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BACKGROUND CD73 is an ectonucleotidase producing the immunosuppressor mediator adenosine. Elevated levels of circulating CD73 in patients with cancer have been associated with disease progression and poor response to immunotherapy. Immunosuppressive pathways associated with exosomes can affect T-cell function and the therapeutic efficacy of anti-programmed cell-death protein 1 (anti-PD-1) therapy. Here,we conducted a retrospective pilot study to evaluate levels of exosomal CD73 before and early during treatment with anti-PD-1 agents in patients with melanoma and its potential contribution to affect T-cell functions and to influence the clinical outcomes of anti-PD-1 monotherapy. METHODS Exosomes were isolated by mini size exclusion chromatography from serum of patients with melanoma (n=41) receiving nivolumab or pembrolizumab monotherapy. Expression of CD73 and programmed death-ligand 1 (PD-L1) were evaluated on exosomes enriched for CD63 by on-bead flow cytometry. The CD73 AMPase activity was evaluated by mass spectrometry,also in the presence of selective inhibitors of CD73. Interferon (IFN)-$\gamma$ production and granzyme B expression were measured in CD3/28 activated T cells incubated with exosomes in presence of the CD73 substrate AMP. Levels of CD73 and PD-L1 on exosomes were correlated with therapy response. Exosomes isolated from healthy subjects were used as control. RESULTS Isolated exosomes carried CD73 on their surface,which is enzymatically active in producing adenosine. Incubation of exosomes with CD3/28 activated T cells in the presence of AMP resulted in a significant reduction of IFN-$\gamma$ release,which was reversed by the CD73 inhibitor APCP or by the selective A2A adenosine receptor antagonist ZM241385. Expression levels of exosomal CD73 from serum of patients with melanoma were not significantly different from those in healthy subjects. Early on-treatment,expression levels of both CD73 and PD-L1 on exosomes isolated from patients receiving pembrolizumab or nivolumab monotherapy were significantly increased compared with baseline. Early during therapy exosomal PD-L1 increased in responders,while exosomal CD73 resulted significantly increased in non-responders. CONCLUSIONS CD73 expressed on exosomes from serum of patients with melanoma produces adenosine and contributes to suppress T-cell functions. Early on-treatment,elevated expression levels of exosomal CD73 might affect the response to anti-PD-1 agents in patients with melanoma who failed to respond to therapy. View Publication

Sepsis remains an important health problem worldwide due to inefficient treatments often resulting in multi-organ failure. Neutrophil recruitment is critical during sepsis. While neutrophils are required to combat invading bacteria,excessive neutrophil recruitment contributes to tissue damage due to their arsenal of molecular weapons that do not distinguish between host and pathogen. Thus,neutrophil recruitment needs to be fine-tuned to ensure bacterial killing,while avoiding neutrophil-inflicted tissue damage. We recently showed that the actin-binding protein HS1 promotes neutrophil extravasation; and hypothesized that HS1 is also a critical regulator of sepsis progression. We evaluated the role of HS1 in a model of lethal sepsis induced by cecal-ligation and puncture. We found that septic HS1-deficient mice had a better survival rate compared to WT mice due to absence of lung damage. Lungs of septic HS1-deficient mice showed less inflammation,fibrosis,and vascular congestion. Importantly,systemic CLP-induced neutrophil recruitment was attenuated in the lungs,the peritoneum and the cremaster in the absence of HS1. Lungs of HS1-deficient mice produced significantly more interleukin-10. Compared to WT neutrophils,those HS1-deficient neutrophils that reached the lungs had increased surface levels of Gr-1,ICAM-1,and L-selectin. Interestingly,HS1-deficient neutrophils had similar F-actin content and phagocytic activity,but they failed to polymerize actin and deform in response to CXCL-1 likely explaining the reduced systemic neutrophil recruitment in HS1-deficient mice. Our data show that HS1 deficiency protects against sepsis by attenuating neutrophil recruitment to amounts sufficient to combat bacterial infection,but insufficient to induce tissue damage. 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

