技术资料

Chronic decreases in the second messenger cyclic AMP (cAMP) occur in numerous settings,but how cells compensate for such decreases is unknown. We have used a unique system-murine dendritic cells (DCs) with a DC-selective depletion of the heterotrimeric GTP binding protein G$\alpha$s-to address this issue. These mice spontaneously develop Th2-allergic asthma and their DCs have persistently lower cAMP levels. We found that phosphodiesterase 4B (PDE4B) is the primary phosphodiesterase expressed in DCs and that its expression is preferentially decreased in G$\alpha$s-depleted DCs. PDE4B expression is dynamic,falling and rising in a protein kinase A-dependent manner with decreased and increased cAMP concentrations,respectively. Treatment of DCs that drive enhanced Th2 immunity with a PDE4B inhibitor ameliorated DC-induced helper T cell response. We conclude that PDE4B is a homeostatic regulator of cellular cAMP concentrations in DCs and may be a target for treating Th2-allergic asthma and other settings with low cellular cAMP concentrations. View Publication

Inhibitory receptors have a critical role in the regulation of immunity. Siglecs are a family of primarily inhibitory receptors expressed by immune cells that recognize specific sialic acid modifications on cell surface glycans. Many tumors have increased sialic acid incorporation. Overexpression of the sialyltransferase ST8Sia6 on tumors led to altered immune responses and increased tumor growth. In this study,we examined the role of ST8Sia6 on immune cells in regulating antitumor immunity. ST8Sia6 knockout mice had an enhanced immune response to tumors. The loss of ST8Sia6 promoted an enhanced intratumoral activation of macrophages and dendritic cells,including upregulation of CD40. Intratumoral regulatory T cells exhibited a more inflammatory phenotype in ST8Sia6 knockout mice. Using adoptive transfer studies,the change in regulatory T cell phenotype was not cell intrinsic and depended on the loss of ST8Sia6 expression in APCs. Thus,ST8Sia6 generates ligands for Siglecs that dampen antitumor immunity. View Publication

Innate lymphoid cells (ILCs) - which include cytotoxic Natural Killer (NK) cells and helper-type ILC - are important regulators of tissue immune homeostasis,with possible roles in tumor surveillance. We analyzed ILC and their functionality in human lymph nodes (LN). In LN,NK cells and ILC3 were the prominent subpopulations. Among the ILC3s,we identified a CD56+/ILC3 subset with a phenotype close to ILC3 but also expressing cytotoxicity genes shared with NK. In tumor-draining LNs (TD-LNs) and tumor samples from breast cancer (BC) patients,NK cells were prominent,and proportions of ILC3 subsets were low. In tumors and TD-LN,NK cells display reduced levels of NCR (Natural cytotoxicity receptors),despite high transcript levels and included a small subset CD127- CD56- NK cells with reduced function. Activated by cytokines CD56+/ILC3 cells from donor and patients LN acquired cytotoxic capacity and produced IFNg. In TD-LN,all cytokine activated ILC populations produced TNF$\alpha$ in response to BC cell line. Analyses of cytotoxic and helper ILC indicate a switch toward NK cells in TD-LN. The local tumor microenvironment inhibited NK cell functions through downregulation of NCR,but cytokine stimulation restored their functionality. View Publication

BACKGROUND AKI is a common sequela of infection with SARS-CoV-2 and contributes to the severity and mortality from COVID-19. Here,we tested the hypothesis that kidney alterations induced by COVID-19-associated AKI could be detected in cells collected from urine. METHODS We performed single-cell RNA sequencing (scRNAseq) on cells recovered from the urine of eight hospitalized patients with COVID-19 with (n=5) or without AKI (n=3) as well as four patients with non-COVID-19 AKI (n=4) to assess differences in cellular composition and gene expression during AKI. RESULTS Analysis of 30,076 cells revealed a diverse array of cell types,most of which were kidney,urothelial,and immune cells. Pathway analysis of tubular cells from patients with AKI showed enrichment of transcripts associated with damage-related pathways compared with those without AKI. ACE2 and TMPRSS2 expression was highest in urothelial cells among cell types recovered. Notably,in one patient,we detected SARS-CoV-2 viral RNA in urothelial cells. These same cells were enriched for transcripts associated with antiviral and anti-inflammatory pathways. CONCLUSIONS We successfully performed scRNAseq on urinary sediment from hospitalized patients with COVID-19 to noninvasively study cellular alterations associated with AKI and established a dataset that includes both injured and uninjured kidney cells. Additionally,we provide preliminary evidence of direct infection of urinary bladder cells by SARS-CoV-2. The urinary sediment contains a wealth of information and is a useful resource for studying the pathophysiology and cellular alterations that occur in kidney diseases. View Publication

