Stat5 opposes the transcription factor Tox and rewires exhausted CD8+ T cells toward durable effector-like states during chronic antigen exposure.

Rewiring exhausted CD8+ T (Tex) cells toward functional states remains a therapeutic challenge. Tex cells are epigenetically programmed by the transcription factor Tox. However, epigenetic remodeling occurs as Tex cells transition from progenitor (Texprog) to intermediate (Texint) and terminal (Texterm) subsets, suggesting development flexibility. We examined epigenetic transitions between Tex cell subsets and revealed a reciprocally antagonistic circuit between Stat5a and Tox. Stat5 directed Texint cell formation and re-instigated partial effector biology during this Texprog-to-Texint cell transition. Constitutive Stat5a activity antagonized Tox and rewired CD8+ T cells from exhaustion to a durable effector and/or natural killer (NK)-like state with superior anti-tumor potential. Temporal induction of Stat5 activity in Tex cells using an orthogonal IL-2:IL2Rß-pair fostered Texint cell accumulation, particularly upon PD-L1 blockade. Re-engaging Stat5 also partially reprogrammed the epigenetic landscape of exhaustion and restored polyfunctionality. These data highlight therapeutic opportunities of manipulating the IL-2-Stat5 axis to rewire Tex cells toward more durably protective states.

Trimodal single-cell profiling reveals a novel pediatric CD8αα+ T cell subset and broad age-related molecular reprogramming across the T cell compartment.

Age-associated changes in the T cell compartment are well described. However, limitations of current single-modal or bimodal single-cell assays, including flow cytometry, RNA-seq (RNA sequencing) and CITE-seq (cellular indexing of transcriptomes and epitopes by sequencing), have restricted our ability to deconvolve more complex cellular and molecular changes. Here, we profile >300,000 single T cells from healthy children (aged 11-13 years) and older adults (aged 55-65 years) by using the trimodal assay TEA-seq (single-cell analysis of mRNA transcripts, surface protein epitopes and chromatin accessibility), which revealed that molecular programming of T cell subsets shifts toward a more activated basal state with age. Naive CD4+ T cells, considered relatively resistant to aging, exhibited pronounced transcriptional and epigenetic reprogramming. Moreover, we discovered a novel CD8αα+ T cell subset lost with age that is epigenetically poised for rapid effector responses and has distinct inhibitory, costimulatory and tissue-homing properties. Together, these data reveal new insights into age-associated changes in the T cell compartment that may contribute to differential immune responses.

In vitro modeling of CD8+ T cell exhaustion enables CRISPR screening to reveal a role for BHLHE40.

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.

The SWI/SNF chromatin remodeling complexes BAF and PBAF differentially regulate epigenetic transitions in exhausted CD8+ T cells.

CD8+ T cell exhaustion (Tex) limits disease control during chronic viral infections and cancer. Here, we investigated the epigenetic factors mediating major chromatin-remodeling events in Tex-cell development. A protein-domain-focused in vivo CRISPR screen identified distinct functions for two versions of the SWI/SNF chromatin-remodeling complex in Tex-cell differentiation. Depletion of the canonical SWI/SNF form, BAF, impaired initial CD8+ T cell responses in acute and chronic infection. In contrast, disruption of PBAF enhanced Tex-cell proliferation and survival. Mechanistically, PBAF regulated the epigenetic and transcriptional transition from TCF-1+ progenitor Tex cells to more differentiated TCF-1- Tex subsets. Whereas PBAF acted to preserve Tex progenitor biology, BAF was required to generate effector-like Tex cells, suggesting that the balance of these factors coordinates Tex-cell subset differentiation. Targeting PBAF improved tumor control both alone and in combination with anti-PD-L1 immunotherapy. Thus, PBAF may present a therapeutic target in cancer immunotherapy.

In Vitro Modeling of CD8 T Cell Exhaustion Enables CRISPR Screening to Reveal a Role for BHLHE40.

Identifying novel molecular mechanisms of exhausted CD8 T cells (T ex ) is a key goal of improving immunotherapy of cancer and other diseases. However, high-throughput interrogation of in vivo T ex can be costly and inefficient. In vitro models of T ex are easily customizable and quickly generate high cellular yield, offering an opportunity to perform 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 T ex . We leveraged this model of in vitro chronic stimulation in combination with pooled CRISPR screening to uncover 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 subsets of T ex . By developing and benchmarking an in vitro model of T ex , we demonstrate the utility of mechanistically annotated in vitro models of T ex , in combination with high-throughput approaches, as a discovery pipeline to uncover novel T ex biology.

Shared and distinct biological circuits in effector, memory and exhausted CD8+ T cells revealed by temporal single-cell transcriptomics and epigenetics.

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.

PD-1 directed immunotherapy alters Tfh and humoral immune responses to seasonal influenza vaccine.

