Anti-CD28 Antibody and Belatacept Exert Differential Effects on Mechanisms of Renal Allograft Rejection.
ABSTRACT: Belatacept is a biologic that targets CD80/86 and prevents its interaction with CD28 and its alternative ligand, cytotoxic T lymphocyte antigen 4 (CTLA-4). Clinical experience in kidney transplantation has revealed a high incidence of rejection with belatacept, especially with intensive regimens, suggesting that blocking CTLA-4 is deleterious. We performed a head to head assessment of FR104 (n=5), a selective pegylated Fab' antibody fragment antagonist of CD28 that does not block the CTLA-4 pathway, and belatacept (n=5) in kidney allotransplantation in baboons. The biologics were supplemented with an initial 1-month treatment with low-dose tacrolimus. In cases of acute rejection, animals also received steroids. In the belatacept group, four of five recipients developed severe, steroid-resistant acute cellular rejection, whereas FR104-treated animals did not. Assessment of regulatory T cell-specific demethylated region methylation status in 1-month biopsy samples revealed a nonsignificant trend for higher regulatory T cell frequencies in FR104-treated animals. Transcriptional analysis did not reveal significant differences in Th17 cytokines but did reveal higher levels of IL-21, the main cytokine secreted by CD4 T follicular helper (Tfh) cells, in belatacept-treated animals. In vitro, FR104 controlled the proliferative response of human preexisting Tfh cells more efficiently than belatacept. In mice, selective CD28 blockade also controlled Tfh memory cell responses to KLH stimulation more efficiently than CD80/86 blockade. Our data reveal that selective CD28 blockade and belatacept exert different effects on mechanisms of renal allograft rejection, particularly at the level of Tfh cell stimulation.
Project description:T cell activation is a complex process that requires multiple cell signaling pathways, including a primary recognition signal and additional costimulatory signals. One of the best-characterized costimulatory pathways includes the Ig superfamily members CD28 and CTLA-4 and their ligands CD80 and CD86. Areas covered: This review discusses past, current and future biological therapies that have been utilized to block the CD28/CTLA-4 cosignaling pathway in the settings of autoimmunity and transplantation, as well the challenges facing successful implementation of these therapies. Expert opinion: The development of CD28 blockers Abatacept and Belatacept provided a more targeted therapy approach for transplant rejection and autoimmune disease relative to calcineurin inhibitors and anti-proliferatives, but overall efficacy may be limited due to their collateral effect of simultaneously blocking CTLA-4 coinhibitory signals. As such, current investigations into the potential of selective CD28 blockade to block the costimulatory potential of CD28 while exploiting the coinhibitory effects of CTLA-4 are promising. However, as selective CD28 blockade inhibits the activity of both effector and regulatory T cells, an important goal for the future is the design of therapies that will maximize the attenuation of effector responses while preserving the suppressive function of T regulatory cells.
Project description:CTLA-4Ig (belatacept) blocks the CD80/CD86 ligands for both CD28 and CTLA-4; thus, in addition to the intended effect of blocking CD28-mediated costimulation, belatacept also has the unintended effect of blocking CTLA-4-mediated coinhibition. Recently, anti-CD28 domain antibodies (dAb) that selectively target CD28 while leaving CTLA-4 intact were shown to more effectively inhibit alloimmune responses and prolong graft survival. However, the impact of selective CD28 blockade on protective immunity has not been extensively investigated. Here, we sought to compare the impact of CTLA-4Ig vs anti-CD28dAb on CD8+ T cell immunity to a transplant-relevant pathogen, a murine homolog of Epstein-Barr virus. Mice were infected with murine gammaherpesvirus-68 (MHV) and treated with vehicle, CTLA-4Ig, or anti-CD28dAb. Although anti-CD28dAb resulted in a decrease in virus-specific CD8+ T cell numbers as compared to CTLA-4Ig, cytolytic function and the expression of markers of high-quality effectors were not different from CTLA-4Ig treated animals. Importantly, MHV-68 viral load was not different between the treatment groups. These results suggest that preserved CTLA-4 coinhibition limits MHV-specific CD8+ T cell accumulation, but the population that remains retains cytolytic function and migratory capacity and is not inferior in its ability to control viral burden relative to T cell responses in CTLA-4Ig-treated animals.
