参考文献

CD8 T cell-mediated autoimmune diseases result from the breakdown of self-tolerance mechanisms in autoreactive CD8 T cells1. How autoimmune T cell populations arise and are sustained,and the molecular programmes defining the autoimmune T cell state,are unknown. In type 1 diabetes,$\beta$-cell-specific CD8 T cells destroy insulin-producing $\beta$-cells. Here we followed the fate of $\beta$-cell-specific CD8 T cells in non-obese diabetic mice throughout the course of type 1 diabetes. We identified a stem-like autoimmune progenitor population in the pancreatic draining lymph node (pLN),which self-renews and gives rise to pLN autoimmune mediators. pLN autoimmune mediators migrate to the pancreas,where they differentiate further and destroy $\beta$-cells. Whereas transplantation of as few as 20 ?»¿autoimmune progenitors induced type 1 diabetes,as many as 100,000 pancreatic autoimmune mediators did not. Pancreatic autoimmune mediators are short-lived,and stem-like ?»¿autoimmune progenitors must continuously seed the pancreas to sustain $\beta$-cell destruction. Single-cell RNA sequencing and clonal analysis revealed that autoimmune CD8 T cells represent unique T cell differentiation states and identified features driving the transition from autoimmune progenitor to autoimmune mediator. Strategies aimed at targeting the stem-like autoimmune progenitor pool could emerge as novel and powerful immunotherapeutic interventions for type 1 diabetes. View Publication

Many solid tumors have low levels of cytotoxic CD56dim natural killer (NK) cells,suggesting that CD56dim NK-cell exclusion from the tumor microenvironment (TME) contributes to the decreased response rate of immunotherapy. Complement component 3a (C3a) is known for its tumor-promoting and immunosuppressive roles in solid tumors. Previous reports have implicated the involvement of the C3a receptor (C3aR) in immune cell trafficking into the TME. C3aR is predominantly expressed on the surface of activated cytotoxic NK cells,but a specific role for C3aR in NK-cell biology has not been investigated. Because solid tumors generate elevated C3a and have decreased NK-cell infiltration,we hypothesized that C3aR might play a role in cytotoxic NK-cell recruitment into the TME. Our results indicate that blocking C3aR signaling in NK cells increased NK-cell infiltration into the TME in mouse models and led to tumor regression. Because the critical lymphocyte trafficking integrin LFA-1 orchestrates the migration of activated NK cells,we wanted to gain insight into the interaction between C3aR signaling and LFA-1. Our results demonstrated that direct interaction between C3aR and LFA-1,which led to a high-affinity LFA-1 conformation,decreased NK-cell infiltration into the TME. We propose that approaches to enhance cytotoxic NK-cell infiltration into the TME,through either disrupting C3a and C3aR interaction or inhibiting the formation of high-affinity LFA-1,represent a new strategy to improve the efficiency of immunotherapy for cancer treatment. View Publication

Extracellular vesicles (EVs) have potential as minimally invasive biomarkers. However,the methods most commonly used for EV retrieval rely on ultracentrifugation,are time-consuming,and unrealistic to translate to standard-of-care. We sought a method suitable for EV separation from blood that could be used in patient care. Sera from breast cancer patients and age-matched controls (n = 27 patients; n = 36 controls) were analysed to compare 6 proposed EV separation methods. The EVs were then characterised on 8 parameters. The selected method was subsequently applied to independent cohorts of sera (n = 20 patients; n = 20 controls),as proof-of-principle,investigating EVs' gremlin-1 cargo. Three independent runs with each method were very reproducible,within each given method. All isolates contained EVs,although they varied in quantity and purity. Methods that require ultracentrifugation were not superior for low volumes of sera typically available in routine standard-of-care. A CD63/CD81/CD9-coated immunobead-based method was most suitable based on EV markers' detection and minimal albumin and lipoprotein contamination. Applying this method to independent sera cohorts,EVs and their gremlin-1 cargo were at significantly higher amounts for breast cancer patients compared to controls. In conclusion,CD63/CD81/CD9-coated immunobeads may enable clinical utility of blood-based EVs as biomarkers. View Publication

