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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

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

T cell antigen-presenting cell (APC) interactions early during chronic viral infection are crucial for determining viral set point and disease outcome,but how and when different APC subtypes contribute to these outcomes is unclear. The TNF receptor superfamily (TNFRSF) member GITR is important for CD4+ T cell accumulation and control of chronic lymphocytic choriomeningitis virus (LCMV). We found that type I interferon (IFN-I) induced TNFSF ligands GITRL,4-1BBL,OX40L,and CD70 predominantly on monocyte-derived APCs and CD80 and CD86 predominantly on classical dendritic cells (cDCs). Mice with hypofunctional GITRL in Lyz2+ cells had decreased LCMV-specific CD4+ T cell accumulation and increased viral load. GITR signals in CD4+ T cells occurred after priming to upregulate OX40,CD25,and chemokine receptor CX3CR1. Thus IFN-I (signal 3) induced a post-priming checkpoint (signal 4) for CD4+ T cell accumulation,revealing a division of labor between cDCs and monocyte-derived APCs in regulating T cell expansion. View Publication

Bone marrow (BM) failure syndrome encompasses a group of disorders characterized by BM stem cell dysfunction,resulting in varying degrees of hypoplasia and blood pancytopenia,and in many patients is autoimmune and inflammatory in nature. The important role of T helper 1 (Th1) polarized CD4+ T cells in driving BM failure has been clearly established in several models. However,animal model data demonstrating a functional role for CD8+ T cells in BM dysfunction is largely lacking and our objective was to test the hypothesis that CD8+ T cells play a non-redundant role in driving BM failure. Clinical evidence implicates a detrimental role for CD8+ T cells in BM failure and a beneficial role for Foxp3+ regulatory T cells (Tregs) in maintaining immune tolerance in the BM. We demonstrate that IL-2-deficient mice,which have a deficit in functional Tregs,develop spontaneous BM failure. Furthermore,we demonstrate a critical role for CD8+ T cells in the development of BM failure,which is dependent on the cytokine,IFNgamma$. CD8+ T cells promote hematopoietic stem cell dysfunction and depletion of myeloid lineage progenitor cells,resulting in anemia. Adoptive transfer experiments demonstrate that CD8+ T cells dramatically expedite disease progression and promote CD4+ T cell accumulation in the BM. Thus,BM dysregulation in IL-2-deficient mice is mediated by a Th1 and IFNgamma$-producing CD8+ T cell (Tc1) response. View Publication

Understanding the mechanisms of CD4 memory T cell (Tmem) differentiation in malaria is critical for vaccine development. However,the metabolic regulation of CD4 Tmem differentiation is not clear,particularly in persistent infections. In this study,we investigated the role of fatty acid synthesis (FAS) in Tmem development in Plasmodium chabaudi chronic mouse malaria infection. We show that T cell-specific deletion and early pharmaceutical inhibition of acetyl CoA carboxylase 1,the rate limiting step of FAS,inhibit generation of early memory precursor effector T cells (MPEC). To compare the role of FAS during early differentiation or survival of Tmem in chronic infection,a specific inhibitor of acetyl CoA carboxylase 1,5-(tetradecyloxy)-2-furoic acid,was administered at different times postinfection. Strikingly,the number of Tmem was only reduced when FAS was inhibited during T cell priming and not during the Tmem survival phase. FAS inhibition during priming increased effector T cell (Teff) proliferation and strongly decreased peak parasitemia,which is consistent with improved Teff function. Conversely,MPEC were decreased,in a T cell-intrinsic manner,upon early FAS inhibition in chronic,but not acute,infection. Early cure of infection also increased mitochondrial volume in Tmem compared with Teff,supporting previous reports in acute infection. We demonstrate that the MPEC-specific effect was due to the higher fatty acid content and synthesis in MPEC compared with terminally differentiated Teff. In conclusion,FAS in CD4 T cells regulates the early divergence of Tmem from Teff in chronic infection. View Publication

