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Regulatory T cells (Tregs) are capable of inhibiting the proliferation,activation and function of T cells and play an important role in impeding the immune response to cancer. In chronic lymphocytic leukemia (CLL) a dysfunctional immune response and elevated percentage of effector-like phenotype Tregs have been described. In this study,using the Eµ-TCL1 mouse model of CLL,we evaluated the changes in the Tregs phenotype and their expansion at different stages of leukemia progression. Importantly,we show that Tregs depletion in DEREG mice triggered the expansion of new anti-leukemic cytotoxic T cell clones leading to leukemia eradication. In TCL1 leukemia-bearing mice we identified and characterized a specific Tregs subpopulation,the phenotype of which suggests its role in the formation of an immunosuppressive microenvironment,supportive for leukemia survival and proliferation. This observation was also confirmed by the gene expression profile analysis of these TCL1-specific Tregs. The obtained data on Tregs are consistent with those described so far,however,above all show that the changes in the Tregs phenotype described in CLL result from the formation of a specific,described in this study Tregs subpopulation. In addition,functional tests revealed the ability of Tregs to inhibit T cells that recognize model antigens expressed by leukemic cells. Moreover,inhibition of Tregs with a MALT1 inhibitor provided a therapeutic benefit,both as monotherapy and also when combined with an immune checkpoint inhibitor. Altogether,activation of Tregs appears to be crucial for CLL progression. View Publication

Diminishing homeostatic proliferation of memory T cells is essential for improving the efficacy of lymphoablation in transplant recipients. Our previous studies in a mouse heart transplantation model established that B lymphocytes secreting proinflammatory cytokines are critical for T cell recovery after lymphoablation. The goal of the current study was to identify mediators of B cell activation following lymphoablation in allograft recipients. Transcriptome analysis revealed that macrophage-inducible C-type lectin (Mincle,Clec4e) expression is up-regulated in B cells from heart allograft recipients treated with murine anti-thymocyte globulin (mATG). Recipient Mincle deficiency diminishes B cell production of pro-inflammatory cytokines and impairs T lymphocyte reconstitution. Mixed bone marrow chimeras lacking Mincle only in B lymphocytes have similar defects in T cell recovery. Conversely,treatment with a synthetic Mincle ligand enhances T cell reconstitution after lymphoablation in non-transplanted mice. Treatment with agonistic CD40 mAb facilitates T cell reconstitution in CD4 T cell-depleted,but not in Mincle-deficient,recipients indicating that CD40 signaling induces T cell proliferation via a Mincle-dependent pathway. These findings are the first to identify an important function of B cell Mincle as a sensor of damage-associated molecular patterns released by the graft and demonstrate its role in clinically relevant settings of organ transplantation. View Publication

BACKGROUND Radiotherapy enhances antitumor immunity. However,it also induces immunosuppressive responses,which are major hurdles for an effective treatment. Thus,targeting the immunosuppressive tumor microenvironment is essential for enhancing the antitumor immunity after radiotherapy. Retrospective studies show that a blockade of PI3K$\delta$ and/or $\gamma$,which are abundant in leukocytes,exhibits antitumor immune response by attenuating activity of immune suppressive cells,however,the single blockade of PI3K$\delta$ or $\gamma$ is not sufficient to completely eliminate solid tumor. METHODS We used BR101801,PI3K$\delta$/$\gamma$ inhibitor in the CT-26 syngeneic mouse model with a subcutaneously implanted tumor. BR101801 was administered daily,and the target tumor site was locally irradiated. We monitored the tumor growth regularly and evaluated the immunological changes using flow cytometry,ELISpot,and transcriptional analysis. RESULTS This study showed that BR101801 combined with irradiation promotes systemic antitumor immunity and abscopal response by attenuating the activity of immune suppressive cells in the CT-26 tumor model. BR101801 combined with irradiation systemically reduced the proliferation of regulatory T cells (Tregs) and enhanced the number of tumor-specific CD8$\alpha$+ T cells in the tumor microenvironment,thereby leading to tumor regression. Furthermore,the high ratio of CD8$\alpha$+ T cells to Tregs was maintained for 14 days after irradiation,resulting in remote tumor regression in metastatic lesions,the so-called abscopal effect. Moreover,our transcriptomic analysis showed that BR101801 combined with irradiation promoted the immune-stimulatory tumor microenvironment,suggesting that the combined therapy converts immunologically cold tumors into hot one. CONCLUSIONS Our data suggest the first evidence that PI3K$\delta$/$\gamma$ inhibition combined with irradiation promotes systemic antitumor immunity against solid tumors,providing the preclinical result of the potential use of PI3K$\delta$/$\gamma$ inhibitor as an immune-regulatory radiosensitizer. 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

