Selective IL-27 production by intestinal regulatory T cells permits gut-specific regulation of TH17 cell immunity.

Regulatory T cells (Treg cells) are instrumental in establishing immunological tolerance. However, the precise effector mechanisms by which Treg cells control a specific type of immune response in a given tissue remains unresolved. By simultaneously studying Treg cells from different tissue origins under systemic autoimmunity, in the present study we show that interleukin (IL)-27 is specifically produced by intestinal Treg cells to regulate helper T17 cell (TH17 cell) immunity. Selectively increased intestinal TH17 cell responses in mice with Treg cell-specific IL-27 ablation led to exacerbated intestinal inflammation and colitis-associated cancer, but also helped protect against enteric bacterial infection. Furthermore, single-cell transcriptomic analysis has identified a CD83+CD62Llo Treg cell subset that is distinct from previously characterized intestinal Treg cell populations as the main IL-27 producers. Collectively, our study uncovers a new Treg cell suppression mechanism crucial for controlling a specific type of immune response in a particular tissue and provides further mechanistic insights into tissue-specific Treg cell-mediated immune regulation.

Tissue-resident memory CD8+ T cells possess unique transcriptional, epigenetic and functional adaptations to different tissue environments.

Tissue-resident memory T cells (TRM cells) provide protective immunity, but the contributions of specific tissue environments to TRM cell differentiation and homeostasis are not well understood. In the present study, the diversity of gene expression and genome accessibility by mouse CD8+ TRM cells from distinct organs that responded to viral infection revealed both shared and tissue-specific transcriptional and epigenetic signatures. TRM cells in the intestine and salivary glands expressed transforming growth factor (TGF)-ß-induced genes and were maintained by ongoing TGF-ß signaling, whereas those in the fat, kidney and liver were not. Constructing transcriptional-regulatory networks identified the transcriptional repressor Hic1 as a critical regulator of TRM cell differentiation in the small intestine and showed that Hic1 overexpression enhanced TRM cell differentiation and protection from infection. Provision of a framework for understanding how CD8+ TRM cells adapt to distinct tissue environments, and identification of tissue-specific transcriptional regulators mediating these adaptations, inform strategies to boost protective memory responses at sites most vulnerable to infection.

A regulatory circuit controlled by extranuclear and nuclear retinoic acid receptor α determines T cell activation and function.

Ligation of retinoic acid receptor alpha (RARα) by RA promotes varied transcriptional programs associated with immune activation and tolerance, but genetic deletion approaches suggest the impact of RARα on TCR signaling. Here, we examined whether RARα would exert roles beyond transcriptional regulation. Specific deletion of the nuclear isoform of RARα revealed an RARα isoform in the cytoplasm of T cells. Extranuclear RARα was rapidly phosphorylated upon TCR stimulation and recruited to the TCR signalosome. RA interfered with extranuclear RARα signaling, causing suboptimal TCR activation while enhancing FOXP3+ regulatory T cell conversion. TCR activation induced the expression of CRABP2, which translocates RA to the nucleus. Deletion of Crabp2 led to increased RA in the cytoplasm and interfered with signalosome-RARα, resulting in impaired anti-pathogen immunity and suppressed autoimmune disease. Our findings underscore the significance of subcellular RA/RARα signaling in T cells and identify extranuclear RARα as a component of the TCR signalosome and a determinant of immune responses.

Small intestine and colon tissue-resident memory CD8+ T cells exhibit molecular heterogeneity and differential dependence on Eomes.

Tissue-resident memory CD8+ T (TRM) cells are a subset of memory T cells that play a critical role in limiting early pathogen spread and controlling infection. TRM cells exhibit differences across tissues, but their potential heterogeneity among distinct anatomic compartments within the small intestine and colon has not been well recognized. Here, by analyzing TRM cells from the lamina propria and epithelial compartments of the small intestine and colon, we showed that intestinal TRM cells exhibited distinctive patterns of cytokine and granzyme expression along with substantial transcriptional, epigenetic, and functional heterogeneity. The T-box transcription factor Eomes, which represses TRM cell formation in some tissues, exhibited unexpected context-specific regulatory roles in supporting the maintenance of established TRM cells in the small intestine, but not in the colon. Taken together, these data provide previously unappreciated insights into the heterogeneity and differential requirements for the formation vs. maintenance of intestinal TRM cells.