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

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

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

Identifying factors secreted by multiple myeloma (MM) cells that may contribute to MM tumor biology and progression is of the utmost importance. In this study,hepatoma-derived growth factor (HDGF) was identified as a protein present in extracellular vesicles (EVs) released from human MM cell lines (HMCLs). Investigation of the role of HDGF in MM cell biology revealed lower proliferation of HMCLs following HDGF knockdown and AKT phosphorylation following the addition of exogenous HDGF. Metabolic analysis demonstrated that HDGF enhances the already high glycolytic levels of HMCLs and significantly lowers mitochondrial respiration,indicating that HDGF may play a role in myeloma cell survival and/or act in a paracrine manner on cells in the bone marrow (BM) tumor microenvironment (ME). Indeed,HDGF polarizes macrophages to an M1-like phenotype and phenotypically alters na{\{i}}ve CD14+ monocytes to resemble myeloid-derived suppressor cells which are functionally suppressive. In summary HDGF is a novel factor in MM biology and may function to both maintain MM cell viability as well as modify the tumor ME." View Publication

BACKGROUND CD47/SIRP$\alpha$ axis is recognized as an innate immune checkpoint and emerging clinical data validate the interest of interrupting this pathway in cancer,particularly in hematological malignancies. In preclinical models,CD47/SIRP$\alpha$ blocking agents have been shown to mobilize phagocytic cells and trigger adaptive immune responses to eliminate tumors. Here,we describe the mechanisms afforded by a CD47xCD19 bispecific antibody (NI-1701) at controlling tumor growth in a mouse xenograft B-cell lymphoma model. METHODS The contribution of immune effector cell subsets behind the antitumor activity of NI-1701 was investigated using flow cytometry,transcriptomic analysis,and in vivo immune-cell depletion experiments. RESULTS We showed that NI-1701 treatment transformed the tumor microenvironment (TME) into a more anti-tumorigenic state with increased NK cells,monocytes,dendritic cells (DC) and MHCIIhi tumor-associated macrophages (TAMs) and decreased granulocytic myeloid-derived suppressor cells. Notably,molecular analysis of isolated tumor-infiltrating leukocytes following NI-1701 administration revealed an upregulation of genes linked to immune activation,including IFN$\gamma$ and IL-12b. Moreover,TAM-mediated phagocytosis of lymphoma tumor cells was enhanced in the TME in the presence of NI-1701,highlighting the role of macrophages in tumor control. In vivo cell depletion experiments demonstrated that both macrophages and NK cells contribute to the antitumor activity. In addition,NI-1701 enhanced dendritic cell-mediated phagocytosis of tumor cells in vitro,resulting in an increased cross-priming of tumor-specific CD8 T cells. CONCLUSIONS The study described the mechanisms afforded by the CD47xCD19 bispecific antibody,NI-1701,at controlling tumor growth in lymphoma mouse model. NI-1701 is currently being evaluated in a Phase I clinical trial for the treatment of refractory or relapsed B-cell lymphoma (NCT04806035). View Publication

Dominant inhibitory receptors for HLA class I (HLA-I) endow NK cells with high intrinsic responsiveness,a process termed licensing or education,but hinder their ability to kill HLA-I+ tumor cells. Cancer immunotherapy with adoptive transfer of NK cells must overcome inhibitory signals by such receptors to promote elimination of HLA-I+ tumor cells. As proof of concept,we show here that a chimeric antigen receptor (CAR) can be engineered to overcome inhibition by receptors for HLA-I and to promote lysis of HLA-I+ tumor cells by CAR-NK cells. The design of this NK-tailored CAR (NK-CAR) relied on the potent NK cell activation induced by the synergistic combination of NK receptors CD28H (CD28 homolog,TMIGD2) and 2B4 (CD244,SLAMF4). An NK-CAR consisting of the single-chain fragment variable (scFv) of a CD19 antibody,the CD28H transmembrane domain,and the fusion of CD28H,2B4,and TCR$\zeta$ signaling domains was compared to a third-generation T-cell CAR with a CD28-41BB-TCR$\zeta$ signaling domain. The NK-CAR delivered stronger activation signals to NK cells and induced more robust tumor cell lysis. Furthermore,such CAR-NK cells could overcome inhibition by HLA-E or HLA-C expressed on tumor cells. Therefore,engineering of CAR-NK cells that could override inhibition by HLA-I in patients undergoing cancer immunotherapy is feasible. This approach offers an attractive alternative to more complex strategies,such as genetic editing of inhibitory receptors in CAR-NK cells or treatment of patients with a combination of CAR-NK cells and checkpoint blockade with antibodies to inhibitory receptors. A significant benefit of inhibition-resistant NK-CARs is that NK cell inhibition would be overcome only during contact with targeted tumor cells and that HLA-I on healthy cells would continue to maintain NK cell responsiveness through licensing. View Publication

