Alloanergization of HLA-Mismatched Human Donor T Cells Results in the Generation of Allosuppressive CD4⁺ T Regulatory Cells Both in Vitro and in Vivo

The alloreactivity of T cells contained in either donor bone marrow (BM) or peripheral blood mononuclear cells (PBMC) can be selectively reduced by alloanergy induction via ex vivo coculture with HLA-mismatched stimulator PBMC in the presence of costimulatory blockade (CSB). Alloanergization of human PBMC via allostimulation and CSB with humanized anti-B7.1 and anti-B7.2 monoclonal antibodies (MoAbs) can reduce alloresponses by 2 logs in vitro without abrogation of virus- or tumor-associated antigenspecific T cell responses. We are currently using this strategy to generate non-alloreactive donor PBMC for use in a Phase I clinical study of delayed infusion of alloanergized donor PBMC to improve immune reconstitution after haploidentical hematopoietic stem cell transplantation (HSCT). In addition to direct induction of anergy (hyporesponsiveness) in alloantigen-specific donor T cells, studies of alloanergization by CSB in both murine and human donor T cells suggest that an additional mechanism of active suppression may contribute to reduced alloresponsiveness. However, the mediators of suppression of human alloresponses after alloanergization have not been fully defined, nor has expansion of allosuppressive cells in vivo after HSCT containing alloanergized donor T cells been demonstrated. We report here that alloanergization of human donor PBMC by HLA-mismatched allostimulation with CSB with anti-B7.1 and -B7.2 MoAbs results in acquisition of potent dose-dependent and specific allosuppressive function. Alloanergized PBMC suppressed alloresponses of allonaïve and alloprimed PBMCs, requiring cell: cell contact for efficacy, whereas proliferative responses to 3rd-party allostimulators and to CMV were not significantly suppressed. Alloanergization of PBMC resulted in an increased frequency of CD4⁺ cells with a FOXP3⁺ CD25^hi CD127^lo T regulatory cell (alloTreg) phenotype, expressing CD45RO, CD62L and CCR4, intracellular CTLA4 but not IL10. Depletion and labeling studies demonstrated that alloTreg originated from the natural T regulatory cell (NTR) pool. Alloanergization increased alloTreg frequency, which was further increased upon repeat allostimulation to up to 20% of CD4⁺ donor T cells. AlloTreg were the primary mediators of the observed allosuppression. Depletion of alloTreg largely abrogated the allosuppressive capacity of alloanergized PBMC. High purity 2-step immunomagnetic-based cell selection was used to directly demonstrate alloTreg allosuppressive function. Although purified CD4⁺CD25^hi CD127^lo NTR from resting donor PBMCs were able to suppress alloresponses, purified alloTreg from allorestimulated alloanergized donor PBMCs demonstrated increased allosuppressive function and specificity on a per cell basis. We therefore investigated the frequency and function of CD4⁺ alloTregs in vivo in 3 patients (pts) who had received large doses of haplo donor T cells contained in BM alloanergized via allostimulation and CSB using humanized anti-B7.1 and -B7.2 MoAbs on a prior clinical study. A marked in vivo expansion of CD4⁺ alloTreg occurred in all pts early post-HSCT (D+30-100) reaching frequencies of up to 30% of CD4⁺ cells, and was associated with rapid reconstitution of absolute CD4⁺ T cells in 2 of the 3 pts assessed. AlloTreg were donor-derived with a phenotype identical to that found in vitro. Finally, alloTreg from 2 pts were purified by flow cytometric sorting, and specifically suppressed recipient-directed alloresponses of untreated donor PBMC. Furthermore, depletion of alloTreg increased recipient-specific alloresponses in donorderived CD4⁺ cells indicating that alloreactive cells had not been deleted at this stage. Despite receiving a median 7 × 10⁷ CD3⁺ haploidentical donor T cells/kg, 0/3 pts developed severe acute GvHD indicating that alloreactivity was controlled in vivo. This is the first data to demonstrate that ex vivo alloanergization of HLA-mismatched donor T cells prior to haploidentical HSCT results in a marked expansion of functional donor-derived alloTreg in vivo. This may represent an active mechanism by which alloresponses can be abrogated after alloanergized HLA-mismatched T cell infusion and suggests alloanergization could be further exploited to generate non-alloreactive donor T cells containing high frequencies of alloTreg for adoptive immunotherapy to improve HLA-mismatched HSCT.