important constraint on the clinical development of combined costimulatory and LFA-1 blockade is the known risks of LFA-1 antagonists themselves, as the report of several cases of progressive multifocal leukoencephalopathy (PML) in patients receiving efalizumab led to its voluntary recall and withdrawal from the market in June 2009 (52–55). Importantly, accumulating evidence suggests that the risk of PML after integrin antagonism is directly related to the duration of therapy (55). Indeed, all patients who developed PML while on efalizumab had received the drug for longer than 3 years (52). Although an extremely short induction regimen of efalizumab would likely not be effective (see Figure S1, SDC,http://links.lww.com/TP/A655), if efalizumab was used solely as an induction agent to protect the graft during its most vulnerable period [i.e., the initial months posttransplant, when the rates of CoB-resistant rejection are highest (5)], the risk of PML in transplant patients might be reduced. Furthermore, the risk-benefit calculus of using efalizumab may be notably different in the setting of transplantation compared with psoriasis. If a combined regimen of belatacept and efalizumab could avert graft loss from CoB-resistant heterologous immune responses, it might justify a nominal absolute risk of PML (56). Future nonhuman primate trials with anti-LFA-1 and belatacept immunosuppression may better evaluate the clinical potential of this promising regimen of combined costimulatory and LFA-1 blockade and to define its potential risks.
MATERIALS AND METHODS
Male 6- to 8-week-old C57BL/6 and BALB/c mice (NCI-Frederick) were obtained. Animals received humane care and treatment in accordance with Emory University Institutional Animal Care and Use Committee guidelines. Viral infections were conducted by intraperitoneal injection of 2×105 pfu lymphocytic choriomeningitis virus Armstrong (gifted by R. Ahmed) and 106 pfu vaccinia virus (gifted by J.R. Bennick).
Skin and Bone Marrow Transplantation
Bone marrow recipients were pretreated with 600 μg of busulfan (GlaxoSmithKline) intraperitoneally. The following day 2×107 BALB/c bone marrow cells (harvested by femur flushing) were adoptively transferred by tail vein injection into the recipients. Full thickness tail skin grafts (∼1 cm2) were transplanted onto the recipient dorsal thorax. Where indicated, transplant recipients were treated with CoB (500 μg each of hamster anti-mouse-CD154 mAb [MR-1, BioXcell, West Lebanon, NH] and human cytotoxic T-lymphocyte antigen 4 Ig [Bristol-Meyers Squibb, New York, NY]), 250 μg of rat anti-mouse-VLA-4 mAb (PS/2, BioXcell), and/or 250 μg of rat anti-mouse-LFA-1 mAb (M17/4, BioXcell). All monoclonal antibodies were administered intraperitoneally on posttransplant days 0, 2, 4, and 6. Integrin antagonists were continued once weekly for the duration of transplant survival.
Flow Cytometric Analyses
Splenocytes, blood, and/or cells obtained from axillary dLNs were stained with H-2Kd-FITC, CD8a-APC, and CD4-V500 (Pharmingen) for analysis on a BD LSRII flow cytometer (BD Biosciences, San Jose, CA). Data were analyzed using FlowJo Software (Tree Star, San Carlos, CA).
Intracellular Cytokine Staining
Splenocyte suspensions from transplant recipients were cocultured with BALB/c splenocyte stimulators at a 1:2 responder-to-stimulator ratio in the presence of 10 μg/mL Brefeldin A (Pharmingen). Replicates with responders alone were also performed. After 5 hr, cells were stained intracellularly with anti-TNF-PE and anti-IFN-γ-AlexaFluor700 (Pharmingen) according to manufacturer’s instructions. For FoxP3 staining, FoxP3-AlexaFluor700 (eBioscience, San Diego, CA) was used per manufacturer protocol.
In Vivo Mixed Lymphocyte Reaction
Splenocytes were harvested on postoperative day 60 from previously sequentially infected graft recipients treated with different immunosuppression regimens. These splenocytes were labeled for 5 min with 10 μM carboxyfluorescein succinimidyl ester, and 2 to 3×107 of these labeled responders were adoptively transferred intravenously into irradiated BALB/c mice (700 rads). Splenocytes were harvested after 72 hr and analyzed by flow cytometry to assess the carboxyfluorescein succinimidyl ester dilution and thus proliferation of H-2Kd-negative (responder) T cells.
Human Allostimulation Assay
After receiving informed consent, PBMCs were obtained from six human donors to form three responder-stimulator pairings. A 1:1 mixture of responders and irradiated stimulators (3500 cGy) was prepared in triplicate (∼106 total cells/well). Cells were either left untreated or were treated with belatacept (100 μg/mL, provided by Bristol-Myers Squibb) and anti-human-LFA-1 (250 μg/mL, clone TS-1 [BioXcell]). After 6 hr, intracellular cytokine staining was performed as described.
Explanted skin grafts were fixed in optimal cutting temperature compound and frozen. Hematoxylin and eosin staining was used to visualize rejection. Sections were stained with anti-CD3e mAb and developed with horseradish peroxidase. Representative images (of at least four transplants per group) are magnified 20×.
Skin graft experiments are presented on Kaplan-Meier survival curves and compared with log-rank test. All other assays were compared with Mann-Whitney nonparametric tests. Statistical analyses used GraphPad Prism (La Jolla, CA).
The authors thank C.P. Larsen, A.D. Kirk, and A.B. Adams for their experimental and technical advice.
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