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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 (TRM), the developmental origins and transcriptional regulation of CD4+ TRM remain largely undefined. Here, we investigated the phenotypic, functional, and transcriptional profiles of CD4+ TRM in the small intestine (SI) responding to acute viral infection, revealing a shared gene expression program and chromatin accessibility profile with circulating TH1 and the progressive acquisition of a mature TRM program. Single-cell RNA sequencing identified heterogeneity among established CD4+ TRM, 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. TH1-associated Blimp1 and Id2 and TFH-associated Bcl6 were required for early TRM formation and development of a mature TRM population in the SI. These results demonstrate a developmental relationship between TH1 effector cells and the establishment of early TRM, as well as highlighted differences in CD4+ versus CD8+ TRM populations, providing insights into the mechanisms underlying the origins, differentiation, and persistence of CD4+ TRM in response to viral infection.

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.

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)-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 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.

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.

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.

T cells are essential mediators of immune responses against infectious diseases and provide long-lived protection from reinfection. The differentiation of naive to effector T cells and the 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. In this study, we investigated a role for Usp1 in posttranslational control of Id2 and Id3 in murine 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 T cell expansion or differentiation following a primary infection. Usp1 deficiency resulted in a gradual loss of memory CD8+ T cells over time and reduced Id2 protein levels and proliferation of effector CD8+ T cell following reinfection. 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 differential regulation of Id2/3 proteins between immune versus nonimmune cell types.

In response to infection, pathogen-specific CD8 T cells differentiate into functionally diverse effector and memory T cell populations critical for resolving disease and providing durable immunity. Through small-molecule inhibition, RNAi studies, and induced genetic deletion, we reveal an essential role for the chromatin modifier and BET family member BRD4 in supporting the differentiation and maintenance of terminally fated effector CD8 T cells during infection. BRD4 bound diverse regulatory regions critical to effector T cell differentiation and controlled transcriptional activity of terminal effector-specific super-enhancers in vivo. Consequentially, induced deletion of Brd4 or small molecule-mediated BET inhibition impaired maintenance of a terminal effector T cell phenotype. BRD4 was also required for terminal differentiation of CD8 T cells in the tumor microenvironment in murine models, which we show has implications for immunotherapies. Taken together, these data reveal an unappreciated requirement for BRD4 in coordinating activity of cis regulatory elements to control CD8 T cell fate and lineage stability.

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.

Inflammatory bowel disease (IBD) encompasses a spectrum of gastrointestinal disorders driven by dysregulated immune responses against gut microbiota. We integrated single-cell RNA and antigen receptor sequencing to elucidate key components, cellular states, and clonal relationships of the peripheral and gastrointestinal mucosal immune systems in health and ulcerative colitis (UC). UC was associated with an increase in IgG1+ plasma cells in colonic tissue, increased colonic regulatory T cells characterized by elevated expression of the transcription factor ZEB2, and an enrichment of a γδ T cell subset in the peripheral blood. Moreover, we observed heterogeneity in CD8+ tissue-resident memory T (TRM) cells in colonic tissue, with four transcriptionally distinct states of differentiation observed across health and disease. In the setting of UC, there was a marked shift of clonally related CD8+ TRM cells toward an inflammatory state, mediated, in part, by increased expression of the T-box transcription factor Eomesodermin. Together, these results provide a detailed atlas of transcriptional changes occurring in adaptive immune cells in the context of UC and suggest a role for CD8+ TRM cells in IBD.

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.

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.

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.

Tissue-resident memory CD8+ T cells (TRM) are localized in non-lymphoid tissues throughout the body where they mediate long-lived protective immunity at common sites of pathogen exposure. As the signals controlling TRM differentiation are uncovered, it is becoming apparent that the dynamic activities of numerous transcription factors are intricately involved in TRM formation. Here, we highlight known transcriptional regulators of TRM differentiation and discuss how understanding the transcriptional programming of CD8+ T cell residency in non-lymphoid tissues can be leveraged to prevent or treat disease.

