The roles of individual microRNAs in the CD8+ T cell response remain mostly unexplored. Katsikis and colleagues show that miR-155 regulates type I interferon responsiveness and CD8+ T cell responses to pathogens in vivo. We found upregulation of expression of the microRNA miR-155 in primary effector and effector memory CD8+ T cells, but low miR-155 expression in naive and central memory cells. Antiviral CD8+ T cell responses and viral clearance were impaired in miR-155-deficient mice, and this defect was intrinsic to CD8+ T cells, as miR-155-deficient CD8+ T cells mounted greatly diminished primary and memory responses. Conversely, miR-155 overexpression augmented antiviral CD8+ T cell responses in vivo. Gene-expression profiling showed that miR-155-deficient CD8+ T cells had enhanced type I interferon signaling and were more susceptible to interferon's antiproliferative effect. Inhibition of the type I interferon–associated transcription factors STAT1 or IRF7 resulted in enhanced responses of miR-155-deficient CD8+ T cells in vivo. We have thus identified a previously unknown role for miR-155 in regulating responsiveness to interferon and CD8+ T cell responses to pathogens in vivo.

Despite the important role that microRNAs (miRNAs) play in immunity and inflammation, their involvement in systemic sclerosis (SSc) remains poorly characterized. miRNA-155 (miR-155) plays a role in pulmonary fibrosis and its expression can be induced with interleukin (IL)-1β. SSc fibroblasts have activated inflammasomes that are integrally involved in mediating the myofibroblast phenotype. In light of this, we investigated whether miR-155 played a role in SSc and if its expression was dependent on inflammasome activation. miR-155 expression was confirmed in SSc dermal and lung fibroblasts by quantitative polymerase chain reaction (PCR). Wild-type and NLRP3-deficient murine fibroblasts were utilized to explore the regulation of miR-155 during inflammasome activation. miR-155-deficient fibroblasts and retroviral transductions with a miR-155 expression or control vectors were used to understand the contribution of miR-155 in fibrosis. miR-155 was significantly increased and the highest expressing miRNA in SSc lung fibroblasts. Its expression was dependent on inflammasome activation as miR-155 expression could be blocked when inflammasome signaling was inhibited. In the absence of miR-155, inflammasome-mediated collagen synthesis could not be induced but was restored when miR-155 was expressed in miR-155-deficient fibroblasts. miR-155 is upregulated in SSc. These results suggest that the inflammasome promotes the expression of miR-155 and that miR-155 is a critical miRNA that drives fibrosis.

Persistent viral infections and tumors drive development of exhausted T (TEX) cells. In these settings, TEX cells establish an important host-pathogen or host-tumor stalemate. However, TEX cells erode over time, leading to loss of pathogen or cancer containment. We identified microRNA (miR)-155 as a key regulator of sustained TEX cell responses during chronic lymphocytic choriomeningitis virus (LCMV) infection. Genetic deficiency of miR-155 ablated CD8 T cell responses during chronic infection. Conversely, enhanced miR-155 expression promoted expansion and long-term persistence of TEX cells. However, rather than strictly antagonizing exhaustion, miR-155 promoted a terminal TEX cell subset. Transcriptional profiling identified coordinated control of cell signaling and transcription factor pathways, including the key AP-1 family member Fosl2. Overexpression of Fosl2 reversed the miR-155 effects, identifying a link between miR-155 and the AP-1 transcriptional program in regulating TEX cells. Thus, we identify a mechanism of miR-155 regulation of TEX cells and a key role for Fosl2 in T cell exhaustion.

The increasing risk of incidental exposure to nanomaterials has led to mounting concerns regarding nanotoxicity. Zinc oxide nanoparticles (ZnO NPs) are produced in large quantities and have come under scrutiny due to their capacity to cause cytotoxicity in vitro and potential to cause harm in vivo. Recent evidence has indicated that ZnO NPs promote autophagy in cells; however, the signaling pathways and the role of ion release inducing toxicity remain unclear. In this study, we report that ZnO NPs are immunotoxic to primary and immortalized immune cells. Importantly, such immunotoxicity is observed in mice in vivo, since death of splenocytes is seen after intranasal exposure to ZnO NPs. We determined that ZnO NPs release free Zn(2+) that can be taken up by immune cells, resulting in cell death. Inhibiting free Zn(2+) ions in solution with EDTA or their uptake with CaCl2 abrogates ZnO NP-induced cell death. ZnO NP-mediated immune cell death was associated with increased levels of intracellular reactive oxygen species (ROS). ZnO NP death was not due to apoptosis, necroptosis or pyroptosis. Exposure of immune cells to ZnO NPs resulted in autophagic death and increased levels of LC3A, an essential component of autophagic vacuoles. Accordingly, ZnO NP-mediated upregulation of LC3A and induction of immune cell death were inhibited by blocking autophagy and ROS production. We conclude that release of Zn(2+) from ZnO NPs triggers the production of excessive intracellular ROS, resulting in autophagic death of immune cells. Our findings suggest that exposure to ZnO NPs has the potential to impact host immunity.

