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Tissue-resident memory T cells (T(RM) cells) provide protective immunity, but the contributions of specific tissue environments to T(RM) cell differentiation and homeostasis are not well understood. In the present study, the diversity of gene expression and genome accessibility by mouse CD8(+) T(RM) cells from distinct organs that responded to viral infection revealed both shared and tissue-specific transcriptional and epigenetic signatures. T(RM) cells in the intestine and salivary glands expressed transforming growth factor (TGF)-β-induced genes and were maintained by ongoing TGF-β 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 T(RM) cell differentiation in the small intestine and showed that Hic1 overexpression enhanced T(RM) cell differentiation and protection from infection. Provision of a framework for understanding how CD8(+) T(RM) 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.

After resolution of infection, T cells differentiate into long-lived memory cells that recirculate through secondary lymphoid organs or establish residence in tissues. In contrast to CD8 + tissue-resident memory T cells (T RM ), the developmental origins and transcriptional regulation of CD4 + T RM remain largely undefined. Here, we investigated the phenotypic, functional, and transcriptional profiles of CD4 + T RM in the small intestine (SI) responding to acute viral infection, revealing a shared gene expression program and chromatin accessibility profile with circulating T H 1 and the progressive acquisition of a mature T RM program. Single-cell RNA sequencing identified heterogeneity among established CD4 + T RM , which were predominantly located in the lamina propria, and revealed a population of cells that coexpressed both effector- and memory-associated genes, including the transcriptional regulators Blimp1, Id2, and Bcl6. T H 1-associated Blimp1 and Id2 and T FH -associated Bcl6 were required for early T RM formation and development of a mature T RM population in the SI. These results demonstrate a developmental relationship between T H 1 effector cells and the establishment of early T RM , as well as highlighted differences in CD4 + versus CD8 + T RM populations, providing insights into the mechanisms underlying the origins, differentiation, and persistence of CD4 + T RM in response to viral infection.

PyMT tumor versus spleen fold change, acute infection day 6 versus spleen fold change.

Selected phosphatases up-regulated in PyMT tumor CD8+ T cells.

PyMT tumor CD8+ versus day 5 effector OT-I gene expression and fold change.

Despite clinical potential and recent advances, durable immunotherapeutic ablation of solid tumors is not routinely achieved. IL15 expands natural killer cell (NK), natural killer T cell (NKT) and CD8+ T-cell numbers and engages the cytotoxic program, and thus is under evaluation for potentiation of cancer immunotherapy. We found that short-term therapy with IL15 bound to soluble IL15 receptor α–Fc (IL15cx; a form of IL15 with increased half-life and activity) was ineffective in the treatment of autochthonous PyMT murine mammary tumors, despite abundant CD8+ T-cell infiltration. Probing of this poor responsiveness revealed that IL15cx only weakly activated intratumoral CD8+ T cells, even though cells in the lung and spleen were activated and dramatically expanded. Tumor-infiltrating CD8+ T cells exhibited cell-extrinsic and cell-intrinsic resistance to IL15. Our data showed that in the case of persistent viral or tumor antigen, single-agent systemic IL15cx treatment primarily expanded antigen-irrelevant or extratumoral CD8+ T cells. We identified exhaustion, tissue-resident memory, and tumor-specific molecules expressed in tumor-infiltrating CD8+ T cells, which may allow therapeutic targeting or programming of specific subsets to evade loss of function and cytokine resistance, and, in turn, increase the efficacy of IL2/15 adjuvant cytokine therapy. Cancer Immunol Res; 4(9); 799–811. ©2016 AACR.

PyMT tumor CD8+ versus day 1 effector OT-I gene expression and fold change.