Anti-programmed death-1 (anti-PD-1) immunotherapy reinvigorates CD8 T cell responses in patients with cancer but PD-1 is also expressed by other immune cells, including follicular helper CD4 T cells (Tfh) which are involved in germinal centre responses. Little is known, however, about the effects of anti-PD-1 immunotherapy on noncancer immune responses in humans. To investigate this question, we examined the impact of anti-PD-1 immunotherapy on the Tfh-B cell axis responding to unrelated viral antigens. Following influenza vaccination, a subset of adults receiving anti-PD-1 had more robust circulating Tfh responses than adults not receiving immunotherapy. PD-1 pathway blockade resulted in transcriptional signatures of increased cellular proliferation in circulating Tfh and responding B cells compared with controls. These latter observations suggest an underlying change in the Tfh-B cell and germinal centre axis in a subset of immunotherapy patients. Together, these results demonstrate dynamic effects of anti-PD-1 therapy on influenza vaccine responses and highlight analytical vaccination as an approach that may reveal underlying immune predisposition to adverse events.

Autoreactive CD8+ T cells are restrained by an exhaustion-like program that is maintained by LAG3.

Impaired chronic viral and tumor clearance has been attributed to CD8+ T cell exhaustion, a differentiation state in which T cells have reduced and altered effector function that can be partially reversed upon blockade of inhibitory receptors. The role of the exhaustion program and transcriptional networks that control CD8+ T cell function and fate in autoimmunity is not clear. Here we show that intra-islet CD8+ T cells phenotypically, transcriptionally, epigenetically and metabolically possess features of canonically exhausted T cells, yet maintain important differences. This 'restrained' phenotype can be perturbed and disease accelerated by CD8+ T cell-restricted deletion of the inhibitory receptor lymphocyte activating gene 3 (LAG3). Mechanistically, LAG3-deficient CD8+ T cells have enhanced effector-like functions, trafficking to the islets, and have a diminished exhausted phenotype, highlighting a physiological role for an exhaustion program in limiting autoimmunity and implicating LAG3 as a target for autoimmune therapy.

MicroRNA-29a attenuates CD8 T cell exhaustion and induces memory-like CD8 T cells during chronic infection.

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.

Human epigenetic and transcriptional T cell differentiation atlas for identifying functional T cell-specific enhancers.

The clinical benefit of T cell immunotherapies remains limited by incomplete understanding of T cell differentiation and dysfunction. We generated an epigenetic and transcriptional atlas of T cell differentiation from healthy humans that included exhausted CD8 T cells and applied this resource in three ways. First, we identified modules of gene expression and chromatin accessibility, revealing molecular coordination of differentiation after activation and between central memory and effector memory. Second, we applied this healthy molecular framework to three settings-a neoadjuvant anti-PD1 melanoma trial, a basal cell carcinoma scATAC-seq dataset, and autoimmune disease-associated SNPs-yielding insights into disease-specific biology. Third, we predicted genome-wide cis-regulatory elements and validated this approach for key effector genes using CRISPR interference, providing functional annotation and demonstrating the ability to identify targets for non-coding cellular engineering. These studies define epigenetic and transcriptional regulation of human T cells and illustrate the utility of interrogating disease in the context of a healthy T cell atlas.

Signaling Through FcγRIIA and the C5a-C5aR Pathway Mediate Platelet Hyperactivation in COVID-19.

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.

Epigenetic scarring of exhausted T cells hinders memory differentiation upon eliminating chronic antigenic stimulation.

Exhausted CD8 T cells (TEX) are a distinct state of T cell differentiation associated with failure to clear chronic viruses and cancer. Immunotherapies such as PD-1 blockade can reinvigorate TEX cells, but reinvigoration is not durable. A major unanswered question is whether TEX cells differentiate into functional durable memory T cells (TMEM) upon antigen clearance. Here, using a mouse model, we found that upon eliminating chronic antigenic stimulation, TEX cells partially (re)acquire phenotypic and transcriptional features of TMEM cells. These 'recovering' TEX cells originated from the T cell factor (TCF-1+) TEX progenitor subset. Nevertheless, the recall capacity of these recovering TEX cells remained compromised as compared to TMEM cells. Chromatin-accessibility profiling revealed a failure to recover core memory epigenetic circuits and maintenance of a largely exhausted open chromatin landscape. Thus, despite some phenotypic and transcriptional recovery upon antigen clearance, exhaustion leaves durable epigenetic scars constraining future immune responses. These results support epigenetic remodeling interventions for TEX cell-targeted immunotherapies.

Vaccine-induced ICOS+CD38+ circulating Tfh are sensitive biosensors of age-related changes in inflammatory pathways.

Humoral immune responses are dysregulated with aging, but the cellular and molecular pathways involved remain incompletely understood. In particular, little is known about the effects of aging on T follicular helper (Tfh) CD4 cells, the key cells that provide help to B cells for effective humoral immunity. We performed transcriptional profiling and cellular analysis on circulating Tfh before and after influenza vaccination in young and elderly adults. First, whole-blood transcriptional profiling shows that ICOS+CD38+ cTfh following vaccination preferentially enriches in gene sets associated with youth versus aging compared to other circulating T cell types. Second, vaccine-induced ICOS+CD38+ cTfh from the elderly had increased the expression of genes associated with inflammation, including tumor necrosis factor-nuclear factor κB (TNF-NF-κB) pathway activation. Finally, vaccine-induced ICOS+CD38+ cTfh display strong enrichment for signatures of underlying age-associated biological changes. These data highlight the ability to use vaccine-induced cTfh as cellular "biosensors" of underlying inflammatory and/or overall immune health.

