BATF regulates progenitor to cytolytic effector CD8+ T cell transition during chronic viral infection.

During chronic viral infection, CD8+ T cells develop into three major phenotypically and functionally distinct subsets: Ly108+TCF-1+ progenitors, Ly108-CX3CR1- terminally exhausted cells and the recently identified CX3CR1+ cytotoxic effector cells. Nevertheless, how CX3CR1+ effector cell differentiation is transcriptionally and epigenetically regulated remains elusive. Here, we identify distinct gene regulatory networks and epigenetic landscapes underpinning the formation of these subsets. Notably, our data demonstrate that CX3CR1+ effector cells bear a striking similarity to short-lived effector cells during acute infection. Genetic deletion of Tbx21 significantly diminished formation of the CX3CR1+ subset. Importantly, we further identify a previously unappreciated role for the transcription factor BATF in maintaining a permissive chromatin structure that allows the transition from TCF-1+ progenitors to CX3CR1+ effector cells. BATF directly bound to regulatory regions near Tbx21 and Klf2, modulating their enhancer accessibility to facilitate the transition. These mechanistic insights can potentially be harnessed to overcome T cell exhaustion during chronic infection and cancer.

Tfh-cell-derived interleukin 21 sustains effector CD8+ T cell responses during chronic viral infection.

CD4+ T cell-derived interleukin 21 (IL-21) sustains CD8+ T cell responses during chronic viral infection, but the helper subset that confers this protection remains unclear. Here, we applied scRNA and ATAC-seq approaches to determine the heterogeneity of IL-21+CD4+ T cells during LCMV clone 13 infection. CD4+ T cells were comprised of three transcriptionally and epigenetically distinct populations: Cxcr6+ Th1 cells, Cxcr5+ Tfh cells, and a previously unrecognized Slamf6+ memory-like (Tml) subset. T cell differentiation was specifically redirected toward the Tml subset during chronic, but not acute, LCMV infection. Although this subset displayed an enhanced capacity to accumulate and some developmental plasticity, it remained largely quiescent, which may hinder its helper potential. Conversely, mixed bone marrow chimera experiments revealed that Tfh cell-derived IL-21 was critical to sustain CD8+ T cell responses and viral control. Thus, strategies that bolster IL-21+Tfh cell responses may prove effective in enhancing CD8+ T cell-mediated immunity.

Lymphocytic Choriomeningitis Virus Clone 13 Infection Results in CD8 T Cell-Mediated Host Mortality in Diacylglycerol Kinase α-Deficient Mice.

Diacylglycerol is a potent element of intracellular secondary signaling cascades whose production is enhanced by cell-surface receptor agonism and function is regulated by enzymatic degradation by diacylglycerol kinases (DGKs). In T cells, stringent regulation of the activity of this second messenger maintains an appropriate balance between effector function and anergy. In this article, we demonstrate that DGKα is an indispensable regulator of TCR-mediated activation of CD8 T cells in lymphocytic choriomeningitis virus Clone 13 viral infection. In the absence of DGKα, Clone 13 infection in a murine model results in a pathologic, proinflammatory state and a multicellular immunopathologic host death that is predominantly driven by CD8 effector T cells.

E2A-regulated epigenetic landscape promotes memory CD8 T cell differentiation.

During an acute viral infection, CD8 T cells encounter a myriad of antigenic and inflammatory signals of variable strength, which sets off individual T cells on their own differentiation trajectories. However, the developmental path for each of these cells will ultimately lead to one of only two potential outcomes after clearance of the infection-death or survival and development into memory CD8 T cells. How this cell fate decision is made remains incompletely understood. In this study, we explore the transcriptional changes during effector and memory CD8 T cell differentiation at the single-cell level. Using single-cell, transcriptome-derived gene regulatory network analysis, we identified two main groups of regulons that govern this differentiation process. These regulons function in concert with changes in the enhancer landscape to confer the establishment of the regulatory modules underlying the cell fate decision of CD8 T cells. Furthermore, we found that memory precursor effector cells maintain chromatin accessibility at enhancers for key memory-related genes and that these enhancers are highly enriched for E2A binding sites. Finally, we show that E2A directly regulates accessibility of enhancers of many memory-related genes and that its overexpression increases the frequency of memory precursor effector cells and accelerates memory cell formation while decreasing the frequency of short-lived effector cells. Overall, our results suggest that effector and memory CD8 T cell differentiation is largely regulated by two transcriptional circuits, with E2A serving as an important epigenetic regulator of the memory circuit.

