Marilen Benner

CHAPTER 4 86 C D25 C D4+ HLA-DR+ dim C D 2 5 + C D 8 + B c e l l s C D 1 4 + N K Hi bright N K C D 3 + T cells NK cells G H PB MMC 1st 2nd Term 0 20 40 60 80 100 %CD3+CD56- of CD45+ PB MMC 1st 2nd Term 0 20 40 60 80 100 %CD56+CD3- of CD45+ E B ^^ ^^^ ^^^ *** *** *** ** *** *** ** ** ^^ ^^^ ^^^ Regulatory T cells ^^ ** ** PB MMC 1st 2nd Term 0 5 10 15 %Foxp3+Helios+ CD25+CD127low of CD4+ F A PB MMC 1st 2nd Term 0 1 2 3 4 5 5 10 15 %CD3-CD19+HLA-DR+ of lymphocytes 1st trim MMC PB 2nd trim Term CD19 HLA-DR CD3- CD3- CD3- CD3- CD3- C D ^^^ ^^^ ^^^ *** *** ** B cells A 12ml 6ml MMC 1st 2nd Term 1) Selection tissue of interest 2) Isolation mucosal lymphocytes 3) Supervised / Unsupervised analysis Immune environment Decidual B cells A B C F D E Term 2nd 1st MMC Cluster frequency relative to total events per sample Cluster A 0 .001 0.010 0.100 1.000 Cluster B Cluster C Term 2nd 1st MMC Cluster D Cluster E Cluster F 0 .001 0.010 0.100 1.000 0 .001 0.010 0.100 1.000 0 .001 0.010 0.100 1.000 0 .001 0.010 0.100 1.000 0 .001 0.010 0.100 1.000 CD56 0.12 1.39 2.97 4.55 6.14 Figure 1. The dynamic immune environment of mucosal tissue from menstrual blood to term decidua ( A ) Representation of samples chosen and workflow to study decidual lymphocytes of varying time points in absence of, and during, pregnancy. ( B - D ) Frequencies of NK cells ( B ), T cells ( C ), and regulatory T cells (D) of single cell suspensions. Gating strategies are shown in Figure S1A and B. Peripheral blood (PB) n=20, menstrual blood (MMC) n=20, 1 st trimester n=16, 2 nd trimester n=11, term pregnancy n=9). ( E and F ) CD19 + HLA-DR + B cells could be found in MMC, 1 st trimester, 2 nd trimester, and term decidua. ( G and H ) Stratification of menstrual blood-derived and lymphocytes based on hierarchical clustering using CD3, CD4, CD8, CD14, CD16, CD19, CD25, CD56 and HLA-DR to identify populations of similar marker expression and their abundance using the CITRUS tool by Cytobank™. 5000 events (gating strategy Figure S2A) were sampled per file and PAM predictive clustering was applied to identify clusters contributing to gestational-age dependent, stratifying signatures (model error rate is shown in Figure S2B). The color scale illustrates the relative marker expression per cluster while size of each node represents event frequency. As an example, CD56 expression is shown ( G ), super-clusters indicates frequency of each marker per cluster (Figure S2C+D). Relative frequency of clusters compared to total lymphocyte count identified as most stratifying between menstrual blood, 1 st , 2 nd trimester and term decidua samples, ordered from A to F decreasing in their contribution to stratification ( H ). Samples <5000 lymphocytes were excluded from analysis (MMC n=20, 1 st trimester n=40, 2 nd trimester n=16, term pregnancy n=9). ^ p<0.05, ^^ p<0.01, ^^^ p<0.001 Kruskal-Wallis and post-hoc Dunn’s multiple comparison test to assess difference between mucosa and peripheral blood; * p<0.05, ** p<0.01, *** p<0.001 Kruskal-Wallis; to compare menstrual blood mononuclear cells, 1st trimester, 2 nd trimester and term decidua. Data are represented as mean ± SEM.