Percentage of major immune cells types in blood and PF samples and expression of functional markers. up study. Table S6. Related to Fig.?6 and Experimental Procedures. Antibodies used in the T cell panel. Table S7. Related to Fig.?6. Information of 11 patient samples used in T cell panel study. Physique S1. Graphic workflow of CyTOF study comparing PFCs and PBCs. Physique S2 and Physique S3. Related to Fig.?1, Fig.?2 and Fig.?6. Manual gating of cells subsets and functional markers. Physique S4. Related to Fig.?1. Clustering of PF and blood samples by PCA. Physique S5. Related to Fig.?1. Phenotypic mapping of PBCs. Physique S6. Related to Fig.?2. Percentage of major immune cells types in blood and PF samples and expression of functional markers. Physique S7. Related to Fig.?2. Cell counts show changes of major cell populations in PF compared to peripheral blood. Physique S8. Related to Fig.?3. Differential expression of CD69 in Oxypurinol endometriosis was not affected by menstruation or hormone. Physique S9. Related to Fig.?4. Cell counts of major cell subtypes in PFCs at disease stages and evaluation of confounding effects from menstrual cycle and hormones. Physique Oxypurinol S10. Related to Fig.?4. A. PCA separates endometriosis (Endo) and Oxypurinol control in PF but not blood samples. Physique S11. Related to Fig.?6. ViSNE plot showing composition of T cells and comparison of CD69 abundance on T cell lineages between control and endometriosis samples from PF. 12916_2019_1470_MOESM1_ESM.pdf (1.5M) GUID:?D6346007-2A94-41D5-81E2-BC6662052F1F Additional file 2. Related to Fig.?1. Patient-by-patient minimum spanning tree plots showing cell clustering of PF and blood samples. 12916_2019_1470_MOESM2_ESM.pdf (670K) GUID:?06EF2F39-63E4-450C-9331-7DD2C5B6C2E1 Data Availability StatementData supporting the findings of this study are available in supplementary information. Original mass cytometry data are available Oxypurinol from the corresponding author upon affordable request. Abstract Background Endometriosis is usually a gynaecological condition characterised by immune cell infiltration and distinct inflammatory signatures found in the peritoneal cavity. In this study, we aim to characterise the immune microenvironment in samples isolated from the peritoneal cavity in patients with endometriosis. Methods We applied mass cytometry (CyTOF), a recently developed multiparameter single-cell technique, in order to characterise and quantify the immune cells found in peritoneal fluid and peripheral blood from endometriosis and control patients. Results Our results demonstrate the presence of more than 40 different distinct immune cell types within the peritoneal cavity. This suggests that there is a complex and highly heterogeneous inflammatory microenvironment underpinning the pathology of endometriosis. Stratification by clinical disease stages reveals a dynamic spectrum of cell signatures suggesting that adaptations in the inflammatory system occur due to the severity of the disease. Notably, among the inflammatory microenvironment in peritoneal fluid (PF), the presence of CD69+ T cell subsets is usually increased in endometriosis when compared to control patient samples. On these CD69+ cells, the expression of markers associated with T cell function are reduced in PF samples compared to blood. Comparisons between CD69+ and CD69? populations reveal distinct phenotypes across peritoneal T cell lineages. Taken together, our results suggest that both the innate and the adaptive immune system play functions in endometriosis. Conclusions This study provides a systematic characterisation of the specific immune environment in the peritoneal cavity and identifies cell immune signatures associated with endometriosis. Overall, our results provide novel insights into the specific cell phenotypes governing inflammation in patients with endometriosis. This prospective study offers a useful resource for understanding disease pathology and opportunities for identifying therapeutic targets. (CyTOF), is usually a recently developed technique that enables multiparametric single-cell analysis. Using stable metal isotopes as reporters, this approach overcomes many limitations of traditional flow cytometry and currently detects up to 40 parameters in a single sample [28], making it particularly powerful in studies with patient samples [29, 30]. The goal of this study was to identify clinically relevant immune cell subtypes implicated in endometriosis. Using a panel of antibodies KITH_VZV7 antibody to label major haematopoietic cell types, we present a single-cell investigation in which we characterise the peritoneal immune cell composition in patients with and without endometriosis. The study offers a systematic view of immune cell signatures found in the peritoneal cavity and Oxypurinol reveals CD69+ T cell populations that are associated with endometriosis. Methods Sample collection Matched peritoneal fluid and peripheral blood samples from consented endometriosis patients and non-endometriosis controls were collected as part of the ENDOX study from patients undergoing laparoscopic surgery at the Womens Centre, John Radcliffe Hospital, Oxford, UK (REC reference 09/H0604/58). Venous blood samples were drawn from patients in the morning on the day of surgery. Peritoneal fluid was collected during laparoscopic surgery before any surgical procedure was performed to avoid.