Self-Renewing Islet TCF1+ CD8 T Cells Undergo IL-27-Controlled Differentiation to Become TCF1- Terminal Effectors during the Progression of Type 1 Diabetes.

In type 1 diabetes (T1D) autoreactive CD8 T cells infiltrate pancreatic islets and destroy insulin-producing ß cells. Progression to T1D onset is a chronic process, which suggests that the effector activity of ß-cell autoreactive CD8 T cells needs to be maintained throughout the course of disease development. The mechanism that sustains diabetogenic CD8 T cell effectors during the course of T1D progression has not been completely defined. Here we used single-cell RNA sequencing to gain further insight into the phenotypic complexity of islet-infiltrating CD8 T cells in NOD mice. We identified two functionally distinct subsets of activated CD8 T cells, CD44highTCF1+CXCR6- and CD44highTCF1-CXCR6+, in islets of prediabetic NOD mice. Compared with CD44highTCF1+CXCR6- CD8 T cells, the CD44highTCF1-CXCR6+ subset expressed higher levels of inhibitory and cytotoxic molecules and was more prone to apoptosis. Adoptive cell transfer experiments revealed that CD44highTCF1+CXCR6- CD8 T cells, through continuous generation of the CD44highTCF1-CXCR6+ subset, were more capable than the latter population to promote insulitis and the development of T1D. We further showed that direct IL-27 signaling in CD8 T cells promoted the generation of terminal effectors from the CD44highTCF1+CXCR6- population. These results indicate that islet CD44highTCF1+CXCR6- CD8 T cells are a progenitor-like subset with self-renewing capacity, and, under an IL-27-controlled mechanism, they differentiate into the CD44highTCF1-CXCR6+ terminal effector population. Our study provides new insight into the sustainability of the CD8 T cell response in the pathogenesis of T1D.

Single Cell RNAseq Analysis of Cytokine-Treated Human Islets: Association of Cellular Stress with Impaired Cytokine Responsiveness.

Pancreatic ß-cells are essential for survival, being the only cell type capable of insulin secretion. While they are believed to be vulnerable to damage by inflammatory cytokines such as interleukin-1 beta (IL-1ß) and interferon-gamma, we have recently identified physiological roles for cytokine signaling in rodent ß-cells that include the stimulation of antiviral and antimicrobial gene expression and the inhibition of viral replication. In this study, we examine cytokine-stimulated changes in gene expression in human islets using single-cell RNA sequencing. Surprisingly, the global responses of human islets to cytokine exposure were remarkably blunted compared to our previous observations in the mouse. The small population of human islet cells that were cytokine responsive exhibited increased expression of IL-1ß-stimulated antiviral guanylate-binding proteins, just like in the mouse. Most human islet cells were not responsive to cytokines, and this lack of responsiveness was associated with high expression of genes encoding ribosomal proteins. We further correlated the expression levels of RPL5 with stress response genes, and when expressed at high levels, RPL5 is predictive of failure to respond to cytokines in all endocrine cells. We postulate that donor causes of death and isolation methodologies may contribute to stress of the islet preparation. Our findings indicate that activation of stress responses in human islets limits cytokine-stimulated gene expression, and we urge caution in the evaluation of studies that have examined cytokine-stimulated gene expression in human islets without evaluation of stress-related gene expression.

Clonal lineage tracing reveals mechanisms skewing CD8+ T cell fate decisions in chronic infection.

Although recent evidence demonstrates heterogeneity among CD8+ T cells during chronic infection, developmental relationships and mechanisms underlying their fate decisions remain incompletely understood. Using single-cell RNA and TCR sequencing, we traced the clonal expansion and differentiation of CD8+ T cells during chronic LCMV infection. We identified immense clonal and phenotypic diversity, including a subset termed intermediate cells. Trajectory analyses and infection models showed intermediate cells arise from progenitor cells before bifurcating into terminal effector and exhausted subsets. Genetic ablation experiments identified that type I IFN drives exhaustion through an IRF7-dependent mechanism, possibly through an IFN-stimulated subset bridging progenitor and exhausted cells. Conversely, Zeb2 was critical for generating effector cells. Intriguingly, some T cell clones exhibited lineage bias. Mechanistically, we identified that TCR avidity correlates with an exhausted fate, whereas SHP-1 selectively restricts low-avidity effector cell accumulation. Thus, our work elucidates novel mechanisms underlying CD8+ T cell fate determination during persistent infection and suggests two potential pathways leading to exhaustion.

