Marilen Benner

CHAPTER 4 88 cluster representing CD45 + CD3 - CD19 + HLA-DR + B cells was present in both MMC and decidual tissue of all gestational ages (Figure 1G). Pregnancy is accompanied by a gain in decidual B cell memory Having established that B cells are present in decidua throughout pregnancy, we investigated their phenotype in more detail. Little is known on their phenotype within the uterine mucosa. Supervised analysis revealed that similar to peripheral blood, MMC derived B cells were mainly naive (IgD + CD27 - , Figure 2A). Their frequency was lower upon initiation of pregnancy, and further dropped towards the 2 nd trimester (Figure 2B). Switched memory B cells (IgD - CD27 + ) increased from MMC throughout gestation (Figure 2C). Abundance of non-switched memory B cells (IgD + CD27 + ) was comparable to peripheral blood and remained steady during gestation (Figure 2D). The frequency of plasmablasts (CD27 + CD38 +/hi IgD - ) was low in MMC and 1 st trimester decidua compared to peripheral blood (Figure 2E). Plasmablast abundance increased from MMC to 2 nd trimester and term decidua. The overall frequency of B cells positive for CD27, a memory marker, increased upon pregnancy (Figure 2F) (21, 22). PB MMC 1st 2nd Term 0 20 40 60 80 100 %CD27+IgD- of CD19+ A B E C D CD27 IgD CD3-CD19+ Naive 17% 3% 46% Switched mem Non switched mem Naive B cells Switched memory B cells ** ** ** * ^^^ ^ 1st PB MMC 1st 2nd Term 0 20 40 60 80 100 %CD27-IgD+ of CD19+ PB MMC 1st 2nd Term 0 20 40 60 80 100 %CD27+ of CD19+ CD27+ B cells CD3-CD19+ 39% 61% CD20 CD27 ** ** PB MMC 1st 2nd Term 0 20 40 60 80 100 %CD27+IgD+ of CD19+ Non-switched memory B cells 1st 1st F CD27 CD38 Switched memory 4% Plasma blast 12% Memory Plasmablasts *** * **c ^^^ ^^^ PB MMC 1st 2nd Term 0 10 20 30 40 50 %CD27+CD38+IgD- of CD19+ Figure 2. Differential expression of B cell subsets in samples of decidua (A - D) Gating strategy (A) for characterization of naive (B) and switched memory (C) and non-switched memory B cells (D) based on IgD and CD27 expression (within B cells, gating strategy Figure S3A) in peripheral blood (PB, n=25), menstrual blood (MMC, n=11), 1st (n=39) and 2nd (n=17) trimester as well as term decidua (n=8). (E) CD27 + IgD - cells were further classified as plasmablasts in case of additional CD38 staining. (F) CD27 positivity of CD19 + B cells. ^p<0.05, ^^p<0.01, ^^^p<0.001 Kruskal-Wallis and Dunn’s multiple comparison test to assess difference between isolated mucosal tissues and peripheral blood; *p<0.05, **p<0.01, ***p<0.001 to compare MMC, 1 st trimester, 2 nd trimester and term decidua. Data are shown as mean±SEM.