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IL11 is a member of the IL6 family of cytokines and signals through its cognate receptor subunits, IL11RA and glycoprotein 130 (GP130), to elicit biological responses via the JAK/STAT signaling pathway. IL11 contributes to cancer progression by promoting the survival and proliferation of cancer cells, but the potential immunomodulatory properties of IL11 signaling during tumor development have thus far remained unexplored. Here, we have characterized a role for IL11 in regulating CD4+ T cell–mediated antitumor responses. Absence of IL11 signaling impaired tumor growth in a sporadic mouse model of colon cancer and syngeneic allograft models of colon cancer. Adoptive bone marrow transfer experiments and in vivo depletion studies demonstrated that the tumor-promoting activity of IL11 was mediated through its suppressive effect on host CD4+ T cells in the tumor microenvironment. Indeed, when compared with Il11ra-proficient CD4+ T cells associated with MC38 tumors, their Il11ra-deficient counterparts displayed elevated expression of mRNA encoding the antitumor mediators IFNγ and TNFα. Likewise, IL11 potently suppressed the production of proinflammatory cytokines (IFNγ, TNFα, IL6, and IL12p70) by CD4+ T cells in vitro, which we corroborated by RNAscope analysis of human colorectal cancers, where IL11RAhigh tumors showed less IFNG and CD4 expression than IL11RAlow tumors. Therefore, our results ascribe a tumor cell–extrinsic immunomodulatory role to IL11 during colon cancer development that could be amenable to an anticytokine-based therapy.See related Spotlight by van der Burg, p. 724.

Supplementary Figure 1. Gating strategy and representative flow cytometric plots of MC38 tumors from WT and Il11ra-/- hosts. Supplementary Figure 2. IL-11 has no effect on tumor-infiltrating immune cells in MC38 and CT26 tumors from WT and Il11ra-/- hosts. Supplementary Figure 3. IL-11 has no effect on PMA/Iono-induced IFNγ and TNFα production by IL-11RA-deficient CD4+ T cells. Supplementary Figure 4. Single cell profiling of IL11RA and IFNG expression in intra-tumoral CD4+ T cells from colon cancer patients.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a low 5-year survival rate and is associated with poor response to therapy. Elevated expression of the myeloid-specific hematopoietic cell kinase (HCK) is observed in PDAC and correlates with reduced patient survival. To determine whether aberrant HCK signaling in myeloid cells is involved in PDAC growth and metastasis, we established orthotopic and intrasplenic PDAC tumors in wild-type and HCK knockout mice. Genetic ablation of HCK impaired PDAC growth and metastasis by inducing an immune-stimulatory endotype in myeloid cells, which in turn reduced the desmoplastic microenvironment and enhanced cytotoxic effector cell infiltration. Consequently, genetic ablation or therapeutic inhibition of HCK minimized metastatic spread, enhanced the efficacy of chemotherapy, and overcame resistance to anti-PD1, anti-CTLA4, or stimulatory anti-CD40 immunotherapy. Our results provide strong rationale for HCK to be developed as a therapeutic target to improve the response of PDAC to chemo- and immunotherapy.

Although gastric cancer is a leading cause of cancer-related deaths, systemic treatment strategies remain scarce. Here, we report the pro-tumorigenic properties of the crosstalk between intestinal tuft cells and type 2 innate lymphoid cells (ILC2) that is evolutionarily optimized for epithelial remodeling in response to helminth infection. We demonstrate that tuft cell-derived interleukin 25 (IL25) drives ILC2 activation, inducing the release of IL13 and promoting epithelial tuft cell hyperplasia. While the resulting tuft cell - ILC2 feed-forward circuit promotes gastric metaplasia and tumor formation, genetic depletion of tuft cells or ILC2s, or therapeutic targeting of IL13 or IL25 alleviates these pathologies in mice. In gastric cancer patients, tuft cell and ILC2 gene signatures predict worsening survival in intestinal-type gastric cancer where ~40% of the corresponding cancers show enriched co-existence of tuft cells and ILC2s. Our findings suggest novel roles for ILC2 and tuft cells, along with their associated cytokine IL13 and IL25 as gatekeepers and enablers of metaplastic transformation and gastric tumorigenesis, thereby providing novel therapeutic vulnerabilities of for the treatment of these pathologies.