Clone Wars: Survival of the Fittest CD8+ Trm Cells.

In this issue of Immunity, Hirai et al. illuminate competition for the cytokine TGFß as a key regulator of CD8+ tissue-resident memory T cell persistence and occupancy in the skin epidermal niche.

Early transcriptional and epigenetic regulation of CD8+ T cell differentiation revealed by single-cell RNA sequencing.

During microbial infection, responding CD8+ T lymphocytes differentiate into heterogeneous subsets that together provide immediate and durable protection. To elucidate the dynamic transcriptional changes that underlie this process, we applied a single-cell RNA-sequencing approach and analyzed individual CD8+ T lymphocytes sequentially throughout the course of a viral infection in vivo. Our analyses revealed a striking transcriptional divergence among cells that had undergone their first division and identified previously unknown molecular determinants that controlled the fate specification of CD8+ T lymphocytes. Our findings suggest a model for the differentiation of terminal effector cells initiated by an early burst of transcriptional activity and subsequently refined by epigenetic silencing of transcripts associated with memory lymphocytes, which highlights the power and necessity of single-cell approaches.

Epigenetic landscapes reveal transcription factors that regulate CD8+ T cell differentiation.

Dynamic changes in the expression of transcription factors (TFs) can influence the specification of distinct CD8+ T cell fates, but the observation of equivalent expression of TFs among differentially fated precursor cells suggests additional underlying mechanisms. Here we profiled the genome-wide histone modifications, open chromatin and gene expression of naive, terminal-effector, memory-precursor and memory CD8+ T cell populations induced during the in vivo response to bacterial infection. Integration of these data suggested that the expression and binding of TFs contributed to the establishment of subset-specific enhancers during differentiation. We developed a new bioinformatics method using the PageRank algorithm to reveal key TFs that influence the generation of effector and memory populations. The TFs YY1 and Nr3c1, both constitutively expressed during CD8+ T cell differentiation, regulated the formation of terminal-effector cell fates and memory-precursor cell fates, respectively. Our data define the epigenetic landscape of differentiation intermediates and facilitate the identification of TFs with previously unappreciated roles in CD8+ T cell differentiation.

Heterogenous Populations of Tissue-Resident CD8+ T Cells Are Generated in Response to Infection and Malignancy.

Tissue-resident memory CD8+ T cells (Trm) provide host protection through continuous surveillance of non-lymphoid tissues. Using single-cell RNA-sequencing (scRNA-seq) and genetic reporter mice, we identified discrete lineages of intestinal antigen-specific CD8+ T cells, including a Blimp1hiId3lo tissue-resident effector cell population most prominent in the early phase of acute viral and bacterial infections and a molecularly distinct Blimp1loId3hi tissue-resident memory population that subsequently accumulated at later infection time points. These Trm populations exhibited distinct cytokine production, secondary memory potential, and transcriptional programs including differential roles for transcriptional regulators Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal Trm. Extending our analysis to malignant tissue, we also identified discrete populations of effector-like and memory-like CD8+ T cell populations with tissue-resident gene-expression signatures that shared features of terminally exhausted and progenitor-exhausted T cells, respectively. Our findings provide insight into the development and functional heterogeneity of Trm cells, which has implications for enhancing vaccination and immunotherapy approaches.

Metabolic programs of T cell tissue residency empower tumour immunity.

