Acute Gvhd Induce the Critical Depletion of Naïve Pool in Regulatory T Cells: Implication for Linked Pathogenesis into Chronic Gvhd

CD4+Foxp3+ regulatory T cells (Treg) play a central role in establishing immune tolerance after allogeneic hematopoietic stem cell transplantation (HSCT). We previously reported that the long-term severe lymphopenia could result in the collapse of Treg homeostasis leading to the onset of chronic GVHD (Matsuoka et al. JCI 2010). We recently found that, not only in the chronic phase but also in the acute phase, the homeostasis of Treg is more susceptible to the post-transplant environment as compared to other lymphocyte subsets (Yoshioka et al. ASH 2014). However, the impact of acute GVHD on Treg homeostasis and the pathogenesis of following chronic GVHD has not been well studied. In this study, we examined Treg reconstitution in the early phase after transplant in patients with or without acute GVHD. For the purpose, we obtained peripheral blood samples at 2, 4, 8 and 12 weeks after transplant from 52 patients who received allogeneic HSCT, and then analyzed CD4+CD25med-highCD127lowFoxp3+ Treg comparing with CD4+CD25neg-lowCD127highFoxp3- conventional T cell (Tcon) and CD8+ T cells. CD4 T cell subsets are further divided into subpopulations by the expression of CD45RA and CD31. The expressions of Helios, Ki-67 and Bcl-2 on these subsets were also examined. After transplant, total lymphocyte counts in examined patients were significantly lower than the counts before the start of conditioning (median lymphocytes 95/ul at 2 weeks and 302/ul at 4 weeks vs 600/ul before conditioning, P<0.01 and P<0.01, respectively). As we reported before, Treg showed the active proliferation without diminishing Bcl-2 levels in the severe lymphopenia, resulted in the increased %Treg of CD4 T cells at 4 weeks after transplant (%Treg of CD4 T cells; 12.2% at 4 weeks, 4.6% in healthy controls, P<0.005). 18 patients who developed acute GVHD were studied Treg homeostasis before and after the onset of GVHD more in detail. Before the onset of acute GVHD, % Ki-67+ cells in Treg and Tcon were in the equivalent levels in these patients. After the onset of acute GVHD, % Ki-67+ cells in Treg was dramatically increased from 19.1% to 61.2% (median) and this was significantly higher than % Ki-67+ cells in Tcon after acute GVHD (P<0.05). %Treg of total CD4 T cells were significantly increased after GVHD (% Treg; Median 7.2% vs 12.2%, P<0.004). Expanded Treg after acute GVHD showed a predominant Helios+CD45RA-CD31- effector/memory phenotype with the lower level of Bcl-2 expression as compared to CD45RA+ naïve Treg. As a consequence, naïve Treg pool including CD45RA+CD31+ recent thymic emigrant Treg (RTE-Treg) were critically decreased during acute GVHD (%CD45RA+ cells; 12.7% into 6.5%, P<0.004: CD45RA+CD31+ cells; 3.6% into 2.1%, P<0.003). In contrast, Tcon still retained a relatively higher level of naïve pool (%CD45RA+ cells; 20.5%, % CD45RA+CD31+ cells; 10.9%) after acute GVHD. These data indicated that Treg proliferation was rapidly promoted in face with the inflammatory condition during acute GVHD and this appears to contribute the amelioration of developing GVHD. However, the prompt reaction resulted in the depletion of naïve Treg pool which is important to maintain stable Treg homeostasis in the long period. In conclusion, our findings suggest that acute GVHD drives aggressive Treg proliferation resulting in the increased percentage of this subset but this also induce the severe alteration of Treg homeostasis by depleting naïve Treg, which may provide the linked pathogenesis of the subsequent onset of chronic GVHD. The careful monitoring of Treg from the point of view might provide important information to promote immune tolerance.