A spatial sequencing atlas of age-induced changes in the lung during influenza infection.

Influenza virus infection causes increased morbidity and mortality in the elderly. Aging impairs the immune response to influenza, both intrinsically and because of altered interactions with endothelial and pulmonary epithelial cells. To characterize these changes, we performed single-cell RNA sequencing (scRNA-seq), spatial transcriptomics, and bulk RNA sequencing (bulk RNA-seq) on lung tissue from young and aged female mice at days 0, 3, and 9 post-influenza infection. Our analyses identified dozens of key genes differentially expressed in kinetic, age-dependent, and cell type-specific manners. Aged immune cells exhibited altered inflammatory, memory, and chemotactic profiles. Aged endothelial cells demonstrated characteristics of reduced vascular wound healing and a prothrombotic state. Spatial transcriptomics identified novel profibrotic and antifibrotic markers expressed by epithelial and non-epithelial cells, highlighting the complex networks that promote fibrosis in aged lungs. Bulk RNA-seq generated a timeline of global transcriptional activity, showing increased expression of genes involved in inflammation and coagulation in aged lungs. Our work provides an atlas of high-throughput sequencing methodologies that can be used to investigate age-related changes in the response to influenza virus, identify novel cell-cell interactions for further study, and ultimately uncover potential therapeutic targets to improve health outcomes in the elderly following influenza infection.

BATF is Required for Treg Homeostasis and Stability to Prevent Autoimmune Pathology.

Regulatory T (Treg) cells are inevitable to prevent deleterious immune responses to self and commensal microorganisms. Treg function requires continuous expression of the transcription factor (TF) FOXP3 and is divided into two major subsets: resting (rTregs) and activated (aTregs). Continuous T cell receptor (TCR) signaling plays a vital role in the differentiation of aTregs from their resting state, and in their immune homeostasis. The process by which Tregs differentiate, adapt tissue specificity, and maintain stable phenotypic expression at the transcriptional level is still inconclusivei. In this work, the role of BATF is investigated, which is induced in response to TCR stimulation in naïve T cells and during aTreg differentiation. Mice lacking BATF in Tregs developed multiorgan autoimmune pathology. As a transcriptional regulator, BATF is required for Treg differentiation, homeostasis, and stabilization of FOXP3 expression in different lymphoid and non-lymphoid tissues. Epigenetically, BATF showed direct regulation of Treg-specific genes involved in differentiation, maturation, and tissue accumulation. Most importantly, FOXP3 expression and Treg stability require continuous BATF expression in Tregs, as it regulates demethylation and accessibility of the CNS2 region of the Foxp3 locus. Considering its role in Treg stability, BATF should be considered an important therapeutic target in autoimmune disease.

Delineating the transcriptional landscape and clonal diversity of virus-specific CD4+ T cells during chronic viral infection.

Although recent evidence indicates that CD4+ T cells responding to chronic viral infection are functionally heterogenous, our understanding of the developmental relationships between these subsets, and a determination of how their transcriptional landscape compares to their acute infection counterparts remains unclear. Additionally, whether cell-intrinsic factors such as TCR usage influence CD4+ T cell fate commitment during persistent infection has not previously been studied. Herein, we perform single-cell RNA sequencing (scRNA-seq) combined with single-cell T cell receptor sequencing (scTCR-seq) on virus-specific CD4+ T cells isolated from mice infected with chronic lymphocytic choriomeningitis virus (LCMV) infection. We identify several transcriptionally distinct states among the Th1, Tfh, and memory-like T cell subsets that form at the peak of infection, including the presence of a previously unrecognized Slamf7+ subset with cytolytic features. We further show that the relative distribution of these populations differs substantially between acute and persistent LCMV infection. Moreover, while the progeny of most T cell clones displays membership within each of these transcriptionally unique populations, overall supporting a one cell-multiple fate model, a small fraction of clones display a biased cell fate decision, suggesting that TCR usage may impact CD4+ T cell development during chronic infection. Importantly, comparative analyses further reveal both subset-specific and core gene expression programs that are differentially regulated between CD4+ T cells responding to acute and chronic LCMV infection. Together, these data may serve as a useful framework and allow for a detailed interrogation into the clonal distribution and transcriptional circuits underlying CD4+ T cell differentiation during chronic viral infection.

