技术资料

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

PURPOSE Chronic inflammation contributes to tumor initiation,progression,and immune escape. Neutrophils are the major component of inflammatory response and participate in the tumorigenesis process. However,compared to other immune cells in the tumor microenvironment of laryngeal squamous cell carcinoma (LSCC),neutrophils,especially the tumor-associated neutrophils (TANs),have not yet been comprehensively explored. The mechanism for regulating the crosstalk between TANs and tumor cells still remains unclear. MATERIALS AND METHODS The distribution profiles and phenotypic features of neutrophils and other inflammatory immune cell populations from a large LSCC patient cohort were systemically analyzed. Co-culturing of peripheral blood associated neutrophils (PANs) and TANs with PBMCs was performed,and the immunosuppression effect on T-cells was examined. RESULTS LSCC microenvironment is highly inflammatory with remarkable TANs infiltration,which is often associated with unfavorable prognosis and advanced clinical stage. We find that TANs in LSCC display morphologically immature and lower apoptosis,exhibit distinctively immunosuppressive phenotype of high PD-L1,and suppress CD8+ T lymphocytes proliferation and activation. We subsequently discover that PD-L1+TANs induced by LSCC-derived GM-CSF potently impair CD8+ T-cells proliferation and cytokines production function,which are partially blocked by a PD-L1-neutralizing antibody. Clinical data further support GM-CSF as an unfavorable prognostic biomarker and reveal a potential association with inflammatory immune cell infiltration,in particular neutrophils. CONCLUSION Tumor-infiltrating PD-L1+ neutrophils induced by LSCC-derived GM-CSF suppress T cell proliferation and activation in the inflammatory microenvironment of LSCC and predict unfavorable prognosis. These TANs cripple antitumor T cell immunity and promote tumor progression. Our findings provide a basis for targeting PD-L1+TANs or GM-CSF as a new immunotherapeutic strategy for LSCC. View Publication

BACKGROUND B1a and B1b lymphocytes produce IgM that inactivates oxidation-specific epitopes (IgMOSE) on LDL (low-density lipoprotein) and protects against atherosclerosis. Loss of ID3 (inhibitor of differentiation 3) in B cells selectively promotes B1b but not B1a cell numbers,leading to higher IgMOSE production and reduction in atherosclerotic plaque formation. Yet,the mechanism underlying this regulation remains unexplored. METHODS Bulk RNA sequencing was utilized to identify differentially expressed genes in B1a and B1b cells from Id3KO and Id3WT mice. CRISPR/Cas9 and lentiviral genome editing coupled with adoptive transfer were used to identify key Id3-dependent signaling pathways regulating B1b cell proliferation and the impact on atherosclerosis. Biospecimens from humans with advanced coronary artery disease imaging were analyzed to translate murine findings to human subjects with coronary artery disease. RESULTS Through RNA sequencing,P62 was found to be enriched in Id3KO B1b cells. Further in vitro characterization reveals a novel role for P62 in mediating BAFF (B-cell activating factor)-induced B1b cell proliferation through interacting with TRAF6 (tumor necrosis factor receptor 6) and activating NF-$\kappa$B (nuclear factor kappa B),leading to subsequent C-MYC (C-myelocytomatosis) upregulation. Promoter-reporter assays reveal that Id3 inhibits the E2A protein from activating the P62 promoter. Mice adoptively transferred with B1 cells overexpressing P62 exhibited an increase in B1b cell number and IgMOSE levels and were protected against atherosclerosis. Consistent with murine mechanistic findings,P62 expression in human B1 cells was significantly higher in subjects harboring a function-impairing single nucleotide polymorphism (SNP) at rs11574 position in the ID3 gene and directly correlated with plasma IgMOSE levels. CONCLUSIONS This study unveils a novel role for P62 in driving BAFF-induced B1b cell proliferation and IgMOSE production to attenuate diet-induced atherosclerosis. Results identify a direct role for Id3 in antagonizing E2A from activating the p62 promoter. Moreover,analysis of putative human B1 cells also implicates these pathways in coronary artery disease subjects,suggesting P62 as a new immunomodulatory target for treating atherosclerosis. View Publication

Pre-leukemic clones carrying DNMT3A mutations have a selective advantage and an inherent chemoresistance,however the basis for this phenotype has not been fully elucidated. Mutations affecting the gene TP53 occur in pre-leukemic hematopoietic stem/progenitor cells (preL-HSPC) and lead to chemoresistance. Many of these mutations cause a conformational change and some of them were shown to enhance self-renewal capacity of preL-HSPC. Intriguingly,a misfolded P53 was described in AML blasts that do not harbor mutations in TP53,emphasizing the dynamic equilibrium between wild-type (WT) and pseudo-mutant" conformations of P53. By combining single cell analyses and P53 conformation-specific monoclonal antibodies we studied preL-HSPC from primary human DNMT3A-mutated AML samples. We found that while leukemic blasts express mainly the WT conformation in preL-HSPC the pseudo-mutant conformation is the dominant. HSPC from non-leukemic samples expressed both conformations to a similar extent. In a mouse model we found a small subset of HSPC with a dominant pseudo-mutant P53. This subpopulation was significantly larger among DNMT3AR882H-mutated HSPC suggesting that while a pre-leukemic mutation can predispose for P53 misfolding additional factors are involved as well. Treatment with a short peptide that can shift the dynamic equilibrium favoring the WT conformation of P53 specifically eliminated preL-HSPC that had dysfunctional canonical P53 pathway activity as reflected by single cell RNA sequencing. Our observations shed light upon a possible targetable P53 dysfunction in human preL-HSPC carrying DNMT3A mutations. This opens new avenues for leukemia prevention." View Publication