Critical Role of CD4+Foxp3+ T Cells In Gvhd Prevention with High-Dose Posttransplant Cyclophosphamide (Cy).

Abstract 3749

Regulatory T cells (Tregs) contribute to the maintenance of self-tolerance and have been shown to ameliorate acute GVHD both in murine models and in the clinical setting. However, translation of results and mechanistic insights from animal models to the clinic is difficult due to inherent incongruities in the use of post-grafting immunosuppression for prevention of GVHD. Administration of high-dose cyclophosphamide (Cy) in mouse models of allogeneic bone marrow transplantation (BMT) and its identical use as a short course single agent prophylaxis for GVHD in a HLA-matched setting provides an ideal model for studying the interaction between postgrafting immunosuppression and Tregs. We explored the hypothesis that Tregs play a critical role in prevention of GVHD in a MHC-matched mouse model of alloBMT using C57Bl/6-Foxp3-DTR mice (H-2^b, CD45.2⁺) as donors, in which Foxp3+ Tregs can be selectively depleted in vivo by administration of diptheria toxin (DT; 2 doses on days 0 and +1). Donor mice were further crossed with those expressing luciferase (luc+) so that the in vivo expansion and engraftment of transplanted T cells could be noninvasively tracked by bioluminescent imaging (BLI) in addition to standard metrics of GVHD severity. We transplanted 1.2×10⁷ splenocytes (or purified conventional T cells) from C57Bl/6-luc+-Foxp3-DTR mice (H-2^b, CD45.2⁺) plus 1×10⁷ bone marrow (BM) cells from B6-Ly5.2 (H-2^b, CD45.1⁺) donors into myeloablatively conditioned allogeneic Balb/b recipients (H-2^b, CD45.2⁺, 775 cGy) to induce acute GVHD. BLI on day 4 post-BMT revealed a significantly higher bioluminescent signal (photons/second/mouse) in animals treated with DT compared to PBS recipients (p < 0.001). Treatment of chimeras with Cy on day+3 led to a significantly reduced expansion of alloreactive luc+ T cells on days 4, 7, 14 and 21 (p < 0.05; Cy vs DT-treated chimeras and controls). Conversely, BLI of chimeras that received DT on days 0 and +1 followed by Cy on day +3 demonstrated a moderate increase in proliferation of donor derived luc+ T-cells in comparison to Cy recipients suggesting that the depletion of CD4+Foxp3+ Tregs early after transplant was detrimental to the protective action of the drug. Clinical severity of GVHD was significantly increased in chimeras with depleted Foxp3+ Tregs (P < 0.05, compared to PBS-treated chimeras), while Cy administration completely prevented manifestation of GVHD (P< 0.01, Cy vs PBS- and DT-treated chimeras). Consistent with BLI findings, depletion of Foxp3+ Tregs early after transplant abrogated the protective effect of Cy on GVHD, reflected in increased morbidity and mortality. To determine the effect of Cy on Foxp3 expression, in vitro experiments were conducted with CD4+Foxp3-GFP^neg T cells co-cultured with allogeneic DCs and treated with mafosfamide. While exposure to increasing doses of the drug (2.5 and 12.5 μg/ml) caused a proportionally higher cell kill, the surviving T cells showed a marked induction of GFP expression in comparison to cells in the untreated culture. Taken together, these results suggest that Tregs are critical for prevention of GVHD in the context of GVHD prophylaxis with single agent high-dose Cy and that this approach besides limiting alloreactive T cell expansion may also foster conversion of naïve peripheral CD4+ T cells into CD4+Foxp3+ Tregs.