Characterization of regulatory T cell (Treg) subsets after allogeneic hematopoietic cell transplantation

Caroline Ritacco1, Grégory Ehx1, Céline Grégoire1,2, Ludovic Belle1, Evelyne Willems1,2, Sophie Servais1,2, Yves Beguin1,2, Stéphanie Humblet-Baron3, Frédéric Baron1,2
1GIGA-I3, laboratory of hematology, University of Liège, Liège, Belgium; 2Clinical Hematology, CHU of Liège, Liège, Belgium; 3VIB Center for Brain & Disease Research, Leuven, Belgium.

There is strong evidence that homeostasis of regulatory T cells (Treg) is abnormal in patients with chronic graft-versus-host disease (cGVHD). This has been attributed to decreased IL-2 / IL-7 ratio that favour expansion of conventional T cells (Tconv) over Treg. Recent studies have demonstrated that Treg represent a heterogeneous population and that several different Treg subsets can be identified by flow-cytometry. Here, we first assessed the impact of IL-2 and IL-7 on Treg subset activation in vitro. Then we compared Treg subsets in healthy donors (n=26) and in patients beyond 6 months after allogeneic hematopoietic cell transplantation (allo-HCT; having or not cGVHD) (n=72).

To assess the impact of IL-2 and IL-7 on T-cell subsets, T cells were first stained with extracellular antibodies then stimulated 15 minutes with recombinant human IL-2 or IL-7. They were then fixed and stained with intracellular antibodies and analysed by flow cytometry. To compare Treg subtypes in controls and in allo-HTC patients with or without cGVHD, PBMC were isolated from blood sample by density gradient centrifugation. CD3 positive cells were isolated from PBMC by immunomagnetic negative selection. T cells were then subjected to extracellular and intracellular staining with 15 markers in order to analyse them by flow cytometry. 

T-cell (including Treg) subsets responded differently to in vitro stimulation by IL-2 or IL-7. Specifically, activated Treg were more sensitive to IL-2 than naïve Treg while Tconv were more sensitive to IL-7 than to IL-2. There were marked differences in the relative frequencies of Treg subsets in healthy controls and allo-HCT patients. Specifically, effector Treg subpopulations were more frequent while the naive Treg subpopulation was less frequent in allo-HCT recipients. Comparison of Treg subsets in patients with or without cGVHD will be presented.

 Our data show that IL-2 and IL-7 have a different impact in various Treg subsets and that there are important alterations in the relative frequencies of Treg subsets after allo-HCT.