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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

Allergic airway inflammation is a universal airway disease that is driven by hyperresponsiveness to inhaled allergens. Group 2 innate lymphoid cells (ILC2s) produce copious amounts of type 2 cytokines,which lead to allergic airway inflammation. Here,we discovered that both peripheral blood of human and mouse lung ILC2s express the endothelin-A receptor (ETAR),and the expression level of ETAR was dramatically induced upon interleukin-33 (IL-33) treatment. Subsequently,both preventive and therapeutic effects of BQ123,an ETAR antagonist,on allergic airway inflammation were observed,which were associated with decreased proliferation and type 2 cytokine productions by ILC2s. Furthermore,ILC2s from BQ123 treatment were found to be functionally impaired in response to an interleukin IL-33 challenged. And BQ123 treatment also affected the phosphorylation level of the extracellular signal-regulated kinase (ERK),as well as the level of GATA binding protein 3 (GATA3) in activated ILC2s. Interestingly,after BQ123 treatment,both mouse and human ILC2s in vitro exhibited decreased function and downregulation of ERK signaling and GATA3 stability. These observations imply that ETAR is an important regulator of ILC2 function and may be involved in ILC2-driven pulmonary inflammation. Therefore,blocking ETAR may be a promising therapeutic strategy for allergic airway inflammation. View Publication

Immunological non-responders (InRs) are HIV-infected individuals in whom the administration of combination antiretroviral therapy (cART),although successful in suppressing viral replication,cannot properly reconstitute patient circulating CD4+ T-cell number to immunocompetent levels. The causes for this immunological failure remain elusive,and no therapeutic strategy is available to restore a proper CD4+ T-cell immune response in these individuals. We have recently demonstrated that platelets harboring infectious HIV are a hallmark of InR,and we now report on a causal connection between HIV-containing platelets and T-cell dysfunctions. We show here that in vivo,platelet-T-cell conjugates are more frequent among CD4+ T cells in InRs displaying HIV-containing platelets (<350 CD4+ T cells/$\mu$l blood for >1 year) as compared with healthy donors or immunological responders (IRs; >350 CD4+ T cells/$\mu$l). This contact between platelet containing HIV and T cell in the conjugates is not infectious for CD4+ T cells,as coculture of platelets from InRs containing HIV with healthy donor CD4+ T cells fails to propagate infection to CD4+ T cells. In contrast,when macrophages are the target of platelets containing HIV from InRs,macrophages become infected. Differential transcriptomic analyses comparing InR and IR CD4+ T cells reveal an upregulation of genes involved in both aerobic and anaerobic glycolysis in CD4+ T cells from InR vs. IR individuals. Accordingly,InR platelets containing HIV induce a dysfunctional increase in glycolysis-mediated energy production in CD4+ T cells as compared with T cells cocultured with IR platelets devoid of virus. In contrast,macrophage metabolism is not affected by platelet contact. Altogether,this brief report demonstrates a direct causal link between presence of HIV in platelets and T-cell dysfunctions typical of InR,contributing to devise a platelet-targeted therapy for improving immune reconstitution in these individuals. View Publication

