Bone Marrow-Derived Mesenchymal Stromal Cells Promote Thymopoiesis and Function Recovery in Acute Graft-Versus-Host Disease Murine Models

Background: The thymus is the primary lymphoid organ of T-cells development and plays a crucial role for immune tolerance following allogeneic hematopoietic stem cell transplantation (allo-HSCT). The toxicity of chemoradiotherapy conditioning regimen and acute graft-versus-host disease (aGVHD) damage the thymic structure and function, which result in the production of autoreactive T-cells and the reduction of natural regulatory T cells (nTregs) to aggravate the development of aGVHD. Nowadays, mesenchymal stromal cells (MSCs) as multipotent stem cells, have been widely recognized as a promising therapeutic strategy for aGVHD. However, the well-known mechanisms of MSCs ameliorating aGVHD mainly focus on their immunoregulatory effect on peripheral immune compartments. The contribution of MSCs to thymus, the central immunological organs, is rarely reported.

Methods: In this study, we carried out studies in a murine aGVHD models of fully MHC-mismatched myeloablative bone marrow transplantation (C57BL/6 to BALB/c). We randomly divided aGVHD mice into two groups, including MSCs group and control group without MSCs treatment. In MSCs group, aGVHD mice were intravenously administrated GFP-labelled bone marrow (BM) derived MSCs at a dose of 5×10⁵ cells/mice at 7 day after allo-HSCT. The mice were killed at 1d, 2d, 4d and 20d after MSCs injection in MSCs group and the corresponding time in control group, respectively. Thymic tissues were collected and manufactured into frozen sections or thymic single-cell suspension. And then we analyzed the distribution of GFP-MSCs in thymus and thymic size, the expression of thymic T cells subsets (CD4⁺CD8^-, CD4^-CD8⁺, CD4⁺CD8⁺ T, CD4⁺CD25⁺Foxp3⁺Tregs), thymic epithelial cells (TECs) substes (CD45^-CD326⁺Ly51⁺ cortical TECs and CD45^-CD326⁺UEA-1⁺ medullary TECs) and the level of T cell receptor rearrangement excision circles (TRECs) in thymus between the two groups to evaluate the repair effect of MSCs for thymus.

Results: We found that MSCs treated mice have longer survival and alleviate the clinical signs of aGVHD compared to the control group (P<0.05). Next, we observed that GFP-MSCs can home to thymic tissue and mainly locate in the corticomedullary boundary at 2d, 4da after MSCs infusion. MSCs treated thymus exhibited a more organized corticomedullary architecture, compared with the conrol group. Significant increases in thymus size and weight, as well as the number of total thymocytes were seen in MSCs group. To further investigate whether MSCs promote thymopoiesis and ameliorate thymic functions after allo-HSCT, we found that in MSCs group the proportion of thymic T cells subets composition (CD4+CD8-, CD4-CD8+, CD4+CD8+), especially Foxp3+ Tregs, and the expression of TECs, especially cTECs, were significantly higher than control group at 20d after the MSCs infusion (P<0.001). Furthermore, MSCs administration resulted in a remarkable increase in the levels of TRECs in the thymocyte at 20d after the MSCs infusion compared with the control group (P=0.007).

Conclusions: Collectively, our data proposed a new mechanism for MSCs ameliorating aGVHD by central immune organ, including promoting thymopoiesis and enhancing thymic output.