Alexandra P. Turner

mTOR regulates memory CD8 T-cell differentiation

Memory CD8 T cells are a critical component of protective immunity, and inducing effective memory T-cell responses is a major goal of vaccines against chronic infections and tumours. Considerable effort has gone into designing vaccine regimens that will increase the magnitude of the memory response, but there has been minimal emphasis on developing strategies to improve the functional qualities of memory T cells. Here we show that mTOR (mammalian target of rapamycin, also known as FRAP1) is a major regulator of memory CD8 T-cell differentiation, and in contrast to what we expected, the immunosuppressive drug rapamycin has immunostimulatory effects on the generation of memory CD8 T cells. Treatment of mice with rapamycin following acute lymphocytic choriomeningitis virus infection enhanced not only the quantity but also the quality of virus-specific CD8 T cells. Similar effects were seen after immunization of mice with a vaccine based on non-replicating virus-like particles. In addition, rapamycin treatment also enhanced memory T-cell responses in non-human primates following vaccination with modified vaccinia virus Ankara. Rapamycin was effective during both the expansion and contraction phases of the T-cell response; during the expansion phase it increased the number of memory precursors, and during the contraction phase (effector to memory transition) it accelerated the memory T-cell differentiation program. Experiments using RNA interference to inhibit expression of mTOR, raptor (also known as 4932417H02Rik) or FKBP12 (also known as FKBP1A) in antigen-specific CD8 T cells showed that mTOR acts intrinsically through the mTORC1 (mTOR complex 1) pathway to regulate memory T-cell differentiation. Thus these studies identify a molecular pathway regulating memory formation and provide an effective strategy for improving the functional qualities of vaccine- or infection-induced memory T cells.

Alefacept promotes co-stimulation blockade based allograft survival in nonhuman primates

Memory T cells promote allograft rejection particularly in co-stimulation blockade–based immunosuppressive regimens. Here we show that the CD2-specific fusion protein alefacept (lymphocyte function–associated antigen-3–Ig; LFA -3–Ig) selectively eliminates memory T cells and, when combined with a co-stimulation blockade–based regimen using cytotoxic T lymphocyte antigen-4 (CTLA-4)-Ig, a CD80- and CD86-specific fusion protein, prevents renal allograft rejection and alloantibody formation in nonhuman primates. These results support the immediate translation of a regimen for the prevention of allograft rejection without the use of calcineurin inhibitors, steroids or pan–T cell depletion.

LFA-1–specific therapy prolongs allograft survival in rhesus macaques

Outcomes in transplantation have been limited by suboptimal long-term graft survival and toxicities associated with current immunosuppressive approaches. T cell costimulation blockade has shown promise as an alternative strategy to avoid the side effects of conventional immunosuppressive therapies, but targeting CD28-mediated costimulation alone has proven insufficient to prevent graft rejection in primates. Donor-specific memory T (TM) cells have been implicated in costimulation blockade-resistant transplant rejection, due to their enhanced effector function and decreased reliance on costimulatory signaling. Thus, we have tested a potential strategy to overcome TM cell-driven rejection by targeting molecules preferentially expressed on these cells, such as the adhesion molecule lymphocyte function-associated antigen 1 (LFA-1). Here, we show that short-term treatment (i.e., induction therapy) with the LFA-1-specific antibody TS-1/22 in combination with either basiliximab (an IL-2Rα-specific mAb) and sirolimus (a mammalian target of rapamycin inhibitor) or belatacept (a high-affinity variant of the CD28 costimulation-blocker CTLA4Ig) prolonged islet allograft survival in nonhuman primates relative to control treatments. Moreover, TS-1/22 masked LFA-1 on TM cells in vivo and inhibited the generation of alloproliferative and cytokine-producing effector T cells that expressed high levels of LFA-1 in vitro. These results support the use of LFA-1-specific induction therapy to neutralize costimulation blockade-resistant populations of T cells and further evaluation of LFA-1-specific therapeutics for use in transplantation.

Sirolimus enhances the magnitude and quality of viral-specific CD8+ T cell responses to vaccinia virus vaccination in rhesus macaques

Sirolimus is a potent antiproliferative agent used clinically to prevent renal allograft rejection. However, little is known about the effects of maintenance immunosuppressive agents on the immune response to potentially protective vaccines. Here we show that sirolimus paradoxically increases the magnitude and quality of the CD8+ T‐cell response to vaccinia vaccination in nonhuman primates, fostering more robust recall responses compared to untreated and tacrolimus‐treated controls. Enhancement of both the central and effector memory compartments of the vaccinia‐specific CD8+ T‐cell response was observed. These data elucidate new mechanistic characteristics of sirolimus and suggest immune applications extending beyond its role as an immunosuppressant.

Nondepleting Anti‐CD40‐Based Therapy Prolongs Allograft Survival in Nonhuman Primates

Costimulation blockade of the CD40/CD154 pathway has been effective at preventing allograft rejection in numerous transplantation models. This strategy has largely depended on mAbs directed against CD154, limiting the potential for translation due to its association with thromboembolic events. Though targeting CD40 as an alternative to CD154 has been successful at preventing allograft rejection in preclinical models, there have been no reports on the effects of CD40‐specific agents in human transplant recipients. This delay in clinical translation may in part be explained by the presence of cellular depletion with many CD40‐specific mAbs. As such, the optimal biologic properties of CD40‐directed immunotherapy remain to be determined. In this report, we have characterized 3A8, a human CD40‐specific mAb and evaluated its efficacy in a rhesus macaque model of islet cell transplantation. Despite partially agonistic properties and the inability to block CD40 binding of soluble CD154 (sCD154) in vitro, 3A8‐based therapy markedly prolonged islet allograft survival without depleting B cells. Our results indicate that the allograft‐protective effects of CD40‐directed costimulation blockade do not require sCD154 blockade, complete antagonism or cellular depletion, and serve to support and guide the continued development of CD40‐specific agents for clinical translation.

