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Study Question: Do distinct subpopulations of decidual stromal cells (DSCs) exist and if so, are given subpopulations enriched in recurrent miscarriage (RM)?Summary Answer: Three subpopulations of DSCs were identified from which inflammatory DSCs (iDSCs) and glycolytic DSCs (glyDSCs) are significantly enriched in RM, with implicated roles in driving decidual inflammation and immune dysregulation.What Is Known Already: DSCs play crucial roles in establishing and maintaining a successful pregnancy; dysfunction of DSCs has been considered as one of the key reasons for the development of RM.Study Design, Size, Duration: We collected 15 early decidual samples from five healthy donors (HDs) and ten RM patients to perform single-cell RNA sequencing (scRNA-seq). A total of 43 RM patients and 37 HDs were enrolled in the validation cohort.Participants/materials, Setting, Methods: Non-immune cells and immune cells of decidual tissues were sorted by flow cytometry to perform scRNA-seq. We used tissue microarrays (TMA) to validate three distinct subpopulations of DSCs. The expression of inflammatory and glycolytic proteins by DSCs was validated by immunohistochemistry (IHC) and multiplex immunohistochemistry (mIHC). Different subsets of decidual NK (dNK) cells and macrophages were also validated by multicolor flow cytometry and mIHC. Cell ligand-receptor and spatial analyses between DSCs and immune cells were analyzed by mIHC.Main Results and the Role Of Chance: We classify the DSCs into three subtypes based on scRNA-seq data: myofibroblastic (myDSCs), inflammatory (iDSCs) and glycolytic (glyDSCs), with the latter two being significantly enriched in RM patients. The distribution patterns of DSC subtypes in the RM and HD groups were validated by mIHC. Single-cell analyses indicate that the differentiation of iDSCs and glyDSCs may be coupled with the degrees of hypoxia. Consequently, we propose a pathological model in which a vicious circle is formed and fueled by hypoxic stress, uncontrolled inflammation and aberrant glycolysis. Furthermore, our results show that the inflammatory SPP1+ macrophages and CD18+ dNK cells are preferentially increased in the decidua of RM patients. Cell ligand-receptor and mIHC spatial analyses uncovered close interactions between pathogenic DSCs and inflammatory SPP1+ macrophages and CD18+ NK cells in RM patients.Large Scale Data: The raw single-cell sequence data reported in this paper were deposited at the National Omics Data Encyclopedia (www.biosino.org), under the accession number OEP002901.Limitations, Reasons For Caution: The number of decidual samples for scRNA-seq was limited and in-depth functional studies on DSCs are warranted in future studies.Wider Implications Of the Findings: Identification of three DSC subpopulations opens new avenues for further investigation of their roles in RM patients.Study Funding/competing Interest(s): This study was supported by the Strategic Priority Research Program (No. XDB29030302), Frontier Science Key Research Project (QYZDB-SSW-SMC036), Chinese Academy of Sciences; National Key Research and Development Program of China (2021YFE0200600), National Natural Science Foundation of China (No. 31770960), Shanghai Municipal Science and Technology Major Project (No. 2019SHZDZX02, HS2021SHZX001), and Shanghai Committee of Science and Technology (17411967800). All authors report no conflict of interest. [ABSTRACT FROM AUTHOR]

Intrahepatic cholangiocarcinoma (iCCA) is a highly heterogeneous cancer with limited understanding of its classification and tumor microenvironment. Here, by performing single-cell RNA sequencing on 144,878 cells from 14 pairs of iCCA tumors and non-tumor liver tissues, we find that S100P and SPP1 are two markers for iCCA perihilar large duct type (iCCAphl) and peripheral small duct type (iCCApps). S100P + SPP1− iCCAphl has significantly reduced levels of infiltrating CD4+ T cells, CD56+ NK cells, and increased CCL18+ macrophages and PD1+CD8+ T cells compared to S100P-SPP1 + iCCApps. The transcription factor CREB3L1 is identified to regulate the S100P expression and promote tumor cell invasion. S100P-SPP1 + iCCApps has significantly more SPP1+ macrophage infiltration, less aggressiveness and better survival than S100P + SPP1− iCCAphl. Moreover, S100P-SPP1 + iCCApps harbors tumor cells at different status of differentiation, such as ALB + hepatocyte differentiation and ID3+ stemness. Our study extends the understanding of the diversity of tumor cells in iCCA. The molecular classification and tumour microenvironment in intrahepatic cholangiocarcinoma (iCCA) need further characterisation. Here, the authors perform single cell RNA-sequencing from 14 pairs of iCCA tumours and non-tumour liver tissues and propose S100P and SPP1 as markers for patient classification. [ABSTRACT FROM AUTHOR]

