CD4+CD25+Foxp3+ regulatory T cells (Treg) are known to play a central role in the maintenance of self-tolerance and immune homeostasis. After allogeneic stem cell transplantation, impaired recovery of Treg is associated with the development of cGVHD. Interleukin-2 (IL-2) is a critical regulator of Treg development, expansion and survival and lack of IL-2 results in Treg deficiency. In patients with cGVHD, we previously demonstrated that Treg proliferate at high levels but this subset is also highly susceptible to apoptosis leading to inadequate Treg numbers (Matsuoka et al. JCI 2010). We also reported that low-dose IL-2 administration resulted in selective expansion of Treg in vivo and clinical improvement of cGVHD (Koreth et al. NEJM 2011). To identify mechanisms responsible for increased Treg susceptibility to apoptosis in cGVHD 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 assessment allowed us to compare BH3 peptide-induced mitochondrial membrane depolarization (“priming”) in different T cell subsets, including CD4 Treg, conventional CD4 T cells (CD4 Tcon), and CD8 T cells. Expression of Bcl-2, CD95 and Ki67 were also studied in each T cell subset. We studied peripheral blood samples from 36 patients with hematologic malignancies (median age 59 yr) who are > 2 years post HSCT (27 patients with cGVHD and 9 patients without cGVHD) and 15 patients who received daily subcutaneous IL-2 for 8 weeks for treatment of steroid-refractory cGvHD. Severity of cGVHD was classified according to NIH criteria.
In patients without cGVHD, BH3 priming was similar in all 3 T cell subsets (CD4 Treg, CD4 Tcon and CD8). In patients with cGVHD, CD4 Treg were more primed than CD4 Tcon when challenged with BIM, BAD, PUMA, BMF and the combination of BAD + NOXA peptides (p<0.01 – 0.0001). Treg were more primed than CD8 T cells when challenged with PUMA peptide (p<0.0001), but priming in Treg and CD8 T cells was similar for other BH3 peptides in patients with cGVHD. We also compared BH3 priming of each T cell subset in patients with different grades of cGVHD. When challenged with BH3 peptides, Treg, Tcon and CD8 T cells were less primed in patients with severe cGVHD. In patients with cGVHD, Treg expressed higher levels of Ki-67, higher levels of CD95 and lower levels of Bcl-2 than Tcon. Expression of CD95 did not vary with severity of GVHD in any T cell subset, but expression of Bcl-2 was significantly increased in all subsets in patients with severe cGVHD. Increased BH3 priming and high expression of CD95 indicate that Treg are more susceptible to apoptosis than Tcon in cGVHD. However, both Treg and Tcon become less primed and Bcl-2 levels increase in severe cGVHD suggesting that these cells are less susceptible to mitochondrial pathway apoptosis. Since the total number of Treg and Tcon are significantly reduced in patients with cGVHD, these findings suggest that the remaining circulating cells are relatively resistant to mitochondrial pathway apoptosis. CD95 expression in Treg remains high indicating no change in death receptor pathway apoptosis. Daily treatment with low-dose IL-2 for 8 weeks selectively expands Treg in vivo in patients with severe cGVHD. As the number of Treg increase, BH3 profiling shows that these cells gradually become more primed and therefore more susceptible to mitochondrial pathway apoptosis. Taken together, 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.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.