Characteristics Of Decidual Tregs And The Role Of CCR8~+ Treg Subset In Maintaining Maternal-fetal Immune Tolerance During Early Pregnancy

Part Ⅰ Landscape of decidual CD4+ T cells during early pregnancy revealed by single-cell sequencingBackground:The microenvironmental homeostasis at the maternal-fetal interface plays an important role in the maintenance of normal pregnancy.The maternal immune system undergoes a series of adaptive changes after conception to establish appropriate immune recognition,response and tolerance to paternal-derived allogeneic antigens of the fetus in order to maintain normal pregnancy and promote fetal growth and development.Once the maternal-fetal immune tolerance is broken,adverse pregnancy outcomes such as miscarriage,preterm delivery and stillbirth may occur.A large number of decidual immune cells,mainly NK cells(～70%),macrophages(～20%)and T cells(-10%),are enriched at the maternal-fetal interface and have unique phenotypes and functions to maintain the homeostasis of the decidual microenvironment.Currently,studies on decidual CD4+ T cells,especially the important immune regulatory subset Tregs,are limited.Additionally,there is a lack of data to reveal the expression profile of decidua-specific Tregs.Compared with peripheral blood,decidual Tregs are enriched throughout the gestation period,and may play vital roles in maintaining maternal-fetal immune tolerance.However,the origin,tissue-specific phenotype,heterogeneity and functional characteristics of decidual Tregs remain to be investigated.In the first part of this study,single-cell transcriptome and immune repertoire sequencing was performed through the 10x Genomics platform to investigate the characteristics of decidual CD4+T cells,with a focus on decidual Tregs during early pregnancy.Objective:To explore the landscape of decidual CD4+T cells by single-cell sequencing during early pregnancy and clarify the phenotypic characteristics of decidual-specific Tregs.Methods:Decidual tissues and matched peripheral blood samples from eight women with normal early pregnancy were collected.The CD4+T cells were enriched by FACS for subsequent 10x Genomics 5’-transcriptome and TCR V(D)J sequencing.Cell Ranger was used for data quality control and filtering,and Seurat for screening of highly variable genes for dimension reduction and clustering and analysis of differentially expressed genes.Flow cytometry was used for verification and investigation of phenotypic characteristics of CD4+T subsets at protein level.STARTRAC algorithm was applied for analysis of T cell clonal expansion.Results:(1)Landscape of decidual CD4+T cells during early pregnancyA total of 17 subsets of CD4+T cells from decidua and matched peripheral blood were identified,mainly including naive T cells,decidual quiescent T cells,effector T cells,Th1-like cells,Treg cells,cytotoxic T cells,NKT cells,and Tr1 cells,etc.The gene expression pattern of cells from decidua and peripheral blood differed significantly,and the RO/E values indicated that most subsets had obvious source preference.(2)Phenotypic characteristics of decidual-specific TregsAnalysis of differentially expressed genes showed that decidual Tregs significantly overexpressed 158 genes such as TNFRSF4,ICOS,TNFRSF18,TIGIT,CTLA4,etc.,and significantly down-regulated 25 genes including SELL,LEF1,TXNIP,TCF7,NOSIP,etc.compared to peripheral Tregs.The STARTRAC expansion index indicated active clonal expansion of decidual Tregs.The results of flow cytometry showed that decidual Tregs expressed significantly less naive marker(CD45RA and SELL),and more co-stimulatory molecules(TNFRSF4,ICOS and TNFRSF18),co-inhibitory molecules(TIGIT and PD1),transcription factors(IKZF4,TOX and BATF),and activation-related molecules(CD25 and CD39)compared to peripheral Tregs.About 91.7%of the core signature genes of decidual Tregs were found to overlap with characteristics of multiple tumor-infiltrating Tregs.(3)Verification of the characteristics of decidual non-Treg subsetsDecidual samples from women with normal early pregnancy were collected to verify the characteristics of Th cells,NKT cells and cytotoxic T cells.Decidual Th cells were dominated by T-bet+IFN-y+Thl type cells during early pregnancy(6 to 8 weeks).Phenotype of NKT cells was similar to decidual specific NK cell.EOMES+GZMK+ cytotoxic T cells expressed high levels of molecules related to T cell exhaustion including TIGIT,PD1 and TOX.Conclusion:Decidual CD4+T subsets exhibited pregnancy-associated phenotypic characteristics during early pregnancy.Decidual Tregs showed a specific feature with enhanced suppressive phenotype similar to tumor-infiltrating Tregs,indicating the potential role in maintaining maternal-fetal immune tolerance.Part Ⅱ Identification and phenotypic characterization of a decidual CCR8+Treg subsetBackground:In the first part of the study,we analyzed the feature of decidual-specific Tregs,indicating their potential importance in maternal-fetal immune tolerance.Tregs are highly heterogeneous and the subsets with different phenotype could have distinct functions on the physiology of various tissues and organs.However,due to the limited quantity of decidual Tregs,there is still a lack of investigations on heterogeneous subsets of decidual Tregs.Based on the previous results,two subsets of decidual Tregs existed with different gene expression profile,and further