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Naïve CD8+ T cells can differentiate into effector (Teff), memory (Tmem) or exhausted (Tex) T cells. These developmental pathways are associated with distinct transcriptional and epigenetic changes that endow cells with different functional capacities and therefore therapeutic potential. The molecular circuitry underlying these developmental trajectories and the extent of heterogeneity within Teff, Tmem and Tex populations remain poorly understood. Here, we used the lymphocytic choriomeningitis virus model of acute-resolving and chronic infection to address these gaps by applying longitudinal single-cell RNA-sequencing (scRNA-seq) and single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq) analyses. These analyses uncovered new subsets, including a subpopulation of Tex cells expressing natural killer cell-associated genes that is dependent on the transcription factor Zeb2, as well as multiple distinct TCF-1+ stem/progenitor-like subsets in acute and chronic infection. These data also revealed insights into the reshaping of Tex subsets following programmed death 1 (PD-1) pathway blockade and identified a key role for the cell stress regulator, Btg1, in establishing the Tex population. Finally, these results highlighted how the same biological circuits such as cytotoxicity or stem/progenitor pathways can be used by CD8+ T cell subsets with highly divergent underlying chromatin landscapes generated during different infections.

CD8 T cells mediate protection against intracellular pathogens and tumors. However, persistent antigen during chronic infections or cancer leads to T cell exhaustion, suboptimal functionality, and reduced protective capacity. Despite considerable work interrogating the transcriptional regulation of exhausted CD8 T cells (TEX), the posttranscriptional control of TEX remains poorly understood. Here, we interrogated the role of microRNAs (miRs) in CD8 T cells responding to acutely resolved or chronic viral infection and identified miR-29a as a key regulator of TEX. Enforced expression of miR-29a improved CD8 T cell responses during chronic viral infection and antagonized exhaustion. miR-29a inhibited exhaustion-driving transcriptional pathways, including inflammatory and T cell receptor signaling, and regulated ribosomal biogenesis. As a result, miR-29a fostered a memory-like CD8 T cell differentiation state during chronic infection. Thus, we identify miR-29a as a key regulator of TEX and define mechanisms by which miR-29a can divert exhaustion toward a more beneficial memory-like CD8 T cell differentiation state.

Current paradigms of CD8+ T cell-mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8+ T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (TRMs). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8+ T cells in LTs also resemble TRMs Moreover, high frequencies of HIV-specific CD8+ TRMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific TRMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-TRMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8+ T cell responses resident within LTs.

T follicular helper cells (Tfh), a subset of [CD4.sup.+] T cells, provide requisite help to B cells in the germinal centers (GC) of lymphoid tissue. GC Tfh are identified by high expression of the chemokine receptor CXCR5 and the inhibitory molecule PD-1. Although more accessible, blood contains lower frequencies of [CXCR5.sup.+] and [PD-1.sup.+] cells that have been termed circulating Tfh (cTfh). However, it remains unclear whether GC Tfh exit lymphoid tissues and populate this cTfh pool. To examine exiting cells, we assessed the phenotype of Tfh present within the major conduit of efferent lymph from lymphoid tissues into blood, the human thoracic duct. Unlike what was found in blood, we consistently identified a CXCR5-bright PD-1-bright ([CXCR5.sup.Br][PD-1.sup.Br]) Tfh population in thoracic duct lymph (TDL). These [CXCR5.sup.Br][PD-1.sup.Br] TDL Tfh shared phenotypic and transcriptional similarities with GC Tfh. Moreover, components of the epigenetic profile of GC Tfh could be detected in [CXCR5.sup.Br][PD-1.sup.Br] TDL Tfh and the transcriptional imprint of this epigenetic signature was enriched in an activated cTfh subset known to contain vaccine-responding cells. Together with data showing shared TCR sequences between the [CXCR5.sup.Br][PD-1.sup.Br] TDL Tfh and cTfh, these studies identify a population in TDL as a circulatory intermediate connecting the biology of Tfh in blood to Tfh in lymphoid tissue., Introduction T follicular helper cells (Tfh) are a subset of [CD4.sup.+] T cells that provide B cell help within the follicles of secondary lymphoid tissues. Tfh promote B cell class [...]

Exhausted CD8.sup.+ T (T.sub.ex) cells in chronic infections and cancer have limited effector function, high co-expression of inhibitory receptors and extensive transcriptional changes compared with effector (T.sub.eff) or memory (T.sub.mem) CD8.sup.+ T cells. T.sub.ex cells are important clinical targets of checkpoint blockade and other immunotherapies. Epigenetically, T.sub.ex cells are a distinct immune subset, with a unique chromatin landscape compared with T.sub.eff and T.sub.mem cells. However, the mechanisms that govern the transcriptional and epigenetic development of T.sub.ex cells remain unknown. Here we identify the HMG-box transcription factor TOX as a central regulator of T.sub.ex cells in mice. TOX is largely dispensable for the formation of T.sub.eff and T.sub.mem cells, but it is critical for exhaustion: in the absence of TOX, T.sub.ex cells do not form. TOX is induced by calcineurin and NFAT2, and operates in a feed-forward loop in which it becomes calcineurin-independent and sustained in T.sub.ex cells. Robust expression of TOX therefore results in commitment to T.sub.ex cells by translating persistent stimulation into a distinct T.sub.ex cell transcriptional and epigenetic developmental program. The transcription factor TOX is a central regulator of the transcriptional and epigenetic development of exhausted T cells., Author(s): Omar Khan [sup.1] [sup.2] [sup.3] [sup.14] , Josephine R. Giles [sup.1] [sup.2] [sup.3] , Sierra McDonald [sup.4] [sup.5] [sup.6] , Sasikanth Manne [sup.1] [sup.2] , Shin Foong Ngiow [sup.1] [...]