Tissue-resident memory T (TRM) cells are non-recirculating cells that exist throughout the body. Although TRM cells in various organs rely on common transcriptional networks to establish tissue residency, location-specific factors adapt these cells to their tissue of lodgment. Here we analyze TRM cell heterogeneity between organs and find that the different environments in which these cells differentiate dictate TRM cell function, durability and malleability. We find that unequal responsiveness to TGFβ is a major driver of this diversity. Notably, dampened TGFβ signaling results in CD103- TRM cells with increased proliferative potential, enhanced function and reduced longevity compared with their TGFβ-responsive CD103+ TRM counterparts. Furthermore, whereas CD103- TRM cells readily modified their phenotype upon relocation, CD103+ TRM cells were comparatively resistant to transdifferentiation. Thus, despite common requirements for TRM cell development, tissue adaptation of these cells confers discrete functional properties such that TRM cells exist along a spectrum of differentiation potential that is governed by their local tissue microenvironment.

IL11 is a member of the IL6 family of cytokines and signals through its cognate receptor subunits, IL11RA and glycoprotein 130 (GP130), to elicit biological responses via the JAK/STAT signaling pathway. IL11 contributes to cancer progression by promoting the survival and proliferation of cancer cells, but the potential immunomodulatory properties of IL11 signaling during tumor development have thus far remained unexplored. Here, we have characterized a role for IL11 in regulating CD4+ T cell–mediated antitumor responses. Absence of IL11 signaling impaired tumor growth in a sporadic mouse model of colon cancer and syngeneic allograft models of colon cancer. Adoptive bone marrow transfer experiments and in vivo depletion studies demonstrated that the tumor-promoting activity of IL11 was mediated through its suppressive effect on host CD4+ T cells in the tumor microenvironment. Indeed, when compared with Il11ra-proficient CD4+ T cells associated with MC38 tumors, their Il11ra-deficient counterparts displayed elevated expression of mRNA encoding the antitumor mediators IFNγ and TNFα. Likewise, IL11 potently suppressed the production of proinflammatory cytokines (IFNγ, TNFα, IL6, and IL12p70) by CD4+ T cells in vitro, which we corroborated by RNAscope analysis of human colorectal cancers, where IL11RAhigh tumors showed less IFNG and CD4 expression than IL11RAlow tumors. Therefore, our results ascribe a tumor cell–extrinsic immunomodulatory role to IL11 during colon cancer development that could be amenable to an anticytokine-based therapy. See related Spotlight by van der Burg, p. 724.

Although immunotherapy has revolutionized cancer treatment, many immunogenic tumors remain refractory to treatment. This can be largely attributed to an immunologically “cold” tumor microenvironment characterized by an accumulation of immunosuppressive myeloid cells and exclusion of activated T cells. Here, we demonstrate that genetic ablation or therapeutic inhibition of the myeloid-specific hematopoietic cell kinase (HCK) enables activity of antagonistic anti–programmed cell death protein 1 (anti-PD1), anti-CTLA4, or agonistic anti-CD40 immunotherapies in otherwise refractory tumors and augments response in treatment-susceptible tumors. Mechanistically, HCK ablation reprograms tumor-associated macrophages and dendritic cells toward an inflammatory endotype and enhances CD8 + T cell recruitment and activation when combined with immunotherapy in mice. Meanwhile, therapeutic inhibition of HCK in humanized mice engrafted with patient-derived xenografts counteracts tumor immunosuppression, improves T cell recruitment, and impairs tumor growth. Collectively, our results suggest that therapeutic targeting of HCK activity enhances response to immunotherapy by simultaneously stimulating immune cell activation and inhibiting the immunosuppressive tumor microenvironment.