Supplemental Figure 1: Five day IL-15cx treatment results in splenic CD8+ T cell activation and up-regulation of cytotoxic molecule expression in PyMT tumor-bearing mice. Supplemental Figure 2: PyMT tumor single-cell suspensions induce IL-2 / IL-15 resistance in CD8+ T cells. Supplemental Figure 3: Abundance of IL-2 / IL-15 cytokine receptor chains in PyMT tumor, acute/persistent infection, and TRM CD8+ T cells. Supplemental Figure 4: Gene expression in tumor versus acute infection CD8+ T cells. Supplemental Figure 5: Differentially-regulated transcripts in PyMT tumor CD8+ T cells versus naive, early effector, peak effector and memory T cells. Supplemental Figure 6: Human breast cancer CD8+ T cell infiltrate. Supplemental Figure 7: TRM "core signature" gene expression and transcriptional regulator TOX correlation with IL-15 resistance. Supplemental Figure 8: IL-15cx treatment of persistent virus-infected mice results in decreased relative abundance of exhausted antigen-specific CD8+ T cells. Supplemental Figure 9: Combined in vivo IL-15cx and αPD-L1 antibody treatment does not rescue PyMT CD8+ T cell cytokine non-responsiveness. Supplemental Figure 10: Expression of CD80 and CD200R on CD8+ T cells from PyMT tumor and persistent viral infection. Supplemental Figure 11: Activated and expanded tumor-specific CD8+ T cells adoptively transferred to tumor-bearing mice localize to tumor, but rapidly up-regulate PD-1 and CD244.

Transcripts up-regulated {greater than or equal to}2-fold in PyMT tumor versus splenic CD8+ T cells.

PyMT tumor CD8+ versus naive gene expression and fold change.

Supplementary Figure Legends 1-4 from Interleukin-15/Interleukin-15Rα Complexes Promote Destruction of Established Tumors by Reviving Tumor-Resident CD8+ T Cells

Supplementary Figure 3 from Interleukin-15/Interleukin-15Rα Complexes Promote Destruction of Established Tumors by Reviving Tumor-Resident CD8+ T Cells

Supplementary Figures 1-4 from Macrophage Expression of Hypoxia-Inducible Factor-1α Suppresses T-Cell Function and Promotes Tumor Progression

T cells can inhibit tumor growth, but their function in the tumor microenvironment is often suppressed. Many solid tumors exhibit abundant macrophage infiltration and low oxygen tension, yet how hypoxic conditions may affect innate immune cells and their role in tumor progression is poorly understood. Targeted deletion of the hypoxia-responsive transcription factor hypoxia-inducible factor-1α (HIF-1α) in macrophages in a progressive murine model of breast cancer resulted in reduced tumor growth, although vascular endothelial growth factor-A levels and vascularization were unchanged. Tumor-associated macrophages can suppress tumor-infiltrating T cells by several mechanisms, and we found that hypoxia powerfully augmented macrophage-mediated T-cell suppression in vitro in a manner dependent on macrophage expression of HIF-1α. Our findings link the innate immune hypoxic response to tumor progression through induction of T-cell suppression in the tumor microenvironment. Cancer Res; 70(19); 7465–75. ©2010 AACR.

Supplementary Figure 1 from Interleukin-15/Interleukin-15Rα Complexes Promote Destruction of Established Tumors by Reviving Tumor-Resident CD8+ T Cells

Tumors often escape immune-mediated destruction by suppressing lymphocyte infiltration or effector function. New approaches are needed that overcome this suppression and thereby augment the tumoricidal capacity of tumor-reactive lymphocytes. The cytokine interleukin-15 (IL-15) promotes proliferation and effector capacity of CD8+ T cells, natural killer (NK) cells, and NKT cells; however, it has a short half-life and high doses are needed to achieve functional responses in vivo. The biological activity of IL-15 can be dramatically increased by complexing this cytokine to its soluble receptor, IL-15Rα. Here, we report that in vivo delivery of IL-15/IL-15Rα complexes triggers rapid and significant regression of established solid tumors in two murine models. Despite a marked expansion of IL-2/IL-15Rβ+ cells in lymphoid organs and peripheral blood following treatment with IL-15/IL-15Rα complexes, the destruction of solid tumors was orchestrated by tumor-resident rather than newly infiltrating CD8+ T cells. Our data provide novel insights into the use of IL-15/IL-15Rα complexes to relieve tumor-resident T cells from functional suppression by the tumor microenvironment and have significant implications for cancer immunotherapy and treatment of chronic infections. [Cancer Res 2008;68(8):2972–83]

Supplementary Figure 2 from Interleukin-15/Interleukin-15Rα Complexes Promote Destruction of Established Tumors by Reviving Tumor-Resident CD8+ T Cells

In this issue of JEM, Qiu et al. (2023. J. Exp. Med. https://doi.org/10.1084/jem.20210923) show that retinoic acid signaling during priming in the mesenteric lymph node licenses CD8+ T cells to develop into small intestinal tissue-resident memory cells, a finding that provides key insights into tissue-specific vaccination strategies.

