Amniotic Membrane Mesenchymal Cells-Derived Factors Skew T Cell Polarization Toward Treg and Downregulate Th1 and Th17 Cells Subsets

We previously demonstrated that cells derived from the mesenchymal layer of the human amniotic membrane (hAMSC) and their conditioned medium (CM-hAMSC) modulate lymphocyte proliferation in a dose-dependent manner. In order to understand the mechanisms involved in immune regulation exerted by hAMSC, we analyzed the effects of CM-hAMSC on T-cell polarization towards Th1, Th2, Th17, and T-regulatory (Treg) subsets. We show that CM-hAMSC equally suppresses the proliferation of both CD4+ T-helper (Th) and CD8+ cytotoxic T-lymphocytes. Moreover, we prove that the CM-hAMSC inhibitory ability affects both central (CD45RO+CD62L+) and effector memory (CD45RO+CD62L−) subsets. We evaluated the phenotype of CD4+ cells in the MLR setting and showed that CM-hAMSC significantly reduced the expression of markers associated to the Th1 (T-bet+CD119+) and Th17 (RORγt+CD161+) populations, while having no effect on the Th2 population (GATA3+CD193+/GATA3+CD294+cells). T-cell subset modulation was substantiated through the analysis of cytokine release for 6 days during co-culture with alloreactive T-cells, whereby we observed a decrease in specific subset-related cytokines, such as a decrease in pro-inflammatory, Th1-related (TNFα, IFNγ, IL-1β), Th2 (IL-5, IL-6), Th9 (IL-9), and Th17 (IL-17A, IL-22). Furthermore, CM-hAMSC significantly induced the Treg compartment, as shown by an induction of proliferating CD4+FoxP3+ cells, and an increase of CD25+FoxP3+ and CD39+FoxP3+ Treg in the CD4+ population. Induction of Treg cells was corroborated by the increased secretion of TGF-β. Taken together, these data strengthen the findings regarding the immunomodulatory properties of CM-hAMSC derived from human amniotic membrane MSC, and in particular provide insights into their effect on regulation of T cell polarization.