Enteric pathogens including Salmonella enteric serovar Typhimurium can breach the epithelial barrier of the host and spread to systemic tissues. In response to infection,the host activates innate immune receptors via the signaling molecule MyD88,which induces protective inflammatory and antimicrobial responses. Most of these innate immune responses have been studied in hematopoietic cells,but the role of MyD88 signaling in other cell types remains poorly understood. Surprisingly,we found that Dermo1-Cre;Myd88fl/fl mice with mesenchymal cell-specific deficiency of MyD88 were less susceptible to orogastric and i.p. STyphimurium infection than their Myd88fl/fl littermates. The reduced susceptibility of Dermo1-Cre;Myd88fl/fl mice to infection was associated with lower loads of S. Typhimurium in the liver and spleen. Mutant analyses revealed that S. Typhimurium employs its virulence type III secretion system 2 to promote its growth through MyD88 signaling pathways in mesenchymal cells. Inflammatory monocytes function as a major cell population for systemic dissemination of S. Typhimurium Mechanistically,mesenchymal cell-specific MyD88 signaling promoted CCL2 production in the liver and spleen and recruitment of inflammatory monocytes to systemic organs in response to STyphimurium infection. Consistently,MyD88 signaling in mesenchymal cells enhanced the number of phagocytes including Ly6ChiLy6G- inflammatory monocytes harboring STyphimurium in the liver. These results suggest that S. Typhimurium promotes its systemic growth and dissemination through MyD88 signaling pathways in mesenchymal cells. View Publication

PURPOSE The outgrowth of antigen-negative variants is a significant challenge for adoptive therapy with T cells that target a single specificity. Chimeric antigen receptors (CAR) are typically designed with one or two scFvs that impart antigen specificity fused to activation and costimulation domains of T-cell signaling molecules. We designed and evaluated the function of CARs with up to three specificities for overcoming tumor escape using Designed Ankyrin Repeat Proteins (DARPins) rather than scFvs for tumor recognition. EXPERIMENTAL DESIGN A monospecific CAR was designed with a DARPin binder (E01) specific for EGFR and compared with a CAR designed using an anti-EGFR scFv. CAR constructs in which DARPins specific for EGFR,EpCAM,and HER2 were linked together in a single CAR were then designed and optimized to achieve multispecific tumor recognition. The efficacy of CAR-T cells bearing a multispecific DARPin CAR for treating tumors with heterogeneous antigen expression was evaluated in vivo. RESULTS The monospecific anti-EGFR E01 DARPin conferred potent tumor regression against EGFR+ targets that was comparable with an anti-EGFR scFv CAR. Linking three separate DARPins in tandem was feasible and in an optimized format generated a single tumor recognition domain that targeted a mixture of heterogeneous tumor cells,each expressing a single antigen,and displayed synergistic activity when tumor cells expressed more than one target antigen. CONCLUSIONS DARPins can serve as high-affinity recognition motifs for CAR design,and their robust architecture enables linking of multiple binders against different antigens to achieve functional synergy and reduce antigen escape. View Publication

Long-term management for leukemia is challenging due to the painful and invasive procedure of bone marrow (BM) biopsy. At present,non-invasive liquid (blood) biopsy is not utilized for leukemia,due to lower counts of leukemia blast cells in the blood. Here,we described a robust system for the simultaneous detection and enrichment of rare blast cells. Enrichment of blast cells was achieved from blood with a one-step microfluidic blast cell biochip (BCB) sorting system,without specific targeting of proteins by antibodies. Non-target cells encountered a differential net force as compared to stiffer blast cells and were removed. The efficiency of the BCB promotes high detection sensitivity (1 in 106 cells) even from patients with minimal residual disease. The procedure was validated using actual blast cells from patients with various types of leukemia. Outcomes were compared to current evaluation standards,such as flow cytometry,using BM aspirates. Blast cell detection efficiency was higher in 55.6{\%} of the patients using the BCB as compared to flow cytometry,despite the lower concentrations of blast cells in liquid biopsy. These studies promote early-stage detection and routine monitoring for minimal residual disease in patients. View Publication