New approaches beyond PD-1/PD-L1 inhibition are required to target the immunologically diverse tumor microenvironment (TME) in high-grade serous ovarian cancer (HGSOC). In this study,we explored the immunosuppressive effect of B7-H3 (CD276) via the CCL2-CCR2-M2 macrophage axis and its potential as a therapeutic target. Transcriptome analysis revealed that B7-H3 is highly expressed in PD-L1-low,nonimmunoreactive HGSOC tumors,and its expression negatively correlated with an IFN$\gamma$ signature,which reflects the tumor immune reactivity. In syngeneic mouse models,B7-H3 (Cd276) knockout (KO) in tumor cells,but not in stromal cells,suppressed tumor progression,with a reduced number of M2 macrophages and an increased number of IFN$\gamma$+CD8+ T cells. CCL2 expression was downregulated in the B7-H3 KO tumor cell lines. Inhibition of the CCL2-CCR2 axis partly negated the effects of B7-H3 suppression on M2 macrophage migration and differentiation,and tumor progression. In patients with HGSOC,B7-H3 expression positively correlated with CCL2 expression and M2 macrophage abundance,and patients with B7-H3-high tumors had fewer tumoral IFN$\gamma$+CD8+ T cells and poorer prognosis than patients with B7-H3-low tumors. Thus,B7-H3 expression in tumor cells contributes to CCL2-CCR2-M2 macrophage axis-mediated immunosuppression and tumor progression. These findings provide new insights into the immunologic TME and could aid the development of new therapeutic approaches against the unfavorable HGSOC phenotype. View Publication

Successful implantation requires the coordinated migration and invasion of trophoblast cells from out of the blastocyst and into the endometrium. This process relies on signals produced by cells in the maternal endometrium. However,the relative contribution of stroma cells remains unclear. The study of human implantation has major technical limitations,therefore the need of in vitro models to elucidate the molecular mechanisms. Using a recently described 3D in vitro models we evaluated the interaction between trophoblasts and human endometrial stroma cells (hESC),we assessed the process of trophoblast migration and invasion in the presence of stroma derived factors. We demonstrate that hESC promotes trophoblast invasion through the generation of an inflammatory environment modulated by TNF-?. We also show the role of stromal derived IL-17 as a promoter of trophoblast migration through the induction of essential genes that confer invasive capacity to cells of the trophectoderm. In conclusion,we describe the characterization of a cellular inflammatory network that may be important for blastocyst implantation. Our findings provide a new insight into the complexity of the implantation process and reveal the importance of inflammation for embryo implantation. View Publication

Although critical for host defense,innate immune cells are also pathologic drivers of acute respiratory distress syndrome (ARDS). Innate immune dynamics during Coronavirus Disease 2019 (COVID-19) ARDS,compared to ARDS from other respiratory pathogens,is unclear. Moreover,mechanisms underlying the beneficial effects of dexamethasone during severe COVID-19 remain elusive. Using single-cell RNA sequencing and plasma proteomics,we discovered that,compared to bacterial ARDS,COVID-19 was associated with expansion of distinct neutrophil states characterized by interferon (IFN) and prostaglandin signaling. Dexamethasone during severe COVID-19 affected circulating neutrophils,altered IFNactive neutrophils,downregulated interferon-stimulated genes and activated IL-1R2+ neutrophils. Dexamethasone also expanded immunosuppressive immature neutrophils and remodeled cellular interactions by changing neutrophils from information receivers into information providers. Male patients had higher proportions of IFNactive neutrophils and preferential steroid-induced immature neutrophil expansion,potentially affecting outcomes. Our single-cell atlas (see 'Data availability' section) defines COVID-19-enriched neutrophil states and molecular mechanisms of dexamethasone action to develop targeted immunotherapies for severe COVID-19. View Publication

Peptidylarginine deiminase 4 (PAD4) is a key regulator of inflammation but its function in infections remains incompletely understood. We investigate PAD4 in the context of malaria and demonstrate a role in regulation of immune cell trafficking and chemokine production. PAD4 regulates liver immunopathology by promoting neutrophil trafficking in a Plasmodium chabaudi mouse malaria model. In human macrophages,PAD4 regulates expression of CXCL chemokines in response to stimulation with TLR ligands and P. falciparum. Using patient samples,we show that CXCL1 may be a biomarker for severe malaria. PAD4 inhibition promotes disease tolerance and may represent a therapeutic avenue in malaria. View Publication

