技术资料

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

Current antiretroviral therapy (ART) for HIV/AIDS slows disease progression by reducing viral loads and increasing CD4 counts. Yet ART is not curative due to the persistence of CD4+ T-cell proviral reservoirs that chronically resupply active virus. Elimination of these reservoirs through the administration of synergistic combinations of latency reversing agents (LRAs),such as histone deacetylase (HDAC) inhibitors and protein kinase C (PKC) modulators,provides a promising strategy to reduce if not eradicate the viral reservoir. Here,we demonstrate that largazole and its analogues are isoform-targeted histone deacetylase inhibitors and potent LRAs. Significantly,these isoform-targeted HDAC inhibitors synergize with PKC modulators,namely bryostatin-1 analogues (bryologs). Implementation of this unprecedented LRA combination induces HIV-1 reactivation to unparalleled levels and avoids global T-cell activation within resting CD4+ T-cells. View Publication

Among different cancer immunotherapy approaches,bispecific antibodies (BsAbs) are of great interest due to their ability to recruit immune cells to kill tumor cells directly. Various BsAbs against Her2 tumor cells have been proposed with potent cytotoxic activities. However,most of these formats require extensive processing to obtain heterodimeric bispecific antibodies. In this study,we describe a bispecific antibody,BiHC (bispecific Her2-CD3 antibody),constructed with a single-domain anti-Her2 and a single-chain Fv (variable fragment) of anti-CD3 in an IgG-like format. In contrast to most IgG-like BsAbs,the two arms in BiHC have different molecular weights,making it easier to separate hetero- or homodimers. BiHC can be expressed in Escherichia coli and purified via Protein A affinity chromatography. The purified BiHC can recruit T cells and induce specific cytotoxicity of Her2-expressing tumor cells in vitro. The BiHC can also efficiently inhibit the tumor growth in vivo. Thus,BiHC is a promising candidate for the treatment of Her2-positive cancers. View Publication

A key determinant of the therapeutic potency of adoptive T-cell transfer is the extent to which infused cells can persist and expand in vivo. Ex vivo propagated virus-specific and chimeric antigen receptor (CAR)-redirected antitumor CD8 effector T cells derived from CD45RA(-) CD62L(+) central memory (TCM) precursors engraft long-term and reconstitute functional memory after adoptive transfer. Here,we describe a clinical scale,closed system,immunomagnetic selection method to isolate CD8(+) T(CM) from peripheral blood mononuclear cells (PBMC). This method uses the CliniMACS device to first deplete CD14(+),CD45RA(+),and CD4(+) cells from PBMC,and then to positively select CD62L(+) cells. The average purity and yield of CD8(+) CD45RA(-) CD62L TCM obtained in full-scale qualification runs were 70% and 0.4% (of input PBMC),respectively. These CD8(+) T(CM) are responsive to anti-CD3/CD28 bead stimulation,and can be efficiently transduced with CAR encoding lentiviral vectors,and undergo sustained expansion in interleukin (IL)-2/IL-15 over 3-6 weeks. The resulting CD8(+) T(CM)-derived effectors are polyclonal,retain expression of CD62L and CD28,exhibit CAR-redirected antitumor effector function,and are capable of huIL-15-dependent in vivo homeostatic engraftment after transfer to immunodeficient NOD/Scid IL-2RgCnull mice. Adoptive therapy using purified T(CM) cells is now the subject of a Food and Drug Administration-authorized clinical trial for the treatment of CD19(+) B-cell malignancies,and 3 clinical cell products expressing a CD19-specific CAR for IND 14645 have already been successfully generated from lymphoma patients using this manufacturing platform. View Publication

Proteasome inhibitors benefit patients with multiple myeloma and B cell-dependent autoimmune disorders but exert toxicity from inhibition of proteasomes in other cells. Toxicity should be minimized by reversible inhibition of the immunoproteasome β5i subunit while sparing the constitutive β5c subunit. Here we report β5i-selective inhibition by asparagine-ethylenediamine (AsnEDA)-based compounds and present the high-resolution cryo-EM structural analysis of the human immunoproteasome. Despite inhibiting noncompetitively,an AsnEDA inhibitor binds the active site. Hydrophobic interactions are accompanied by hydrogen bonding with β5i and β6 subunits. The inhibitors are far more cytotoxic for myeloma and lymphoma cell lines than for hepatocarcinoma or non-activated lymphocytes. They block human B-cell proliferation and promote apoptotic cell death selectively in antibody-secreting B cells,and to a lesser extent in activated human T cells. Reversible,β5i-selective inhibitors may be useful for treatment of diseases involving activated or neoplastic B cells or activated T cells. View Publication

