技术资料

The early events that determine the decision between lymphocyte tolerance and activation are not well-understood. Using a model of systemic self-antigen recognition by CD4(+) T cells,we show,using single-cell biochemical analyses,that tolerance is characterized by transient signaling events downstream of T-cell receptor engagement in the mammalian target of rapamycin (mTOR) and NF-κB pathways. Parallel studies done by live cell imaging show that the key difference between tolerance and activation is the duration of the T cell-antigen presenting cell (APC) interaction,as revealed by stable T-cell immobilization on antigen encounter. Brief T cell-APC interactions result in tolerance,and prolonged interactions are associated with activation and the development of effector cells. These studies show that the duration of T cell-APC interactions and magnitude of associated TCR-mediated signaling are key determinants of lymphocyte tolerance vs. activation. View Publication

Natural T regulatory cells (nTregs) play a key role in inducing and maintaining immunological tolerance. Cell-based therapy using purified nTregs is under consideration for several conditions,but procedures employed to date have resulted in cell populations that are contaminated with cytokine secreting effector cells. We have established a method for isolation and ex vivo expansion of human nTregs from healthy blood donors for cellular therapy aimed at preventing allograft rejection in organ transplants. The Robosep instrument was used for initial nTreg isolation and rapamycin was included in the expansion phase of cell cultures. The resulting cell population exhibited a stable CD4(+)CD25(++bright)Foxp3(+) phenotype,had potent functional ability to suppress CD4(+)CD25(negative) T cells without evidence of conversion to effector T cells including TH17 cells,and manifested little to no production of pro-inflammatory cytokines upon in vitro stimulation. Boolean gating analysis of cytokine-expressing cells by flow cytometry for 32 possible profile end points revealed that 96% of expanded nTregs did not express any cytokine. From a single buffy coat,approximately 80 million pure nTregs were harvested after expansion under cGMP conditions; these cell numbers are adequate for infusion of approximately one million cells kg�?�¹ for cell therapy in clinical trials. View Publication

The adaptive immune response involves T cell differentiation and migration to sites of inflammation. T cell trafficking is initiated by rolling on inflamed endothelium. Tethers and slings,discovered in neutrophils,facilitate cell rolling at high shear stress. Here,we demonstrate that the ability to form tethers and slings during rolling is highly inducible in T helper 1 (Th1),Th17,and regulatory T (Treg) cells but less in Th2 cells. In vivo,endogenous Treg cells rolled stably in cremaster venules at physiological shear stress. Quantitative dynamic footprinting nanoscopy of Th1,Th17,and Treg cells uncovered the formation of multiple tethers per cell. Human Th1 cells also showed tethers and slings. RNA sequencing (RNA-seq) revealed the induction of cell migration and cytoskeletal genes in sling-forming cells. We conclude that differentiated CD4 T cells stabilize rolling by inducible tether and sling formation. These phenotypic changes approximate the adhesion phenotype of neutrophils and support CD4 T cell access to sites of inflammation. View Publication

Improved understanding of expression of immune checkpoint receptors (ICR) on tumor-infiltrating lymphocytes (TIL) may facilitate more effective immunotherapy in head and neck cancer (HNC) patients. A higher frequency of PD-1+ TIL has been reported in human papillomavirus (HPV)+ HNC patients,despite the role of PD-1 in T-cell exhaustion. This discordance led us to hypothesize that the extent of PD-1 expression more accurately defines T-cell function and prognostic impact,because PD-1high T cells may be more exhausted than PD-1low T cells and may influence clinical outcome and response to anti-PD-1 immunotherapy. In this study,PD-1 expression was indeed upregulated on HNC patient TIL,and the frequency of these PD-1+ TIL was higher in HPV+ patients (P = 0.006),who nonetheless experienced significantly better clinical outcome. However,PD-1high CD8+ TILs were more frequent in HPV- patients and represented a more dysfunctional subset with compromised IFN-γ secretion. Moreover,HNC patients with higher frequencies of PD-1high CD8+ TIL showed significantly worse disease-free survival and higher hazard ratio for recurrence (P < 0.001),while higher fractions of PD-1low T cells associated with HPV positivity and better outcome. In a murine HPV+ HNC model,anti-PD-1 mAb therapy differentially modulated PD-1high/low populations,and tumor rejection associated with loss of dysfunctional PD-1high CD8+ T cells and a significant increase in PD-1low TIL. Thus,the extent of PD-1 expression on CD8+ TIL provides a potential biomarker for anti-PD-1-based immunotherapy. Cancer Res; 77(22); 6353-64. textcopyright2017 AACR. 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

