Stability of Foxp3 Expression Is a Critical Factor In the Ability of Regulatory T Cells to Mitigate Graft Versus Host Disease

Abstract 731

Graft versus host disease (GVHD) is a proinflammatory T cell-mediated syndrome that is the major complication of allogeneic bone marrow transplantation (BMT). During the course of GVHD, there is a progressive loss of regulatory T cells (Tregs), leading to an imbalance between the effector and regulatory arms of the immune system. Tregs have been subdivided into two distinct subsets, termed natural and induced, which have overlapping yet unique characteristics. While the role of natural regulatory T cells (nTregs) in GVHD biology has been extensively examined, the role of induced regulatory T cells (iTregs) remains largely unknown. An attractive aspect of the latter cell population is that they can be differentiated in vitro from conventional T cells and expanded in large numbers making them a potential source for regulatory T cell therapy in vivo. To determine whether in vitro-expanded iTregs were able to suppress alloreactive donor T cell responses and to compare the efficacy of these cells relative to nTregs, studies were performed using an MHC-incompatible murine BMT model (B6[H−2^b]−Balb/c[H−2^d]). In initial studies, purified CD4⁺ Foxp3^EGFP– T cells obtained from B6 Foxp3^EGFP reporter mice were cultured with anti-CD3 and anti-CD28 antibodies in the presence of IL-2 and TGF-b. After three days in culture, approximately 60–70% of cells were Foxp3⁺, expressed GITR, CD25, and CD103, and were equally suppressive to nTregs in mixed lymphocyte cultures. To determine if iTregs were suppressive in vivo, lethally irradiated Balb/c mice were transplanted with either B6 BM alone, B6 BM and spleen cells, or B6 BM/spleen cells and in vitro-expanded iTregs. In contrast to in vitro results, adoptive transfer of iTregs failed to protect mice from lethal GVHD even when administered at high Treg: effector T cell ratios (5:1) and were much less effective than equivalent doses of nTregs at abrogating GVHD pathology. iTregs also had no additive effect when co-administered with nTregs. Notably, we observed that whereas transferred nTregs persisted for up to 60 days in transplanted animals, iTregs were undetectable after only 14 days in liver, lung, colon and spleen, indicating that reduced in vivo survival was a potential explanation for the lack of protection. Further examination, however, revealed that the inability to detect iTregs was primarily attributable to the loss of Foxp3 expression and the subsequent in vivo reversion of these cells to a proinflammatory phenotype characterized by the secretion of interferon-gamma. In prior studies (Chen et al, Blood, 2009), we demonstrated that blockade of IL-6 signaling augmented reconstitution of nTregs and reduced overall GVHD severity. To determine whether inhibition of IL-6 could stabilize Foxp3 expression and prevent phenotypic reversion of iTregs, lethally irradiated Balb/c recipients were transplanted with B6 BM and spleen cells along with in vitro-differentiated iTregs and then treated with either isotype control or anti-IL-6R-specific antibody. Analysis of cells obtained from spleen, liver, lung and colon revealed that blockade of IL-6 signaling did not prevent loss of Foxp3 expression or reversion of iTregs to a Th1 cytokine phenotype. While Tregs can be converted from conventional T cells in vitro, they can also be generated in vivo during inflammatory syndromes. We therefore examined whether in vivo induction of iTregs occurred during GVHD and the extent to which blockade of IL-6 signaling affected iTreg expansion and overall GVHD protection. To address this question, lethally irradiated Balb/c mice were transplanted with B6 Rag-1 BM cells and purified CD4⁺ Foxp3^EGFP– T cells, and then treated with either anti-IL-6R or control antibody. We observed that in vivo conversion of Tregs was negligible in control animals (<1%), but that administration of anti-IL-6R antibody significantly increased the relative and absolute number of iTregs in GVHD target tissues with a commensurate reduction in overall pathological damage. Thus, blockade of IL-6 signaling was able to enhance reconstitution of iTregs in vivo, but had no discernible affect on adoptively transferred iTregs. In summary, these studies demonstrate that the stability of Foxp3 expression is a critical factor in the maintenance of transplantation tolerance and that instability of expression limits the utility of adoptively transferred iTregs as a source of cellular therapy for the abrogation of GVHD.