Mandy L. Ford

Specificity, magnitude, and kinetics of MOG‐specific CD8+ T cell responses during experimental autoimmune encephalomyelitis

Experimental autoimmune encephalomyelitis (EAE) has traditionally been thought to be almost exclusively mediated by CD4+ effector T cells. Here, we provide evidence for the existence of mouse CD8+ T cells that are specific for an epitope of the myelin oligodendrocyte glycoprotein (MOG). Using a panel of truncated MOG peptides, we have identified the minimal epitope recognized by these T cells as MOG 37–46. This peptide, while possessing relatively low affinity for H‐2Db, efficiently stimulates IFN‐γ production from MOG‐specific CD8+ T cell lines in vitro and induces EAE in vivo. To further characterize the magnitude and kinetics of expansion of the MOG‐specific CD8+ T cell population in vivo, we used MOG 37–50/H‐2Db MHC tetramers to visualize MOG‐specific CD8+ effectors in the peripheral lymphoid organs and central nervous system during the course of EAE induction and progression. Our results identify MOG‐specific CD8+ T cells in the central nervous system prior to and after the onset of disease, suggesting that CD8+ T cells are a possible target for therapeutic intervention during EAE.

Translating costimulation blockade to the clinic: lessons learned from three pathways

Summary:  As the recognition that costimulatory signals are critical for optimal T‐cell activation, proliferation, and differentiation, there has been an explosion in the study of costimulatory molecules and their roles in enhancing anti‐donor T‐cell responses following transplantation. Here, we focus on the bench‐to‐beside translation of blocking agents designed to target three critical costimulatory pathways: the CD28/CD80/CD86 pathway, the CD154/CD40 pathway, and the lymphocyte function associated antigen‐1/intercellular adhesion molecule pathway. While blockade of each of these pathways proved promising in inhibiting donor‐reactive T‐cell responses and promoting long‐term graft survival in murine models of transplantation, the progression of development of therapeutic agents to block these pathways has each taken a slightly different course. Both logistical and biological pitfalls have accompanied the translation of blockers of all three pathways into clinically applicable therapies, and the development of costimulatory blockade as a substitute for current standard‐of‐care calcineurin inhibitors has by no means reached completion. Collaboration between both the basic and clinical arenas will further propel the development of costimulation blockers currently in the pipeline, as well as of novel methods to target these critical pathways during transplantation.

Antigen-specific precursor frequency impacts T cell proliferation, differentiation, and requirement for costimulation

After a brief period of antigenic stimulation, T cells become committed to a program of autonomous expansion and differentiation. We investigated the role of antigen-specific T cell precursor frequency as a possible cell-extrinsic factor impacting T cell programming in a model of allogeneic tissue transplantation. Using an adoptive transfer system to incrementally raise the precursor frequency of antigen-specific CD8+ T cells, we found that donor-reactive T cells primed at low frequency exhibited increased cellular division, decreased development of multifunctional effector activity, and an increased requirement for CD28- and CD154-mediated costimulation relative to those primed at high frequency. The results demonstrated that recipients with low CD4+ and CD8+ donor-reactive T cell frequencies exhibited long-term skin graft survival upon CD28/CD154 blockade, whereas simultaneously raising the frequency of CD4+ T cells to ∼0.5% and CD8+ T cells to ∼5% precipitated graft rejection despite CD28/CD154 blockade. Antigenic rechallenge of equal numbers of cells stimulated at high or low frequency revealed that cells retained an imprint of the frequency at which they were primed. These results demonstrate a critical role for initial precursor frequency in determining the CD8+ T cell requirement for CD28- and CD154-mediated costimulatory signals during graft rejection.

Selective Targeting of Human Alloresponsive CD8+ Effector Memory T Cells Based on CD2 Expression

Costimulation blockade (CoB), specifically CD28/B7 inhibition with belatacept, is an emerging clinical replacement for calcineurin inhibitor‐based immunosuppression in allotransplantation. However, there is accumulating evidence that belatacept incompletely controls alloreactive T cells that lose CD28 expression during terminal differentiation. We have recently shown that the CD2‐specific fusion protein alefacept controls costimulation blockade‐resistant allograft rejection in nonhuman primates. Here, we have investigated the relationship between human alloreactive T cells, costimulation blockade sensitivity and CD2 expression to determine whether these findings warrant potential clinical translation. Using polychromatic flow cytometry, we found that CD8+ effector memory T cells are distinctly high CD2 and low CD28 expressors. Alloresponsive CD8+CD2hiCD28− T cells contained the highest proportion of cells with polyfunctional cytokine (IFNγ, TNF and IL‐2) and cytotoxic effector molecule (CD107a and granzyme B) expression capability. Treatment with belatacept in vitro incompletely attenuated allospecific proliferation, but alefacept inhibited belatacept‐resistant proliferation. These results suggest that highly alloreactive effector T cells exert their late stage functions without reliance on ongoing CD28/B7 costimulation. Their high CD2 expression increases their susceptibility to alefacept. These studies combined with in vivo nonhuman primate data provide a rationale for translation of an immunosuppression regimen pairing alefacept and belatacept to human renal transplantation.