Cytokine and Nitric Oxide-Dependent Gene Regulation in Islet Endocrine and Nonendocrine Cells.

While exposure to inflammatory cytokines is thought to contribute to pancreatic ß-cell damage during diabetes, primarily because cytokine-induced nitric oxide impairs ß-cell function and causes cell death with prolonged exposure, we hypothesize that there is a physiological role for cytokine signaling that protects ß-cells from a number of environmental stresses. This hypothesis is derived from the knowledge that ß-cells are essential for survival even though they have a limited capacity to replicate, yet they are exposed to high cytokine levels during infection as most of the pancreatic blood flow is directed to islets. Here, mouse islets were subjected to single-cell RNA sequencing following 18-h cytokine exposure. Treatment with IL-1ß and IFN-γ stimulates expression of inducible nitric oxide synthase (iNOS) mRNA and antiviral and immune-associated genes as well as repression of islet identity factors in a subset of ß- and non-ß-endocrine cells in a nitric oxide-independent manner. Nitric oxide-dependent expression of genes encoding heat shock proteins was observed in both ß- and non-ß-endocrine cells. Interestingly, cells with high expression of heat shock proteins failed to increase antiviral and immune-associated gene expression, suggesting that nitric oxide may be an internal "off switch" to prevent the negative effects of prolonged cytokine signaling in islet endocrine cells. We found no evidence for pro-apoptotic gene expression following 18-h cytokine exposure. Our findings suggest that the primary functions of cytokines and nitric oxide are to protect islet endocrine cells from damage, and only when regulation of cytokine signaling is lost does irreversible damage occur.

Autoreactive CD8 T cells in NOD mice exhibit phenotypic heterogeneity but restricted TCR gene usage.

Type 1 diabetes (T1D) is an autoimmune disorder defined by CD8 T cell-mediated destruction of pancreatic ß cells. We have previously shown that diabetogenic CD8 T cells in the islets of non-obese diabetic mice are phenotypically heterogeneous, but clonal heterogeneity remains relatively unexplored. Here, we use paired single-cell RNA and T-cell receptor sequencing (scRNA-seq and scTCR-seq) to characterize autoreactive CD8 T cells from the islets and spleens of non-obese diabetic mice. scTCR-seq demonstrates that CD8 T cells targeting the immunodominant ß-cell epitope IGRP206-214 exhibit restricted TCR gene usage. scRNA-seq identifies six clusters of autoreactive CD8 T cells in the islets and six in the spleen, including memory and exhausted cells. Clonal overlap between IGRP206-214-reactive CD8 T cells in the islets and spleen suggests these cells may circulate between the islets and periphery. Finally, we identify correlations between TCR genes and T-cell clonal expansion and effector fate. Collectively, our work demonstrates that IGRP206-214-specific CD8 T cells are phenotypically heterogeneous but clonally restricted, raising the possibility of selectively targeting these TCR structures for therapeutic benefit.

Spatial transcriptomics demonstrates the role of CD4 T cells in effector CD8 T cell differentiation during chronic viral infection.

CD4 T cell help is critical to sustain effector CD8 T cell responses during chronic infection, notably via T follicular helper (Tfh)-derived interleukin-21 (IL-21). Conversely, CD4 depletion results in severe CD8 T cell dysfunction and lifelong viremia despite CD4 T cell reemergence following transient depletion. These observations suggest that repopulating CD4 subsets are functionally or numerically insufficient to orchestrate a robust CD8 response. We utilize spatial transcriptomics and single-cell RNA sequencing (scRNA-seq) to investigate CD4 T cell heterogeneity under CD4-replete and -deplete conditions and explore cellular interactions during chronic infection. Although IL-21-producing Tfh cells repopulate following transient CD4 depletion, they are outnumbered by immunomodulatory CD4 T cells. Moreover, the splenic architecture appears perturbed, with decreases in white pulp regions, coinciding with germinal center losses. These disruptions in splenic architecture are associated with diminished Tfh and progenitor CD8 T cell colocalization, providing a potential mechanism for impaired progenitor-to-effector CD8 T cell differentiation during "un-helped" conditions.