SUMMARYThe differentiation of naïve CD8+cytotoxic T lymphocytes (CTLs) into effector and memory states results in large scale changes in transcriptional and phenotypic profiles. Little is known about how large-scale changes in genome organisation reflect or underpin these transcriptional programs. We utilised Hi-C to map changes in the spatial organisation of long-range genome contacts within naïve, effector and memory virus-specific CD8+T cells. We observed that the architecture of the naive CD8+T cell genome was distinct from effector and memory genome configurations with extensive changes within discrete functional chromatin domains. However, deletion of the BACH2 or SATB1 transcription factors was sufficient to remodel the naïve chromatin architecture and engage transcriptional programs characteristic of differentiated cells. This suggests that the chromatin architecture within naïve CD8+T cells is preconfigured to undergo autonomous remodelling upon activation, with key transcription factors restraining differentiation by actively enforcing the unique naïve chromatin state.One Sentence SummaryCD8+T cell naïvety is actively maintained by transcription factors that enforce a distinct, naïve chromatin architecture.HighlightsCD8+T cell differentiation states are underscored by distinct chromatin looping architectures.Chromatin loops juxtapose CTL state appropriate enhancers, transcription factors and genes.Effector and memory CTLs have similar genome architectures, explaining rapid memory recall.CTL differentiation is restrained by BACH2 and SATB1, which enforce a naïve loop architecture.

CD8+T cells with stem cell-like properties (TSCM) sustain adaptive immunity to intracellular pathogens and tumors. However, the developmental origins and chromatin regulatory factors (CRFs) that establish their differentiation are unclear. Using an RNA interference screen of all CRFs we discovered the histone methylase Mll1 was required during T cell receptor (TCR) stimulation for development of a TSCMprecursor state and mature memory (TMEM) cells, but not short-lived or transitory effector cell-like states, in response to viral infections and tumors. Mll1 was essential for widespreadde novodeposition of histone H3 lysine 4 trimethylation (H3K4me3) upon TCR stimulation, which accounted for 70% of all activation-induced sites in mature TMEMcells. Mll1 promoted both H3K4me3 deposition and reduced TCR-induced Pol II pausing at genes whose single-cell transcriptional dynamics explained trajectories into nascent TSCMprecursor states during viral infection. Our results suggest Mll1-dependent control of Pol II elongation and H3K4me3 establishes and maintains differentiation of CD8+TSCMcell states.

Memory CD4

T follicular helper (TFH) cells are a distinct type of CD4+ T cells that are essential for most antibody and B lymphocyte responses. TFH cell regulation and dysregulation is involved in a range of diseases. Bcl-6 is the lineage defining transcription factor of TFH cells and its activity is essential for TFH cell differentiation and function. However, how Bcl-6 controls TFH biology has largely remained unclear, at least in part due to intrinsic challenges of connecting repressors to gene upregulation in complex cell types with multiple possible differentiation fates. Multiple competing models were tested here by a series of experimental approaches to determine that Bcl-6 exhibited negative autoregulation and controlled pleiotropic attributes of TFH differentiation and function, including migration, costimulation, inhibitory receptors, and cytokines, via multiple repressor-of-repressor gene circuits.