Regulatory T cells (Tregs) are crucial for the maintenance of peripheral tolerance, but they also limit beneficial responses through cancer‐induced immunoediting. The roles of Treg subsets in cervical squamous cell carcinoma (CSCC) are currently unknown. Here, we aimed to perform an extensive study with an increased resolution of the Treg compartment in the peripheral blood and tumor tissues of CSCC patients. We first identified that an HLADRhi Treg population in the peripheral blood was significantly increased in CSCC patients compared to precancer patients and healthy donors. We found that HLADRhi Tregs express high levels of a panel of inhibition and activation markers and the TCR‐responsive transcription factors BATF and IRF4. However, this Treg subset showed reduced calcium influx after TCR crosslinking. In addition, HLADRhi Tregs are highly proliferative and vulnerable to apoptosis. Further studies demonstrated that the HLADRhi Tregs display high levels of suppressive activity. Quantitative multiplexed immunohistochemistry revealed that an increase in the number of tumor‐infiltrating HLADRhi Tregs is associated with unfavorable classical risk parameters of advanced disease stage and stromal invasion. Context‐based quantification revealed that a high frequency of stromal HLADRhi Tregs in patients is significantly associated with worse progression‐free survival. In the current study, we characterized a population of highly activated and immunosuppressive HLADRhi Tregs in CSCC patients. An increased HLADRhi Treg frequency may be a potential biomarker to stratify CSCC patients and evaluate therapeutic efficacies in personalized immuno‐oncology studies. What's new? While regulatory T cells (Tregs) are essential for immune homeostasis, they pose a major barrier to the generation of effective antitumor immunity. The latter effect may be attributed to functionally diverse Treg subsets, though how these different subsets impact antitumor immunity remains unknown. Here, the authors identified an expanded population of highly activated human leukocyte antigen‐DR (HLADRhi) Tregs with potent immunosuppressive activity in the blood and tumor tissues of cervical squamous cell carcinoma (CSCC) patients. Importantly, increased numbers of HLADRhi Tregs within tumors correlated with unfavorable outcomes. Thus, HLADRhi Tregs represent a potential biomarker and immune intervention target in CSCC. [ABSTRACT FROM AUTHOR]

Background: CD8+ T cells differentiate into exhausted status within tumors, including hepatocellular carcinoma (HCC), which constitutes a solid barrier to effective anti-tumor immunity. A detailed characterization of exhausted T cells and their prognostic value in HCC is lacking. Methods: We collected fresh tumor tissues with adjacent non-tumor liver tissues and blood specimens of 56 HCC patients, as well as archived samples from two independent cohorts of HCC patients (n = 358 and n = 254), who underwent surgical resection. Flow cytometry and multiplex immunostaining were used to characterize CD8+ T cells. Patient prognosis was evaluated by Kaplan-Meier analysis and Cox regression analysis. Results: CD8+ T cells were classified into three distinct subpopulations: PD1Hi, PD1Int and PD1−. PD1Hi CD8+ T cells were significantly enriched in tumor compared to adjacent non-tumor liver tissues. PD1Hi CD8+ T cells highly expressed exhaustion-related inhibitory receptors (TIM3, CTLA-4, etc.) and transcription factors (Eomes, BATF, etc.). In addition, PD1Hi CD8+ T cells expressed low levels of cytotoxic molecules and displayed a compromised capacity to produce pro-inflammatory cytokines while the expression of anti-inflammatory IL-10 was up-regulated following mitotic stimulation. Furthermore, PD1Hi CD8+ T cells shared features with tissue resident memory T cells and were also characterized in an aberrantly activated status with an apoptosis-prone potential. In two independent cohorts of HCC patients (n = 358 and n = 254), we demonstrated that PD1Hi or TIM3+PD1Hi CD8+ T cells were significantly correlated with poor prognosis, and the latter was positioned in close proximity to PD-L1+ tumor associated macrophages. Conclusion: The current study unveils the unique features of PD1Hi CD8+ exhausted T cells in HCC, and also suggests that exhausted T cells could act as a biomarker to select the most care-demanding patients for tailored therapies. [ABSTRACT FROM AUTHOR]

