Mechanisms of Regulatory T Cell Recruitment By Mesenchymal Stem Cells in Vitro

Regulatory T cells (Treg) play a critical role in the maintenance of immune tolerance. The scarcity of clinical trials using adoptive Treg therapy in humans has been largely due to the difficulty in obtaining sufficient Treg numbers under GMP conditions. Due to the paucity of Treg in the peripheral blood, several groups are investigating the possibility of expanding these cells in vitro .

We sought to investigate the potential of mesenchymal stem cells (MSC) to recruit Treg in vitro . Previous studies suggest that MSC-mediated immunomodulation may be partly driven by Treg induction and/or expansion. However, the mechanisms of Treg recruitment by MSC are so far poorly understood.

We performed co-cultures of human peripheral blood mononuclear cells (PBMC) from healthy donors with allogeneic bone marrow (BM)-derived MSC expanded in xeno-free conditions. Our data show an increase in the absolute counts and frequency within the T cell pool of CD4⁺ CD25^high Foxp3⁺ CD127^low Treg cells (4 and 6fold, respectively) after a 14day coculture, both compared to PBMC cultured alone and to Day 0. This increase appears to be specific for Treg, as the levels of conventional CD4 T cells (Tcon) and CD8 T cells are not increased in the presence of MSC. Co-culture with MSC does not induce Treg proliferation, as assessed by Ki67 expression. In terms of susceptibility to apoptosis, MSC increase Bcl-2 levels within Treg but not within Tcon, further suggesting a preferential impact on Treg.

The observation of increased Treg numbers after MSC co-culture in the absence of Treg proliferation suggests that MSC recruit Treg by inducing Tcon cells to acquire a Treg-like phenotype. In order to determine the mechanism of MSC-mediated Treg recruitment, we co-cultured either purified CFSE-labelled Treg or Tcon with MSC. In Treg:MSC co-cultures, cell numbers are maintained but Treg do not proliferate. In Tcon:MSC co-cultures, only the cells that acquire a Treg-like phenotype proliferate, whereas the cells that maintain a Tcon phenotype do not. These results indicate that the increase in Treg numbers after co-culture of PBMC with MSC is mainly due to the induction of Tcon-derived Treg-like cells, rather than to Treg expansion.

In order to determine if these Treg-like cells induced by MSC also resemble Treg in terms of functional ability, we co-cultured PBMC or purified Tcon with MSC and FACSorted the resulting Treg-like populations after 10 days. The suppressive ability of these purified cells, as well as of unsorted cells from the bulk co-cultures, was assessed in suppression assays using autologous fresh Tcon labelled with CFSE as responder cells. We further compared their suppressive ability to that of Treg co-cultured with MSC, as well as of fresh Treg. Our data suggest that bulk co-cultures of PBMC:MSC, Tcon:MSC and Treg:MSC significantly suppress Tcon proliferation (% divided cells at 1:1 Tcon:Treg ratio compared to Tcon alone, Mann Whitney test: P=0.0022, P=0.0043 and P=0.0303, respectively). As for the functional ability of Treg purified after MSC co-culture, both induced Treg-like cells isolated from Tcon:MSC co-cultures and natural Treg isolated from Treg:MSC co-cultures significantly suppress Tcon proliferation (Mann Whitney test: P=0.0095 for both).

We also assessed the expression of functional markers that are associated with Treg induction and/or expansion. After Tcon:MSC co-culture, Treg-like cells express higher levels of PD-1 and CD15s than the cells that maintain a Tcon phenotype, whereas the expression of GITR was similar within Treg-like and Tcon cells. Interestingly, the frequency of MSC expressing PD-1 ligand (PDL-1) increases after co-culture, both compared to Day 0 and to MSC cultured alone.

In order to investigate which cytokines may potentially be driving the induction of Treg-like cells by MSC, we measured cytokine levels in co-culture supernatants. The anti-inflammatory cytokines IL-10 and TGF-β are increased, whilst the pro-inflammatory cytokines IFN-γ and TNF-α are reduced in PBMS:MSC co-cultures when compared to PBMC cultured alone.

Overall, our data suggest that Treg recruitment by MSC is due to Tcon conversion into Treg-like cells, rather than Treg expansion. Our results demonstrate that MSC induce the generation of a Treg-like population, both in terms of the phenotypic hallmarks of Treg cells and of the functional ability to suppress autologous Tcon proliferation.