Immunostimulatory agents such as agonistic anti-CD137 and interleukin (IL)-2 generate effective anti-tumor immunity but also elicit serious toxicities,hampering their clinical application. Here we show that combination therapy with anti-CD137 and an IL-2-Fc fusion achieves significant initial anti-tumor activity,but also lethal immunotoxicity deriving from stimulation of circulating leukocytes. To overcome this toxicity,we demonstrate that anchoring IL-2 and anti-CD137 on the surface of liposomes allows these immune agonists to rapidly accumulate in tumors while lowering systemic exposure. In multiple tumor models,immunoliposome delivery achieves anti-tumor activity equivalent to free IL-2/anti-CD137 but with the complete absence of systemic toxicity. Immunoliposomes stimulated tumor infiltration by cytotoxic lymphocytes,cytokine production,and granzyme expression,demonstrating equivalent immunostimulatory effects to the free drugs in the local tumor microenvironment. Thus,surface-anchored particle delivery may provide a general approach to exploit the potent stimulatory activity of immune agonists without debilitating systemic toxicities. 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

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

Regions of the normal arterial intima predisposed to atherosclerosis are sites of ongoing monocyte trafficking and also contain resident myeloid cells with features of dendritic cells. However,the pathophysiological roles of these cells are poorly understood. Here we found that intimal myeloid cells underwent reverse transendothelial migration (RTM) into the arterial circulation after systemic stimulation of pattern-recognition receptors (PRRs). This process was dependent on expression of the chemokine receptor CCR7 and its ligand CCL19 by intimal myeloid cells. In mice infected with the intracellular pathogen Chlamydia muridarum,blood monocytes disseminated infection to the intima. Subsequent CCL19-CCR7-dependent RTM was critical for the clearance of intimal C. muridarum. This process was inhibited by hypercholesterolemia. Thus,RTM protects the normal arterial intima,and compromised RTM during atherogenesis might contribute to the intracellular retention of pathogens in atherosclerotic lesions. View Publication

Redirecting T cells to attack cancer using engineered chimeric receptors provides powerful new therapeutic capabilities. However,the effectiveness of therapeutic T cells is constrained by the endogenous T cell response: certain facets of natural response programs can be toxic,whereas other responses,such as the ability to overcome tumor immunosuppression,are absent. Thus,the efficacy and safety of therapeutic cells could be improved if we could custom sculpt immune cell responses. Synthetic Notch (synNotch) receptors induce transcriptional activation in response to recognition of user-specified antigens. We show that synNotch receptors can be used to sculpt custom response programs in primary T cells: they can drive a la carte cytokine secretion profiles,biased T cell differentiation,and local delivery of non-native therapeutic payloads,such as antibodies,in response to antigen. SynNotch T cells can thus be used as a general platform to recognize and remodel local microenvironments associated with diverse diseases. View Publication

In several clinical contexts,the measurement of ATP concentration in T lymphocytes has been proposed as a biomarker of immune status,predictive of secondary infections. However,the use of such biomarker in lymphopenic patients requires some adaptations in the ATP dosage protocol. We used blood from healthy volunteers to determine the optimal experimental settings. We investigated technical aspects such as the type of anticoagulant for blood sampling,the effect of freeze and thaw cycles,the reagent and sample mixing sequence,and the optimal dilution buffer. We also shortened the incubation time to 8h,and even showed that a 30min incubation may be sufficient. To evaluate the ATP rise upon lymphocyte activation,the optimal dose of stimulant was defined to be 4μg/mL of phytohaemagglutinin. Lastly,we determined that the number of T cells needed for this measurement was as low as 50,000,which is compatible with the existing lymphopenia in clinical settings. This optimized protocol appears ready to be assessed in lymphopenic patients to further investigate the interconnection between T lymphocyte metabolism and impaired phenotype and functions. View Publication

Spatial organization of the genome plays a central role in gene expression,DNA replication,and repair. But current epigenomic approaches largely map DNA regulatory elements outside of the native context of the nucleus. Here we report assay of transposase-accessible chromatin with visualization (ATAC-see),a transposase-mediated imaging technology that employs direct imaging of the accessible genome in situ,cell sorting,and deep sequencing to reveal the identity of the imaged elements. ATAC-see revealed the cell-type-specific spatial organization of the accessible genome and the coordinated process of neutrophil chromatin extrusion,termed NETosis. Integration of ATAC-see with flow cytometry enables automated quantitation and prospective cell isolation as a function of chromatin accessibility,and it reveals a cell-cycle dependence of chromatin accessibility that is especially dynamic in G1 phase. The integration of imaging and epigenomics provides a general and scalable approach for deciphering the spatiotemporal architecture of gene control. View Publication