技术资料

PURPOSE Recent advances in immunotherapy of advanced human cancers underscored the need to address and eliminate tumor immune evasion. The myeloid-derived suppressor cells (MDSC) are important inhibitors of T-cell responses in solid tumors,such as prostate cancers. However,targeting MDSCs proved challenging due to their phenotypic heterogeneity. EXPERIMENTAL DESIGN Myeloid cell populations were evaluated using flow cytometry on blood samples,functional assays,and immunohistochemical/immunofluorescent stainings on specimens from healthy subjects,localized and metastatic castration-resistant prostate cancer patients. RESULTS Here,we identify a population of Lin(-)CD15(HI)CD33(LO) granulocytic MDSCs that accumulate in patients' circulation during prostate cancer progression from localized to metastatic disease. The prostate cancer-associated MDSCs potently inhibit autologous CD8(+) T cells' proliferation and production of IFNγ and granzyme-B. The circulating MDSCs have high levels of activated STAT3,which is a central immune checkpoint regulator. The granulocytic pSTAT3(+) cells are also detectable in patients' prostate tissues. We previously generated an original strategy to silence genes specifically in Toll-like Receptor-9 (TLR9) positive myeloid cells using CpG-siRNA conjugates. We demonstrate that human granulocytic MDSCs express TLR9 and rapidly internalize naked CpG-STAT3siRNA,thereby silencing STAT3 expression. STAT3 blocking abrogates immunosuppressive effects of patients-derived MDSCs on effector CD8(+) T cells. These effects depended on reduced expression and enzymatic activity of Arginase-1,a downstream STAT3 target gene and a potent T-cell inhibitor. CONCLUSIONS Overall,we demonstrate the accumulation of granulocytic MDSCs with prostate cancer progression and the feasibility of using TLR9-targeted STAT3siRNA delivery strategy to alleviate MDSC-mediated immunosuppression. View Publication

Exaggerated inflammatory responses during influenza A virus (IAV) infection are typically associated with severe disease. Neutrophils are among the immune cells that can drive this excessive and detrimental inflammation. In moderation,however,neutrophils are necessary for optimal viral control. In this study,we explore the role of the nucleotide-binding domain leucine-rich repeat containing receptor family member Nlrp12 in modulating neutrophilic responses during lethal IAV infection. Nlrp12-/- mice are protected from lethality during IAV infection and show decreased vascular permeability,fewer pulmonary neutrophils,and a reduction in levels of neutrophil chemoattractant CXCL1 in their lungs compared with wild-type mice. Nlrp12-/- neutrophils and dendritic cells within the IAV-infected lungs produce less CXCL1 than their wild-type counterparts. Decreased CXCL1 production by Nlrp12-/- dendritic cells was not due to a difference in CXCL1 protein stability,but instead to a decrease in Cxcl1 mRNA stability. Together,these data demonstrate a previously unappreciated role for Nlrp12 in exacerbating the pathogenesis of IAV infection through the regulation of CXCL1-mediated neutrophilic responses. View Publication

Typhoid fever caused by Salmonella enterica serovar (S.) Typhi differs in its clinical presentation from gastroenteritis caused by S. Typhimurium and other non-typhoidal Salmonella serovars. The different clinical presentations are attributed in part to the virulence-associated capsular polysaccharide (Vi antigen) of S. Typhi,which prevents phagocytes from triggering a respiratory burst by preventing antibody-mediated complement activation. Paradoxically,the Vi antigen is absent from S. Paratyphi A,which causes a disease that is indistinguishable from typhoid fever. Here,we show that evasion of the phagocyte respiratory burst by S. Paratyphi A required very long O antigen chains containing the O2 antigen to inhibit antibody binding. We conclude that the ability to avoid the phagocyte respiratory burst is a property distinguishing typhoidal from non-typhoidal Salmonella serovars that was acquired by S. Typhi and S. Paratyphi A independently through convergent evolution. 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

BACKGROUND Atopy and viral respiratory tract infections synergistically promote asthma exacerbations. IgE cross-linking inhibits critical virus-induced IFN-α responses of plasmacytoid dendritic cells (pDCs),which can be deficient in patients with allergic asthma. OBJECTIVE We sought to determine whether reducing IgE levels in vivo with omalizumab treatment increases pDC antiviral IFN-α responses in inner-city children with asthma. METHODS PBMCs and pDCs isolated from children with exacerbation-prone asthma before and during omalizumab treatment were stimulated ex vivo with rhinovirus and influenza in the presence or absence of IgE cross-linking. IFN-α levels were measured in supernatants,and mRNA expression of IFN-α pathway genes was determined by using quantitative RT-PCR (qRT-PCR) in cell pellets. FcεRIα protein levels and mRNA expression were measured in unstimulated cells by using flow cytometry and qRT-PCR,respectively. Changes in these outcomes and associations with clinical outcomes were analyzed,and statistical modeling was used to identify risk factors for asthma exacerbations. RESULTS Omalizumab treatment increased rhinovirus- and influenza-induced PBMC and rhinovirus-induced pDC IFN-α responses in the presence of IgE cross-linking and reduced pDC surface FcεRIα expression. Omalizumab-induced reductions in pDC FcεRIα levels were significantly associated with a lower asthma exacerbation rate during the outcome period and correlated with increases in PBMC IFN-α responses. PBMC FcεRIα mRNA expression measured on study entry significantly improved an existing model of exacerbation prediction. CONCLUSIONS These findings indicate that omalizumab treatment augments pDC IFN-α responses and attenuates pDC FcεRIα protein expression and provide evidence that these effects are related. These results support a potential mechanism underlying clinical observations that allergic sensitization is associated with increased susceptibility to virus-induced asthma exacerbations. View Publication

