Title: Origin and homeostasis of regulatory T cells in chronic Graft-versus-Host Disease

1. Objectives

Chronic graft-versus-Host disease (cGvHD) is a frequent complication of allogeneic hematopoietic stem cell transplantation that is basically caused by the donor-derived T cells immune response against recipient tissues. Within cGvHD, general defects in central and peripheral tolerance have been respectively attributed to damages to the thymus and an alteration in Treg homeostasis. Hence, the aim of the project consists both in assessing thymic function upon the injection of different doses of T cells, and in determining how T cells subsets evolve. 

2. Methods

On the background of the C57BL/6 (H2-Kb) → BALB/C (H2-Kd) mismatched model of cGvHD, we developed a new three strains-based model (Figure 1) that allows to trace the different origin of T cells after transplantation by flow cytometry. BALB/C mice were lethally irradiated (8.5 Gy) and injected with 2.5・106 C57BL/6 bone marrow cells, different doses of C57BL/6 sorted Treg (0.3 and 1・105 CD45.1+/Luc+ Treg) and splenic αβ-TCR+ Tconv (0.3 and 0.4∙106 CD45.2+/FoxP3-DTR+, respectively). The CD45.2+ Treg fraction was depleted upon the I.P. administration of diphteria toxin (DT; 5 μg/Kg) to all the mice at day +1 and +3 post-transplant. The origin of T cells is determined by the differential expression of CD45 isoforms.

3. Results

The T cells subsets showed functional and quantitative variations among the cGvHD mice injected with different doses of lymphocytes within one and two months from the transplant and within all the assessed organs. Interestingly, also the origin of the different T cells subsets showed an organ-dependent variation according to the number injected cells.

4. Conclusions

Upon the injection of a greater dose of Treg, we noticed a substantial normalization in the T cells activation markers and a different contribution of T cells fractions to the disease, thus confirming their preponderant role in the disease. Unexpectedly, variations were present within the thymus as well, revealing a potentially relevant role of this organ in maintaining the Tconv and Treg pools in a cGVHD setting.