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Emerging evidence suggests an immunosuppressive role of altered tumor glycosylation due to downregulation of innate immune responses via immunoregulatory Siglecs. In contrast,human T cells,a major anticancer effector cell,only rarely express Siglecs. However,here,we report that the majority of intratumoral,but not peripheral blood,cytotoxic CD8+ T cells expressed Siglec-9 in melanoma. We identified Siglec-9+ CD8+ T cells as a subset of effector memory cells with high functional capacity and signatures of clonal expansion. This cytotoxic T-cell subset was functionally inhibited in the presence of Siglec-9 ligands or by Siglec-9 engagement by specific antibodies. TCR signaling pathways and key effector functions (cytotoxicity,cytokine production) of CD8+ T cells were suppressed by Siglec-9 engagement,which was associated with the phosphorylation of the inhibitory protein tyrosine phosphatase SHP-1,but not SHP-2. Expression of cognate Siglec-9 ligands was observed on the majority of tumor cells in primary and metastatic melanoma specimens. Targeting the tumor-restricted,glycosylation-dependent Siglec-9 axis may unleash this intratumoral T-cell subset,while confining T-cell activation to the tumor microenvironment. View Publication

The types and magnitude of Ag-specific immune responses can be determined by the functional plasticity of dendritic cells (DCs). However,how DCs display functional plasticity and control host immune responses have not been fully understood. In this study,we report that ligation of DC-asialoglycoprotein receptor (DC-ASGPR),a C-type lectin receptor (CLR) expressed on human DCs,resulted in rapid activation of Syk,followed by PLCgamma2 and PKCdelta engagements. However,different from other Syk-coupled CLRs,including Dectin-1,signaling cascade through DC-ASGPR did not trigger NF-kappaB activation. Instead,it selectively activated MAPK ERK1/2 and JNK. Rapid and prolonged phosphorylation of ERK1/2 led to sequential activation of p90RSK and CREB,which consequently bound to IL10 promoter and initiated cytokine expression. In addition,DC-ASGPR ligation activated Akt,which differentially regulated the activities of GSK-3alpha/beta and beta-catenin and further contributed to IL-10 expression. Our observations demonstrate that DC-ASGPR induces IL-10 expression via an intrinsic signaling pathway,which provides a molecular explanation for DC-ASGPR-mediated programing of DCs to control host immune responses. View Publication

PURPOSE Targeting nonspecific,tumor-associated antigens (TAA) with chimeric antigen receptors (CAR) requires specific attention to restrict possible detrimental on-target/off-tumor effects. A reduced affinity may direct CAR-engineered T (CAR-T) cells to tumor cells expressing high TAA levels while sparing low expressing normal tissues. However,decreasing the affinity of the CAR-target binding may compromise the overall antitumor effects. Here,we demonstrate the prime importance of the type of intracellular signaling on the function of low-affinity CAR-T cells. EXPERIMENTAL DESIGN We used a series of single-chain variable fragments (scFv) with five different affinities targeting the same epitope of the multiple myeloma-associated CD38 antigen. The scFvs were incorporated in three different CAR costimulation designs and we evaluated the antitumor functionality and off-tumor toxicity of the generated CAR-T cells in vitro and in vivo. RESULTS We show that the inferior cytotoxicity and cytokine secretion mediated by CD38 CARs of very low-affinity (Kd {\textless} 1.9 × 10-6 mol/L) bearing a 4-1BB intracellular domain can be significantly improved when a CD28 costimulatory domain is used. Additional 4-1BB signaling mediated by the coexpression of 4-1BBL provided the CD28-based CD38 CAR-T cells with superior proliferative capacity,preservation of a central memory phenotype,and significantly improved in vivo antitumor function,while preserving their ability to discriminate target antigen density. CONCLUSIONS A combinatorial costimulatory design allows the use of very low-affinity binding domains (Kd {\textless} 1 mumol/L) for the construction of safe but also optimally effective CAR-T cells. Thus,very-low-affinity scFvs empowered by selected costimulatory elements can enhance the clinical potential of TAA-targeting CARs. View Publication

The upper respiratory tract is continuously exposed to a vast array of potentially pathogenic viruses and bacteria. Influenza A virus (IAV) has particular synergism with the commensal bacterium Streptococcus pneumoniae in this niche,and co-infection exacerbates pathogenicity and causes significant mortality. However,it is not known whether this synergism is associated with a direct interaction between the two pathogens. We have previously reported that co-administration of a whole-inactivated IAV vaccine (gamma-Flu) with a whole-inactivated pneumococcal vaccine (gamma-PN) enhances pneumococcal-specific responses. In this study,we show that mucosal co-administration of gamma-Flu and gamma-PN similarly augments IAV-specific immunity,particularly tissue-resident memory cell responses in the lung. In addition,our in vitro analysis revealed that S. pneumoniae directly interacts with both gamma-Flu and with live IAV,facilitating increased uptake by macrophages as well as increased infection of epithelial cells by IAV. These observations provide an additional explanation for the synergistic pathogenicity of IAV and S. pneumoniae,as well as heralding the prospect of exploiting the phenomenon to develop better vaccine strategies for both pathogens. View Publication

