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Summary Persistent viral infections and tumors drive development of exhausted T (T EX ) cells. In these settings, T EX cells establish an important host-pathogen or host-tumor stalemate. However, T EX cells erode over time, leading to loss of pathogen or cancer containment. We identified microRNA (miR)-155 as a key regulator of sustained T EX 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 T EX cells. However, rather than strictly antagonizing exhaustion, miR-155 promoted a terminal T EX 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 T EX cells. Thus, we identify a mechanism of miR-155 regulation of T EX cells and a key role for Fosl2 in T cell exhaustion. [ABSTRACT FROM AUTHOR]

Acute respiratory tract viral infections (ARTIs) cause significant morbidity and mortality. CD8 T cells are fundamental to host responses, but transcriptional alterations underlying anti-viral mechanisms and links to clinical characteristics remain unclear. CD8 T cell transcriptional circuitry in acutely ill pediatric patients with influenza-like illness was distinct for different viral pathogens. Although changes included expected upregulation of interferon-stimulated genes (ISGs), transcriptional downregulation was prominent upon exposure to innate immune signals in early IFV infection. Network analysis linked changes to severity of infection, asthma, sex, and age. An influenza pediatric signature (IPS) distinguished acute influenza from other ARTIs and outperformed other influenza prediction gene lists. The IPS allowed a deeper investigation of the connection between transcriptional alterations and clinical characteristics of acute illness, including age-based differences in circuits connecting the STAT1/2 pathway to ISGs. A CD8 T cell-focused systems immunology approach in pediatrics identified age-based alterations in ARTI host response pathways. [ABSTRACT FROM AUTHOR]

Summary 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. [ABSTRACT FROM AUTHOR]

The transcription factors T-bet and Eomesodermin (Eomes) regulate CD8 T cell exhaustion through undefined mechanisms. Here, we show that the subcellular localization of T-bet and Eomes dictate their regulatory activity in exhausted T cells (T EX s). T EX s had a higher ratio of nuclear Eomes:T-bet than memory T cells (T MEM s) during chronic lymphocytic choriomeningitis virus (LCMV) infection in preclinical cancer models and in human tumors. Biochemically, T-bet and Eomes compete for the same DNA sequences, including the Pdcd1 T-box. High nuclear T-bet strongly represses Pdcd1 transcription in T MEM , whereas low nuclear T-bet in T EX leads to a dominant effect of Eomes that acts as a weaker repressor of Pdcd1. Blocking PD-1 signaling in T EX s increases nuclear T-bet, restoring stronger repression of Pdcd1 , and driving T-bet-associated gene expression programs of chemotaxis, homing, and activation. These data identify a mechanism whereby the T-bet-Eomes axis regulates exhaustion through their nuclear localization, providing insights into how these transcription factors regulate T EX biology. [Display omitted] • The relative amounts of nuclear T-bet and Eomes T cells partially define exhaustion • PD1 blockade increases nuclear T-bet and upregulates T cell activation and homing genes • T-bet and Eomes recognize and bind to the same T-box domain in the Pdcd1 promoter • Eomes is a weak transcriptional repressor of Pdcd1 in T EX s McLane et al. demonstrate that T-bet and Eomes expression contributes to exhaustion, but also their nuclear localization, and therefore functional activity, plays a key role. PD-1 blockade restores nuclear T-bet and promotes T cell homing and activation through direct competition with Eomes at gene promoters, such as Pdcd1. [ABSTRACT FROM AUTHOR]

Combination checkpoint blockade with anti-PD-1 and anti-CTLA-4 antibodies has shown promising efficacy in melanoma. However, the underlying mechanism in humans remains unclear. Here, we perform paired single-cell RNA and T cell receptor (TCR) sequencing across time in 36 patients with stage IV melanoma treated with anti-PD-1, anti-CTLA-4, or combination therapy. We develop the algorithm Cyclone to track temporal clonal dynamics and underlying cell states. Checkpoint blockade induces waves of clonal T cell responses that peak at distinct time points. Combination therapy results in greater magnitude of clonal responses at 6 and 9 weeks compared to single-agent therapies, including melanoma-specific CD8+ T cells and exhausted CD8+ T cell (T EX) clones. Focused analyses of T EX identify that anti-CTLA-4 induces robust expansion and proliferation of progenitor T EX , which synergizes with anti-PD-1 to reinvigorate T EX during combination therapy. These next generation immune profiling approaches can guide the selection of drugs, schedule, and dosing for novel combination strategies. [Display omitted] • A novel algorithm Cyclone uncovers patterns of clonal trajectories across time • Immune checkpoint blockade induces distinct waves of clonal responses after each dose • Melanoma-specific CD8+ T cell responses are present at 3 and 6 weeks after treatment • CTLA-4 blockade induces robust progenitor-exhausted CD8+ T cell responses Wang et al. analyze longitudinal blood from melanoma patients on checkpoint blockade using paired single-cell RNA and TCR sequencing and uncover waves of clonal responses that peak at different time points. By studying anti-CTLA-4, anti-PD-1, and in combination, they show that anti-CTLA-4 reinvigorates progenitor exhausted CD8+ T cells while anti-PD-1 drives their differentiation. [ABSTRACT FROM AUTHOR]