Single-cell immune profiling reveals a developmentally distinct CD4+ GM-CSF+ T-cell lineage that induces GI tract GVHD.

Gastrointestinal (GI) tract involvement is a major determinant for subsequent morbidity and mortality arising during graft-versus-host disease (GVHD). CD4+ T cells that produce granulocyte-macrophage colony stimulating factor (GM-CSF) have emerged as central mediators of inflammation in this tissue site as GM-CSF serves as a critical cytokine link between the adaptive and innate arms of the immune system. However, cellular heterogeneity within the CD4+ GM-CSF+ T-cell population due to the concurrent production of other inflammatory cytokines has raised questions as to whether these cells have a common ontology or if a unique CD4+ GM-CSF+ subset exists that differs from other defined T helper subtypes. Using single-cell RNA sequencing analysis (scRNAseq), we identified two CD4+ GM-CSF+ T-cell populations that arose during GVHD and were distinguishable according to the presence or absence of interferon-γ (IFN-γ) coexpression. CD4+ GM-CSF+ IFN-γ- T cells, which emerged preferentially in the colon, had a distinct transcriptional profile, used unique gene regulatory networks, and possessed a nonoverlapping T-cell receptor repertoire compared with CD4+ GM-CSF+ IFN-γ+ T cells as well as all other transcriptionally defined CD4+ T-cell populations in the colon. Functionally, this CD4+ GM-CSF+ T-cell population contributed to pathologic damage in the GI tract that was critically dependent on signaling through the interleukin-17 (IL-7) receptor but was independent of type 1 interferon signaling. Thus, these studies help to unravel heterogeneity within CD4+ GM-CSF+ T cells that arise during GVHD and define a developmentally distinct colitogenic T helper subtype GM-CSF+ subset that mediates immunopathology.

BATF promotes group 2 innate lymphoid cell-mediated lung tissue protection during acute respiratory virus infection.

Activated group 2 innate lymphoid cells (ILC2s) accumulate and promote inflammatory resolution and tissue repair in host defense against acute respiratory viral infections. However, the heterogeneity of ILC2s in the lung and the mechanisms by which ILC2 cells contribute to tissue repair remain elusive. Using single-cell RNA sequencing, we identify a transcriptionally distinct ILC2 subset that showed enrichment for wound healing signature genes and the transcription factor BATF. BATF promotes the proliferation and function of ILC2s and restricts their plasticity during infection with influenza virus. In the absence of BATF, ILC2s lose their immune protective properties and acquire pathogenic ILC3-like functions, leading to persistent neutrophil infiltration, tissue damage, and respiratory failure. Mechanistically, BATF directly binds to the cis-regulatory elements of wound healing genes, maintains their chromatin accessibility, and promotes their expression. Last, BATF plays an important role in an IL-33­ST2 feed-forward loop that supports ILC2 cell identity and function. Collectively, our findings shed light on a BATF-dependent ILC2 program, thereby providing a potential therapeutic target for terminating detrimental inflammation during acute viral infection.

Group 3 innate lymphoid cells require BATF to regulate gut homeostasis in mice.

Group 3 innate lymphoid cells (ILC3s) are crucial for the maintenance of host-microbiota homeostasis in gastrointestinal mucosal tissues. The mechanisms that maintain lineage identity of intestinal ILC3s and ILC3-mediated orchestration of microbiota and mucosal T cell immunity are elusive. Here, we identified BATF as a gatekeeper of ILC3 homeostasis in the gut. Depletion of BATF in ILC3s resulted in excessive interferon-γ production, dysbiosis, aberrant T cell immune responses, and spontaneous inflammatory bowel disease (IBD), which was considerably ameliorated by the removal of adaptive immunity, interferon-γ blockade, or antibiotic treatment. Mechanistically, BATF directly binds to the cis-regulatory elements of type 1 effector genes, restrains their chromatin accessibility, and inhibits their expression. Conversely, BATF promotes chromatin accessibility of genes involved in MHCII antigen processing and presentation pathways, which in turn directly promotes the transition of precursor ILC3s to MHCII+ ILC3s. Collectively, our findings reveal that BATF is a key transcription factor for maintaining ILC3 stability and coordinating ILC3-mediated control of intestinal homeostasis.