exploration for the specific markers to identify the decidual Treg subsets is essential.Therefore,in this part of the study,we investigated the distinct markers and functional phenotypes of decidual Treg subsets and analyzed their origin and differentiation pathways,which contributed to the in-depth study of the decidual microenvironment and maternal-fetal immune tolerance.Objective:To investigate the specific markers and features of decidual Treg subsets and analyze their origin and differentiation pathways.Methods:Decidua and peripheral blood samples from women with normal early pregnancy were collected to screen the markers of decidual Treg subsets by flow cytometry.Various functional molecules were detected as well.STARTRAC algorithm was used to analyze the expansion,migration and transition by TCR V(D)J data,and RNA velocity was used to predict the origin and differentiation pathway of decidual Treg subsets.Results:(1)Identification of the new decidual CCR8+Treg subsetThe analysis of differentially expressed genes revealed that a subset of decidual Tregs specifically overexpressed the CCR8 gene and a series of molecules related to the activation and suppressive function of Tregs including TNFRSF4、TNFRSF9、TNFRSF18、TIGIT and CTLA4.It was further verified by flow cytometry that CCR8 could be used as a distinct marker to identify decidual Treg subset,and the CCR8+Treg subset was specifically enriched in decidua tissue during early pregnancy.Compared with CCR8-Tregs and Tconvs,decidual CCR8+Tregs expressed significantly less naive marker SELL,and more co-stimulatory molecules(TNFRSF4,ICOS and TNFRSF18),co-inhibitory molecules(TIGIT,PD1 and CTLA4),transcription factors(IKZF4 and TOX),activation-related molecules(CD25 and CD39),and chemokine receptors(CCR4 and CXCR6).(2)Analysis of the origin and differentiation pathways of decidual CCR8+TregsThe STARTRAC migration index showed that decidual CCR8+Tregs were tissue-resident.The transition index showed that CCR8+Tregs were actively transited with CCR8-Tregs and had certain relationship with peripheral Tregs and decidual non-Treg cells.It was predicted by RNA velocity that decidual CCR8+Treg might originate from peripheral recruitment or be induced by decidual resting T cells.Conclusion:Decidual CCR8+Treg subset was newly identified and it might represent a "driver"subset of decidual Tregs with enhanced immunosuppressive function.The decidual CCR8+Tregs might be recruited from periphery or induced by decidual resting T cells.Part Ⅲ The role of decidual CCR8+Treg subset in maintaining maternal-fetal immune toleranceBackground:CCR8 molecules are specifically enriched in tumor-infiltrating Tregs.The CCR8+Treg subset is considered as the "dominant" Treg subpopulation in tumor tissues.The enhanced immunosuppressive function of tumor-infiltrating CCR8+Tregs is involved in mediating immune escape of tumor cells,and targeting CCR8 could significantly inhibit tumor growth in mice models.Currently,there are no studies on decidual CCR8+Tregs,and its role in the maintenance of maternal-fetal immune tolerance remains unclear.In the second part of the study,we identified the decidual CCR8+Treg subset with enhanced immunosuppressive phenotype.To further investigate whether decidual CCR8+Tregs participate in maintaining maternal-fetal immune tolerance,we included patients with recurrent pregnancy loss(RPL)and established related animal models to clarify the underlying mechanisms.Objective:To clarify the role of decidual CCR8+Tregs in maintaining maternal-fetal immune tolerance.Methods:The proportion of decidual CCR8+Tregs was detected between RPL patients and women with normal pregnancy by flow cytometry.The CBA/J abortion-prone and normal pregnancy mice models were constructed.The decidual CCR8+Tregs were detected as well.Anti-CCR8 antibody was injected to normal pregnant mice intra-peritoneally.The rate of embryo resorption,the number and weight of live fetuses were observed.Moreover,the dynamic changes of cytotoxic NK cells in decidual microenvironment were examined as well.Results:(1)Deficiency of decidual CCR8+Tregs in RPL patients and abortion-prone pregnant miceThe proportion of decidual CCR8+Tregs was significantly lower in RPL patients compared to healthy controls.There was no significant difference of the expression of functional molecules in decidual CCR8+Tregs between RPL patients and healthy controls.Consistently,mice in abortion-prone group had significantly lower decidual CCR8+Tregs compared to normal pregnancy group,with no significant differences in the functional molecular expression.(2)Increasing abortion rate and alteration of decidual microenvironment of normal pregnant mice caused by anti-CCR8 treatmentIn the anti-CCR8 treatment group,decidual CCR8+Tregs were specifically reduced.Meanwhile,the embryo resorption rate was significantly higher and the number of live fetuses was significantly lower in the anti-CCR8 treated mice.The detection of other lymphocytes revealed a significant increase of decidual cytotoxic NK cells and CD8+T cells in the anti-CCR8 treatment group.Conclusion:Decidual CCR8+Tregs participate in maintaining maternal-fetal immune tolerance.The deficiency of decidual CCR8+Tregs is associated with the occurrence of RPL.Decreased decidual CCR8+Tregs may disrupt the maternal-fetal immune tolerance by increasing cytotoxic immune cells and cause pregnancy failure.