Tissue resident memory CD8+ T (TRM) cells offer rapid and long-term protection at sites of reinfection1. Tumour-infiltrating lymphocytes with characteristics of TRM cells maintain enhanced effector functions, predict responses to immunotherapy and accompany better prognoses2,3. Thus, an improved understanding of the metabolic strategies that enable tissue residency by T cells could inform new approaches to empower immune responses in tissues and solid tumours. Here, to systematically define the basis for the metabolic reprogramming supporting TRM cell differentiation, survival and function, we leveraged in vivo functional genomics, untargeted metabolomics and transcriptomics of virus-specific memory CD8+ T cell populations. We found that memory CD8+ T cells deployed a range of adaptations to tissue residency, including reliance on non-steroidal products of the mevalonate-cholesterol pathway, such as coenzyme Q, driven by increased activity of the transcription factor SREBP2. This metabolic adaptation was most pronounced in the small intestine, where TRM cells interface with dietary cholesterol and maintain a heightened state of activation4, and was shared by functional tumour-infiltrating lymphocytes in diverse tumour types in mice and humans. Enforcing synthesis of coenzyme Q through deletion of Fdft1 or overexpression of PDSS2 promoted mitochondrial respiration, memory T cell formation following viral infection and enhanced antitumour immunity. In sum, through a systematic exploration of TRM cell metabolism, we reveal how these programs can be leveraged to fuel memory CD8+ T cell formation in the context of acute infections and enhance antitumour immunity.

Molecular regulation of effector and memory T cell differentiation.

Immunological memory is a cardinal feature of adaptive immunity and an important goal of vaccination strategies. Here we highlight advances in the understanding of the diverse T lymphocyte subsets that provide acute and long-term protection from infection. These include new insights into the transcription factors, and the upstream 'pioneering' factors that regulate their accessibility to key sites of gene regulation, as well as metabolic regulators that contribute to the differentiation of effector and memory subsets; ontogeny and defining characteristics of tissue-resident memory lymphocytes; and origins of the remarkable heterogeneity exhibited by activated T cells. Collectively, these findings underscore progress in delineating the underlying pathways that control diversification in T cell responses but also reveal gaps in the knowledge, as well as the challenges that arise in the application of this knowledge to rationally elicit desired T cell responses through vaccination and immunotherapy.

Early specification of CD8+ T lymphocyte fates during adaptive immunity revealed by single-cell gene-expression analyses.

T lymphocytes responding to microbial infection give rise to effector cells that mediate acute host defense and memory cells that provide long-lived immunity, but the fundamental question of when and how these cells arise remains unresolved. Here we combined single-cell gene-expression analyses with 'machine-learning' approaches to trace the transcriptional 'roadmap' of individual CD8(+) T lymphocytes throughout the course of an immune response in vivo. Gene-expression signatures predictive of eventual fates could be discerned as early as the first T lymphocyte division and may have been influenced by asymmetric partitioning of the receptor for interleukin 2 (IL-2Rα) during mitosis. Our findings emphasize the importance of single-cell analyses in understanding fate determination and provide new insights into the specification of divergent lymphocyte fates early during an immune response to microbial infection.

Early transcriptional and epigenetic divergence of CD8+ T cells responding to acute versus chronic infection.

During a microbial infection, responding CD8+ T cells give rise to effector cells that provide acute host defense and memory cells that provide sustained protection. An alternative outcome is exhaustion, a state of T cell dysfunction that occurs in the context of chronic infections and cancer. Although it is evident that exhausted CD8+ T (TEX) cells are phenotypically and molecularly distinct from effector and memory CD8+ T cells, the factors regulating the earliest events in the differentiation process of TEX cells remain incompletely understood. Here, we performed single-cell RNA-sequencing and single-cell ATAC-sequencing of CD8+ T cells responding to LCMV-Armstrong (LCMV-Arm) or LCMV-Clone 13 (LCMV-Cl13), which result in acute or chronic infections, respectively. Compared to CD8+ T cells that had undergone their first division in response to LCMV-Arm (Div1ARM) cells, CD8+ T cells that had undergone their first division in response to LCMV-Cl13 (Div1CL13) expressed higher levels of genes encoding transcription factors previously associated with exhaustion, along with higher levels of Ezh2, the catalytic component of the Polycomb Repressive Complex 2 (PRC2) complex, which mediates epigenetic silencing. Modulation of Ezh2 resulted in altered expression of exhaustion-associated molecules by CD8+ T cells responding to LCMV-Cl13, though the specific cellular and infectious contexts, rather than simply the level of Ezh2 expression, likely determine the eventual outcome. Taken together, these findings suggest that the differentiation paths of CD8+ T cells responding to acute versus chronic infections may diverge earlier than previously appreciated.