Exhaustion is a dysfunctional state of cytotoxic CD8+ T cells (CTL) observed in chronic infection and cancer. Current in vivo models of CTL exhaustion using chronic viral infections or cancer yield very few exhausted CTL, limiting the analysis that can be done on these cells. Establishing an in vitro system that rapidly induces CTL exhaustion would therefore greatly facilitate the study of this phenotype, identify the truly exhaustion-associated changes and allow the testing of novel approaches to reverse or prevent exhaustion. Here we show that repeat stimulation of purified TCR transgenic OT-I CTL with their specific peptide induces all the functional (reduced cytokine production and polyfunctionality, decreased in vivo expansion capacity) and phenotypic (increased inhibitory receptors expression and transcription factor changes) characteristics of exhaustion. Importantly, in vitro exhausted cells shared the transcriptomic characteristics of the gold standard of exhaustion, CTL from LCMV cl13 infections. Gene expression of both in vitro and in vivo exhausted CTL was distinct from T cell anergy. Using this system, we show that Tcf7 promoter DNA methylation contributes to TCF1 downregulation in exhausted CTL. Thus this novel in vitro system can be used to identify genes and signaling pathways involved in exhaustion and will facilitate the screening of reagents that prevent/reverse CTL exhaustion. In this manuscript, we describe an in vitro method that rapidly establishes large numbers of exhausted CD8+ T cells. The exhaustion of CTL induced by this method has been fully validated by multiple approaches (cytokine production, polyfunctionality, cytotoxicity, in vivo proliferation, inhibitory receptors, transcription factors, RNAseq and DNA methylation). This method will facilitate not only the study of T cell exhaustion but also the screening of drugs. As proof of point, we use this method to show that TCF-1 downregulation in terminally exhausted T cells is accompanied by Tcf7 DNA promoter methylation and show that a transmethylase inhibitor can prevent TCF-1 downregulation. Our method presents a critical resource for the study of CTL exhaustion and the screening of drugs and interventions.

The role of Type I interferon (IFN) during pathogenic HIV and SIV infections remains unclear, with conflicting observations suggesting protective versus immunopathological effects. We therefore examined the effect of IFNα/β on T cell death and viremia in HIV infection. Ex vivo analysis of eight pro- and anti-apoptotic molecules in chronic HIV-1 infection revealed that pro-apoptotic Bak was increased in CD4+ T cells and correlated directly with sensitivity to CD95/Fas-mediated apoptosis and inversely with CD4+ T cell counts. Apoptosis sensitivity and Bak expression were primarily increased in effector memory T cells. Knockdown of Bak by RNA interference inhibited CD95/Fas-induced death of T cells from HIV-1-infected individuals. In HIV-1-infected patients, IFNα-stimulated gene expression correlated positively with ex vivo T cell Bak levels, CD95/Fas-mediated apoptosis and viremia and negatively with CD4+ T cell counts. In vitro IFNα/β stimulation enhanced Bak expression, CD95/Fas expression and CD95/Fas-mediated apoptosis in healthy donor T cells and induced death of HIV-specific CD8+ T cells from HIV-1-infected patients. HIV-1 in vitro sensitized T cells to CD95/Fas-induced apoptosis and this was Toll-like receptor (TLR)7/9- and Type I IFN-dependent. This sensitization by HIV-1 was due to an indirect effect on T cells, as it occurred in peripheral blood mononuclear cell cultures but not purified CD4+ T cells. Finally, peak IFNα levels and viral loads correlated negatively during acute SIV infection suggesting a potential antiviral effect, but positively during chronic SIV infection indicating that either the virus drives IFNα production or IFNα may facilitate loss of viral control. The above findings indicate stage-specific opposing effects of Type I IFNs during HIV-1 infection and suggest a novel mechanism by which these cytokines contribute to T cell depletion, dysregulation of cellular immunity and disease progression. Type I interferons (IFNα/β) are innate immune mediators that are produced by cells in response to viral infections. Although the protective effects of IFNα/β are well-established, it is not clear whether these cytokines are beneficial or deleterious during HIV-1 infection. We report that HIV-1 infection renders T cells more prone to undergo programmed death and that this enhanced apoptosis susceptibility is associated with abnormal expression of pro- and anti-apoptotic molecules. Importantly, IFNα/β increases the expression level of the pro-apoptotic protein, Bak, a major gatekeeper of the mitochondrial apoptosis machinery. Exposure of healthy donor T cells to IFNα/β increased Bak expression and induced an apoptosis sensitivity that is similar to what is observed in HIV-1-infected patient T cells. Furthermore, elevated IFNα production and Bak expression correlated with heightened T cell apoptosis, low CD4+ T cell numbers and high viral loads in patients. In a primate model of HIV-1 infection, we observed that increased IFNα was associated with low peak viral loads in early infection, but high viremia and decreased CD4+ T cell counts during chronic infection. These studies identify a novel mechanism by which Type I IFN may induce immune dysfunction in HIV-1 infection.