Following allogeneic hematopoietic stem cell transplantation (allo-HSCT), the homeostasis of the T-cell pool is deeply challenged, requiring years to recover. Frequently, this recovery is dysfunctional, accompanied by loss of T-cell tolerance and subsequent development of chronic Graft-versus-Host Disease (cGvHD), leading to significant morbidity and mortality. With this work we investigated the mechanisms involved in the restoration of peripheral T-cell homeostasis following allo-HSCT, particularly the role of CD4+ regulatory T-cells (Tregs), critical mediators of peripheral T-cell tolerance. We performed a comprehensive analysis of the reconstitution of peripheral blood CD4+ and CD8+ T-cells in patients undergoing allo-HSCT for the treatment of hematologic malignancies. We took advantage of being able to study three distinct transplant settings, differing primarily in the conditioning regimen, GvHD prophylaxis and HLA (Human Leukocyte Antigen) matching. We consistently found a significantly lower number of Tregs at nine months post-transplant in patients developing cGvHD, coinciding with decreased Treg:Tcon (conventional T-cells) and Treg:CD8 ratios. This unbalance resulted from significantly delayed proliferation of naïve Tregs, and increased expansion of memory Tcon and CD8+ T-cell, observed in all cohorts. The expansion of memory subsets, likely involved in cGvHD pathogenesis, together with impaired thymic generation of de novo naïve CD4+ T-cells, resulted in significantly decreased diversity of the CD4+ TCR repertoire in cGvHD patients. Importantly, we were able study for the first time the contribution of true naïve and naïve-derived T memory stem cells (TSCM) to the development of cGvHD in humans. An early and transient increase of naïve T-cells was observed in two cohorts, anticipating the development of cGvHD, while the sustained increase of TSCM CD8 was observed in the cohort where allo-HSCT was performed with concomitant T-cell depletion . The heterogeneity of these results, reflect the effect of the different transplant settings, particularly in the recovery of donor-infused naïve T-cells and their progeny, in the early post-transplant period. In the final part of this work, we sought to determine the mechanisms involved in cGvHD amelioration after the administration of low-dose rIL-2 (interleukin 2) to patients with steroid dependent/refractory cGvHD. In this setting, the expansion of novel or low frequency Treg clones was the only variable associated to cGvHD improvement. This finding enhances the relevance of antigen specificity for adequate Treg suppression and induction of tolerance post allo-HSCT. Overall, the data obtained reiterates that achieving an adequate balance between effector and regulatory T-cells post allo-HSCT is of critical importance to avoid the development of cGvHD in the post transplantation period. Hence, we not only confirm the association between impaired Treg homeostasis and cGvHD, but further validate the contribution of naïve T-cells to the initiation of cGvHD. In addition, we describe for the first time, a role for TSCM in cGvHD pathogenesis in humans, and stress the importance to clarify the role of antigen-specificity to efficient Treg function
