Marilen Benner

THREE TYPES OF TREG CONTROL PLACENTAL INFLAMMATION 59 3 low expression of cytokines by CD25 HI cells and high expression of IL-10 by PD1 HI cells (Figure S5C). Thus, besides suppressing Teff proliferation, both CD25 HI and PD1 HI Tregs also modulated the production of cytokines by Teffs. CD25 HI FOXP3+, PD1 HI , and TIGIT+ Tregs from decidua and blood express distinct transcriptional profiles To further investigate the molecular mechanisms the three Treg types may utilize to modulate T cell proliferation and cytokine production, RNA was isolated from CD25 HI FOXP3 + , PD1 HI , and TIGIT + Tregs purified from peripheral blood, first-trimester decidua (6–12 weeks), and term pregnancy d.parietalis (>37 weeks). The BioMark Fluidigm 96x96 QPCR chip was used to detect gene expression. Sixty-six primer pairs had detectable CT values and melting curves (gene and primer list are included in Table S1). CT values were normalized against GAPDH ( Δ CT), and the fold change (FC) was calculated relative to the median Δ CT of the blood CD4 + Teffs. Expression levels of CD25, FOXP3, PD1, and TIGIT confirmed the purity of the Treg fractions (Figure S6A). K-means cluster analysis of gene expression in the four T cell populations visualized two separate gene clusters in blood and decidua. Cluster I identified a set of genes, including CD25, FOXP3, TIGIT, CD39, LRRC32 (GARP), ST2, BATF, and CCR8, which are highly expressed by CD25 HI FOXP3 + , low in PD1 HI , and intermediate in TIGIT + Tregs (Figure 4A). Cluster II identified a set of genes, including PD1, IFN ɣ , IL-10, CCR5, and CXCR3, that are upregulated by PD1 HI Tregs (Figure 4B). Heatmaps depicting the differentially expressed genes identified in these clusters are shown (Figures 4C - 4E). These data further support that CD25 HI FOXP3 + , PD1 HI , and TIGIT + are separate Treg types that may utilize distinct molecular mechanisms of immune modulation. Additional K-means cluster analysis revealed several key regulatory genes that differ throughout gestation and between decidual and blood Tregs (Figures S6B and S6C). Decidual CD25 HI FOXP3 + Tregs show increased expression of CCR5, ST2, CD25, BATF, IL10, GITR, LRRC32 (GARP), and CCR8, compared to blood CD25 HI FOXP3 + Tregs, while decidual PD1 HI Tregs had increased expression of IL-10, IFN ɣ , and CCR5, compared to blood PD1 HI Tregs. Interestingly, first- trimester decidual TIGIT + Tregs increased expression of IL- 10, IFN ɣ , LRRC32, and GZMA, while term pregnancy TIGIT + Tregs had increased expression of, for example, VEGFA, IFN ɣ , GITR, and CD39 (Figures S6B and S6C). Flow cytometric analysis of freshly isolated Tregs from all tissues confirmed differential protein expression of key mRNAs identified here (Figure S7). The increased expression of a variety of cytokines (e.g., IL-10, IFN ɣ ), chemokines (e.g., CCR5, CCR8), activation markers (e.g., HLA-DR, GZMA), and co-inhibitory genes and molecules (e.g., CD39, TIM3, LRRC32) on decidual Tregs may suggest increased Treg activation and suppressive function in decidual tissue to regulate inflammatory responses at the maternal fetal interface.