Marilen Benner

THREE TYPES OF TREG CONTROL PLACENTAL INFLAMMATION 63 3 (A) CD4+ T cells were cultured alone or in the presence of EVTs or decidual macrophages for 3 days. Cells were analyzed for the percentage of FOXP3+, HELIOS+, PD1HI, and TIGIT+ cells as described in Figures S1A and S1B. (B) CD4+ T cells were cultured alone, with EVTs, or with EVTs separated by a transwell membrane for 3 days and analyzed for the percentage of FOXP3+, HELIOS+, and PD1HI cells. (C) CD4+ T cells were cultured alone, with decidual macrophages, or with decidual macrophages separated by a transwell membrane for 3 days and analyzed for the percentage of FOXP3+ cells. (D–F) CD4+ T cells were cultured alone or with EVTs or decidual macrophages in the presence of IgG, or blocking antibodies for CD3, HLA-C, ILT2, HLA-G, PDL1, TGFb, and IL-10R and analyzed for the percentage of FOXP3+ (D and E) and PD1HI (F) cells. (G) CD4+ T cells were cultured alone or with EVTs or HCMV-infected EVTs and analyzed for the percentage of FOXP3+ and PD1HI cells. Bars represent median and interquartile range; n = 8–14; *p < 0.05; **p < 0.01; ***p < 0.005. DISCUSSION Modulation of co-inhibitory molecules and Tregs function has exceptional therapeutic potential for treatment of a wide variety of inflammatory disorders, including cancer, chronic infection, autoimmune disease, and pregnancy complications. This study has refined our view of Treg populations at the maternal-fetal interface by presenting phenotypic and functional data of three Treg populations, CD25 HI FOXP3 + , PD1 HI FOXP3 - IL-10 + , and TIGIT + FOXP3 dim Tregs, found in decidual tissues of human first-trimester and term pregnancy. Functional suppression assays confirmed that decidual CD25 HI FOXP3 + Tregs suppress prolifer ation and production of IFN ɣ and TNF α by CD4 + and CD8 + Teffs. Decidual PD1 HI Tregs were shown to suppress proliferation of CD4 + (but not CD8 + ) Teffs in an IL-10-dependent manner. PD1 HI Tregs also increased expression of IL-10 in Teffs, possibly resulting in a positive feedback loop sustaining Teff suppression while inducing additional IL-10 secreting Tregs. Decidual TIGIT + cells significantly inhibited CD4 + T cell proliferation but did not influence CD8 + Teff proliferation or cytokine production, suggesting TIGIT + Tregs only have limited capacity to suppress T cells, but their ability to suppress other cell types (e. g. APCs) was not investigated here. Further analysis of gene and protein expression of all Treg types in blood and decidua clearly separated the three Treg types and revealed that many immune regulatory molecules (e. g. CTLA4, ST2, LRRC32, GITR, IFN ɣ , and IL-10) have increased expression in decidua compared to blood. Thus, decidual Tregs are highly activated and have the potential to influence immune responses through a variety of molecular pathways and cellular targets. Of importance here is further investigation into the contribution of decidual Treg types on the modulation of decidual CD8 + effector-memory T cells, which were shown to have signatures of T cell activation and dysfunction (41). Most interesting here is the discovery of the role of HLA-C in the induction of PD1 HI Tregs by EVT. EVTs, but not decidual macrophages, have the capacity to directly increase PD1 HI Tregs through cell-cell contact. Blocking of the TCR co-receptor CD3 or HLA-C in these EVT and CD4 T cell co- cultures significantly reduced this increase, suggesting that antigen-specificity may be involved.