BACKGROUND Beh{\c{c}}et's disease (BD) is a multisystem autoinflammatory disease characterised by mucosal ulceration,ocular,neural,joint and skin inflammation. The cause of BD is not known but there is a strong genetic association with HLA-B*51,IL10 and IL23R. Neutrophils are a first line of defence against invading pathogens and have been described as activated in patients with BD. Neutrophils can now be separated into different subsets,such as low density (LDN) and normal density (NDN) that have diverse functional roles. We wished to address neutrophil heterogeneity in patients with BD. METHODS Peripheral blood neutrophils were obtained from 32 BD patients and 37 healthy aged-matched controls. Percoll isolation was used to isolate all neutrophils,while Ficol-Hypaque was used to obtain LDN and NDN. Phagocytic capacity and production of reactive oxygen species (ROS),and neutrophil extracellular traps (NET) stimulated with phorbol 12-myristate 13-acetate (PMA) and Escherichia coli (E.coli) were assessed in both groups. RESULTS We have demonstrated reduced phagocytic capacity and ROS production but greater NET production by total neutrophils stimulated with PMA or E.coli from BD patients in comparison with healthy controls. Patients with BD had elevated numbers of LDN and lower number of NDN compared with healthy controls. However,both neutrophil subsets showed the same reduced ROS production and phagocytic function as total neutrophils in both groups. CONCLUSION Our novel findings indicate that the neutrophil population in BD is heterogeneous and the increased number of LDN in combination with greater NET production may contribute to the inflammatory response and pathogenesis. View Publication

The use of lipid-formulated RNA vaccines for cancer or COVID-19 is associated with dose-limiting systemic inflammatory responses in humans that were not predicted from preclinical studies. Here,we show that the 'interleukin 1 (IL-1)-interleukin 1 receptor antagonist (IL-1ra)' axis regulates vaccine-mediated systemic inflammation in a host-specific manner. In human immune cells,RNA vaccines induce production of IL-1 cytokines,predominantly IL-1$\beta$,which is dependent on both the RNA and lipid formulation. IL-1 in turn triggers the induction of the broad spectrum of pro-inflammatory cytokines (including IL-6). Unlike humans,murine leukocytes respond to RNA vaccines by upregulating anti-inflammatory IL-1ra relative to IL-1 (predominantly IL-1$\alpha$),protecting mice from cytokine-mediated toxicities at >1,000-fold higher vaccine doses. Thus,the IL-1 pathway plays a key role in triggering RNA vaccine-associated innate signaling,an effect that was unexpectedly amplified by certain lipids used in vaccine formulations incorporating N1-methyl-pseudouridine-modified RNA to reduce activation of Toll-like receptor signaling. View Publication

Immune checkpoint blockade has revolutionized the field of oncology,inducing durable anti-tumour immunity in solid tumours. In patients with advanced prostate cancer,immunotherapy treatments have largely failed1-5. Androgen deprivation therapy is classically administered in these patients to inhibit tumour cell growth,and we postulated that this therapy also affects tumour-associated T cells. Here we demonstrate that androgen receptor (AR) blockade sensitizes tumour-bearing hosts to effective checkpoint blockade by directly enhancing CD8 T cell function. Inhibition of AR activity in CD8 T cells prevented T cell exhaustion and improved responsiveness to PD-1 targeted therapy via increased IFN$\gamma$ expression. AR bound directly to Ifng and eviction of AR with a small molecule significantly increased cytokine production in CD8 T cells. Together,our findings establish that T cell intrinsic AR activity represses IFN$\gamma$ expression and represents a novel mechanism of immunotherapy resistance. View Publication