CD8+ T cells responding to chronic infections or tumors acquire an 'exhausted' state associated with elevated expression of inhibitory receptors, including PD-1, and impaired cytokine production. Exhausted T cells are continuously replenished by T cells with precursor characteristics that self-renew and depend on the transcription factor TCF1; however, their developmental requirements are poorly understood. In the present study, we demonstrate that high antigen load promoted the differentiation of precursor T cells, which acquired hallmarks of exhaustion within days of infection, whereas early effector cells retained polyfunctional features. Early precursor T cells showed epigenetic imprinting characteristic of T cell receptor-dependent transcription factor binding and were restricted to the generation of cells displaying exhaustion characteristics. Transcription factors BACH2 and BATF were key regulators with opposing functions in the generation of early precursor T cells. Overall, we demonstrate that exhaustion manifests first in TCF1+ precursor T cells and is propagated subsequently to the pool of antigen-specific T cells.

Differentiation and homeostasis of Foxp3+ regulatory T (Treg) cells are strictly controlled by T-cell receptor (TCR) signals; however, molecular mechanisms that govern these processes are incompletely understood. Here we show that Bach2 is an important regulator of Treg cell differentiation and homeostasis downstream of TCR signaling. Bach2 prevents premature differentiation of fully suppressive effector Treg (eTreg) cells, limits IL-10 production and is required for the development of peripherally induced Treg (pTreg) cells in the gastrointestinal tract. Bach2 attenuates TCR signaling-induced IRF4-dependent Treg cell differentiation. Deletion of IRF4 promotes inducible Treg cell differentiation and rescues pTreg cell differentiation in the absence of Bach2. In turn, loss of Bach2 normalizes eTreg cell differentiation of IRF4-deficient Treg cells. Mechanistically, Bach2 counteracts the DNA-binding activity of IRF4 and limits chromatin accessibility, thereby attenuating IRF4-dependent transcription. Thus, Bach2 balances TCR signaling induced transcriptional activity of IRF4 to maintain homeostasis of thymically-derived and peripherally-derived Treg cells., The transcription factor Bach2 is critical for T cell differentiation, but how it functions in Treg cells is unclear. Here the authors use a Treg-specific mouse model to show that Bach2 controls homeostasis and function of Treg cells by limiting DNA accessibility and activity of IRF4 in response to TCR signaling.

Background RNA sequencing is currently the method of choice for genome-wide profiling of gene expression. A popular approach to quantify expression levels of genes from RNA-seq data is to map reads to a reference genome and then count mapped reads to each gene. Gene annotation data, which include chromosomal coordinates of exons for tens of thousands of genes, are required for this quantification process. There are several major sources of gene annotations that can be used for quantification, such as Ensembl and RefSeq databases. However, there is very little understanding of the effect that the choice of annotation has on the accuracy of gene expression quantification in an RNA-seq analysis. Results In this paper, we present results from our comparison of Ensembl and RefSeq human annotations on their impact on gene expression quantification using a benchmark RNA-seq dataset generated by the SEQC consortium. We show that the use of RefSeq gene annotation models led to better quantification accuracy, based on the correlation with ground truths including expression data from >800 real-time PCR validated genes, known titration ratios of gene expression and microarray expression data. We also found that the recent expansion of the RefSeq annotation has led to a decrease in its annotation accuracy. Finally, we demonstrated that the RNA-seq quantification differences observed between different annotations were not affected by the use of different normalization methods. Conclusion In conclusion, our study found that the use of the conservative RefSeq gene annotation yields better RNA-seq quantification results than the more comprehensive Ensembl annotation. We also found that, surprisingly, the recent expansion of the RefSeq database, which was primarily driven by the incorporation of sequencing data into the gene annotation process, resulted in a reduction in the accuracy of RNA-seq quantification.

Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1∆ or chs3∆ or the vps23∆chs3∆ double knockout strain were unable to rescue the yeast cells as compared to vps23∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling.

Interleukin-36 gamma (IL-36G) is a member of the IL-36 subfamily of cytokines and acts as a potent driver of inflammation. IL-36G has been extensively characterized in the pathogenesis of psoriasis and has been recently described to play roles in wound healing particularly in the gastrointestinal tract. However, the effects of IL-36G during cancer development including gastric cancer remain unexplored. Here, we show that IL-36G induced ERK1/2 activation in AGS, MKN1 and MKN45 human gastric cancer cell lines. Moreover, IL-36G induced colony formation, migration and invasion of these gastric cancer cell lines that was inhibited by the natural antagonist, IL-36 receptor antagonist (RA). Interrogation of TCGA stomach adenocarcinoma patient datasets revealed highly elevated IL-36G gene expression in human gastric cancer compared to normal tissue independent of tumor stage, and high IL-36G expression corresponded with poorer patient survival. Collectively, our results indicate for the first time that IL-36G supports a neoplastic phenotype in human gastric cancer cells.