OBJECTIVE The prostate is metabolically unique: it produces high levels of citrate for secretion via a truncated tricarboxylic acid (TCA) cycle to maintain male fertility. In prostate cancer (PCa),this phenotype is reprogrammed,making it an interesting therapeutic target. However,how the truncated prostate TCA cycle works is still not completely understood. METHODS We optimized targeted metabolomics in mouse and human organoid models in ex vivo primary culture. We then used stable isotope tracer analyses to identify the pathways that fuel citrate synthesis. RESULTS First,mouse and human organoids were shown to recapitulate the unique citrate-secretory program of the prostate,thus representing a novel model that reproduces this unusual metabolic profile. Using stable isotope tracer analysis,several key nutrients were shown to allow the completion of the prostate TCA cycle,revealing a much more complex metabolic profile than originally anticipated. Indeed,along with the known pathway of aspartate replenishing oxaloacetate,glutamine was shown to fuel citrate synthesis through both glutaminolysis and reductive carboxylation in a GLS1-dependent manner. In human organoids,aspartate entered the TCA cycle at the malate entry point,upstream of oxaloacetate. Our results demonstrate that the citrate-secretory phenotype of prostate organoids is supported by the known aspartate-oxaloacetate-citrate pathway,but also by at least three additional pathways: glutaminolysis,reductive carboxylation,and aspartate-malate conversion. CONCLUSIONS Our results add a significant new dimension to the prostate citrate-secretory phenotype,with at least four distinct pathways being involved in citrate synthesis. Better understanding this distinctive citrate metabolic program will have applications in both male fertility as well as in the development of novel targeted anti-metabolic therapies for PCa. View Publication

We investigated genomic and transcriptomic changes in paired tumor samples of 29 in-house multiple myeloma (MM) patients and 28 patients from the MMRF CoMMpass study before and after treatment. A change in clonal composition was found in 46/57 (82%) of patients,and single-nucleotide variants (SNVs) increased from median 67 to 86. The highest increase in prevalence of genetic aberrations was found in RAS genes (60% to 72%),amp1q21 (18% to 35%),and TP53 (9% to 18%). The SBS-MM1 mutation signature was detected both in patients receiving high and low dose melphalan. A total of 2589 genes were differentially expressed between early and late samples (FDR?? View Publication

Extracellular acidification occurs in inflamed tissue and the tumor microenvironment; however,a systematic study on how pH sensing contributes to tissue homeostasis is lacking. In the present study,we examine cell type-specific roles of the pH sensor G protein-coupled receptor 65 (GPR65) and its inflammatory disease-associated Ile231Leu-coding variant in inflammation control. GPR65 Ile231Leu knock-in mice are highly susceptible to both bacterial infection-induced and T cell-driven colitis. Mechanistically,GPR65 Ile231Leu elicits a cytokine imbalance through impaired helper type 17 T cell (TH17 cell) and TH22 cell differentiation and interleukin (IL)-22 production in association with altered cellular metabolism controlled through the cAMP-CREB-DGAT1 axis. In dendritic cells,GPR65 Ile231Leu elevates IL-12 and IL-23 release at acidic pH and alters endo-lysosomal fusion and degradation capacity,resulting in enhanced antigen presentation. The present study highlights GPR65 Ile231Leu as a multistep risk factor in intestinal inflammation and illuminates a mechanism by which pH sensing controls inflammatory circuits and tissue homeostasis. View Publication