Precise control of Wnt signaling is necessary for immune system development. In this study,we detected severely impaired development of all lymphoid lineages in mice,resulting from an N-ethyl-N-nitrosourea-induced mutation in the limb region 1-like gene (Lmbr1l),which encodes a membrane-spanning protein with no previously described function in immunity. The interaction of LMBR1L with glycoprotein 78 (GP78) and ubiquitin-associated domain-containing protein 2 (UBAC2) attenuated Wnt signaling in lymphocytes by preventing the maturation of FZD6 and LRP6 through ubiquitination within the endoplasmic reticulum and by stabilizing destruction complex" proteins. LMBR1L-deficient T cells exhibited hallmarks of Wnt/beta-catenin activation and underwent apoptotic cell death in response to proliferative stimuli. LMBR1L has an essential function during lymphopoiesis and lymphoid activation acting as a negative regulator of the Wnt/beta-catenin pathway." View Publication

Multiple myeloma (MM) is a tumor of plasma cells (PCs). Due to the intense immunoglobulin secretion,PCs are prone to endoplasmic reticulum stress and activate several stress-managing pathways,including autophagy. Indeed,autophagy deregulation is maladaptive for MM cells,resulting in cell death. CK1alpha,a pro-survival kinase in MM,has recently been involved as a regulator of the autophagic flux and of the transcriptional competence of the autophagy-related transcription factor FOXO3a in several cancers. In this study,we investigated the role of CK1alpha in autophagy in MM. To study the autophagic flux we generated clones of MM cell lines expressing the mCherry-eGFP-LC3B fusion protein. We observed that CK1 inhibition with the chemical ATP-competitive CK1 alpha/delta inhibitor D4476 resulted in an impaired autophagic flux,likely due to an alteration of lysosomes acidification. However,D4476 caused the accumulation of the transcription factor FOXO3a in the nucleus,and this was paralleled by the upregulation of mRNA coding for autophagic genes. Surprisingly,silencing of CK1alpha by RNA interference triggered the autophagic flux. However,FOXO3a did not shuttle into the nucleus and the transcription of autophagy-related FOXO3a-dependent genes was not observed. Thus,while the chemical inhibition with the dual CK1alpha/delta inhibitor D4476 induced cell death as a consequence of an accumulation of ineffective autophagic vesicles,on the opposite,CK1alpha silencing,although it also determined apoptosis,triggered a full activation of the early autophagic flux,which was then not supported by the upregulation of autophagic genes. Taken together,our results indicate that the family of CK1 kinases may profoundly influence MM cells survival also through the modulation of the autophagic pathway. View Publication

A proposed strategy to cure HIV uses latency-reversing agents (LRAs) to reactivate latent proviruses for purging HIV reservoirs. A variety of LRAs have been identified,but none has yet proven effective in reducing the reservoir size in vivo. Nanocarriers could address some major challenges by improving drug solubility and safety,providing sustained drug release,and simultaneously delivering multiple drugs to target tissues and cells. Here,we formulated hybrid nanocarriers that incorporate physicochemically diverse LRAs and target lymphatic CD4+ T cells. We identified one LRA combination that displayed synergistic latency reversal and low cytotoxicity in a cell model of HIV and in CD4+ T cells from virologically suppressed patients. Furthermore,our targeted nanocarriers selectively activated CD4+ T cells in nonhuman primate peripheral blood mononuclear cells as well as in murine lymph nodes,and substantially reduced local toxicity. This nanocarrier platform may enable new solutions for delivering anti-HIV agents for an HIV cure. View Publication

Chronic hepatitis C virus (HCV) infection causes generalized CD8+ T cell impairment,not limited to HCV-specific CD8+ T-cells. Liver-infiltrating monocyte-derived macrophages (MDMs) contribute to the local micro-environment and can interact with and influence cells routinely trafficking through the liver,including CD8+ T-cells. MDMs can be polarized into M1 (classically activated) and M2a,M2b,and M2c (alternatively activated) phenotypes that perform pro- and anti-inflammatory functions,respectively. The impact of chronic HCV infection on MDM subset functions is not known. Our results show that M1 cells generated from chronic HCV patients acquire M2 characteristics,such as increased CD86 expression and IL-10 secretion,compared to uninfected controls. In contrast,M2 subsets from HCV-infected individuals acquired M1-like features by secreting more IL-12 and IFN-gamma. The severity of liver disease was also associated with altered macrophage subset differentiation. In co-cultures with autologous CD8+ T-cells from controls,M1 macrophages alone significantly increased CD8+ T cell IFN-gamma expression in a cytokine-independent and cell-contact-dependent manner. However,M1 macrophages from HCV-infected individuals significantly decreased IFN-gamma expression in CD8+ T-cells. Therefore,altered M1 macrophage differentiation in chronic HCV infection may contribute to observed CD8+ T-cell dysfunction. Understanding the immunological perturbations in chronic HCV infection will lead to the identification of therapeutic targets to restore immune function in HCV+ individuals,and aid in the mitigation of associated negative clinical outcomes. View Publication