Measles virus envelope pseudotyped LV (MV-LV) can achieve high B cell transduction rates (up to 50%),but suffers from low titers. To overcome current limitations,we developed an optimized MV-LV production protocol that achieved consistent B cell transduction efficiency up to 75%. We detail this protocol along with analytical assays to assess the results of MV-LV mediated B cell transduction,including flow cytometry for B cell phenotypic characterization and measurement of transduction efficiency,and ddPCR for VCN analysis. View Publication

BACKGROUND Seminal plasma contains signaling molecules capable of modulating the maternal immune environment to support implantation and pregnancy. Prior studies indicated that seminal plasma induces changes in gene transcription of maternal immune cells. Reduced immune suppressive capacity may lead to pregnancy loss. The aim of this study was to investigate the immunomodulating effects of seminal plasma on T cells and monocytes in the context of recurrent pregnancy loss (RPL). METHODS Female T cells and monocytes were incubated with seminal plasma of 20 males in unexplained RPL couples (RPL males) and of 11 males whose partners had ongoing pregnancies (control males). The effect of seminal plasma on messenger RNA (mRNA) expression of immune cells was measured. Levels of mRNA expression were related to key signaling molecules present in the seminal plasma. Agglomerative hierarchical cluster analysis was performed on seminal plasma expression profiles and on mRNA expression profiles. RESULTS Expression of CD25 and anti-inflammatory IL-10 by female T cells was significantly lower after stimulation with seminal plasma of RPL males compared to control males. Female monocytes treated with seminal plasma of RPL males showed an immune activation signature of relatively elevated HLA-DR expression. Expression of these T cell and monocyte components was particularly correlated with the amounts of TGF-$\beta$ and VEGF in the seminal plasma. CONCLUSION Our findings indicate that seminal plasma has immunomodulating properties on female immune cells compatible with the induction of a more regulatory phenotype,which may be impaired in cases of unexplained RPL. View Publication

Macrophages are mediators of inflammation having an important role in the pathogenesis of cardiovascular diseases. Recently,a pro-inflammatory subpopulation,known as metabolically activated macrophages (MMe),has been described in conditions of obesity and metabolic syndrome where they are known to release cytokines that can promote insulin resistance. Dyslipidemia represents an important feature in metabolic syndrome and corresponds to one of the main modifiable risk factors for the development of cardiovascular diseases. Circulating monocytes can differentiate into macrophages under certain conditions. They correspond to a heterogeneous population,which include inflammatory and anti-inflammatory subsets; however,there is a wide spectrum of phenotypes. Therefore,we decided to investigate whether the metabolic activated monocyte (MoMe) subpopulation is already present under dyslipidemia conditions. Secondly,we assessed whether different levels of cholesterol and triglycerides play a role in the polarization towards the metabolic phenotype (MMe) of macrophages. Our results indicate that MoMe cells are found in both healthy and dyslipidemia patients,with cells displaying the following metabolic phenotype: CD14varCD36+ABCA1+PLIN2+. Furthermore,the percentages of CD14++CD68+CD80+ pro-inflammatory monocytes are higher in dyslipidemia than in healthy subjects. When analysing macrophage differentiation,we observed that MMe percentages were higher in the dyslipidemia group than in healthy subjects. These MMe have the ability to produce high levels of IL-6 and the anti-inflammatory cytokine IL-10. Furthermore,ABCA1 expression in MMe correlates with LDL serum levels. Our study highlights the dynamic contributions of metabolically activated macrophages in dyslipidemia,which may have a complex participation in low-grade inflammation due to their pro- and anti-inflammatory function. View Publication

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

T cell inhibitory receptors can regulate the proliferation or function of T cells by binding to their ligands and present a unique opportunity to manage destructive immune responses during porcine islet xenotransplantation. We applied ex vivo porcine islet xenotransplantation and in vitro mixed lymphocyte-islet reaction models to assess immune checkpoint receptor expression profiles in recipient T cells,investigated whether CTLA4 or VISTA immunoglobulin (Ig) combination therapy alone could suppress porcine islet xenograft rejection and further analyzed its potential immune tolerance mechanism. Recipient T cells expressed moderate to high levels of CTLA4,PD-1,TIGIT and VISTA,and the frequency of CTLA4+ CD4+,TIGIT+ CD4+,VISTA+ CD4+ and VISTA+ CD8+ T cells was positively correlated with porcine islet xenograft survival time in xenotransplant recipients. Combined treatment with CTLA4Ig and VISTAIg selectively inhibited recipient CD4+ T cell hyper-responsiveness and proinflammatory cytokine production and significantly delayed xenograft rejection. SOCS1 deficiency in CD4+ T cells stimulated by xenogeneic islets facilitated hyper-responsiveness and abolished the suppressive effect of combination therapy on recipient T cell-mediated porcine islet damage in vivo and in vitro. Further mechanistic studies revealed that combined treatment significantly induced SOCS1 expression and inhibited the Jak-STAT signalling pathway in wild-type recipient CD4+ T cells stimulated by xenogeneic islets,whereas SOCS1 deficiency resulted in Jak-STAT signalling pathway activation in recipient CD4+ T cells. We demonstrated a major role for CTLA4 and VISTA as key targets in CD4+ T cell hyper-responsiveness and porcine islet xenograft rejection. The selective inhibition of CD4+ T cell immunity by CTLA4Ig/VISTAIg is based on SOCS1-dependent signalling. View Publication