Global chemical effects of the microbiome include new bile-acid conjugations.

A mosaic of cross-phylum chemical interactions occurs between all metazoans and their microbiomes. A number of molecular families that are known to be produced by the microbiome have a marked effect on the balance between health and disease1-9. Considering the diversity of the human microbiome (which numbers over 40,000 operational taxonomic units10), the effect of the microbiome on the chemistry of an entire animal remains underexplored. Here we use mass spectrometry informatics and data visualization approaches11-13 to provide an assessment of the effects of the microbiome on the chemistry of an entire mammal by comparing metabolomics data from germ-free and specific-pathogen-free mice. We found that the microbiota affects the chemistry of all organs. This included the amino acid conjugations of host bile acids that were used to produce phenylalanocholic acid, tyrosocholic acid and leucocholic acid, which have not previously been characterized despite extensive research on bile-acid chemistry14. These bile-acid conjugates were also found in humans, and were enriched in patients with inflammatory bowel disease or cystic fibrosis. These compounds agonized the farnesoid X receptor in vitro, and mice gavaged with the compounds showed reduced expression of bile-acid synthesis genes in vivo. Further studies are required to confirm whether these compounds have a physiological role in the host, and whether they contribute to gut diseases that are associated with microbiome dysbiosis.

Regulation of CD8 T Cell Differentiation by the RNA-Binding Protein DDX5.

The RNA-binding protein DEAD-box protein 5 (DDX5) is a polyfunctional regulator of gene expression, but its role in CD8+ T cell biology has not been extensively investigated. In this study, we demonstrate that deletion of DDX5 in murine CD8+ T cells reduced the differentiation of terminal effector, effector memory T, and terminal effector memory cells while increasing the generation of central memory T cells, whereas forced expression of DDX5 elicited the opposite phenotype. DDX5-deficient CD8+ T cells exhibited increased expression of genes that promote central memory T cell differentiation, including Tcf7 and Eomes. Taken together, these findings reveal a role for DDX5 in regulating the differentiation of effector and memory CD8+ T cell subsets in response to microbial infection.

Systems-level identification of key transcription factors in immune cell specification.

Transcription factors (TFs) are crucial for regulating cell differentiation during the development of the immune system. However, the key TFs for orchestrating the specification of distinct immune cells are not fully understood. Here, we integrated the transcriptomic and epigenomic measurements in 73 mouse and 61 human primary cell types, respectively, that span the immune cell differentiation pathways. We constructed the cell-type-specific transcriptional regulatory network and assessed the global importance of TFs based on the Taiji framework, which is a method we have previously developed that can infer the global impact of TFs using integrated transcriptomic and epigenetic data. Integrative analysis across cell types revealed putative driver TFs in cell lineage-specific differentiation in both mouse and human systems. We have also identified TF combinations that play important roles in specific developmental stages. Furthermore, we validated the functions of predicted novel TFs in murine CD8+ T cell differentiation and showed the importance of Elf1 and Prdm9 in the effector versus memory T cell fate specification and Kdm2b and Tet3 in promoting differentiation of CD8+ tissue resident memory (Trm) cells, validating the approach. Thus, we have developed a bioinformatic approach that provides a global picture of the regulatory mechanisms that govern cellular differentiation in the immune system and aids the discovery of novel mechanisms in cell fate decisions.