The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors., Dietary extracellular vesicles (EVs) could potentially be absorbed by the intestinal tract of the host and exert multiple phenotypic changes. Here, the authors isolate and characterize EVs from raw and commercial bovine milk and show orally administered EVs to have a context specific role in promoting or suppressing primary tumor growth and metastasis in multiple mouse tumor models.

Antigen-specific CD8+ T cells in chronic viral infections and tumors functionally deteriorate, a process known as exhaustion. Exhausted T cells are sustained by precursors of exhausted (Tpex) cells that self-renew while continuously generating exhausted effector (Tex) cells. However, it remains unknown how Tpex cells maintain their functionality. Here, we demonstrate that Tpex cells sustained mitochondrial fitness, including high spare respiratory capacity, while Tex cells deteriorated metabolically over time. Tpex cells showed early suppression of mTOR kinase signaling but retained the ability to activate this pathway in response to antigen receptor signals. Early transient mTOR inhibition improved long-term T cell responses and checkpoint inhibition. Transforming growth factor-β repressed mTOR signaling in exhausted T cells and was a critical determinant of Tpex cell metabolism and function. Overall, we demonstrate that the preservation of cellular metabolism allows Tpex cells to retain long-term functionality to sustain T cell responses during chronic infection.

CD8

Adipose tissue is an energy store and a dynamic endocrine organ1,2. In particular, visceral adipose tissue (VAT) is critical for the regulation of systemic metabolism3,4. Impaired VAT function-for example, in obesity-is associated with insulin resistance and type 2 diabetes5,6. Regulatory T (Treg) cells that express the transcription factor FOXP3 are critical for limiting immune responses and suppressing tissue inflammation, including in the VAT7-9. Here we uncover pronounced sexual dimorphism in Treg cells in the VAT. Male VAT was enriched for Treg cells compared with female VAT, and Treg cells from male VAT were markedly different from their female counterparts in phenotype, transcriptional landscape and chromatin accessibility. Heightened inflammation in the male VAT facilitated the recruitment of Treg cells via the CCL2-CCR2 axis. Androgen regulated the differentiation of a unique IL-33-producing stromal cell population specific to the male VAT, which paralleled the local expansion of Treg cells. Sex hormones also regulated VAT inflammation, which shaped the transcriptional landscape of VAT-resident Treg cells in a BLIMP1 transcription factor-dependent manner. Overall, we find that sex-specific differences in Treg cells from VAT are determined by the tissue niche in a sex-hormone-dependent manner to limit adipose tissue inflammation.

Colorectal cancer (CRC) is the third most common form of cancer and has the fourth highest mortality rate in the world. To understand the origin and progression of this disease, biomedical researchers undertake global analyses of omics data of CRC patient samples and representative cell lines. However, due to the heterogeneity and high dimensionality nature of `omics’ data, traditional tools for analysing this sort of data are inadequate and the heterogeneous nature of cancer makes the process of identifying essential genes very difficult. ‘Omics’ is a term that is used to refer to areas of study in biology that end with the ending ‘omics’ such as genomics, proteomics and metabolomics. This paper uses network theory-based methods to address the problem of high dimensionality in omics datasets and applies network propagation to address the problem of heterogeneity in both omics datasets and cancer in identifying the essential genes. The method successfully identifies known essential genes in CRC as well as a new set of genes that are likely to be essential in the study of CRC.