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion,however,can be obstructed by transmembrane proteins (pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44 an abundant transmembrane protein capable of indirect association with F-actin hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages where receptor mobility was minimal. Conversely receptors were most mobile at the leading edge where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier enabling receptors to engage their targets. View Publication

There is compelling evidence for the role of the leucine-rich repeat kinase 2 (LRRK2) and in particular its kinase function in Parkinson's disease. Orally bioavailable,brain penetrant and potent LRRK2 kinase inhibitors are in the later stages of clinical development. Here,we describe a facile and robust assay to quantify LRRK2 kinase pathway activity by measuring LRRK2-mediated phosphorylation of Rab10 in human peripheral blood neutrophils. We use the selective MJFF-pRab10 monoclonal antibody recognising the Rab10 Thr73 phospho-epitope that is phosphorylated by LRRK2. We highlight the feasibility and practicability of using our assay in the clinical setting by studying a few patients with G2019S LRRK2 associated and sporadic Parkinson's as well as healthy controls. We suggest that peripheral blood neutrophils are a valuable resource for LRRK2 research and should be considered for inclusion in Parkinson's bio-repository collections as they are abundant,homogenous and express relatively high levels of LRRK2 as well as Rab10. In contrast,the widely used peripheral blood mononuclear cells are heterogeneous and only a minority of cells (monocytes and contaminating neutrophils) express LRRK2. While our LRRK2 kinase pathway assay could assist in patient stratification based on LRRK2 kinase activity,we envision that it may find greater utility in pharmacodynamic and target engagement studies in future LRRK2 inhibitor trials. View Publication

Lung macrophage subpopulations have been identified based on size. We investigated characteristics of small and large macrophages in the alveolar spaces and lung interstitium of COPD patients and controls. Alveolar and interstitial cells were isolated from lung resection tissue from 88 patients. Macrophage subpopulation cell-surface expression of immunological markers and phagocytic ability were assessed by flow cytometry. Inflammatory related gene expression was measured. Alveolar and interstitial macrophages had subpopulations of small and large macrophages based on size and granularity. Alveolar macrophages had similar numbers of small and large cells; interstitial macrophages were mainly small. Small macrophages expressed significantly higher cell surface HLA-DR,CD14,CD38 and CD36 and lower CD206 compared to large macrophages. Large alveolar macrophages showed lower marker expression in COPD current compared to ex-smokers. Small interstitial macrophages had the highest pro-inflammatory gene expression levels,while large alveolar macrophages had the lowest. Small alveolar macrophages had the highest phagocytic ability. Small alveolar macrophage CD206 expression was lower in COPD patients compared to smokers. COPD lung macrophages include distinct subpopulations; Small interstitial and small alveolar macrophages with more pro-inflammatory and phagocytic function respectively,and large alveolar macrophages with low pro-inflammatory and phagocytic 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

A capability for analyzing complex cellular communication among tissues is important in drug discovery and development,and in vitro technologies for doing so are required for human applications. A prominent instance is communication between the gut and the liver,whereby perturbations of one tissue can influence behavior of the other. Here,we present a study on human gut-liver tissue interactions under normal and inflammatory contexts,via an integrative multi-organ platform comprising human liver (hepatocytes and Kupffer cells),and intestinal (enterocytes,goblet cells,and dendritic cells) models. Our results demonstrated long-term (>2 weeks) maintenance of intestinal (e.g.,barrier integrity) and hepatic (e.g.,albumin) functions in baseline interaction. Gene expression data comparing liver in interaction with gut,versus isolation,revealed modulation of bile acid metabolism. Intestinal FGF19 secretion and associated inhibition of hepatic CYP7A1 expression provided evidence of physiologically relevant gut-liver crosstalk. Moreover,significant non-linear modulation of cytokine responses was observed under inflammatory gut-liver interaction; for example,production of CXCR3 ligands (CXCL9,10,11) was synergistically enhanced. RNA-seq analysis revealed significant upregulation of IFNα/β/γ signaling during inflammatory gut-liver crosstalk,with these pathways implicated in the synergistic CXCR3 chemokine production. Exacerbated inflammatory response in gut-liver interaction also negatively affected tissue-specific functions (e.g.,liver metabolism). These findings illustrate how an integrated multi-tissue platform can generate insights useful for understanding complex pathophysiological processes such as inflammatory organ crosstalk. Biotechnol. Bioeng. 2017;114: 2648-2659. textcopyright 2017 Wiley Periodicals,Inc. View Publication