Regulation of Apoptotic Pathways in Human Regulatory T Cells in Chronic Graft Versus Host Disease (cGVHD) After Allogeneic Hematopoietic Stem Cell Transplantation (HSCT)

CD4+CD25+Foxp3+ regulatory T cells (Treg) play a central role in the maintenance of immune tolerance. Impaired recovery of Treg after HSCT is associated with the development of cGVHD. In patients with cGVHD, Treg proliferate at high levels but these cells are also highly susceptible to apoptosis, leading to decreased survival and relative Treg deficiency. To identify mechanisms that regulate Treg susceptibility to apoptosis we used a new flow cytometry-based assay to measure mitochondrial membrane depolarization in response to a panel of pro-apoptotic BH3 peptides (BIM, BID, BAD, NOXA, PUMA, BMF, HRK). This allowed us to compare BH3 peptide-induced mitochondrial membrane depolarization (“priming”) in different T cell subsets; CD4 Treg, conventional CD4 T cells (CD4 Tcon), and CD8 T cells. We also examined cytoplasmic expression of Bcl-2, Mcl1, BclxL, Bim and cell surface expression of CD95 death receptor (Fas). Functional susceptibility to apoptosis was measured following in vitro stimulation with staurosporine or anti-CD95. We studied peripheral blood from 54 patients with hematologic malignancies >2 years post HSCT (39 with cGVHD; 15 without cGVHD). In patients without cGVHD, BH3 priming was similar in Treg, Tcon and CD8. In contrast, Treg and CD8 were more primed than Tcon in cGVHD when challenged with BIM, BAD, PUMA, BMF and BAD + NOXA peptides. In patients with cGVHD, Treg expressed higher levels of Bim, BclxL and CD95 and lower levels of Bcl-2, but there was no significant difference in Mcl1. Consistent with these observations, Treg were more susceptible than Tcon and CD8 to both mitochondrial and death receptor pathway apoptosis in vitro. Increased Treg susceptibility to apoptosis in cGVHD therefore appears to be mediated primarily by increased expression of CD95 and Bim and lower levels of Bcl-2. Although Treg priming was increased overall in cGVHD, examination of Treg in severe cGVHD actually revealed less priming in this subset. This correlated with increased Treg expression of Bcl-2 and Mcl1. Since the total number of Treg is significantly reduced in severe cGVHD, these findings suggest that the remaining Treg are relatively resistant to the mitochondrial pathway of apoptosis. However, CD95 expression in Treg remains high in severe cGVHD indicating persistent high-level apoptosis mediated through the death receptor pathway. Administration of low dose IL-2 to patients with severe cGVHD led to increased expression of Bcl-2 and less susceptibility to both mitochondrial and death receptor pathway apoptosis. These studies help define the complex and distinct pathways that regulate survival in different T cell subsets, and changes in these pathways that occur in patients with chronic GVHD. These pathways play important roles in the maintenance of T cell homeostasis and targeting these complex pathways can provide new opportunities to promote immune tolerance after allogeneic HSCT.