IL11 is a member of the IL6 family of cytokines and signals through its cognate receptor subunits, IL11RA and glycoprotein 130 (GP130), to elicit biological responses via the JAK/STAT signaling pathway. IL11 contributes to cancer progression by promoting the survival and proliferation of cancer cells, but the potential immunomodulatory properties of IL11 signaling during tumor development have thus far remained unexplored. Here, we have characterized a role for IL11 in regulating CD4

Extracellular vesicles (EVs) are membranous vesicles that are released by cells. In this study, the role of the Endosomal Sorting Complex Required for Transport (ESCRT) machinery in the biogenesis of yeast EVs was examined. Knockout of components of the ESCRT machinery altered the morphology and size of EVs as well as decreased the abundance of EVs. In contrast, strains with deletions in cell wall biosynthesis genes, produced more EVs than wildtype. Proteomic analysis highlighted the depletion of ESCRT components and enrichment of cell wall remodelling enzymes, glucan synthase subunit Fks1 and chitin synthase Chs3, in yeast EVs. Interestingly, EVs containing Fks1 and Chs3 rescued the yeast cells from antifungal molecules. However, EVs from fks1∆ or chs3∆ or the vps23∆chs3∆ double knockout strain were unable to rescue the yeast cells as compared to vps23∆ EVs. Overall, we have identified a potential role for yeast EVs in cell wall remodelling., Kening Zhao et al. show that yeast extracellular vesicles are depleted of ESCRT proteins but enriched with Fks1 and Chs3. The toxic effect of antifungal agents can be diminished by exposure to the Fks1- and Chs3- rich extracellular vesicles, suggesting a role for yeast extracellular vesicles in cell wall remodelling.

In order to advance our understanding of colorectal cancer (CRC) development and progression, biomedical researchers have generated large amounts of OMICS data from CRC patient samples and representative cell lines. However, these data are deposited in various repositories or in supplementary tables. A database which integrates data from heterogeneous resources and enables analysis of the multidimensional data sets, specifically pertaining to CRC is currently lacking. Here, we have developed Colorectal Cancer Atlas (http://www.colonatlas.org), an integrated web-based resource that catalogues the genomic and proteomic annotations identified in CRC tissues and cell lines. The data catalogued to-date include sequence variations as well as quantitative and non-quantitative protein expression data. The database enables the analysis of these data in the context of signaling pathways, protein-protein interactions, Gene Ontology terms, protein domains and post-translational modifications. Currently, Colorectal Cancer Atlas contains data for >13 711 CRC tissues, >165 CRC cell lines, 62 251 protein identifications, >8.3 million MS/MS spectra, >18 410 genes with sequence variations (404 278 entries) and 351 pathways with sequence variants. Overall, Colorectal Cancer Atlas has been designed to serve as a central resource to facilitate research in CRC.

Exosomes are extracellular vesicles secreted by multiple cell types into the extracellular space. They contain cell-state specific cargos which often reflects the (patho)physiological condition of the cells/organism. Milk contains high amounts of exosomes and it is unclear whether their cargo is altered based on the lactation stage of the organism. Here, we isolated exosomes from bovine milk that were obtained at various stages of lactation and examined the content by quantitative proteomics. Exosomes were isolated by OptiPrep density gradient centrifugation from milk obtained from cow after 24, 48 and 72 h post calving. As control, exosomes were also isolated from cows during mid-lactation period which has been referred to as mature milk (MM). Biochemical and biophysical characterization of exosomes revealed the high abundance of exosomes in colostrum and MM samples. Quantitative proteomics analysis highlighted the change in the proteomic cargo of exosomes based on the lactation state of the cow. Functional enrichment analysis revealed that exosomes from colostrum are significantly enriched with proteins that can potentially regulate the immune response and growth. This study highlights the importance of exosomes in colostrum and hence opens up new avenues to exploit these vesicles in the regulation of the immune response and growth.

Bioinformatics tools are imperative for the in depth analysis of heterogeneous high-throughput data. Most of the software tools are developed by specific laboratories or groups or companies wherein they are designed to perform the required analysis for the group. However, such software tools may fail to capture "what the community needs in a tool". Here, we describe a novel community-driven approach to build a comprehensive functional enrichment analysis tool. Using the existing FunRich tool as a template, we invited researchers to request additional features and/or changes. Remarkably, with the enthusiastic participation of the community, we were able to implement 90% of the requested features. FunRich enables plugin for extracellular vesicles wherein users can download and analyse data from Vesiclepedia database. By involving researchers early through community needs software development, we believe that comprehensive analysis tools can be developed in various scientific disciplines. We would like to thank Tejaswee P. Shah for her valuable input in the development of FunRich. We would like to thank Jan Lotvall for helping us in this effort by circulating the email within his group. We thanks many other users who requested for features/changes in FunRich though the e-mail and the online forum. Suresh Mathivanan is supported by Australian Research Council DECRA (DE150101777) and Award U54- DA036134 supported by the NIH Common Fund through the Office of Strategic Coordination/Office of the NIH Director.The funders had no role in study design, data collection andanalysis, decision to publish, or preparation of the manuscript. Sí

