Marilen Benner

CHAPTER 3 54 controversial. The nTregs are generated in the thymus, are specific for self-antigens, and are responsible for preventing anti-self (autoimmune) responses (25, 26). In contrast, induced Tregs (iTregs) are generated in the periphery and can be specific for a large variety of antigens, including allo-antigens and viral-antigens (27-29). A well-characterized type of iTregs are Tr1 cells that secrete high levels of IL-10; express PD1; co-express CD49b and LAG3, but do not express FOXP3; and are important in the control of alloimmune responses (30, 31). Other iTregs include TIGIT + cells that modulate antigen-presenting cells (APC) through interaction with CD155 on APCs and Tr35 cells that function through secretion of IL-35, an immune suppressive cytokine (32, 33). A large variety of other markers have been used to identify distinct iTreg populations (including but not limited to FOXP3, CD25, GITR, TIM3, CD39, LRRC32 (also known as GARP), LAP, and CCR8) (34-37). None of these markers are truly specific for iTregs, as they can also be expressed on activated T cells. Thus, to identify iTregs, functional assays are required to demonstrate their capacity to suppress immune responses such as proliferation, cytokine secretion, and cytotoxicity (29, 38). In this study, we provide extensive phenotypic and functional characterization of three types of decidual CD4 + Tregs in uncomplicated human pregnancies and investigate the ability of HLA-G + HLA-C + EVTs and decidual macrophages, the main APCs at the maternal-fetal interface, to increase Treg proportions. RESULTS Distinct CD4+ T cell types with a regulatory phenotype are present in decidual tissue FACS analysis on freshly isolated peripheral blood CD4 + T cells (CD4 + pTs) and decidual CD4 + T cells (CD4 + dTs) isolated from first-trimester decidua (gestational age 6–12 weeks) and term placenta decidua basalis (d.basalis) and decidua parietalis (d.parietalis) (gestational age > 37 weeks) was performed to determine cell surface expression of CD45, CD4, CD25, PD1, TIGIT, CD127, CD45RA, CD49b, and LAG3 and intracellular expression of FOXP3 and HELIOS. A clear population of activated nTregs was identified in all tissues based on the high expression of CD25, FOXP3, and HELIOS and the lack of CD45RA and CD127 (Figure 1; Figures S1A-S1D) (25). While the percentage of FOXP3 + and HELIOS + cells within this CD25 HI population significantly decreased in term pregnancy decidua, the proportion of HELIOS + cells within CD25 HI FOXP3 + cells remained relatively stable (Figure 1D, right panel). A second T cell population was identified based on the high expression of PD1, the lack of FOXP3 and HELIOS, and low CD25. LAG3 and CD49b were not expressed by CD4 + dTs. While both CD25 HI and PD1 HI cells co-expressed high levels of TIGIT, a third population of CD4 + dTs also expressed high levels of TIGIT and low levels of FOXP3, HELIOS, PD1, and CD25 (Figure 1; Figures S1A S1D). FOXP3 expression in TIGIT + cells was significantly lower than in CD25 HI cells (Figure S1E). The t-Distributed Stochastic Neighbor Embedding (t-SNE) analysis confirmed the separation of these three T cell populations (Figure S2) that hereafter will be named (1) CD25 HI FOXP3 + , (2) PD1 HI , and (3) TIGIT + .