The T-cell surface molecule TIGIT is an immune checkpoint molecule that inhibits T-cell responses,but its roles in cancer are little understood. In this study,we evaluated the role TIGIT checkpoint plays in the development and progression of gastric cancer. We show that the percentage of CD8 T cells that are TIGIT+ was increased in gastric cancer patients compared with healthy individuals. These cells showed functional exhaustion with impaired activation,proliferation,cytokine production,and metabolism,all of which were rescued by glucose. In addition,gastric cancer tissue and cell lines expressed CD155,which bound TIGIT receptors and inactivated CD8 T cells. In a T cell-gastric cancer cell coculture system,gastric cancer cells deprived CD8 T cells of glucose and impaired CD8 T-cell effector functions; these effects were neutralized by the additional glucose or by TIGIT blockade. In gastric cancer tumor cells,CD155 silencing increased T-cell metabolism and IFNγ production,whereas CD155 overexpression inhibited T-cell metabolism and IFNγ production; this inhibition was neutralized by TIGIT blockade. Targeting CD155/TIGIT enhanced CD8 T-cell reaction and improved survival in tumor-bearing mice. Combined targeting of TIGIT and PD-1 further enhanced CD8 T-cell activation and improved survival in tumor-bearing mice. Our results suggest that gastric cancer cells inhibit CD8 T-cell metabolism through CD155/TIGIT signaling,which inhibits CD8 T-cell effector functions,resulting in hyporesponsive antitumor immunity. These findings support the candidacy of CD155/TIGIT as a potential therapeutic target in gastric cancer. Cancer Res; 77(22); 6375-88. textcopyright2017 AACR. View Publication

Regulatory T cells (Tregs) play a pivotal role in maintaining immunological tolerance,but they can also play a detrimental role by preventing antitumor responses. Here,we characterized T helper (Th)-like Treg subsets to further delineate their biological function and tissue distribution,focusing on their possible contribution to disease states. RNA sequencing and functional assays revealed that Th2-like Tregs displayed higher viability and autocrine interleukin-2 (IL-2)-mediated activation than other subsets. Th2-like Tregs were preferentially found in tissues rather than circulation and exhibited the highest migratory capacity toward chemokines enriched at tumor sites. These cellular responses led us to hypothesize that this subset could play a role in maintaining a tumorigenic environment. Concurrently,Th2-like Tregs were enriched specifically in malignant tissues from patients with melanoma and colorectal cancer compared to healthy tissue. Overall,our results suggest that Th2-like Tregs may contribute to a tumorigenic environment due to their increased cell survival,higher migratory capacity,and selective T-effector suppressive ability. View Publication

MicroRNAs play an important role in T cell responses. However,how microRNAs regulate CD8 T cell memory remains poorly defined. Here,we found that miR-150 negatively regulates CD8 T cell memory in vivo. Genetic deletion of miR-150 disrupted the balance between memory precursor and terminal effector CD8 T cells following acute viral infection. Moreover,miR-150-deficient memory CD8 T cells were more protective upon rechallenge. A key circuit whereby miR-150 repressed memory CD8 T cell development through the transcription factor c-Myb was identified. Without miR-150,c-Myb was upregulated and anti-apoptotic targets of c-Myb,such as Bcl-2 and Bcl-xL,were also increased,suggesting a miR-150-c-Myb survival circuit during memory CD8 T cell development. Indeed,overexpression of non-repressible c-Myb rescued the memory CD8 T cell defects caused by overexpression of miR-150. Overall,these results identify a key role for miR-150 in memory CD8 T cells through a c-Myb-controlled enhanced survival circuit. View Publication

There is emerging evidence that the commensal microbiota has a role in the pathogenesis of multiple sclerosis (MS),a putative autoimmune disease of the CNS. Here,we compared the gut microbial composition of 34 monozygotic twin pairs discordant for MS. While there were no major differences in the overall microbial profiles,we found a significant increase in some taxa such as Akkermansia in untreated MS twins. Furthermore,most notably,when transplanted to a transgenic mouse model of spontaneous brain autoimmunity,MS twin-derived microbiota induced a significantly higher incidence of autoimmunity than the healthy twin-derived microbiota. The microbial profiles of the colonized mice showed a high intraindividual and remarkable temporal stability with several differences,including Sutterella,an organism shown to induce a protective immunoregulatory profile in vitro. Immune cells from mouse recipients of MS-twin samples produced less IL-10 than immune cells from mice colonized with healthy-twin samples. IL-10 may have a regulatory role in spontaneous CNS autoimmunity,as neutralization of the cytokine in mice colonized with healthy-twin fecal samples increased disease incidence. These findings provide evidence that MS-derived microbiota contain factors that precipitate an MS-like autoimmune disease in a transgenic mouse model. They hence encourage the detailed search for protective and pathogenic microbial components in human MS. View Publication