Project description:Costimulation blockade with the B7-CD28 pathway-specific agent belatacept is now used in clinical kidney transplantation, but its efficacy remains imperfect. Numerous alternate costimulatory pathways have been proposed as targets to synergize with belatacept, one of which being the inducible costimulator (ICOS)-ICOS ligand (ICOS-L) pathway. Combined ICOS-ICOS-L and CD28-B7 blockade has been shown to prevent rejection in mice, but has not been studied in primates. We therefore tested a novel ICOS-Ig human Fc-fusion protein in a nonhuman primate (NHP) kidney transplant model alone and in combination with belatacept. ICOS-Ig did not prolong rejection-free survival as a monotherapy or in combination with belatacept. In ICOS-Ig alone treated animals, most graft-infiltrating CD4(+) and CD8(+) T cells expressed ICOS, and ICOS(+) T cells were present in peripheral blood to a lesser degree. Adding belatacept reduced the proportion of graft-infiltrating ICOS(+) T cells and virtually eliminated their presence in peripheral blood. Graft-infiltrating T cells in belatacept-resistant rejection were primarily CD8(+) CD28(-) , but importantly, very few CD8(+) CD28(-) T cells expressed ICOS. We conclude that ICOS-Ig, alone or combined with belatacept, does not prolong renal allograft survival in NHPs. This may relate to selective loss of ICOS with CD28 loss.
Project description:Recently, newer therapies have been designed to more specifically target rejection in an effort to improve efficacy and limit unwanted toxicity. Belatacept, a CD28-CD80/86 specific reagent, is associated with superior patient survival and graft function compared with traditional therapy, but its adoption as a mainstay immunosuppressive therapy has been tempered by increased rejection rates. It is essential that the underlying mechanisms associated with this rejection be elucidated before belatacept is more widely used. To that end, we designed a study in a nonhuman primate kidney transplant model where animals were treated with either a belatacept- or a tacrolimus-based immunosuppressive regimen. Interestingly, we found that elevated pretransplant frequencies of CD28+ CD8+ TEMRA cells are associated with rejection on belatacept but not tacrolimus treatment. Further analysis showed that the CD28+ CD8+ TEMRA cells rapidly lose CD28 expression after transplant in those animals that go on to reject with the allograft infiltrate being predominantly CD28- . These data suggest that CD28+ memory T cells may be resistant to belatacept, capable of further differentiation including loss of CD28 expression while maintaining effector function. The unique signaling requirements of CD28+ memory T cells provide opportunities for the development of targeted therapies, which may synergize with belatacept to prevent costimulation-independent rejection.
Project description:The CD28/cytotoxic T-lymphocyte antigen 4 (CTLA-4)blocker belatacept selectively inhibits alloreactive T cell responses but is associated with a high incidence of acute rejection following renal transplantation,which led us to investigate the etiology of belatacept–resistant graft rejection. T cells can differentiate into functionally distinct subsets of memory T cellsthat collectively enable protection against diverse classes of pathogens and can cross-react with allogeneicantigen and mediate graft rejection. T helper 17(Th17) cells are a pro-inflammatory CD4+ lineage that provides immunity to pathogens and are pathogenic in autoimmune disease. We found that T helper 1 (Th1)and Th17 memory compartments contained a similar frequency of divided cells following allogeneic stimulation.Compared to Th1 cells, Th17 memory cells expressed significantly higher levels of the coinhibitory molecule CTLA-4. Stimulation in the presence of belatacept inhibited Th1 responses but augmented Th17 cells due to greater sensitivity to coinhibition by CTLA-4. Th17 cells from renal transplant recipients were resistant to ex vivo CD28/CTLA-4 blockade with belatacept, and an elevated frequency of Th17 memory cells was associated with acute rejection during belatacept therapy. These data highlight important differences in costimulatory and coinhibitory requirements of CD4+ memory subsets, and demonstrate that the heterogeneity of pathogen-derived memory has implications for immunomodulation strategies.
Project description: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.