BACKGROUND With the rapid development of immune checkpoint inhibitors and neoantigen (NeoV)-based personalized tumor vaccines,tumor immunotherapy has shown promising therapeutic results. However,the limited efficacy of available tumor vaccines impedes the development of personalized tumor immunotherapy. In this study,we developed a novel tumor vaccine system and proposed combined therapeutic strategies for improving treatment effects. METHODS We developed a novel tumor vaccine system comprising a newly synthesized peptidic microarchitecture (PMA) with high assembly efficacy. The PMA-trapped neoantigen vaccine was developed to codeliver tumor neoantigen and the Toll-like receptor 9 agonist CpG (NeoV),abbreviated as PMA-NeoV. A microfluidic chip was used to produce PMA particles in a uniform and precise manner. Vaccine effectiveness was investigated both in vitro and in vivo. The combined immunotherapeutic effect of PMA-NeoV with anti-programmed cell death ligand 1 antibody (aPD-L1) or with the phosphatidylinositol 3?‘kinase $\gamma$ (PI3K$\gamma$) inhibitor IPI-549 was further tested in MC38 mouse tumor model. RESULTS PMA-NeoV not only promoted codelivery of the tumor vaccine but also potentiated vaccine immunogenicity. Moreover,compared with free NeoV,PMA-NeoV significantly increased the number of tumor-infiltrating lymphocytes,promoted the neoantigen-specific systemic immune response,and suppressed murine colon MC38 tumor growth. Furthermore,PMA-NeoV increased the expression of programmed cell death receptor-1 on T lymphocytes,and in combination with aPD-L1 eradicated seven of eight MC38 tumors by rescuing exhausted T lymphocytes. Moreover,we combined the PMA-NeoV with the IPI-549,a molecular switch that controls immune suppression,and found that this combination significantly suppressed tumor growth and eradicated five of eight inoculated tumors,by switching suppressive macrophages to their active state and activating T cells to prime a robust tumor immune microenvironment. CONCLUSIONS We developed a tumor vaccine delivery system and presented a promising personalized tumor vaccine-based therapeutic regimen in which a tumor vaccine delivery system is combined with an aPD-L1 or PI3K$\gamma$ inhibitor to improve tumor immunotherapy outcomes. View Publication

Immunosuppressive factors in the tumor microenvironment (TME) impair T cell function and limit the antitumor immune response. T cell surface receptors and surface proteins that influence interactions and function in the TME are proven targets for cancer immunotherapy. However,how the entire surface proteome remodels in primary human T cells in response to specific suppressive factors in the TME remains to be broadly and systematically characterized. Here,using a reductionist cell culture approach with primary human T cells and stable isotopic labeling with amino acids in cell culture-based quantitative cell surface capture glycoproteomics,we examined how two immunosuppressive TME factors,regulatory T cells (Tregs) and hypoxia,globally affect the activated CD8+ surface proteome (surfaceome). Surprisingly,coculturing primary CD8+ T cells with Tregs only modestly affected the CD8+ surfaceome but did partially reverse activation-induced surfaceomic changes. In contrast,hypoxia drastically altered the CD8+ surfaceome in a manner consistent with both metabolic reprogramming and induction of an immunosuppressed state. The CD4+ T cell surfaceome similarly responded to hypoxia,revealing a common hypoxia-induced surface receptor program. Our surfaceomics findings suggest that hypoxic environments create a challenge for T cell activation. These studies provide global insight into how Tregs and hypoxia remodel the T cell surfaceome and we believe represent a valuable resource to inform future therapeutic efforts to enhance T cell function. View Publication

Intrasynovial flexor tendon lacerations of the hand are clinically problematic,typically requiring operative repair and extensive rehabilitation. The small-molecule connective tissue growth factor (CTGF) mimics,oxotremorine M (Oxo-M) and 4-PPBP maleate (4-PPBP),have been shown to improve tendon healing in small animal models by stimulating the expansion and differentiation of perivascular CD146+ cells. To enhance intrasynovial flexor tendon healing,small-molecule CTGF mimics were delivered to repaired canine flexor tendons via porous sutures. In vitro studies demonstrated that Oxo-M and 4-PPBP retained their bioactivity and could be released from porous sutures in a sustained manner. However,in vivo delivery of the CTGF mimics did not improve intrasynovial tendon healing. Histologic analyses and expression of tenogenic,extracellular matrix,inflammation,and remodeling genes showed similar outcomes in treated and untreated repairs across two time points. Although in vitro experiments revealed that CTGF mimics stimulated robust responses in extrasynovial tendon cells,there was no response in intrasynovial tendon cells,explaining the lack of in vivo effects. The results of the current study indicate that therapeutic strategies for tendon repair must carefully consider the environment and cellular makeup of the particular tendon for improving the healing response. View Publication