Marilen Benner

B CELLS CONTRIBUTE TO DECIDUAL IMMUNITY 89 4 Decidual B cells are distinct from peripheral blood B cells Recent studies emphasize the diversity in B cell subsets, including populations with regulatory capacities (Breg), especially when comparing systemic to tissue-specific immunity (23, 24). To gain more in-depth insight in B cell subsets in decidual tissues versus peripheral blood, we employed a CITRUS based multidimensional unsupervised clustering analysis. While considered a common B cell marker (25), we included CD20 in our clustering analysis for assessment of its possible differential expression. The decidual B cell population contained four subsets that were absent in peripheral blood (Cluster I-IV, Figure 3A; marker expression 3C,D); cluster I, comprising CD24 hi CD27 + CD38 - cells, and cluster II, comprising CD24 + CD27 + CD38 + cells (cells of both clusters were further characterized as IgD low IgM low CD20 + ), cluster III, comprising IgD - IgM low CD20 - CD24 hi CD27 - CD38 hi cells, and cluster IV, comprising IgD + IgM low CD20 + CD24 + CD27 - CD38 + cells (Figure 3A). Cluster III was not observed in term decidua. Two additional clusters were more abundant in peripheral blood: cluster V, resembling plasmablasts (IgD - IgM low CD20 - CD24 - CD27 hi CD38 hi cells) and cluster VI (IgD + IgM + CD20 + CD24 low CD27 low CD38 low cells) (Figure 3B, marker expression 3C,D). Decidual B cells have in vitro IL-10 producing capacity and are found in clusters Mouse studies revealed a role for regulatory B cells (Breg) in healthy pregnancy (26-28). For human B cells, regulatory properties have been ascribed to a number of phenotypically distinct B cell subsets, united in their ability to produce IL-10 (29). To examine the presence of possible Breg in the different decidual tissues, we performed in vitro stimulation of decidual and peripheral blood mononuclear cells (PBMC) in the absence or presence of CpG (TLR9 activation) and CD40L (mimicking T cell dependent triggering of CD40) (30, 31). Upon stimulation, higher frequencies of decidua derived IL-10 expressing B cells compared to peripheral blood derived ones were found (Figure 4 A,B). In the absence of additional CpG and CD40L stimulation, 1 st and 2 nd trimester decidua derived B cells already expressed IL-10 (Figure 4C). Information on decidual B cell localization is scarce. Immunohistochemical analysis on 1 st and 2 nd trimester decidua revealed that B cells were located closely together with T cells (Figure 4D). As several studies in humans and mice have pointed out the importance of IL-10 secreting B cells for regulatory T cell induction (32-35), we investigated whether these decidual lymphocyte clusters also harbored Treg. Additional staining of Foxp3, a key transcription factor of Treg, showed the presence of Foxp3 + CD3 + T cells in close proximity to the B cells (Figure 4E, S3C).