BACKGROUND During the COVID-19 pandemic,thousands of pregnant women have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The implications of maternal SARS-CoV-2 infection on fetal and childhood well-being need to be characterized. We aimed to characterize the fetal immune response to maternal SARS-CoV-2 infection. METHODS We performed single-cell RNA-sequencing and T cell receptor sequencing on cord blood mononuclear cells (CBMCs) from newborns of mothers infected with SARS-CoV-2 in the third trimester (cases) or without SARS-CoV-2 infection (controls). RESULTS We identified widespread gene expression changes in CBMCs from cases,including upregulation of interferon-stimulated genes and major histocompatibility complex genes in CD14+ monocytes,transcriptional changes suggestive of activation of plasmacytoid dendritic cells,and activation and exhaustion of natural killer cells. Lastly,we observed fetal T cell clonal expansion in cases compared to controls. CONCLUSIONS As none of the infants were infected with SARS-CoV-2,our results suggest that maternal SARS-CoV-2 infection might modulate the fetal immune system in the absence of vertical transmission. IMPACT The implications of maternal SARS-CoV-2 infection in the absence of vertical transmission on fetal and childhood well-being are poorly understood. Maternal SARS-CoV-2 infection might modulate the fetal immune system in the absence of vertical transmission. This study raises important questions about the untoward effects of maternal SARS-CoV-2 on the fetus,even in the absence of vertical transmission. View Publication

Immunogenicity following inactivated SARS-CoV-2 vaccination among solid organ transplant recipients has not been assessed. Seventy-five patients (37 kidney transplant [KT] recipients and 38 healthy controls) received two doses,at 4-week intervals,of an inactivated whole-virus SARS-CoV-2 vaccine. SARS-CoV-2-specific humoral (HMI) and cell-mediated immunity (CMI) were measured before,4 weeks post-first dose,and 2 weeks post-second dose. The median (IQR) age of KT recipients was 50 (42-54) years and 89% were receiving calcineurin inhibitors/mycophenolate/corticosteroid regimens. The median (IQR) time since transplant was 4.5 (2-9.5) years. Among 35 KT patients,the median (IQR) of anti-RBD IgG level measured by CLIA after vaccination was not different from baseline,but was significantly lower than in controls (2.4 [1.1-3.7] vs. 1742.0 [747.7-3783.0] AU/ml,p < .01) as well as percentages of neutralizing antibody inhibition measured by surrogate viral neutralization test (0 [0-0] vs. 71.2 [56.8-92.2]%,p < .01). However,the median (IQR) of SARS-CoV-2 mixed peptides-specific T cell responses measured by ELISpot was significantly increased compared with baseline (30 [4-120] vs. 12 [0-56] T cells/106  PBMCs,p = .02) and not different from the controls. Our findings revealed weak HMI but comparable CMI responses in fully vaccinated KT recipients receiving inactivated SARS-CoV-2 vaccination compared to immunocompetent individuals (Thai Clinical Trials Registry,TCTR20210226002). View Publication

BACKGROUND Thrombocytopenia leading to life-threatening excessive bleeding can be treated by platelet transfusion. Currently,such treatments are totally dependent on donor-derived platelets. To support future applications in the use of in vitro-derived platelets,we sought to identify genes whose manipulation might improve the efficiency of megakaryocyte production and resulting hemostatic effectiveness. Disruption of Lyn kinase has previously been shown to improve cell survival,megakaryocyte ploidy and TPO-mediated activation in mice,but its role in human megakaryocytes and platelets has not been examined. METHODS To analyze the role of Lyn at defined differentiation stages during human megakaryocyte differentiation,conditional Lyn-deficient cells were generated using CRISPR/Cas9 technology in iPS cells. The efficiency of Lyn-deficient megakaryocytes to differentiate and become activated in response to a range of platelet agonists was analyzed in iPSC-derived megakaryocytes. RESULTS Temporally controlled deletion of Lyn improved the in vitro differentiation of hematopoietic progenitor cells into mature megakaryocytes,as measured by the rate and extent of appearance of CD41+ CD42+ cells. Lyn-deficient megakaryocytes also demonstrated improved hemostatic effectiveness,as reported by their ability to mediate clot formation in rotational thromboelastometry. Finally,Lyn-deficient megakaryocytes produced increased numbers of platelet-like particles (PLP) in vitro. CONCLUSIONS Conditional deletion of Lyn kinase increases the hemostatic effectiveness of megakaryocytes and their progeny as well as improving their yield. Adoption of this system during generation of in vitro-derived platelets may contribute to both their efficiency of production and their ability to support hemostasis. View Publication

