Marilen Benner

B CELLS CONTRIBUTE TO DECIDUAL IMMUNITY 91 4 24h CpG+CD40L(+PIB) Day 0 PIB only D F E B C FixViab SS Int CD3 CD19 FMO CD19 IL-10 CD19 8,4% PB 1st 2nd 0 2 4 6 8 10 %IL10+ of CD19+ PB 1st 2nd 0 5 10 15 20 25 * %IL10+ of CD19+ A CD19 CD3 B c e l l T c e l l IL-10 T r e g Healthy pregnancy CD3 CD19 Foxp3 DAPI Figure 4. B cells in decidua secrete IL-10 (A) Representative gating strategy for assessment of intracellular IL-10 in peripheral blood and decidual B cells. Live, CD19 + events were selected. For each IL-10 assessment, a fluorescent minus one (FMO) strategy (using a staining mix excluding IL- 10) was applied to cells subjected to the identical conditions for correct positioning of the gate selecting IL-10 positive cells. (B) 50.000 mononuclear cells were stimulated for 24h with 5µg/ml CpG and 1µg/ml CD40L, or (C) in absence of stimulation (peripheral blood, PB n= 7, 1st trim. n=15, 2nd trim. n=9). (D) Representative immunohistochemistry of 10 week decidua stained for CD19 (brown) and CD3 (red) of decidual lymphocyte clusters (n=8 in 3 biological replicates, 9wk-14.5wk). Original magnification x40, scale bar (black) indicating 200µm. (E) Representative 4-color multiplex staining of 10 week decidua. Original magnification x20, scale bar (white) indicating 50µm. (F) Schematic representation summarizing co-localization of B and T cells. Decidual B cells are able to secrete IL-10, with possible impact on regulatory T cell maintenance and thus healthy pregnancy. PIB, PMA+ionomycin+Brefeldin A. Treg, regulatory T cell. **p<0.01 Kruskal-Wallis and Dunn’s multiple comparison test for comparison of PB, 1 st trimester and 2 nd trimester decidua. Data are shown as mean±SEM.