Effective presentation of antigens by human leukocyte antigen (HLA) class I molecules to CD8

BackgroundIn cancer, CD8+ T cells have the power to target and kill tumor cells with precision, but often fail due to chronic activation from the immunosuppressive tumor microenvironment (TME). T cells that experience prolonged activation in the TME differentiate into a severely dysfunctional cell state known as exhaustion. In healthy tissues, T cells differentiate into tissue-resident memory cells (TRM) in response to infection, which remain lodged in tissues to provide protection from reinfection. When TRM-like cells are found in patient tumors, they are correlated with improved outcomes and responses to immunotherapy. Understanding how to manipulate T cell fates in an effort to prevent exhaustion and favor TRM-characteristics could benefit cancer immunotherapy.MethodsTo explore differences between these cell states, we screened the core TRM gene-expression signatures for genes downregulated as T cells undergo terminal exhaustion. Targets were then overexpressed in antigen-specific T cells and adoptively transferred into tumor-bearing mice for analysis.ResultsInterestingly, many genes related to protein regulation and processing were identified, including a novel gene called Neuralized E3 Ubiquitin Protein Ligase 3 (Neurl3). Neurl3's function is not well described, however, experimentally mutating the RING domain suggests Neurl3 transfers ubiquitin to target proteins for degradation. When Neurl3 was overexpressed in tumor-specific T cells, we found tumor infiltrating lymphocytes still upregulated inhibitory receptors PD1 and Tim3, but showed enhanced anti-tumor function. Neurl3-overexpressing T cells had increased accumulation in the TME, upregulated canonical TRM markers CD69 and CD103, produced more cytokines, controlled tumor growth, and increased mouse survival in B16 melanoma.ConclusionsThese results highlight the understudied field of negative regulation of T cell function by protein degradation in T cell differentiation fate and uncover a potential gene target for immunocellular therapies to favor functional T cell fates in cancer.Ethics ApprovalThe study was approved by UCSD's Institutional Animal Care and Use Committee, protocol number S04105.

Cytotoxic CD8+ T cells play a key role in the elimination of intracellular infections and malignant cells and can provide long-term protective immunity. In the response to infection, CD8+ T cell metabolism is coupled to transcriptional, translational and epigenetic changes that are driven by extracellular metabolites and immunological signals. These programmes facilitate the adaptation of CD8+ T cells to the diverse and dynamic metabolic environments encountered in the circulation and in the tissues. In the setting of disease, both cell-intrinsic and cell-extrinsic metabolic cues contribute to CD8+ T cell dysfunction. In addition, changes in whole-body metabolism, whether through voluntary or disease-induced dietary alterations, can influence CD8+ T cell-mediated immunity. Defining the metabolic adaptations of CD8+ T cells in specific tissue environments informs our understanding of how these cells protect against pathogens and tumours and maintain tissue health at barrier sites. Here, we highlight recent findings revealing how metabolic networks enforce specific CD8+ T cell programmes and discuss how metabolism is integrated with CD8+ T cell differentiation and function and determined by environmental cues.

Upon activation, naive T cells give rise to a heterogeneous cell population of effector and memory T cells that mediate antigen clearance and provide long-lived protection from rechallenge. Many of the transcriptional regulators that direct the differentiation of naive T cells to acquire the range of phenotypic and functional characteristics of effector and memory T cells have been described. However, the active programs that maintain the specific subsets of memory T cells are less clear. Here, we discuss recent studies that suggest effector and memory CD8(+) T cells exist in cellular ‘states’ with inherent plasticity. Further, we consider the newly identified role of active transcriptional and epigenetic programming in maintaining the identity of the distinct subsets within the memory population.

In response to infection, naive CD4+ T-cells proliferate and differentiate into several possible effector subsets, including conventional T helper effector cells (TH 1, TH 2, TH 17), T regulatory cells (Treg ) and T follicular helper cells (TFH ). Once infection is cleared, a small population of long-lived memory cells remains that mediate immune defenses against reinfection. Memory T lymphocytes have classically been categorized into central memory cell (TCM ) and effector memory cell (TEM ) subsets, both of which circulate between blood, secondary lymphoid organs and in some cases non-lymphoid tissues. A third subset of memory cells, referred to as tissue-resident memory cells (TRM ), resides in tissues without recirculation, serving as 'first line' of defense at barrier sites, such as skin, lung and intestinal mucosa, and augmenting innate immunity in the earliest phases of reinfection and recruiting circulating CD4+ and CD8+ T-cells. The presence of multiple CD4+ T helper subsets has complicated studies of CD4+ memory T-cell differentiation, and the mediators required to support their function. In this review, we summarize recent investigations into the origins of CD4+ memory T-cell populations and discuss studies addressing CD4+ TRM differentiation in barrier tissues.

