Ex Vivo Costimulatory Blockade to Generate Regulatory T Cells From Patients Awaiting Kidney Transplantation.

Short-term outcomes of kidney transplantation have improved dramatically, but chronic rejection and regimen-related toxicity continue to compromise overall patient outcomes. Development of regulatory T cells (Tregs) as a means to decrease alloresponsiveness and limit the need for pharmacologic immunosuppression is an active area of preclinical and clinical investigation. Nevertheless, the immunomodulatory effects of end-stage renal disease on the efficacy of various strategies to generate and expand recipient Tregs for kidney transplantation are incompletely characterized. In this study, we show that Tregs can be successfully generated from either freshly isolated or previously cryopreserved uremic recipient (responder) and healthy donor (stimulator) peripheral blood mononuclear cells using the strategy of ex vivo costimulatory blockade with belatacept during mixed lymphocyte culture. Moreover, these Tregs maintain a CD3(+) CD4(+) CD25(+) CD127(lo) surface phenotype, high levels of intracellular FOXP3 and significant demethylation of the FOXP3 Treg-specific demethylation region on allorestimulation with donor stimulator cells. These data support evaluation of this simple, brief Treg production strategy in clinical trials of mismatched kidney transplantation.

Alloanergization of human T cells results in expansion of alloantigen-specific CD8(+) CD28(-) suppressor cells.

Allostimulation with concurrent costimulatory blockade induces alloantigen-specific hyporesponsiveness in responder T cells ("alloanergization"). Alloanergized responder cells also acquire alloantigen-specific suppressive activity, suggesting this strategy induces active immune tolerance. While this acquired suppressive activity is mediated primarily by CD4(+) FOXP3(+) cells, other cells, most notably CD8(+) suppressor cells, have also been shown to ameliorate human alloresponses. To determine whether alloanergization expands CD8(+) cells with allosuppressive phenotype and function, we used mixed lymphocyte cultures in which costimulatory blockade was provided by belatacept, an FDA-approved, second-generation CTLA-4-immunoglobulin fusion protein that blocks CD28-mediated costimulation, as an in vitro model of HLA-mismatched transplantation. This strategy resulted in an eightfold expansion of CD8(+) CD28(-) T cells which potently and specifically suppressed alloresponses of both CD4(+) and CD8(+) T cells without reducing the frequency of a range of functional pathogen-specific T cells. This CD8-mediated allosuppression primarily required cell-cell contact. In addition, we observed expansion of CD8(+) CD28(-) T cells in vivo in patients undergoing alloanergized HLA-mismatched bone marrow transplantation. Use of costimulatory blockade-mediated alloanergization to expand allospecific CD8(+) CD28(-) suppressor cells merits exploration as an approach to inducing or supporting immune tolerance to alloantigens after allogeneic transplantation.