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.

Upon infection with an intracellular pathogen, cytotoxic CD8+ T cells develop diverse differentiation states characterized by function, localization, longevity, and the capacity for self-renewal. The program of differentiation is determined, in part, by FOXO1, a transcription factor known to integrate extrinsic input in order to specify survival, DNA repair, self-renewal, and proliferation. At issue is whether the state of T cell differentiation is specified by initial conditions of activation or is actively maintained. To study the spectrum of T cell differentiation, we have analyzed an infection with mouse cytomegalovirus, a persistent-latent virus that elicits different cytotoxic T cell responses characterized as acute resolving or inflationary. Our results show that FOXO1 is continuously required for all the phenotypic characteristics of memory-effector T cells such that with acute inactivation of the gene encoding FOXO1, T cells revert to a short-lived effector phenotype, exhibit reduced viability, and manifest characteristics of anergy.

Recognition of pathogens by innate and adaptive immune cells instructs rapid alterations of cellular processes to promote effective resolution of infection. To accommodate increased bioenergetic and biosynthetic demands, metabolic pathways are harnessed to maximize proliferation and effector molecule production. In parallel, activation initiates context-specific gene-expression programs that drive effector functions and cell fates that correlate with changes in epigenetic landscapes. Many chromatin- and DNA-modifying enzymes make use of substrates and cofactors that are intermediates of metabolic pathways, providing potential cross talk between metabolism and epigenetic regulation of gene expression. In this review, we discuss recent studies of T cells and macrophages supporting a role for metabolic activity in integrating environmental signals with activation-induced gene-expression programs through modulation of the epigenome and speculate as to how this may influence context-specific macrophage and T cell responses to infection.

Extensive metabolic changes accompany T cell activation, including a switch to glycolytic energy production and increased biosynthesis. Recent studies suggest that subsequent return to reliance on oxidative phosphorylation and increasing spare respiratory capacity are essential for the differentiation of memory CD8+ T cells. In contrast, we found that constitutive glycolytic metabolism and suppression of oxidative phosphorylation in CD8+ T cells, achieved by conditional deletion of hypoxia-inducible factor regulator Vhl, accelerated CD8+ memory cell differentiation during viral infection. Despite sustained glycolysis, CD8+ memory cells emerged that upregulated key memory-associated cytokine receptors and transcription factors and showed a heightened response to secondary challenge. In addition, increased glycolysis not only permitted memory formation, but it also favored the formation of long-lived effector-memory CD8+ T cells. These data redefine the role of cellular metabolism in memory cell differentiation, showing that reliance on glycolytic metabolism does not hinder formation of a protective memory population.

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.

Targeted deletion of BAFF causes severe deficiency of splenic B cells. BAFF-R is commonly thought to signal to nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB)-inducing kinase dependent noncanonical NF-κB RelB. However, RelB-deficient mice have normal B-cell numbers. Recent studies showed that BAFF also signals to the canonical NF-κB pathway, and we found that both RelB and cRel are persistently activated, suggesting BAFF signaling coordinates both pathways to ensure robust B-cell development. Indeed, we report now that combined loss of these 2 NF-κB family members leads to impaired BAFF-mediated survival and development in vitro. Although single deletion of RelB and cRel was dispensable for normal B-cell development, double knockout mice displayed an early B-cell developmental blockade and decreased mature B cells. Despite disorganized splenic architecture in Relb(-/-)cRel(-/-) mice, generation of mixed-mouse chimeras established the developmental phenotype to be B-cell intrinsic. Together, our results indicate that BAFF signals coordinate both RelB and cRel activities to ensure survival during peripheral B-cell maturation.

