Marilen Benner

B CELLS CONTRIBUTE TO DECIDUAL IMMUNITY 87 4 over time, using supervised flowcytometric analysis. For menstrual blood mononuclear cells (MMC), we observed that the NK cell abundance (CD45 + CD3 - CD56 + ) was relatively higher than in peripheral blood (Figure 1B), as previously shown (16, 17). NK cell levels were high in 1 st trimester decidua, stayed at that level in 2 nd trimester and dropped towards term (Figure 1B). Independent of the time point of gestation, the majority of the decidual NK cells were negative for CD16 and showed high expression of CD56 (NK bright , Figure S1C). Compared to peripheral blood, lower T cell (CD45 + CD3 + CD56 - ) frequencies were seen in MMC, decreasing further in 1 st and 2 nd trimester decidua (Figure 1C), and increasing again towards term. In MMC, the distribution of CD4 + and CD8 + cells within the total CD3 + T cell population was similar to that in peripheral blood (Figure S1D). After initiation of pregnancy, within CD3 + T cells, the frequency of CD8 + cells increased locally and remained at that level throughout pregnancy. CD4 + T cell frequency decreased in 1 st trimester decidua (Figure S1D). Overall abundance of CD4 + T cells within all CD3 + T cells remained relatively low over the course of pregnancy compared to MMC or peripheral blood. Previously, the percentage of naive CD4 + (CD45RA + CCR7 + ) T cells was shown to decrease over the course of pregnancy (Feyaerts et al., 2017). Here, we observed that this switch from naive towards a memory/effector phenotype already takes place at the earliest stage of pregnancy in 1 st trimester decidua (Figure S1E,F). A slight drop in CCR4 - CCR6 - CXCR3 + CD4 + T cells (Th1) was observed in 2 nd trimester samples (Figure S1G). CCR4 + CCR6 + CXCR3 - CD4 + (Th17) decreased from 1 st trimester to term (Figure S1G). A prominent role in the delicate balance of immunity vs tolerance at the fetal-maternal interface is ascribed to regulatory T cells (Treg (18). Compared to peripheral blood, (CD4 + Foxp3 + Helios + CD25 Hi CD127 low/- , Figure S1H) Treg levels in MMC were low. In pregnancy, levels rose gradually from 1 st trimester to 2 nd trimester, and remained at this level up to parturition (Figure 1D). While levels of NKT cells (CD45 + CD3 + CD56 + ) in MMC were similar to those in peripheral blood, their abundance in decidual tissue was highest at term (Fig S1I). Samples of all gestational ages contained a small CD3 - CD19 + HLA-DR + B cell population (Figure 1E, F). To further explore phenotypic features of the different cell populations, we employed an unsupervised approach, using the CITRUS (cluster identification, characterization, and regression) tool on the Cytobank™ platform. This hierarchical clustering method stratifies cell subtypes based on similar descriptive features (i.e. marker expression) that correlate with the assigned sample type, in this case, gestational age (19). The six most stratifying clusters to predict gestational age are shown in Figure 1G and 1H. Most predictive clusters were CD4 + T cells (Cluster A, CD45 + CD3 + CD4 + CD8 - ), followed by NK bright (Cluster B, CD45 + CD3 - CD56 + CD16 - ) cells, CD8 + T cells (Cluster E, CD45 + CD3 + CD4 - CD8 + ), and regulatory T cells (Cluster F, CD45 + CD4 + CD25 Hi HLA - DR + ). In line with supervised analysis, T cell clusters A, C, E, F decreased in frequency of overall lymphocytes from MMC to 1 st trimester decidua and increased towards term; NK cell clusters (B, D) showed the reverse pattern. Decidual NK cells expressing CD25 (Cluster D, CD45 + CD3 - CD56 + CD25 + CD16 - ), an NK cell subtype suspected to be upregulated due to trophoblast contact at the fetal-maternal interface (20) was detected. A