Project description:Memory T cells pose a significant problem to successful therapeutic control of unwanted immune responses during autoimmunity and transplantation, as they are differentially controlled by cosignaling receptors such as CD28 and CTLA-4. Treatment with abatacept and belatacept impede CD28 signaling by binding to CD80 and CD86, but they also have the unintended consequence of blocking the ligands for CTLA-4, a process that may inadvertently boost effector responses. Here, we show that a potentially novel anti-CD28 domain antibody (dAb) that selectively blocks CD28 but preserves CTLA-4 coinhibition confers improved allograft survival in sensitized recipients as compared with CTLA-4 Ig. However, both CTLA-4 Ig and anti-CD28 dAb similarly and significantly reduced the accumulation of donor-reactive CD8+ memory T cells, demonstrating that regulation of the expansion of CD8+ memory T cell populations is controlled in part by CD28 signals and is not significantly impacted by CTLA-4. In contrast, selective CD28 blockade was superior to CTLA-4 Ig in inhibiting IFN-?, TNF, and IL-2 production by CD8+ memory T cells, which in turn resulted in reduced recruitment of innate CD11b+ monocytes into allografts. Importantly, this superiority was CTLA-4 dependent, demonstrating that effector function of CD8+ memory T cells is regulated by the balance of CD28 and CTLA-4 signaling.
Project description:Selective blockade of CD28 is a promising therapy to inhibit pathogenic alloimmunity. However, evaluation of this approach in transplantation has been very limited. Using a novel nonactivating single-chain Fv-based reagent (?28scFv), we have investigated the role of CD28 and cytotoxic T lymphocyte antigen 4 (CTLA-4) in a murine cardiac transplant model. Blockade of CD28 for 2 weeks after engraftment promoted allograft survival, and significantly attenuated chronic rejection when combined with transient CD154-blockade or calcineurin inhibition. Graft acceptance was associated with decreased alloantibody production, increased proportion of early graft infiltration by regulatory T cells and increased expression of regulatory dendritic cell genes. Blockade of CTLA-4 during ?28scFv-based treatments led to prompt rejection in all animals and inhibited expression of forkhead box P3 (Foxp3), programmed death (PD)-1 and 2,3-indoleamine dioxygenase (IDO) in the graft. These results show that CD28 signaling during the first weeks after transplant is a pivotal mediator of pathogenic alloimmunity, and that selective CD28 blockade prolongs graft acceptance by at least two immunomodulatory mechanisms. Selective CD28 inhibition while sparing CTLA-4 is thus a promising approach to inhibit pathogenic alloimmunity.
Project description:Regulatory T cells (Treg) are critical regulators of immune tolerance. Both IL-2 and CD28-CD80/CD86 signaling are critical for CD4(+)CD25(+)FOXP3(+) Treg survival in mice. Yet, both belatacept (a second-generation CTLA-4Ig) and basiliximab (an anti-CD25 monoclonal antibody) are among the arsenal of current immunotherapies being used in kidney transplant patients. In this study, we explored the direct effect of basiliximab and belatacept on the Tregs in peripheral blood both in the short term and long term and in kidney biopsies of patients with acute rejection. We report that the combined belatacept/basiliximab therapy has no long-term effect on circulating Tregs when compared to a calcineurin inhibitor (CNI)-treated group. Moreover, belatacept-treated patients had a significantly greater number of FOXP3(+) T cells in graft biopsies during acute rejection as compared to CNI-treated patients. Finally, it appears that the basiliximab caused a transient loss of both FOXP3(+) and FOXP3(-) CD25(+) T cells in the circulation in both treatment groups raising important questions about the use of this therapy in tolerance promoting therapeutic protocols.
Project description:Belatacept is an inhibitor of CD28/B7 costimulation that is clinically indicated as a calcineurin inhibitor (CNI) alternative in combination with mycophenolate mofetil and steroids after renal transplantation. We sought to develop a clinically translatable, nonlymphocyte depleting, belatacept-based regimen that could obviate the need for both CNIs and steroids. Thus, based on murine data showing synergy between costimulation blockade and mTOR inhibition, we studied rhesus monkeys undergoing MHC-mismatched renal allotransplants treated with belatacept and the mTOR inhibitor, sirolimus. To extend prior work on costimulation blockade-resistant rejection, some animals also received CD2 blockade with alefacept (LFA3-Ig). Belatacept and sirolimus therapy successfully prevented rejection in all animals. Tolerance was not induced, as animals rejected after withdrawal of therapy. The regimen did not deplete T cells. Alefecept did not add a survival benefit to the optimized belatacept and sirolimus regimen, despite causing an intended depletion of memory T cells, and caused a marked reduction in regulatory T cells. Furthermore, alefacept-treated animals had a significantly increased incidence of CMV reactivation, suggesting that this combination overly compromised protective immunity. These data support belatacept and sirolimus as a clinically translatable, nondepleting, CNI-free, steroid-sparing immunomodulatory regimen that promotes sustained rejection-free allograft survival after renal transplantation.