Individual naive CD8 T cells activated in lymphoid organs differentiate into functionally diverse and anatomically distributed T cell phylogenies in response to intracellular microbes. During infections that resolve rapidly, including live viral vaccines1, distinct effector (TEFF) and memory (TMEM) cell populations develop that ensure long term immunity2. During chronic infections, responding cells progressively become dysfunctional and “exhaust”3. A diverse taxonomy of TEFF, TMEM and exhausted (TEX) CD8 T cell populations is known, but the initial developmental basis of this phenotypic variation remains unclear4–10. Here, we defined single-cell trajectories and identified chromatin regulators that establish antiviral CD8 T cell heterogeneity using unsupervised analyses of single-cell RNA dynamics11–13 and an in vivo RNAi screen14. Activated naive cells differentiate linearly into uncommitted effector-memory progenitor (EMP) cells, which initially branch into an analogous manifold during either acute or chronic infection. Disparate RNA velocities in single EMP cells initiate divergence into stem, circulating, and tissue-resident memory lineages that generate diverse TMEM and TEX precursor states in specific developmental orders. Interleukin-2 receptor (IL-2R) signals are essential for formation and transcriptional heterogeneity of EMP cells, and promote trajectories toward TEFF rather than TEX states. Nucleosome remodelers Smarca4 and Chd7 differentially promote transcription that delineates divergent TMEM lineages before cooperatively driving terminal TEFF cell differentiation. Thus, the lineage architecture is established by specific chromatin regulators that stabilize diverging transcription in uncommitted progenitors.

Tissue-resident memory T cells (T

BRD4 is a bromodomain-containing protein that binds acetylated histones to regulate transcription. In this issue of JEM, Milner et al. show that BRD4 plays a critical role in the effector function of CD8 T cells responding to infection and cancer., 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.

Effective presentation of antigens by HLA class I molecules to CD8+ T cells is required for viral elimination and generation of long-term immunological memory. In this study, we applied a single-cell, multi-omic technology to generate the first unified ex vivo characterization of the CD8+ T cell response to SARS-CoV-2 across 4 major HLA class I alleles. We found that HLA genotype conditions key features of epitope specificity, TCR α/β sequence diversity, and the utilization of pre-existing SARS-CoV-2 reactive memory T cell pools. Single-cell transcriptomics revealed functionally diverse T cell phenotypes of SARS-CoV-2-reactive T cells, associated with both disease stage and epitope specificity. Our results show that HLA variations influence pre-existing immunity to SARS-CoV-2 and shape the immune repertoire upon subsequent viral exposure.One-Sentence SummaryWe perform a unified, multi-omic characterization of the CD8+ T cell response to SARS-CoV-2, revealing pre-existing immunity conditioned by HLA genotype.

T cells are essential mediators of the immune responses against infectious diseases and provide long-lived protection from reinfection. The differentiation of naive T cells to effector T cells and subsequent differentiation and persistence of memory T cell populations in response to infection is a highly regulated process. E protein transcription factors and their inhibitors, Id proteins, are important regulators of both CD4+ and CD8+ T cell responses; however, their regulation at the protein level has not been explored. Recently, the deubiquitinase USP1 was shown to stabilize Id2 and modulate cellular differentiation in osteosarcomas. Here, we investigated a role for Usp1 in posttranslational control of Id2 and Id3 in T cells. We show that Usp1 was upregulated in T cells following activation in vitro or following infection in vivo, and the extent of Usp1 expression correlated with the degree of T cell expansion. Usp1 directly interacted with Id2 and Id3 following T cell activation. However, Usp1-deficiency did not impact Id protein abundance in effector T cells or alter effector CD8+ T cell expansion or differentiation following a primary infection. Usp1 deficiency did result in a gradual loss of memory cells over time and impaired accumulation and altered differentiation following a secondary infection. Together, these results identify Usp1 as a player in modulating recall responses at the protein level and highlight differences in regulation of T cell responses between primary and subsequent infection encounters. Finally, our observations reveal that differential regulation of Id2/3 proteins between immune vs non-immune cell types.