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.

Cutting Edge: Rapamycin Augments Pathogen-Specific but Not Graft-Reactive CD8+ T Cell Responses

Recent evidence demonstrating that exposure to rapamycin during viral infection increased the quantity and quality of Ag-specific T cells poses an intriguing paradox, because rapamycin is used in transplantation to dampen, rather than enhance, donor-reactive T cell responses. In this report, we compared the effects of rapamycin on the Ag-specific T cell response to a bacterial infection versus a transplant. Using a transgenic system in which the Ag and the responding T cell population were identical in both cases, we observed that treatment with rapamycin augmented the Ag-specific T cell response to a pathogen, whereas it failed to do so when the Ag was presented in the context of a transplant. These results suggest that the environment in which an Ag is presented alters the influence of rapamycin on Ag-specific T cell expansion and highlights a fundamental difference between Ag presented by an infectious agent as compared with an allograft.

Evaluation of human and non-human primate antibody binding to pig cells lacking GGTA1/CMAH/β4GalNT2 genes.

Simultaneous inactivation of pig GGTA1 and CMAH genes eliminates carbohydrate xenoantigens recognized by human antibodies. The β4GalNT2 glycosyltransferase may also synthesize xenoantigens. To further characterize glycan‐based species incompatibilities, we examined human and non‐human primate antibody binding to cells derived from genetically modified pigs lacking these carbohydrate‐modifying genes.

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.

Pre‐transplant antibody screening and anti‐CD154 costimulation blockade promote long‐term xenograft survival in a pig‐to‐primate kidney transplant model

Xenotransplantation has the potential to alleviate the organ shortage that prevents many patients with end‐stage renal disease from enjoying the benefits of kidney transplantation. Despite significant advances in other models, pig‐to‐primate kidney xenotransplantation has met limited success. Preformed anti‐pig antibodies are an important component of the xenogeneic immune response. To address this, we screened a cohort of 34 rhesus macaques for anti‐pig antibody levels. We then selected animals with both low and high titers of anti‐pig antibodies to proceed with kidney transplant from galactose‐α1,3‐galactose knockout/CD55 transgenic pig donors. All animals received T‐cell depletion followed by maintenance therapy with costimulation blockade (either anti‐CD154 mAb or belatacept), mycophenolate mofetil, and steroid. The animal with the high titer of anti‐pig antibody rejected the kidney xenograft within the first week. Low‐titer animals treated with anti‐CD154 antibody, but not belatacept exhibited prolonged kidney xenograft survival (>133 and >126 vs. 14 and 21 days, respectively). Long‐term surviving animals treated with the anti‐CD154‐based regimen continue to have normal kidney function and preserved renal architecture without evidence of rejection on biopsies sampled at day 100. This description of the longest reported survival of pig‐to‐non‐human primate kidney xenotransplantation, now >125 days, provides promise for further study and potential clinical translation.

Targeting co-stimulatory pathways: transplantation and autoimmunity.

The myriad of co-stimulatory signals expressed, or induced, upon T-cell activation suggests that these signalling pathways shape the character and magnitude of the resulting autoreactive or alloreactive T-cell responses during autoimmunity or transplantation, respectively. Reducing pathological T-cell responses by targeting T-cell co-stimulatory pathways has met with therapeutic success in many instances, but challenges remain. In this Review, we discuss the T-cell co-stimulatory molecules that are known to have critical roles during T-cell activation, expansion, and differentiation. We also outline the functional importance of T-cell co-stimulatory molecules in transplantation, tolerance and autoimmunity, and we describe how therapeutic blockade of these pathways might be harnessed to manipulate the immune response to prevent or attenuate pathological immune responses. Ultimately, understanding the interplay between individual co-stimulatory and co-inhibitory pathways engaged during T-cell activation and differentiation will lead to rational and targeted therapeutic interventions to manipulate T-cell responses and improve clinical outcomes.