Marilen Benner

CHAPTER 4 90 PB 1st 2nd Term 0.0 0.1 0.2 0.3 0.4 0.5 CD38 0.33 1.58 3.16 4.73 6.30 Cluster I Cluster II Cluster III Cluster IV Cluster V Cluster VI I II III V VI IV A B C D Overall marker expression Marker expression within cluster IgD IgM CD20 CD24 CD27 CD38 I II III IV V VI ^^^ ^^^ ^^^ ^^^ ^^^ ^^^ ^^^ ^^^ ^^^ ^ ^ ^^^ ^^^ ^^^ ^^ PB 1st 2nd Term 0.0 0.1 0.2 0.3 0.4 0.5 ** * PB 1st 2nd Term 0.0 0.1 0.2 0.3 0.4 0.5 Frequency of total B cells Frequency of total B cells Frequency of total B cells Frequency of total B cells Frequency of total B cells Frequency of total B cells PB 1st 2nd Term 0.00 0.05 0.10 0.15 0.20 PB 1st 2nd Term 0.00 0.05 0.10 0.15 0.20 PB 1st 2nd Term 0.00 0.05 0.10 0.15 0.20 Figure 3. Unsupervised clustering of B cell subsets in decidua (A and B) Decidual B cells (n=33) were stratified by automatic clustering based on IgD, IgM, CD20, CD24, CD27 and CD38 expression using the CITRUS tool by Cytobank™ and compared to peripheral blood (PB, n= 25). 246 B cells as gated in Figure S3A for each sample were used as input. A minimum cluster size of 2% was handled. Clusters of significantly higher (I-IV, A) or lower (V and VI, B) expression in decidua compared to peripheral blood are shown and abundance was separated into the individual gestational time points. Abundance depicts number of cells within a cluster divided by the total number of cells per sample. Samples of <200 B cells were excluded (1 st trimester n=16, 2 nd trimester n=13, and term n=4). Data are represented as mean±SEM. (C) Clustering tree depicting relation of identified clusters. The color scale illustrates relative marker expression per cluster while size of each node represents event frequency. As an example, CD38 expression is shown (remaining markers Figure S3B). (D) Marker expression of each subset in relation to overall expression. ^p<0.05, ^^p<0.01, ^^^p<0.001 Kruskal-Wallis and Dunn’s multiple comparison test; *p<0.05, **p<0.01 to compare 1 st trimester, 2 nd trimester and term decidua. Data are represented as mean±SEM.