Therapeutic IL-12 has demonstrated the ability to reduce local immune suppression in preclinical models,but clinical development has been limited by severe inflammation-related adverse events with systemic administration. Here,we show that potent immunologic tumor control of established syngeneic carcinomas can be achieved by i.t. administration of a tumor-targeted IL-12 antibody fusion protein (NHS-rmIL-12) using sufficiently low doses to avoid systemic toxicity. Single-cell transcriptomic analysis and ex vivo functional assays of NHS-rmIL-12-treated tumors revealed reinvigoration and enhanced proliferation of exhausted CD8+ T lymphocytes,induction of Th1 immunity,and a decrease in Treg number and suppressive capacity. Similarly,myeloid cells transitioned toward inflammatory phenotypes and displayed reduced suppressive capacity. Cell type-specific IL-12 receptor-KO BM chimera studies revealed that therapeutic modulation of both lymphoid and myeloid cells is required for maximum treatment effect and tumor cure. Study of single-cell data sets from human head and neck carcinomas revealed IL-12 receptor expression patterns similar to those observed in murine tumors. These results describing the diverse mechanisms underlying tumor-directed IL-12-induced antitumor immunity provide the preclinical rationale for the clinical study of i.t. NHS-IL-12. View Publication

C-Type lectin receptor 5A (CLEC5A) is a spleen tyrosine kinase- (Syk-) coupled pattern recognition receptor expressed on myeloid cells and involved in the innate immune response to viral and bacterial infections. Activation of the CLEC5A receptor with pathogen-derived antigens leads to a secretion of proinflammatory mediators such as TNF-$\alpha$ and IL-6 that may provoke a systemic cytokine storm,and CLEC5A gene polymorphisms are associated with the severity of DV infection. In addition,the CLEC5A receptor was mentioned in the context of noninfectious disorders like chronic obstructive pulmonary disease (COPD) or arthritis. Altogether,CLEC5A may be considered as an innate immune checkpoint capable to amplify proinflammatory signals,and this way contributes to infection or to aseptic inflammation. In this study,we determined CLEC5A receptor expression on different macrophage subsets (in vitro and ex vivo) and the functional consequences of its activation in aseptic conditions. The CLEC5A surface expression appeared the highest on proinflammatory M1 macrophages while intermediate on tumor-associated phenotypes (M2c or TAM). In contrast,the CLEC5A expression on ex vivo-derived alveolar macrophages from healthy donors or macrophages from ovarian cancer patients was hardly detectable. Targeting CLEC5A on noninflammatory macrophages with an agonistic $\alpha$-CLEC5A antibody triggered a release of proinflammatory cytokines,resembling a response to dengue virus,and led to phenotypic changes in myeloid cells that may suggest their reprogramming towards a proinflammatory phenotype,e.g.,upregulation of CD80 and downregulation of CD163. Interestingly,the CLEC5A agonist upregulated immune-regulatory molecules like CD206,PD-L1,and cytokines like IL-10,macrophage-derived chemokine (MDC/CCL22),and thymus and activation chemokine (TARC/CCL17) which are associated with an anti-inflammatory or a protumorigenic macrophage phenotype. In the absence of concomitant pathogenic or endogenous danger signals,the CLEC5A receptor activation did not amplify an autologous T cell response,which may represent a protective innate mechanism to avoid an undesirable autoimmune adaptive response. View Publication

As a potential source of myofibroblasts,pericytes may play a role in human peritoneal fibrosis. The culture of primary vascular pericytes in animals has previously been reported,most of which are derived from cerebral and retinal microvasculature. Here,in the field of peritoneal dialysis,we describe a method to isolate and characterize mouse peritoneal microvascular pericytes. The mesenteric tissues of five mice were collected and digested by type II collagenase and type I DNase. After cell attachment,the culture fluid was replaced with pericyte-conditioned medium. Pericytes with high purity (99.0%) could be isolated by enzymatic disaggregation combined with conditional culture and magnetic activated cell sorting. The primary cells were triangular or polygonal with protrusions,and confluent cell culture could be established in 3??days. The primary pericytes were positive for platelet-derived growth factor receptor-$\beta$,$\alpha$-smooth muscle actin,neuron-glial antigen 2,and CD13. Moreover,they promoted formation of endothelial tubes,and pericyte-myofibroblast transition occurred after treatment with transforming growth factor-$\beta$1. In summary,we describe here a reproducible isolation protocol for primary peritoneal pericytes,which may be a powerful tool for in??vitro peritoneal fibrosis studies. View Publication