Proton export is often considered a detoxifying process in animal cells,with monocarboxylate symporters coexporting excessive lactate and protons during glycolysis or the Warburg effect. We report a novel mechanism by which lactate/H+ export is sufficient to induce cell growth. Increased intracellular pH selectively activates catalysis by key metabolic gatekeeper enzymes HK1/PKM2/G6PDH,thereby enhancing glycolytic and pentose phosphate pathway carbon flux. The result is increased nucleotide levels,NADPH/NADP+ ratio,and cell proliferation. Simply increasing the lactate/proton symporter monocarboxylate transporter 4 (MCT4) or the sodium-proton antiporter NHE1 was sufficient to increase intracellular pH and give normal hematopoietic cells a significant competitive growth advantage in vivo. This process does not require additional cytokine triggers and is exploited in malignancy,where leukemogenic mutations epigenetically increase MCT4. Inhibiting MCT4 decreased intracellular pH and carbon flux and eliminated acute myeloid leukemia-initiating cells in mice without cytotoxic chemotherapy. Intracellular alkalization is a primitive mechanism by which proton partitioning can directly reprogram carbon metabolism for cell growth. View Publication

BACKGROUND & AIMS An invasive form of intestinal Entamoeba (E.) histolytica infection,which causes amoebic liver abscess,is more common in men than in women. Immunopathological mechanisms are responsible for the more severe outcome in males. Here,we used a mouse model of hepatic amoebiasis to investigate the contribution of hepatic hypoxia-inducible factor (HIF)-1$\alpha$ to T helper 17 (Th17)/regulatory T cell (Treg) responses in the context of the sex-specific outcome of liver damage. METHODS C57BL/6J mice were infected intrahepatically with E. histolytica trophozoites. HIF-1$\alpha$ expression was determined by qPCR,flow cytometry and immunohistochemistry. Tregs and Th17 cells were analysed by immunohistochemistry and flow cytometry. Finally,male and female hepatocyte-specific Hif1$\alpha$ knockout mice were generated,and the effect of HIF-1$\alpha$ on abscess development,the cytokine milieu,and Th17/Treg differentiation was examined. RESULTS E. histolytica infection increased hepatic HIF-1$\alpha$ levels,along with the elevated frequencies of hepatic Th17 and Treg cells. While the Th17 cell population was larger in male mice,Tregs characterised by increased expression of Foxp3 in female mice. Male mice displayed increased IL-6 expression,contributing to immunopathology; this increase in IL-6 expression declined upon deletion of hepatic HIF-1$\alpha$. In both sexes,hepatic deletion of HIF-1$\alpha$ reduced the Th17 cell frequency; however,the percentage of Tregs was reduced in female mice only. CONCLUSIONS Hepatic HIF-1$\alpha$ modulates the sex-specific outcome of murine E. histolytica infection. Our results suggest that in male mice,Th17 cells can be modulated by hepatic HIF-1$\alpha$ via IL-6,indicating marked involvement in the immunopathology underlying abscess development. Strong expression of Foxp3 by hepatic Tregs from female mice suggests a potent immunosuppressive function,leading to initiation of liver regeneration. LAY SUMMARY Infection with the parasite Entamoeba histolytica activates immunopathological mechanisms in male mice,which lead to liver abscesses that are larger than those in female mice. In the absence of the protein HIF-1$\alpha$ in hepatocytes,abscess formation is reduced; moreover,the sex difference in abscess size is abolished. These results suggest that HIF-1$\alpha$ modulates the immune response involved in the induction of immunopathology,resulting in differential disease susceptibility in males and females. View Publication