Inhibiting BRD4 to generate BETter T cell memory.

BRD4 is a bromodomain-containing protein that binds acetylated histones to regulate transcription. In this issue of JEM, Milner et al. (2021. J. Exp. Med.https://doi.org/10.1084/jem.20202512) show that BRD4 plays a critical role in the effector function of CD8 T cells responding to infection and cancer.

Single-Cell Transcriptomics Reveals Core Regulatory Programs That Determine the Heterogeneity of Circulating and Tissue-Resident Memory CD8+ T Cells.

During acute infections, CD8+ T cells form various memory subpopulations to provide long-lasting protection against reinfection. T central memory (TCM), T effector memory (TEM), and long-lived effector (LLE) cells are circulating memory populations with distinct plasticity, migration patterns, and effector functions. Tissue-resident memory (TRM) cells permanently reside in the frontline sites of pathogen entry and provide tissue-specific protection upon reinfection. Here, using single-cell RNA-sequencing (scRNA-seq) and bulk RNA-seq, we examined the different and shared transcriptomes and regulators of TRM cells with other circulating memory populations. Furthermore, we identified heterogeneity within the TRM pool from small intestine and novel transcriptional regulators that may control the phenotypic and functional heterogeneity of TRM cells during acute infection. Our findings provide a resource for future studies to identify novel pathways for enhancing vaccination and immunotherapeutic approaches.

Single-cell RNA sequencing of mouse islets exposed to proinflammatory cytokines.

Exposure to proinflammatory cytokines is believed to contribute to pancreatic ß-cell damage during diabetes development. Although some cytokine-mediated changes in islet gene expression are known, the heterogeneity of the response is not well-understood. After 6-h treatment with IL-1ß and IFN-γ alone or together, mouse islets were subjected to single-cell RNA sequencing. Treatment with both cytokines together led to expression of inducible nitric oxide synthase mRNA (Nos2) and antiviral and immune-associated genes in a subset of ß-cells. Interestingly, IL-1ß alone activated antiviral genes. Subsets of δ- and α-cells expressed Nos2 and exhibited similar gene expression changes as ß-cells, including increased expression of antiviral genes and repression of identity genes. Finally, cytokine responsiveness was inversely correlated with expression of genes encoding heat shock proteins. Our findings show that all islet endocrine cell types respond to cytokines, IL-1ß induces the expression of protective genes, and cellular stress gene expression is associated with inhibition of cytokine signaling.

Harnessing the IL-21-BATF Pathway in the CD8+ T Cell Anti-Tumor Response.

In cancer, CD8+ T cells enter a dysfunctional state which prevents them from effectively targeting and killing tumor cells. Tumor-infiltrating CD8+ T cells consist of a heterogeneous population of memory-like progenitor, effector, and terminally exhausted cells that exhibit differing functional and self-renewal capacities. Our recently published work has shown that interleukin (IL)-21-producing CD4+ T cells help to generate effector CD8+ T cells within the tumor, which results in enhanced tumor control. However, the molecular mechanisms by which CD4+ helper T cells regulate the differentiation of effector CD8+ T cells are not well understood. In this study, we found that Basic Leucine Zipper ATF-Like Transcription Factor (BATF), a transcription factor downstream of IL-21 signaling, is critical to maintain CD8+ T cell effector function within the tumor. Using mixed bone marrow chimeras, we demonstrated that CD8+ T cell-specific deletion of BATF resulted in impaired tumor control. In contrast, overexpressing BATF in CD8+ T cells enhanced effector function and resulted in improved tumor control, bypassing the need for CD4+ helper T cells. Transcriptomic analyses revealed that BATF-overexpressing CD8+ T cells had increased expression of costimulatory receptors, effector molecules, and transcriptional regulators, which may contribute to their enhanced activation and effector function. Taken together, our study unravels a previously unappreciated CD4+ T cell-derived IL-21-BATF axis that could provide therapeutic insights to enhance effector CD8+ T cell function to fight cancer.