Delineation of a molecularly distinct terminally differentiated memory CD8 T cell population.

Memory CD8 T cells provide durable protection against diverse intracellular pathogens and can be broadly segregated into distinct circulating and tissue-resident populations. Paradigmatic studies have demonstrated that circulating memory cells can be further divided into effector memory (Tem) and central memory (Tcm) populations based on discrete functional characteristics. Following resolution of infection, we identified a persisting antigen-specific CD8 T cell population that was terminally fated with potent effector function but maintained memory T cell qualities and conferred robust protection against reinfection. Notably, this terminally differentiated effector memory CD8 T cell population (terminal-Tem) was conflated within the conventional Tem population, prompting redefinition of the classical characteristics of Tem cells. Murine terminal-Tem were transcriptionally, functionally, and developmentally unique compared to Tem cells. Through mass cytometry and single-cell RNA sequencing (RNA-seq) analyses of human peripheral blood from healthy individuals, we also identified an analogous terminal-Tem population of CD8 T cells that was transcriptionally distinct from Tem and Tcm Key findings from this study show that parsing of terminal-Tem from conventionally defined Tem challenge the reported characteristics of Tem biology, including enhanced presence in lymphoid tissues, robust IL-2 production, and recall potential, greater than expected homeostatic fitness, refined transcription factor dependencies, and a distinct molecular phenotype. Classification of terminal-Tem and clarification of Tem biology hold broad implications for understanding the molecular regulation of memory cell states and harnessing immunological memory to improve immunotherapies.

A gp130-Src-YAP module links inflammation to epithelial regeneration.

Inflammation promotes regeneration of injured tissues through poorly understood mechanisms, some of which involve interleukin (IL)-6 family members, the expression of which is elevated in many diseases including inflammatory bowel diseases and colorectal cancer. Here we show in mice and human cells that gp130, a co-receptor for IL-6 cytokines, triggers activation of YAP and Notch, transcriptional regulators that control tissue growth and regeneration, independently of the gp130 effector STAT3. Through YAP and Notch, intestinal gp130 signalling stimulates epithelial cell proliferation, causes aberrant differentiation and confers resistance to mucosal erosion. gp130 associates with the related tyrosine kinases Src and Yes, which are activated on receptor engagement to phosphorylate YAP and induce its stabilization and nuclear translocation. This signalling module is strongly activated upon mucosal injury to promote healing and maintain barrier function.

Elevated A20 promotes TNF-induced and RIPK1-dependent intestinal epithelial cell death.

Intestinal epithelial cell (IEC) death is a common feature of inflammatory bowel disease (IBD) that triggers inflammation by compromising barrier integrity. In many patients with IBD, epithelial damage and inflammation are TNF-dependent. Elevated TNF production in IBD is accompanied by increased expression of the TNFAIP3 gene, which encodes A20, a negative feedback regulator of NF-κB. A20 in intestinal epithelium from patients with IBD coincided with the presence of cleaved caspase-3, and A20 transgenic (Tg) mice, in which A20 is expressed from an IEC-specific promoter, were highly susceptible to TNF-induced IEC death, intestinal damage, and shock. A20-expressing intestinal organoids were also susceptible to TNF-induced death, demonstrating that enhanced TNF-induced apoptosis was a cell-autonomous property of A20. This effect was dependent on Receptor Interacting Protein Kinase 1 (RIPK1) activity, and A20 was found to associate with the Ripoptosome complex, potentiating its ability to activate caspase-8. A20-potentiated RIPK1-dependent apoptosis did not require the A20 deubiquitinase (DUB) domain and zinc finger 4 (ZnF4), which mediate NF-κB inhibition in fibroblasts, but was strictly dependent on ZnF7 and A20 dimerization. We suggest that A20 dimers bind linear ubiquitin to stabilize the Ripoptosome and potentiate its apoptosis-inducing activity.