V-domain immunoglobulin suppressor of T cell activation (VISTA) is a recently discovered immune checkpoint ligand that functions to suppress T cell activity. The therapeutic potential of activating this immune checkpoint pathway to reduce inflammatory responses remains untapped,largely due to the inability to derive agonists targeting its unknown receptor. A dimeric construct of the IgV domain of VISTA (VISTA-Fc) was shown to suppress the activation of T cells in vitro. However,this effect required its immobilization on a solid surface,suggesting that VISTA-Fc may display limited efficacy as a VISTA-receptor agonist in vivo. Herein,we have designed a stable pentameric VISTA construct (VISTA.COMP) by genetically fusing its IgV domain to the pentamerization domain from the cartilage oligomeric matrix protein (COMP). In contrast to VISTA-Fc,VISTA.COMP does not require immobilization to inhibit the proliferation of CD4+ T cells undergoing polyclonal activation. Furthermore,we show that VISTA.COMP,but not VISTA-Fc,functions as an immunosuppressive agonist in vivo capable of prolonging the survival of skin allografts in a mouse transplant model as well as rescuing mice from acute concanavalin-A-induced hepatitis. Collectively,we believe our data demonstrate that VISTA.COMP is a checkpoint receptor agonist and the first agent to our knowledge targeting the putative VISTA-receptor to suppress T cell-mediated immune responses. View Publication

The maintenance of peripheral naive T lymphocytes in humans is dependent on their homeostatic division,not continuing emigration from the thymus,which undergoes involution with age. However,postthymic maintenance of naive T cells is still poorly understood. Previously we reported that recent thymic emigrants (RTEs) are contained in CD31+CD25- naive T cells as defined by their levels of signal joint T cell receptor rearrangement excision circles (sjTRECs). Here,by differential gene expression analysis followed by protein expression and functional studies,we define that the naive T cells having divided the least since thymic emigration express complement receptors (CR1 and CR2) known to bind complement C3b- and C3d-decorated microbial products and,following activation,produce IL-8 (CXCL8),a major chemoattractant for neutrophils in bacterial defense. We also observed an IL-8-producing memory T cell subpopulation coexpressing CR1 and CR2 and with a gene expression signature resembling that of RTEs. The functions of CR1 and CR2 on T cells remain to be determined,but we note that CR2 is the receptor for Epstein-Barr virus,which is a cause of T cell lymphomas and a candidate environmental factor in autoimmune disease. View Publication

Events defining the progression to human type 1 diabetes (T1D) have remained elusive owing to the complex interaction between genetics,the immune system,and the environment. Type 1 interferons (T1-IFN) are known to be a constituent of the autoinflammatory milieu within the pancreas of patients with T1D. However,the capacity of IFNα/β to modulate human activated autoreactive CD8+ T-cell (cytotoxic T lymphocyte) responses within the islets of patients with T1D has not been investigated. Here,we engineer human β-cell-specific cytotoxic T lymphocytes and demonstrate that T1-IFN augments cytotoxicity by inducing rapid phosphorylation of STAT4,resulting in direct binding at the granzyme B promoter within 2 h of exposure. The current findings provide novel insights concerning the regulation of effector function by T1-IFN in human antigen-experienced CD8+ T cells and provide a mechanism by which the presence of T1-IFN potentiates diabetogenicity within the autoimmune islet. View Publication

Fatty acid (FA) metabolism directly influences the functional capabilities of T cells in tumor microenvironments. Thus,developing tools to interrogate FA-uptake by T cell subsets is important for understanding tumor immunosuppression. Herein,we have generated a novel FA-Qdot 605 dye conjugate with superior sensitivity and flexibility to any of the previously commercially available alternatives. For the first time,we demonstrate that this nanoparticle can be used as a specific measure of fatty acid uptake by T cells both in-vitro and in-vivo. Flow cytometric analysis shows that both the location and activation status of T cells determines their FA uptake. Additionally,CD4+ Foxp3+ regulatory T cells (Tregs) uptake FA at a higher rate than effector T cell subsets,supporting the role of FA metabolism for Treg function. Furthermore,we are able to simultaneously detect glucose and fatty acid uptake directly within the tumor microenvironment. Cumulatively,our results suggest that this novel fluorescent probe is a powerful tool to understand FA utilization within the tumor,thereby providing an unprecedented opportunity to study T cell FA metabolism in-vivo. View Publication