BACKGROUND Epidemiological surveys have revealed that low serum vitamin D level was correlated with increased risk of tumors. Dysfunctional T cells in patients with tumor are characterized as exhausted with high levels of immune checkpoint receptors (ICRs). However,whether the reduced level of vitamin D in patients with cancer correlates with cytotoxic T-cell exhaustion is unknown. METHODS Periphery blood samples from 172 patients with non-small cell lung cancer (NSCLC) were prospectively collected. Patients with NSCLC received one course of intravenous docetaxel (75 mg/m2) followed by treatment with or without rocaltrol at a dose of 0.5-2.0 µg/day for total of 3 weeks. We performed phenotypical and functional analysis of T-cell through flow cytometry. Vitamin D receptor (VDR) knockout and overexpression CD8+ and V$\delta$2+ T cells were constructed using Cas9-gRNA targeted and overexpressing approaches to identify 1$\alpha$,25(OH)2D3/VDR-mediated transcription regulation for ICRs or antitumor activity in T cells. RESULTS We show that serum level of vitamin D is negatively correlated with expression of programmed cell death-1 (PD-1),T-cell immunoreceptor with Ig and ITIM domains (TIGIT),and T-cell immunoglobulin and mucin-domain containing-3 (Tim-3),but positively correlated with CD28 expression on CD8+ and V$\gamma$9V$\delta$2+ T cells in patients with NSCLC. 1$\alpha$,25(OH)2D3,the active form of vitamin D,promotes the nuclear translocation of VDR,which binds to the promoter region of Pdcd1,Tim3,and Tigit genes and inhibits their expression. Besides,1$\alpha$,25(OH)2D3 pretreatment also promotes the methylation of CpG island in the promoter region of the Pdcd1 gene and increases H3K27 acetylation at the promoter region of the Cd28 gene,which leads to surface PD-1 downregulation and CD28 upregulation,respectively. We further reveal that VDR-mediated Ca2+ influx enhanced expression of Th1 cytokines via T-cell receptor activation. Functionally,1$\alpha$,25(OH)2D3 pretreated CD8+ T cells or V$\gamma$9V$\delta$2+ T cells showed increased Th1 cytokine production and enhanced antitumor immunity. Finally,oral 1$\alpha$,25(OH)2D3 could also decrease expression of PD-1,Tim-3,TIGIT and increase expression of CD28,resulting in cytokine production (associated with antitumor immunity) by cytotoxic T cells of patients with NSCLC. CONCLUSIONS Our findings uncover the pleiotropic effects of 1$\alpha$,25(OH)2D3 in rescuing the exhausted phenotype of human cytotoxic T cells in patients with tumor and in promoting their antitumor immunity. TRIAL REGISTRATION NUMBER ChiCTR2100051135. View Publication

Early diagnosis of psoriatic arthritis (PSA) is important for successful therapeutic intervention but currently remains challenging due,in part,to the scarcity of non-invasive biomarkers. In this study,we performed single cell profiling of transcriptome and cell surface protein expression to compare the peripheral blood immunocyte populations of individuals with PSA,individuals with cutaneous psoriasis (PSO) alone,and healthy individuals. We identified genes and proteins differentially expressed between PSA,PSO,and healthy subjects across 30 immune cell types and observed that some cell types,as well as specific phenotypic subsets of cells,differed in abundance between these cohorts. Cell type-specific gene and protein expression differences between PSA,PSO,and healthy groups,along with 200 previously published genetic risk factors for PSA,were further used to perform machine learning classification,with the best models achieving AUROC ?? 0.87 when either classifying subjects among the three groups or specifically distinguishing PSA from PSO. Our findings thus expand the repertoire of gene,protein,and cellular biomarkers relevant to PSA and demonstrate the utility of machine learning-based diagnostics for this disease. View Publication

BACKGROUND Myeloid-derived suppressor cells (MDSCs) represent a negative prognostic factor in malignant melanoma. These cells are generated under chronic inflammatory conditions typical of cancer. The transcription factor signal transducer and activator of transcription 3 (STAT3) orchestrates MDSC accumulation and acquisition of immunosuppressive properties. Here we studied STAT3 inhibition by Napabucasin as a way to block MDSC accumulation and activity and its potential to treat malignant melanoma. METHODS In vitro generated murine MDSC and primary MDSC from melanoma-bearing mice were used to investigate the effects of Napabucasin on MDSC in vitro. The RET transgenic mouse model of malignant melanoma was used to examine Napabucasin therapy efficiency and its underlying mechanisms in vivo. Furthermore,STAT3 activation and its correlation with survival were explored in MDSC from 19 patients with malignant melanoma and human in vitro generated monocytic myeloid-derived suppressor cell (M-MDSC) were used to evaluate the effects of Napabucasin. RESULTS Napabucasin was able to abrogate the capacity of murine MDSC to suppress CD8+ T-cell proliferation. The STAT3 inhibitor induced apoptosis in murine MDSC,significantly increased expression of molecules associated with antigen processing and presentation,as well as slightly decreased expression of immunosuppressive factors on these cells. RET transgenic mice treated with Napabucasin showed prolonged survival accompanied by a strong accumulation of tumor-infiltrating antigen-presenting cells and activation of CD8+ and CD4+ T cells. Interestingly,patients with malignant melanoma with high expression of activated STAT3 in circulating M-MDSC showed significantly worse progression-free survival (PFS) than patients with low levels of activated STAT3. In addition,Napabucasin was able to abrogate suppressive capacity of human in vitro generated M-MDSC. CONCLUSION Our findings demonstrate that STAT3 inhibitor Napabucasin completely abrogated the immunosuppressive capacity of murine MDSC and human M-MDSC and improved melanoma-bearing mouse survival. Moreover,patients with malignant melanoma with high expression levels of activated STAT3 in M-MDSC displayed shorter PFS,indicating its role as a promising therapeutic target in patients with malignant melanoma and a predictive marker for their clinical outcome. View Publication