Recent advancements in high-throughput technologies such as mass spectrometry have led to an increase in the rate at which data is generated and accumulated. As a result, standard statistical methods no longer suffice as a way of analyzing such gigantic amounts of data. Network analysis, the evaluation of how nodes relate to one another, has over the years become an integral tool for analyzing high throughput proteomic data as they provide a structure that helps reduce the complexity of the underlying data.Computational tools, including pathway databases and network building tools, have therefore been developed to store, analyze, interpret, and learn from proteomics data. These tools enable the visualization of proteins as networks of signaling, regulatory, and biochemical interactions. In this chapter, we provide an overview of networks and network theory fundamentals for the analysis of proteomics data. We further provide an overview of interaction databases and network tools which are frequently used for analyzing proteomics data.

Recent advancements in high-throughput technologies such as mass spectrometry have led to an increase in the rate at which data is generated and accumulated. As a result, standard statistical methods no longer suffice as a way of analyzing such gigantic amounts of data. Network analysis, the evaluation of how nodes relate to one another, has over the years become an integral tool for analyzing high throughput proteomic data as they provide a structure that helps reduce the complexity of the underlying data.Computational tools, including pathway databases and network building tools, have therefore been developed to store, analyze, interpret, and learn from proteomics data. These tools enable the visualization of proteins as networks of signaling, regulatory, and biochemical interactions. In this chapter, we provide an overview of networks and network theory fundamentals for the analysis of proteomics data. We further provide an overview of interaction databases and network tools which are frequently used for analyzing proteomics data.

Exosomes are membranous vesicles that are released by a variety of cells into the extracellular microenvironment and are implicated in intercellular communication. As exosomes contain RNA, proteins and lipids, there is a significant interest in characterizing the molecular cargo of exosomes. Here, we describe ExoCarta (http://www.exocarta.org), a manually curated Web-based compendium of exosomal proteins, RNAs and lipids. Since its inception, the database has been highly accessed (>54,000 visitors from 135 countries). The current version of ExoCarta hosts 41,860 proteins, >7540 RNA and 1116 lipid molecules from more than 286 exosomal studies annotated with International Society for Extracellular Vesicles minimal experimental requirements for definition of extracellular vesicles. Besides, ExoCarta features dynamic protein-protein interaction networks and biological pathways of exosomal proteins. Users can download most often identified exosomal proteins based on the number of studies. The downloaded files can further be imported directly into FunRich (http://www.funrich.org) tool for additional functional enrichment and interaction network analysis.

CD8+ T cells that respond to chronic viral infections or cancer are characterized by the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) and by the impaired production of cytokines. This state of restrained functionality—which is referred to as T cell exhaustion1,2—is maintained by precursors of exhausted T (TPEX) cells that express the transcription factor T cell factor 1 (TCF1), self-renew and give rise to TCF1− exhausted effector T cells3–6. Here we show that the long-term proliferative potential, multipotency and repopulation capacity of exhausted T cells during chronic infection are selectively preserved in a small population of transcriptionally distinct CD62L+ TPEX cells. The transcription factor MYB is not only essential for the development of CD62L+ TPEX cells and maintenance of the antiviral CD8+ T cell response, but also induces functional exhaustion and thereby prevents lethal immunopathology. Furthermore, the proliferative burst in response to PD-1 checkpoint inhibition originates exclusively from CD62L+ TPEX cells and depends on MYB. Our findings identify CD62L+ TPEX cells as a stem-like population that is central to the maintenance of long-term antiviral immunity and responsiveness to immunotherapy. Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity.