SUMMARY During persistent antigen stimulation, CD8+ T cells show a gradual decrease in effector function, referred to as exhaustion, which impairs responses in the setting of tumors and infections. Here we demonstrate that the transcription factor NFAT controls the program of T cell exhaustion. When expressed in cells, an engineered form of NFAT1 unable to interact with AP-1 transcription factors diminished T cell receptor (TCR) signaling, increased the expression of inhibitory cell surface receptors, and interfered with the ability of CD8+ T cells to protect against Listeria infection and attenuate tumor growth in vivo. We defined the genomic regions occupied by endogenous and engineered NFAT1 in primary CD8+ T cells, and showed that genes directly induced by the engineered NFAT1 overlapped with genes expressed in exhausted CD8+ T cells in vivo. Our data show that NFAT promotes T cell anergy and exhaustion by binding at sites that do not require cooperation with AP-1.

Summary In response to acute infection, naive CD8+ T cells expand, differentiate into effector cells and then contract to a long-lived pool of memory cells after pathogen clearance. During chronic infections or in tumors, CD8+ T cells acquire an “exhausted” phenotype. Here we present genome-wide comparisons of chromatin accessibility and gene expression from endogenous CD8+ T cells responding to acute and chronic viral infection using ATAC-seq and RNA-seq. Acquisition of effector, memory or exhausted phenotypes was associated with stable changes in chromatin accessibility away from the naive T cell state. Regions differentially-accessible between functional subsets in vivo were enriched for binding sites of transcription factors known to regulate these subsets, including E2A, BATF, IRF4, T-bet and TCF1. Exhaustion-specific accessible regions were enriched for consensus binding sites for NFAT and Nr4a family members, indicating that chronic stimulation confers a unique accessibility profile on exhausted cells.

Dynamic changes in the expression of transcription factors (TFs) can influence specification of distinct CD8+ T cell fates, but the observation of equivalent expression of TF among differentially-fated precursor cells suggests additional underlying mechanisms. Here, we profiled genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8+ T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that TF expression and binding contributed to establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal novel TFs influencing the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8+ T cell differentiation, regulated the formation of terminal-effector and memory-precursor cell-fates, respectively. Our data define the epigenetic landscape of differentiation intermediates, facilitating identification of TFs with previously unappreciated roles in CD8+ T cell differentiation.

Dengue virus (DENV) affects millions of people, causing more than 20,000 deaths annually. No effective treatment for the disease caused by DENV infection is currently available, partially due to the lack of knowledge on the basic aspects of the viral life cycle, including the molecular basis of the interaction between viral components and cellular compartments. Here, we characterized the properties of the interaction between the DENV capsid (C) protein and hepatic lipid droplets (LDs), which was recently shown to be essential for the virus replication cycle. Zeta potential analysis revealed a negative surface charge of LDs, with an average surface charge of ؊19 mV. The titration of LDs with C protein led to an increase of the surface charge, which reached a plateau at ؉13.7 mV, suggesting that the viral protein-LD interaction exposes the protein cationic surface to the aqueous environment. Atomic force microscopy (AFM)-based force spectroscopy measurements were performed by using C proteinfunctionalized AFM tips. The C protein-LD interaction was found to be strong, with a single (un)binding force of 33.6 pN. This binding was dependent on high intracellular concentrations of potassium ions but not sodium. The inhibition of Na ؉ /K ؉ -ATPase in DENV-infected cells resulted in the dissociation of C protein from LDs and a 50-fold inhibition of infectious virus production but not of RNA replication, indicating a biological relevance for the potassium-dependent interaction. Limited proteolysis of the LD surface impaired the C protein-LD interaction, and force measurements in the presence of specific antibodies indicated that perilipin 3 (TIP47) is the major DENV C protein ligand on the surface of LDs. Dengue virus (DENV) causes the most important arthropodborne human viral disease, with 2.5 billion people at risk, 100 million infections, and more than 20,000 deaths annually, primarily in tropical developing countries (20). Four genetically distinct serotypes (DENV1 to DENV4) have been identified, which are transmitted among humans through the bite of an infected mosquito of the genus Aedes. DENV belongs to the family Flaviviridae, together with other important human pathogens, such as yellow fever virus (YFV), West Nile virus (WNV), and hepatitis C virus (HCV). The clinical manifestations of DENV infection range from a mild illness to a severe and potentially life-threatening disease for which no treatment is available so far, due at least in part to the limited understanding of the molecular mechanisms that underlie the interaction between DENV and its host cells.The DENV genome is a single-stranded positive-sense RNA molecule of approximately 11 kb that is translated from a single open reading frame, generating a polyprotein associated with the endoplasmic reticulum (ER) membrane (34). The polyprotein is cleaved co-and posttranslationally by cellular and viral proteases into three structural proteins (capsid [C], premembrane [prM], and envelope [E]) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B,...