Fig. 1: CD4T cells in the human endometrium.

a Workflow of methods applied in this study, patients are recruited and single cell preparations of endometrium or PBMC are prepared for either (top panel) flow cytometric analysis of Treg and Tconv cells in human endometrium: FSC-A vs. SSC-A and FSC-H determine lymphocyte singlet cells, ZA^lo/CD45⁺/CD3⁺/CD4⁺ (total CD4T cells) are subpopulated CD25^hi/CD127^lo/FOXP3⁺ (Treg) or CD25⁻/FOXP3⁻ (Tconv), (bottom panel) basic description of method for RNA sequencing. b Flow cytometric analysis of endometrial tissue single cell preparations from proven fertile women (n = 15) showing the proportion of total CD4T cells in the T cell population in the endometrium, and (c) the proportion of Treg in the total CD4T cell population. d % CD45RO expression by endometrial (E) (n = 12) or peripheral blood (PB) (n = 3) Tconv or Treg; bars = median + IQR. e Principal Component Analysis (PCA) of RNASeq data from peripheral blood Treg (PB-Tr) and endometrial Treg (E-Treg) or endometrial Tconv (E-Tconv) from nulliparous (Nul) or parous controls (Par), primary RPL (PRPL) or secondary (SRPL). f Volcano plot depicting differential gene expression (DESeq2) between endometrial Tconv and Treg, top 25 differentially expressed genes are labelled, (g) heatmap of top 30 upregulated and downregulated genes, red = high, blue = low expression, arrows show genes chose for further analysis in (h) using flow cytometry showing mean fluorescence intensity of intracellular FOXP3 and HELIOS (n = 15), and cell surface CTLA4 and TIGIT (n = 15), CCR8 (n = 8), CD39 (n = 12) in Tconv (left/grey bars) and Treg (right/blue bars), dots are individual samples and bars = mean +/− S.E.M.