Early Homeostatic Expansion of Regulatory T Cells with the Predominant Effector/Memory Phenotype May Stabilize Immune Recovery in the First Month after HSCT

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). However, Treg homeostasis in the very early phase after HSCT has not been well studied. To address this issue, we here examined the early lymphocytes reconstitution in total 34 patients who received HSCT. Peripheral blood samples were obtained at 2, 4, 8 and 12 weeks after transplant and analyzed the reconstitution of CD4⁺CD25^med-highCD127^lowFoxp3⁺ Treg comparing with CD4⁺CD25^neg-lowCD127^highFoxp3^- 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, Bcl-2 and C-C chemokine receptor type 4 (CCR4) on these subsets were also examined. These patients were transplanted the grafts from various stem cell sources (7 HLA-matched PBSCT, 12 HLA-matched BMT, 6 HLA-mismatched CBT and 9 HLA-haploidentical PBSCT) and this enables us the opportunity to comparatively evaluate the early lymphocyte reconstitution among the different types of HSCT. After transplant, total lymphocyte counts were significantly lower than the counts before the start of conditioning (median lymphocytes 113/ul at 2 weeks and 223/ul at 4 weeks vs 550/ul before conditioning, P<0.01 and P<0.01, respectively). In the severely lymphopenic condition in the first month after HSCT, all T cell subsets were undergoing aggressive proliferation in this acute phase as compared to proliferation in the chronic phase, however, Treg proliferation was significantly higher than in Tcon at 4 weeks (%Ki-67+ cells; median 56.4%, 23.4%, respectively, P<0.02). %Treg of total CD4 T cells elevated and peaked at 4 weeks post-transplant. At this timepoint, %Treg of CD4 T cells showed the clear inverse correlation with %CD45RA+ of Treg (r²=0.40), suggesting the expansion of Treg in this phase appears to be a result from severe lymphopenia-driven proliferation which involves conversion from naive into memory phenotype. Elevation of %Treg was most evident in the patients who received HLA-haploidentical graft after ATG-containing conditioning (median 8.41% in haplo-HSCT, 5.25% in other groups, P<0.05), again indicating the lymphopenia is critical factor to drive Treg proliferatrion. Expanded Treg showed a predominant Helios⁺CD45RA^-CD31^- effector/memory phenotype with the lower level of Bcl-2 expression as compared to CD45RA⁺ naïve Treg. The elevation of Treg did not sustain and %Treg of CD4 T cells got back to the baseline level by 8 weeks. During the first 3 months after HSCT, CD45RA^- Treg exhibited high level of CCR4 and the recovery of this subset was critically delayed in Adult T-cell Leukemia (ATL) patients treated with anti-CCR4 antibody in the peri-transplant period, resulting in the development of acute graft-versus-host diseases. In conclusion, our findings suggest 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. Post-transplant lymphopenia drives aggressive Treg proliferation resulting in the increased percentage of this subset in the very acute phase which may contribute to stabilize the immune recovery. The careful monitoring of Treg from the point of view might provide important information to promote immune tolerance.