The phenotype of PF IL-17-producing T cells in endometriosis
Several cell surface markers have been applied to distinguish T cell subsets [12, 13]. To characterize the phenotype of PF Th17 cells, we first detected the expression of these markers on IL-17-producing T cells in PF of endometriosis patients. As shown in Figure 1A , a substantial PF CD3+ IL-17-producing T cell population was observed. Further detection revealed that these cells were CD4+CXCR3- (Figure 1B) . Based on the expression of CCR6 and CCR4, these cells can be divided into CCR6+CCR4+ and CCR6+CCR4- subpopulations, respectively (Figure 1B & 1C) . Based on the expression of IL-17RE and CD27, these cells can be divided into IL-17RE+CD27- subset, IL-17RE+CD27+ subset, IL-17RE-CD27+ subset (very few or almost absent in some samples), and IL-17RE-CD27- subset (Figure 1B & 1D) .
Because IL-17-producing T cells might involve both Th17 and Th1/17 cells, we needed to accurately discriminate these two populations to isolate live Th17 cells for further investigations. To this end, we analyzed PF CD3+CD4+ T cells(Figure 2A) for the expression of CCR6, CCR4, CXCR3, and CCR10. As shown in Figure 2B , within PF CD3+CD4+ T cells, four subpopulations were seen based on the expression of CCR4 and CXCR3: CCR4+CXCR3- cells, CCR4+CXCR3+ cells, CCR4-CXCR3+ cells, and CCR4-CXCR3- cells. We then sorted the four subpopulations and measured the expression of T cell subset master regulators, i.e. T-bet (Gene name TBX21), GATA3, and RORγt. As shown in Figure 2C , T-bet was highly expressed in CCR4-CXCR3+ cells and GATA3 was highly expressed in CCR4+CXCR3-cells, whereas RORγt was also significantly CCR4+CXCR3- cells. This suggested that CCR4+CXCR3- cells contained both Th2 and Th17 cells. Interestingly, CCR4-CXCR3+ T cells, which expressed high T-bet, also expressed moderate levels of RORγt, suggesting that this subpopulation harbored Th1 and Th1/17 cells.
We then further dissected CCR4+CXCR3- T cells based on the expression of CCR6 and CCR10. As shown in Figure 2D , four subpopulations were observed: CCR6+CCR10- cells, CCR6+CCR10+ cells, CCR6-CCR10+ cells, and CCR6-CCR10- cells. We then sorted these cells to check the expression of T-bet, GATA3, and RORγt. T-bet expression was ubiquitously low in all four subpopulations, while GATA3 was highly expressed in the CCR6-CCR10- subpopulation(Figure 2E) . RORγt was robustly expressed in the CCR6+CCR10- subpopulation but low in the other three subpopulations (Figure 2E) . Therefore, Th17 cells were CCR6+CCR4+CXCR3-CCR10-.
To precisely study live PF Th17 cells, we needed to introduce more fluorophore-conjugated antibodies to label Th17 cells. However, it is difficult for conventional flow cytometers without ultraviolet lasers to recognize more than 6 fluorophores. Therefore, we came up with a new method to distinguish live Th17 cells. PE-conjugated anti-CD14 antibody, anti-CXCR3 antibody, and anti-CCR10 antibody were used together to exclude CD14+ macrophages, CXCR3+Th1 cells, CXCR3+ Th1/17 cells, and other non-Th2 and non-Th17 T cells (Figure 3A) . Within the CD14-CXCR3-CCR10-cells, CD4+ T cells were divided into four subpopulations according to the expression of CCR6 and CCR4: CCR6+CCR4- cells, CCR6+CCR4+ cells, CCR6-CCR4+ cells, and CCR6+CCR4+ cells (Figure 3A) . Evaluation of T cell subset master regulators indicated that RORγt was exclusively highly expressed in the CCR6+CCR4+ subpopulation while GATA3 was predominantly expressed in the CCR6-CCR4+ subpopulation(Figure 3B) . Consistently, analysis of IL-17A expression confirmed that the CCR6+CCR4+subpopulation was Th17 cells (Figure 3C & 3D) . Previous studies have reported the significance of IL-17RE and CD27 to Th17 activity [14, 15]. Therefore, we further dissected the CCR6+CCR4+ subpopulation based on the expression of IL-17RE and CD27. As shown in Figure 3E , the CCR6+CCR4+ subpopulation was divided into three subsets: IL-17RE+CD27- cells, IL-17RE+CD27+ cells, and IL-17RE-CD27- cells. The IL-17RE+CD27- subset and IL-17RE+CD27+ subset expressed higher IL-17A than the IL-17RE-CD27-subset (Figure 3F) . However, their RORγt expression was comparable (Figure 3G) . Therefore, IL-17RE and CD27 signify the heterogeneity of PF Th17 cells.