B lymphocytes are essential mediators of humoral immunity and play multiple roles in human cancer. To decode the functions of tumor-infiltrating B cells, we generated a B cell blueprint encompassing single-cell transcriptome, B cell-receptor repertoire, and chromatin accessibility data across 20 different cancer types (477 samples, 269 patients). B cells harbored extraordinary heterogeneity and comprised 15 subsets, which could be grouped into two independent developmental paths (extrafollicular versus germinal center). Tumor types grouped into the extrafollicular pathway were linked with worse clinical outcomes and resistance to immunotherapy. The dysfunctional extrafollicular program was associated with glutamine-derived metabolites through epigenetic-metabolic cross-talk, which promoted a T cell-driven immunosuppressive program. These data suggest an intratumor B cell balance between extrafollicular and germinal-center responses and suggest that humoral immunity could possibly be harnessed for B cell-targeting immunotherapy.

Regulatory T cells (Tregs) are crucial for the maintenance of peripheral tolerance, but they also limit beneficial responses through cancer-induced immunoediting. The roles of Treg subsets in cervical squamous cell carcinoma (CSCC) are currently unknown. Here, we aimed to perform an extensive study with an increased resolution of the Treg compartment in the peripheral blood and tumor tissues of CSCC patients. We first identified that an HLADR hi Treg population in the peripheral blood was significantly increased in CSCC patients compared to precancer patients and healthy donors. We found that HLADR hi Tregs express high levels of a panel of inhibition and activation markers and the TCR-responsive transcription factors BATF and IRF4. However, this Treg subset showed reduced calcium influx after TCR crosslinking. In addition, HLADR hi Tregs are highly proliferative and vulnerable to apoptosis. Further studies demonstrated that the HLADR hi Tregs display high levels of suppressive activity. Quantitative multiplexed immunohistochemistry revealed that an increase in the number of tumor-infiltrating HLADR hi Tregs is associated with unfavorable classical risk parameters of advanced disease stage and stromal invasion. Context-based quantification revealed that a high frequency of stromal HLADR hi Tregs in patients is significantly associated with worse progression-free survival. In the current study, we characterized a population of highly activated and immunosuppressive HLADR hi Tregs in CSCC patients. An increased HLADR hi Treg frequency may be a potential biomarker to stratify CSCC patients and evaluate therapeutic efficacies in personalized immuno-oncology studies., (© 2019 UICC.)

Biliary atresia (BA) is a destructive pediatric liver disease and CD4 + T cell activation is demonstrated to play an important role in BA. However, a comprehensive scenario regarding the involvement of CD4 + T cell subsets to the development of BA remains unclear. Here, we aim to explore the infiltration of CD4 + T cell subsets and their clinical significance in BA. In the present study, thirty BA liver samples were collected during surgery and were divided into good (BA1, n = 16) and poor prognosis (BA2, n = 14), with samples from choledochal cyst patients ( n = 8) as control. By using multiplex immunohistochemistry, we evaluated the infiltration level of CD4 + T cell subsets in the portal areas. RT-qPCR and flow cytometry were further applied to explore detailed features of Treg subsets. We revealed that hepatic infiltrating Th1, Th2, Th17, and ICOS + Treg cells were significantly increased in BA patients compared to controls and were negatively associated with prognosis, while high infiltrating ICOS - Tregs showed a favorable outcome. Phenotypic analysis indicated that, in contrast to ICOS + Tregs, ICOS - Tregs were mainly CD45RA hi CD45RO low , and preferentially expressed more CD73. Besides, RT-qPCR revealed elevated expression of CD25, CD73, TGF- β, and BCL-2 genes in ICOS - Tregs. Finally, functional assay confirmed that ICOS - Tregs had a higher suppressive capacity to cytokine secretion and were more resistant to apoptosis in vitro . Collectively, we demonstrate that a mixed immune response is involved in BA pathogenesis, and the globally enhanced effector CD4 + T cell response is associated with unfavorable prognosis, highly suppressive ICOS - Tregs is a protective factor and may serve an important reference to predict prognosis.