Adoptive T cell therapies (ACTs) hold great promise in cancer treatment, but low overall response rates in patients with solid tumors underscore remaining challenges in realizing the potential of this cellular immunotherapy approach. Promoting CD8(+) T cell adaptation to tissue residency represents an underutilized but promising strategy to improve tumor-infiltrating lymphocyte (TIL) function. Here, we report that deletion of the HIF negative regulator von Hippel-Lindau (VHL) in CD8(+) T cells induced HIF-1α/HIF-2α–dependent differentiation of tissue-resident memory–like (Trm-like) TILs in mouse models of malignancy. VHL-deficient TILs accumulated in tumors and exhibited a core Trm signature despite an exhaustion-associated phenotype, which led to retained polyfunctionality and response to αPD-1 immunotherapy, resulting in tumor eradication and protective tissue-resident memory. VHL deficiency similarly facilitated enhanced accumulation of chimeric antigen receptor (CAR) T cells with a Trm-like phenotype in tumors. Thus, HIF activity in CD8(+) TILs promotes accumulation and antitumor activity, providing a new strategy to enhance the efficacy of ACTs.

Cytotoxic CD8(+) T cells play a key role in the elimination of intracellular infections and malignant cells and can provide long-term protective immunity. In the response to infection, CD8(+) T cell metabolism is coupled to transcriptional, translational and epigenetic changes that are driven by extracellular metabolites and immunological signals. These programmes facilitate the adaptation of CD8(+) T cells to the diverse and dynamic metabolic environments encountered in the circulation and in the tissues. In the setting of disease, both cell-intrinsic and cell-extrinsic metabolic cues contribute to CD8(+) T cell dysfunction. In addition, changes in whole-body metabolism, whether through voluntary or disease-induced dietary alterations, can influence CD8(+) T cell-mediated immunity. Defining the metabolic adaptations of CD8(+) T cells in specific tissue environments informs our understanding of how these cells protect against pathogens and tumours and maintain tissue health at barrier sites. Here, we highlight recent findings revealing how metabolic networks enforce specific CD8(+) T cell programmes and discuss how metabolism is integrated with CD8(+) T cell differentiation and function and determined by environmental cues.

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 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 simultaneously 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 RNAseq 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. A key finding of this study was that parsing of terminal-Tem from conventionally defined Tem challenges classical 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.

During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of noncirculating tissue-resident memory (TRM) cells that mediate potent protection within nonlymphoid tissues. Here, we used single-cell RNA sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several stages of differentiation, representing functionally distinct TRM cell subsets and a subset of TRM cell precursors within the tissue early in infection. Together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer.

Unremitting defense against diverse pathogens and malignancies requires a dynamic and durable immune response. Tissue-resident memory CD8+ T cells (Trm) afford robust protection against infection and cancer progression through continuous surveillance of non-lymphoid tissues. Here, we provide insight into how Trm confer potent and persistent immunity through partitioning of distinct cellular subsets differing in longevity, effector function, and multipotency. Antigen-specific CD8+ T cells localized to the epithelium of the small intestine are primarily comprised of a shorter-lived effector population most prominent early following both acute viral and bacterial infections, and a longer-lived Id3hi Trm population that subsequently accumulates at later memory timepoints. We define regulatory gene-programs driving these distinct Trm states, and further clarify roles for Blimp1, T-bet, Id2, and Id3 in supporting and maintaining intestinal Trm heterogeneity during infection. Further, through single-cell RNAseq analysis we demonstrate that tumor-infiltrating lymphocytes broadly differentiate into discrete populations of short-lived and long-lived Trm-like subsets, which share qualities with terminally-exhausted and progenitor-exhausted cells, respectively. As the clinical relevance of Trm continues to widen from acute infections to settings of chronic inflammation and malignancy, clarification of the spectrum of phenotypic and functional states exhibited by CD8+ T cells that reside in non-lymphoid tissues will provide a framework for understanding their regulation and identity in diverse pathophysiological contexts.

