Scientific Resources

Oxidative stress,caused by the imbalance between reactive species generation and the dysfunctional capacity of antioxidant defenses,is one of the characteristic features of cancer. Here,we quantified hydrogen peroxide in the tumor microenvironment (TME) and demonstrated that hydrogen peroxide concentrations are elevated in tumor interstitial fluid isolated from murine breast cancers in vivo,when compared with blood or normal subcutaneous fluid. Therefore,we investigated the effects of increased hydrogen peroxide concentration on immune cell functions. NK cells were more susceptible to hydrogen peroxide than T cells or B cells,and by comparing T,B,and NK cells' sensitivities to redox stress and their antioxidant capacities,we identified peroxiredoxin-1 (PRDX1) as a lacking element of NK cells' antioxidative defense. We observed that priming with IL15 protected NK cells' functions in the presence of high hydrogen peroxide and simultaneously upregulated PRDX1 expression. However,the effect of IL15 on PRDX1 expression was transient and strictly dependent on the presence of the cytokine. Therefore,we genetically modified NK cells to stably overexpress PRDX1,which led to increased survival and NK cell activity in redox stress conditions. Finally,we generated PD-L1-CAR NK cells overexpressing PRDX1 that displayed potent antitumor activity against breast cancer cells under oxidative stress. These results demonstrate that hydrogen peroxide,at concentrations detected in the TME,suppresses NK cell function and that genetic modification strategies can improve CAR NK cells' resistance and potency against solid tumors. View Publication

Asthma and its heterogeneity change with age. Increased airspace neutrophil numbers contribute to severe steroid-resistant asthma exacerbation in the elderly,which correlates with the changes seen in adults with asthma. However,whether that resembles the same disease mechanism and pathophysiology in aged and adults is poorly understood. Here,we sought to address the underlying molecular mechanism of steroid-resistant airway inflammation development and response to corticosteroid (Dex) therapy in aged mice. To study the changes in inflammatory mechanism,we used a clinically relevant treatment model of house-dust mite (HDM)-induced allergic asthma and investigated lung adaptive immune response in adult (20-22 wk old) and aged (80-82 wk old) mice. Our result indicates an age-dependent increase in airway hyperresponsiveness (AHR),mixed granulomatous airway inflammation comprising eosinophils and neutrophils,and Th1/Th17 immune response with progressive decrease in frequencies and numbers of HDM-bearing dendritic cells (DC) accumulation in the draining lymph node (DLn) of aged mice as compared with adult mice. RNA-Seq experiments of the aged lung revealed short palate,lung,and nasal epithelial clone 1 (SPLUNC1) as one of the steroid-responsive genes,which progressively declined with age and further by HDM-induced inflammation. Moreover,we found increased glycolytic reprogramming,maturation/activation of DCs,the proliferation of OT-II cells,and Th2 cytokine secretion with recombinant SPLUNC1 (rSPLUNC1) treatment. Our results indicate a novel immunomodulatory role of SPLUNC1 regulating metabolic adaptation/maturation of DC. An age-dependent decline in the SPLUNC1 level may be involved in developing steroid-resistant airway inflammation and asthma heterogeneity. 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

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

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

OBJECTIVE Tissue-resident memory T cells (TRM) are vital immune sentinels that provide protective immunity. While hepatic CD8+ TRM have been well described,little is known about the location,phenotype and function of CD4+ TRM. DESIGN We used multiparametric flow cytometry,histological assessment and novel human tissue coculture systems to interrogate the ex vivo phenotype,function and generation of the intrahepatic CD4+ T-cell compartment. We also used leukocytes isolated from human leukocyte antigen (HLA)-disparate liver allografts to assess long-term retention. RESULTS Hepatic CD4+ T cells were delineated into three distinct populations based on CD69 expression: CD69-,CD69INT and CD69HI. CD69HICD4+ cells were identified as tissue-resident CD4+ T cells on the basis of their exclusion from the circulation,phenotypical profile (CXCR6+CD49a+S1PR1-PD-1+) and long-term persistence within the pool of donor-derived leukcoocytes in HLA-disparate liver allografts. CD69HICD4+ T cells produced robust type 1 polyfunctional cytokine responses on stimulation. Conversely,CD69INTCD4+ T cells represented a more heterogenous population containing cells with a more activated phenotype,a distinct chemokine receptor profile (CX3CR1+CXCR3+CXCR1+) and a bias towards interleukin-4 production. While CD69INTCD4+ T cells could be found in the circulation and lymph nodes,these cells also formed part of the long-term resident pool,persisting in HLA-mismatched allografts. Notably,frequencies of CD69INTCD4+ T cells correlated with necroinflammatory scores in chronic hepatitis B infection. Finally,we demonstrated that interaction with hepatic epithelia was sufficient to generate CD69INTCD4+ T cells,while additional signals from the liver microenvironment were required to generate liver-resident CD69HICD4+ T cells. CONCLUSIONS High and intermediate CD69 expressions mark human hepatic CD4+ TRM and a novel functionally distinct recirculating population,respectively,both shaped by the liver microenvironment to achieve diverse immunosurveillance. View Publication