Assessment of the expression of other Th17-related cytokines indicated that in comparison with the IL-17RE-CD27- subset, the IL-17RE+CD27- subset and IL-17RE+CD27+ subset produced more GM-CSF (Figure 4A & 4B) as well as IL-22 mRNA (Figure 4C) . Furthermore, evaluation of cell cycle using Hoechst 33342 and Pyronin Y demonstrated that the IL-17RE+CD27- subset and IL-17RE+CD27+ subset had more cells in G1 phase and S-G2/M phase than the IL-17RE-CD27- subset, suggesting that the former two subsets were more proliferative than the latter(Figure 4D) . Therefore, IL-17RE+ Th17 cells were more pro-inflammatory than IL-17RE- Th17 cells. CD27 seemed not profoundly influencing Th17 activity because no significant difference was seen between the IL-17RE+CD27- subset and IL-17RE+CD27+ subset.
To deeply understand the molecular traits of PF IL-17RE+ Th17 cells and IL-17RE-Th17 cells, we sorted these cells to conduct the RNA-Seq analysis(Figure 5A) . Compared with IL-17RE- Th17 cells, a total of 5288 differentially expressed transcripts (DETs) were identified in IL-17RE+ Th17 cells, including 3866 up-regulated DETs and 1422 down-regulated DETs (Figure 5B & 5C) . The GO biological process (BP) enrichment results showed significant associations with respiratory electron transport chain, oxidative phosphorylation, and ATP synthesis coupled transport, etc.(Figure 5D) . GO cellular component (CC) analysis showed notable associations with respiratory chain, ribosome, and ribosomal subunit, etc. (Figure 5E) . GO molecular function (MF) analysis showed remarkable associations with structural molecular activity, structural constituent of ribosome, RNA binding, NADH dehydrogenase (ubiquinone) activity, and other metabolism-related activity (Figure 5F) . KEGG analysis also enriched DETs in certain KEGG terms such as ribosome, oxidative phosphorylation, olfactory transduction, and glycolysis/gluconeogenesis (Figure 5G) . Hence, the expression of genes for OXPHOS and ETC seemed to be remarkably increased in IL-17RE+ Th17 cells relative to IL-17RE- Th17 cells.
Analysis of metabolism-related DETs revealed that in comparison to PF IL-17RE- Th17 cells, PF IL-17RE+Th17 cells up-regulated the expression of genes involved in OXPHOS and ETC, including NDUFB5 ,MT-CO1 ,UQCRC1 , andNDUFS6 , etc. (Figure 6A and Table.1) . The up-regulation of these genes was validated by qRT-PCR (Figure 6B) . Since OXPHOS and ETC are crucial for the generation of ATP and ROS, we quantified ATP and ROS in the two Th17 subsets. As shown in Figure 6C , IL-17RE+ Th17 cells produced more ATP than IL-17RE- Th17 cells. H2DCFDA staining suggested more ROS generation in IL-17RE+ Th17 cells relative to IL-17RE- Th17 cells (Figure 6D) . Therefore, IL-17RE+ Th17 cells and IL-17RE-Th17 cells are metabolically different.