During an immune response to microbial infection, CD8+ T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of non-circulating tissue-resident memory (TRM) cells that mediate potent protection within non-lymphoid tissues. Here, we utilized single-cell RNA-sequencing to examine the gene expression patterns of individual CD8+ T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8+ T cell population at several states of differentiation, representing functionally distinct TRM cell subsets as well as a subset of TRM cell precursors within the tissue early in infection. Taken together, these findings may inform strategies to optimize CD8+ T cell responses to protect against microbial infection and cancer.One sentence summaryHere, we applied single-cell RNA-sequencing to elucidate the gene expression patterns of individual CD8+ T cells differentiating throughout the course of infection in the spleen and small intestinal epithelium, which revealed previously unidentified molecular determinants of tissue-resident T cell differentiation as well as functional heterogeneity within the tissue-resident CD8+ T cell population.

CD8+ T cells responding to infection differentiate into short-lived effector cells destined to die or memory cells that provide long-lived protection. Omilusik et al. demonstrate that commitment to an effector cell fate is not necessarily terminal and that sustained transcriptional regulation is required to maintain subset-specific properties., CD8+ T cells responding to infection differentiate into a heterogeneous population composed of progeny that are short-lived and participate in the immediate, acute response and those that provide long-lasting host protection. Although it is appreciated that distinct functional and phenotypic CD8+ T cell subsets persist, it is unclear whether there is plasticity among subsets and what mechanisms maintain subset-specific differences. Here, we show that continued Id2 regulation of E-protein activity is required to maintain the KLRG1hi CD8+ T cell population after lymphocytic choriomeningitis virus infection. Induced deletion of Id2 phenotypically and transcriptionally transformed the KLRG1hi “terminal” effector/effector-memory CD8+ T cell population into a KLRG1lo memory-like population, promoting a gene-expression program that resembled that of central memory T cells. Our results question the idea that KLRG1hi CD8+ T cells are necessarily terminally programmed and suggest that sustained regulation is required to maintain distinct CD8+ T cell states.

Tissue-resident memory CD8+ T (TRM) cells are found at common sites of pathogen exposure, where they elicit rapid and robust protective immune responses. However, the molecular signals that control TRM cell differentiation and homeostasis are not fully understood. Here we show that mouse TRM precursor cells represent a unique CD8+ T cell subset that is distinct from the precursors of circulating memory cell populations at the levels of gene expression and chromatin accessibility. Using computational and pooled in vivo RNA interference screens, we identify the transcription factor Runx3 as a key regulator of TRM cell differentiation and homeostasis. Runx3 was required to establish TRM cell populations in diverse tissue environments, and supported the expression of crucial tissue-residency genes while suppressing genes associated with tissue egress and recirculation. Furthermore, we show that human and mouse tumour-infiltrating lymphocytes share a core tissue-residency gene-expression signature with TRM cells that is associated with Runx3 activity. In a mouse model of adoptive T cell therapy for melanoma, Runx3-deficient CD8+ tumour-infiltrating lymphocytes failed to accumulate in tumours, resulting in greater rates of tumour growth and mortality. Conversely, overexpression of Runx3 enhanced tumour-specific CD8+ T cell abundance, delayed tumour growth, and prolonged survival. In addition to establishing Runx3 as a central regulator of TRM cell differentiation, these results provide insight into the signals that promote T cell residency in non-lymphoid sites, which could be used to enhance vaccine efficacy or adoptive cell therapy treatments that target cancer.

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.

During an immune response to microbial infection, CD8(+) T cells give rise to distinct classes of cellular progeny that coordinately mediate clearance of the pathogen and provide long-lasting protection against reinfection, including a subset of non-circulating tissue-resident memory (T(RM)) cells that mediate potent protection within non-lymphoid tissues. Here, we utilized single-cell RNA-sequencing to examine the gene expression patterns of individual CD8(+) T cells in the spleen and small intestine intraepithelial lymphocyte (siIEL) compartment throughout the course of their differentiation in response to viral infection. These analyses revealed previously unknown transcriptional heterogeneity within the siIEL CD8(+) T cell population at several stages of differentiation, representing functionally distinct T(RM) cell subsets as well as a subset of T(RM) cell precursors within the tissue early in infection. Taken together, these findings may inform strategies to optimize CD8(+) T cell responses to protect against microbial infection and cancer.