Alternative immunomodulatory strategies for xenotransplantation: CD40/154 pathway-sparing regimens promote xenograft survival.

Immunosuppressive therapies that block the CD40/CD154 costimulatory pathway have proven to be uniquely effective in preclinical xenotransplant models. Given the challenges facing clinical translation of CD40/CD154 pathway blockade, we examined the efficacy and tolerability of CD40/CD154 pathway‐sparing immunomodulatory strategies in a pig‐to‐nonhuman primate islet xenotransplant model. Rhesus macaques were rendered diabetic with streptozocin and given an intraportal infusion of ∼50 000 islet equivalents/kg wild‐type neonatal porcine islets. Base immunosuppression for all recipients included maintenance therapy with belatacept and mycophenolate mofetil plus induction with basiliximab and LFA‐1 blockade. Cohort 1 recipients (n = 3) were treated with the base regimen alone; cohort 2 recipients (n = 5) were additionally treated with tacrolimus induction and cohort 3 recipients (n = 5) were treated with alefacept in place of basiliximab, and more intense LFA‐1 blockade. Three of five recipients in both cohorts 2 and 3 achieved sustained insulin‐independent normoglycemia (median rejection‐free survivals 60 and 111 days, respectively), compared to zero of three recipients in cohort 1. These data show that CD40/CD154 pathway‐sparing regimens can promote xenoislet survival. Further optimization of these strategies is warranted to aid the clinical translation of islet xenotransplantation.

Belatacept and Sirolimus Prolong Nonhuman Primate Islet Allograft Survival: Adverse Consequences of Concomitant Alefacept Therapy

Calcineurin inhibitors (CNI) and steroids are known to promote insulin resistance, and their avoidance after islet transplantation is preferred from a metabolic standpoint. Belatacept, a B7‐specific mediator of costimulation blockade (CoB), is clinically indicated as a CNI alternative in renal transplantation, and we have endeavored to develop a clinically translatable, belatacept‐based regimen that could obviate the need for both CNIs and steroids. Based on the known synergy between CoB and mTOR inhibition, we studied rhesus monkeys undergoing MHC‐mismatched islet allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on CoB‐resistant rejection, some animals also received CD2 blockade with alefacept (LFA3‐Ig). Nine rhesus macaques were rendered diabetic with streptozotocin and underwent islet allotransplantation. All received belatacept and sirolimus; six also received alefacept. Belatacept and sirolimus significantly prolonged rejection‐free graft survival (median 225 days compared to 8 days in controls receiving basiliximab and sirolimus; p = 0.022). The addition of alefacept provided no additional survival benefit, but was associated with Cytomegalovirus reactivation in four of six animals. No recipients produced donor‐specific alloantibodies. The combination of belatacept and sirolimus successfully prevents islet allograft survival in rhesus monkeys, but induction with alefacept provides no survival benefit and increases the risk of viral reactivation.

Induction immunosuppression in liver transplantation: a review.

Antibody therapy for induction is seldom used in liver transplantation in the United States, but continues to be used in approximately 10% of patients. The most commonly used antibody at the current time is basiliximab (Simulect, Novartis) and is used in adults with renal dysfunction at the time of liver transplantation with the intention of delaying introduction of calcineurin‐inhibitors. In children, the same antibody is commonly used in order to reduce rates of acute rejection. Most patients, adult and pediatric, are treated with initially higher levels of tacrolimus rather than antibody induction.

CTLA4Ig Prevents Alloantibody Formation Following Nonhuman Primate Islet Transplantation Using the CD40-Specific Antibody 3A8

Islet transplantation to treat type 1 diabetes has been limited in part by toxicities of current immunosuppression and recipient humoral sensitization. Blockade of the CD28/CD80/86 and CD40/CD154 pathways has shown promise to remedy both these limitations, but translation has been hampered by difficulties in translating CD154‐directed therapies. Prior CD40‐directed regimens have led to prolonged islet survival, but fail to prevent humoral allosensitization. We therefore evaluated the addition of CTLA4Ig to a CD40 blockade‐based regimen in nonhuman primate (NHP) alloislet transplantation. Diabetic rhesus macaques were transplanted allogeneic islets using the CD40‐specific antibody 3A8, basiliximab induction, and sirolimus with or without CTLA4Ig maintenance therapy. Allograft survival was determined by fasting blood glucose levels and flow cytometric techniques were used to test for donor‐specific antibody (DSA) formation. CTLA4Ig plus 3A8, basiliximab and sirolimus was well tolerated and induced long‐term islet allograft survival. The addition of CTLA4Ig prevented DSA formation, but did not facilitate withdrawal of the 3A8‐based regimen. Thus, CTLA4Ig combines with a CD40‐specific regimen to prevent DSA formation in NHPs, and offers a potentially translatable calcineurin inhibitor‐free protocol inclusive of a single investigational agent for use in clinical islet transplantation without relying upon CD154 blockade.