Chronic lymphocytic leukaemia (CLL) exhibits differences between Asians and Caucasians in terms of incidence rate,age at onset,immunophenotype,and genetic profile. We performed genome-wide methylation profiling of CLL in an Asian cohort for the first time. Eight Korean patients without somatic immunoglobulin heavy chain gene hypermutations underwent methyl-CpG-binding domain sequencing (MBD-seq),as did five control subjects. Gene Ontology,pathway analysis,and network-based prioritization of differentially methylated genes were also performed. More regions were hypomethylated (2,062 windows) than were hypermethylated (777 windows). Promoters contained the highest proportion of differentially methylated regions (0.08{\%}),while distal intergenic and intron regions contained the largest number of differentially methylated regions. Protein-coding genes were the most abundant,followed by long noncoding and short noncoding genes. The most significantly over-represented signalling pathways in the differentially methylated gene list included immune/cancer-related pathways and B-cell receptor signalling. Among the top 10 hub genes identified via network-based prioritization,four (UBC,GRB2,CREBBP,and GAB2) had no known relevance to CLL,while the other six (STAT3,PTPN6,SYK,STAT5B,XPO1,and ABL1) have previously been linked to CLL in Caucasians. As such,our analysis identified four novel candidate genes of potential significance to Asian patients with CLL. View Publication

PURPOSE Patients with metastatic colorectal cancer refractory to chemotherapy have limited treatment options. Ensituximab (NEO-102) is a novel chimeric mAb targeting a variant of MUC5AC with specificity to colorectal cancer. PATIENTS AND METHODS Single-arm,phase II trial assessed the efficacy and safety of ensituximab in patients with advanced,refractory cancer who expressed MUC5AC antigen in tumor tissue. Ensituximab was administered intravenously every 2 weeks with 3 mg/kg as recommended phase II dose (RP2D). A minimum sample size of 43 patients was required on the basis of the assumption that ensituximab would improve median overall survival (OS) by 7 months using a one-sided significance level of 10{\%} and 80{\%} power. Written informed consent was obtained from all patients. RESULTS Sixty-three patients with advanced,refractory colorectal cancer were enrolled and 53 subjects were treated in phase II arm. Median age was 58 years and 46{\%} of the patients were female. Among 57 evaluable patients,median OS was 6.8 months. No responses were observed,and stable disease was achieved in 21{\%} of the patients. The most common treatment-related adverse events (AE) at RP2D included fatigue (38{\%}),anemia (30{\%}),nausea (15{\%}),vomiting (11{\%}),increased bilirubin (9{\%}),constipation (8{\%}),decreased appetite (6{\%}),and diarrhea (6{\%}). Serious AEs at least possibly related to ensituximab occurred in 4 patients and included anemia,nausea,increased bilirubin,and hypoxia. No patients discontinued treatment due to drug-related AEs. CONCLUSIONS Ensituximab was well tolerated and demonstrated modest antitumor activity in patients with heavily pretreated refractory colorectal cancer. View Publication

Generating robust CD4+ T-helper cell type 1 (Th1) responses is essential for protective vaccine-induced type 1 immunity. Here,we examine whether immunization formulation associated with enhanced vaccine efficacy promotes antigen targeting and cell recruitment into lymph node (LN) niches associated with optimal type 1 responses. Immunization with antigen and Toll-like receptor agonist emulsified in oil leads to an increased differentiation of IFN$\gamma$/TNF-$\alpha$+ polyfunctional Th1 cells compared to an identical immunization in saline. Oil immunization results in a rapid delivery and persistence of antigen in interfollicular regions (IFRs) of the LN,whereas without oil,antigen is distributed in the medullary region. Following oil immunization,CXCL10-producing inflammatory monocytes accumulate in the IFR,which mobilizes antigen-specific CD4+ T cells into this niche. In this microenvironment,CD4+ T cells are advantageously positioned to encounter arriving IL-12-producing inflammatory dendritic cells (DCs). These data suggest that formulations delivering antigen to the LN IFR create an inflammatory niche that can improve vaccine efficacy. View Publication