Signaling through FcγRIIA and the C5a-C5aR pathway mediates platelet hyperactivation in COVID-19.

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 exhibit higher basal levels of activation measured by P-selectin surface expression, and have a poor functional reserve upon in vitro stimulation. Correlating clinical features to the ability of plasma from COVID-19 patients to stimulate control platelets 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, thus identifying these potentially actionable pathways as central for platelet activation and/or vascular complications 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. These studies have implications for the role of platelet hyperactivation in complications associated with SARS-CoV-2 infection. ONE-SENTENCE SUMMARY: The FcγRIIA and C5a-C5aR pathways mediate platelet hyperactivation in COVID-19.

Role of nuclear localization in the regulation and function of T-bet and Eomes in exhausted CD8 T cells.

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 (TEXs). TEXs had a higher ratio of nuclear Eomes:T-bet than memory T cells (TMEMs) 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 TMEM, whereas low nuclear T-bet in TEX leads to a dominant effect of Eomes that acts as a weaker repressor of Pdcd1. Blocking PD-1 signaling in TEXs 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 TEX biology.

In vivo CD8+ T cell CRISPR screening reveals control by Fli1 in infection and cancer.

Improving effector activity of antigen-specific T cells is a major goal in cancer immunotherapy. Despite the identification of several effector T cell (TEFF)-driving transcription factors (TFs), the transcriptional coordination of TEFF biology remains poorly understood. We developed an in vivo T cell CRISPR screening platform and identified a key mechanism restraining TEFF biology through the ETS family TF, Fli1. Genetic deletion of Fli1 enhanced TEFF responses without compromising memory or exhaustion precursors. Fli1 restrained TEFF lineage differentiation by binding to cis-regulatory elements of effector-associated genes. Loss of Fli1 increased chromatin accessibility at ETS:RUNX motifs, allowing more efficient Runx3-driven TEFF biology. CD8+ T cells lacking Fli1 provided substantially better protection against multiple infections and tumors. These data indicate that Fli1 safeguards the developing CD8+ T cell transcriptional landscape from excessive ETS:RUNX-driven TEFF cell differentiation. Moreover, genetic deletion of Fli1 improves TEFF differentiation and protective immunity in infections and cancer.

Inhibitory signaling sustains a distinct early memory CD8+ T cell precursor that is resistant to DNA damage.

The developmental origins of memory T cells remain incompletely understood. During the expansion phase of acute viral infection, we identified a distinct subset of virus-specific CD8+ T cells that possessed distinct characteristics including expression of CD62L, T cell factor 1 (TCF-1), and Eomesodermin; relative quiescence; expression of activation markers; and features of limited effector differentiation. These cells were a quantitatively minor subpopulation of the TCF-1+ pool and exhibited self-renewal, heightened DNA damage surveillance activity, and preferential long-term recall capacity. Despite features of memory and somewhat restrained proliferation during the expansion phase, this subset displayed evidence of stronger TCR signaling than other responding CD8+ T cells, coupled with elevated expression of multiple inhibitory receptors including programmed cell death 1 (PD-1), lymphocyte activating gene 3 (LAG-3), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), CD5, and CD160. Genetic ablation of PD-1 and LAG-3 compromised the formation of this CD62Lhi TCF-1+ subset and subsequent CD8+ T cell memory. Although central memory phenotype CD8+ T cells were formed in the absence of these cells, subsequent memory CD8+ T cell recall responses were compromised. Together, these results identify an important link between genome integrity maintenance and CD8+ T cell memory. Moreover, the data indicate a role for inhibitory receptors in preserving key memory CD8+ T cell precursors during initial activation and differentiation. Identification of this rare subpopulation within the memory CD8+ T cell precursor pool may help reconcile models of the developmental origin of long-term CD8+ T cell memory.

The Identity of Human Tissue-Emigrant CD8+ T Cells.

Lymphocyte migration is essential for adaptive immune surveillance. However, our current understanding of this process is rudimentary, because most human studies have been restricted to immunological analyses of blood and various tissues. To address this knowledge gap, we used an integrated approach to characterize tissue-emigrant lineages in thoracic duct lymph (TDL). The most prevalent immune cells in human and non-human primate efferent lymph were T cells. Cytolytic CD8+ T cell subsets with effector-like epigenetic and transcriptional signatures were clonotypically skewed and selectively confined to the intravascular circulation, whereas non-cytolytic CD8+ T cell subsets with stem-like epigenetic and transcriptional signatures predominated in tissues and TDL. Moreover, these anatomically distinct gene expression profiles were recapitulated within individual clonotypes, suggesting parallel differentiation programs independent of the expressed antigen receptor. Our collective dataset provides an atlas of the migratory immune system and defines the nature of tissue-emigrant CD8+ T cells that recirculate via TDL.