Identifying molecular mechanisms of exhausted CD8 T cells (Tex) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo Tex can be costly and inefficient. In vitro models of Tex are easily customizable and quickly generate high cellular yield, enabling CRISPR screening and other high-throughput assays. We established an in vitro model of chronic stimulation and benchmarked key phenotypic, functional, transcriptional, and epigenetic features against bona fide in vivo Tex. We leveraged this model of in vitro chronic stimulation in combination with CRISPR screening to identify transcriptional regulators of T cell exhaustion. This approach identified several transcription factors, including BHLHE40. In vitro and in vivo validation defined a role for BHLHE40 in regulating a key differentiation checkpoint between progenitor and intermediate Tex subsets. By developing and benchmarking an in vitro model of Tex, then applying high-throughput CRISPR screening, we demonstrate the utility of mechanistically annotated in vitro models of Tex. Editor's summary: T cell exhaustion represents a distinct cellular state that contributes to immune dysfunction during chronic viral infections and cancer. To overcome limitations of in vivo models, Wu et al. developed and benchmarked an in vitro platform of T cell exhaustion. CD8+ T cells were cultured with dendritic cells repeatedly pulsed with cognate peptide and IL-2. This system reproduced the functional, transcriptomic, and epigenetic features of exhausted T cells generated during chronic lymphocytic choriomeningitis virus infection in mice. Using a pooled CRISPR screen targeting 120 transcription factors, they identified BHLHE40 as a regulator of T cell exhaustion, and genetic silencing of BHLHE40 prevented the development of more differentiated exhausted T cell subsets. Together, these findings provide a scalable and controlled platform for interrogating the biology underlying T cell exhaustion. —Claire Olingy [ABSTRACT FROM AUTHOR]

Immunologic responses to anti-PD-1 therapy in melanoma patients occur rapidly with pharmacodynamic T cell responses detectable in blood by 3 weeks. It is unclear, however, whether these early blood-based observations translate to the tumor microenvironment. We conducted a study of neoadjuvant/adjuvant anti-PD-1 therapy in stage III/IV melanoma. We hypothesized that immune reinvigoration in the tumor would be detectable at 3 weeks and that this response would correlate with disease-free survival. We identified a rapid and potent anti-tumor response, with 8 of 27 patients experiencing a complete or major pathological response after a single dose of anti-PD-1, all of whom remain disease free. These rapid pathologic and clinical responses were associated with accumulation of exhausted CD8 T cells in the tumor at 3 weeks, with reinvigoration in the blood observed as early as 1 week. Transcriptional analysis demonstrated a pretreatment immune signature (neoadjuvant response signature) that was associated with clinical benefit. In contrast, patients with disease recurrence displayed mechanisms of resistance including immune suppression, mutational escape, and/or tumor evolution. Neoadjuvant anti-PD-1 treatment is effective in high-risk resectable stage III/IV melanoma. Pathological response and immunological analyses after a single neoadjuvant dose can be used to predict clinical outcome and to dissect underlying mechanisms in checkpoint blockade. Neoadjuvant PD-1 blockade in patients with resectable melanoma followed by adjuvant maintenance results in early immunological effects driving clinical benefit and reveals transcriptional and genomic mechanisms of response., Author(s): Alexander C. Huang [sup.1] [sup.2] [sup.3] [sup.4] , Robert J. Orlowski [sup.1] [sup.11] , Xiaowei Xu [sup.4] [sup.5] , Rosemarie Mick [sup.3] [sup.4] [sup.6] , Sangeeth M. George [sup.7] [...]

Patients with COVID-19 present with a wide variety of clinical manifestations. Thromboembolic events constitute a significant cause of morbidity and mortality in patients infected with SARS-CoV-2. Severe COVID-19 has been associated with hyperinflammation and pre-existing cardiovascular disease. Platelets are important mediators and sensors of inflammation and are directly affected by cardiovascular stressors. In this report, we found that platelets from severely ill, hospitalized COVID-19 patients exhibited higher basal levels of activation measured by P-selectin surface expression and had poor functional reserve upon in vitro stimulation. To investigate this question in more detail, we developed an assay to assess the capacity of plasma from COVID-19 patients to activate platelets from healthy donors. Platelet activation was a common feature of plasma from COVID-19 patients and correlated with key measures of clinical outcome including kidney and liver injury, and APACHEIII scores. Further, we identified ferritin as a pivotal clinical marker associated with platelet hyperactivation. The COVID-19 plasma-mediated effect on control platelets was highest for patients that subsequently developed inpatient thrombotic events. Proteomic analysis of plasma from COVID-19 patients identified key mediators of inflammation and cardiovascular disease that positively correlated with in vitro platelet activation. Mechanistically, blocking the signaling of the FcγRIIa-Syk and C5a-C5aR pathways on platelets, using antibody-mediated neutralization, IgG depletion or the Syk inhibitor fostamatinib, reversed this hyperactivity driven by COVID-19 plasma and prevented platelet aggregation in endothelial microfluidic chamber conditions. These data identified these potentially actionable pathways as central for platelet activation and/or vascular complications and clinical outcomes in COVID-19 patients. In conclusion, we reveal a key role of platelet-mediated immunothrombosis in COVID-19 and identify distinct, clinically relevant, targetable signaling pathways that mediate this effect. [ABSTRACT FROM AUTHOR]