The engineering of autologous patient T cells for adoptive cell therapies has revolutionized the treatment of several types of cancer1. However,further improvements are needed to increase response and cure rates. CRISPR-based loss-of-function screens have been limited to negative regulators of T cell functions2-4 and raise safety concerns owing to the permanent modification of the genome. Here we identify positive regulators of T cell functions through overexpression of around 12,000 barcoded human open reading frames (ORFs). The top-ranked genes increased the proliferation and activation of primary human CD4+ and CD8+ T cells and their secretion of key cytokines such as interleukin-2 and interferon-$\gamma$. In addition,we developed the single-cell genomics method OverCITE-seq for high-throughput quantification of the transcriptome and surface antigens in ORF-engineered T cells. The top-ranked ORF-lymphotoxin-$\beta$ receptor (LTBR)-is typically expressed in myeloid cells but absent in lymphocytes. When overexpressed in T cells,LTBR induced profound transcriptional and epigenomic remodelling,leading to increased T cell effector functions and resistance to exhaustion in chronic stimulation settings through constitutive activation of the canonical NF-$\kappa$B pathway. LTBR and other highly ranked genes improved the antigen-specific responses of chimeric antigen receptor T cells and ?? T cells,highlighting their potential for future cancer-agnostic therapies5. Our results provide several strategies for improving next-generation T cell therapies by the induction of synthetic cell programmes. View Publication

BACKGROUND Bispecific T-cell engager (BiTE) molecules induce redirected lysis of cancer cells by T cells and are an emerging modality for solid tumor immunotherapy. While signs of clinical activity have been demonstrated,efficacy of T-cell engagers (TCEs) in solid tumors settings,molecular determinants of response,and underlying mechanisms of resistance to BiTE therapy require more investigation. METHODS To uncover cancer cell-intrinsic genetic modifiers of TCE-mediated cytotoxicity,we performed genome-wide CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loss-of-function and CRISPRa (CRISPR activation) gain-of-function screens using TCEs against two distinct tumor-associated antigens (TAAs). By using in vitro T-cell cytotoxicity assays and in vivo efficacy studies,we validated the roles of two common pathways identified in our screen,T-cell costimulation pathway and apoptosis pathway,as key modifiers of BiTE activity. RESULTS Our genetic screens uncovered TAAs-independent cancer cell-intrinsic genes with functions in autophagy,T-cell costimulation,the apoptosis pathway,chromatin remodeling,and cytokine signaling that altered responsiveness to BiTE-mediated killing. Notably,loss of CD58 (the ligand of the CD2 T-cell costimulatory receptor),a gene frequently altered in cancer,led to decreased TCE-mediated cytotoxicity,T-cell activation and antitumor efficacy in vitro and in vivo. Moreover,the effects of CD58 loss were synergistically compounded by concurrent loss of CD80/CD86 (ligands for the CD28 T-cell costimulatory receptor),whereas joint CD2 and CD28 costimulation additively enhanced TCE-mediated killing,indicating non-redundant costimulatory mechanisms between the two pathways. Additionally,loss of CFLAR (Caspase-8 and FADD Like Apoptosis Regulator),BCL2L1,and BID (BH3 Interacting Domain Death Agonist) induced profound changes in sensitivity to TCEs,indicating that key regulators of apoptosis,which are frequently altered in cancer,impact tumor responsiveness to BiTE therapy. CONCLUSIONS This study demonstrates that genetic alterations central to carcinogenesis and commonly detected in cancer samples lead to significant modulation of BiTE antitumor activity in vitro and in vivo,findings with relevance for a better understanding of patient responses to BiTE therapy and novel combinations that enhance TCE efficacy. View Publication