Resolution of infection requires the coordinated response of heterogeneous cell types to a range of physiological and pathological signals to regulate their proliferation, migration, differentiation, and effector functions. One mechanism by which immune cells integrate these signals is through modulating metabolic activity. A well-studied regulator of cellular metabolism is the hypoxia-inducible factor (HIF) family, the highly conserved central regulators of adaptation to limiting oxygen tension. HIF's regulation of cellular metabolism and a variety of effector, signaling, and trafficking molecules has made these transcription factors a recent topic of interest in T cell biology. Low oxygen availability, or hypoxia, increases expression and stabilization of HIF in immune cells, activating molecular programs both unique and common among cell types, including glycolytic metabolism. Notably, numerous oxygen-independent signals, many of which are active in T cells, also result in enhanced HIF activity. Here, we discuss both oxygen-dependent and -independent regulation of HIF activity in T cells and the resulting impacts on metabolism, differentiation, function, and immunity.

ZEB2 is a multi-zinc-finger transcription factor known to play a significant role in early neurogenesis and in epithelial-mesenchymal transition-dependent tumor metastasis. Although the function of ZEB2 in T lymphocytes is unknown, activity of the closely related family member ZEB1 has been implicated in lymphocyte development. Here, we find that ZEB2 expression is up-regulated by activated T cells, specifically in the KLRG1(hi) effector CD8(+) T cell subset. Loss of ZEB2 expression results in a significant loss of antigen-specific CD8(+) T cells after primary and secondary infection with a severe impairment in the generation of the KLRG1(hi) effector memory cell population. We show that ZEB2, which can bind DNA at tandem, consensus E-box sites, regulates gene expression of several E-protein targets and may directly repress Il7r and Il2 in CD8(+) T cells responding to infection. Furthermore, we find that T-bet binds to highly conserved T-box sites in the Zeb2 gene and that T-bet and ZEB2 regulate similar gene expression programs in effector T cells, suggesting that T-bet acts upstream and through regulation of ZEB2. Collectively, we place ZEB2 in a larger transcriptional network that is responsible for the balance between terminal differentiation and formation of memory CD8(+) T cells.

T follicular helper (Tfh) cells are essential in the induction of high-affinity, class-switched antibodies. The differentiation of Tfh cells is a multi-step process that depends upon the co-receptor ICOS and the activation of phosphoinositide-3 kinase leading to the expression of key Tfh cell genes. We report that ICOS signaling inactivates the transcription factor FOXO1, and a Foxo1 genetic deletion allowed for generation of Tfh cells with reduced dependence on ICOS ligand. Conversely, enforced nuclear localization of FOXO1 inhibited Tfh cell development even though ICOS was overexpressed. FOXO1 regulated Tfh cell differentiation through a broad program of gene expression exemplified by its negative regulation of Bcl6. Final differentiation to germinal center Tfh cells (GC-Tfh) was instead FOXO1 dependent as the Foxo1(-/-) GC-Tfh cell population was substantially reduced. We propose that ICOS signaling transiently inactivates FOXO1 to initiate a Tfh cell contingency that is completed in a FOXO1-dependent manner.

Abstract Background Administration of recombinant G-CSF following cytoreductive therapy enhances the recovery of myeloid cells, minimizing the risk of opportunistic infection. Free G-CSF, however, is expensive, exhibits a short half-life, and has poor biological activity in vivo. Methods We evaluated whether the biological activity of G-CSF could be improved by pre-association with anti-G-CSF mAb prior to injection into mice. Results We find that the efficacy of G-CSF therapy can be enhanced more than 100-fold by pre-association of G-CSF with an anti-G-CSF monoclonal antibody (mAb). Compared with G-CSF alone, administration of G-CSF/anti-G-CSF mAb complexes induced the potent expansion of CD11b+Gr-1+ myeloid cells in mice with or without concomitant cytoreductive treatment including radiation or chemotherapy. Despite driving the dramatic expansion of myeloid cells, in vivo antigen-specific CD8+ T cell immune responses were not compromised. Furthermore, injection of G-CSF/anti-G-CSF mAb complexes heightened protective immunity to bacterial infection. As a measure of clinical value, we also found that antibody complexes improved G-CSF biological activity much more significantly than pegylation. Conclusions Our findings provide the first evidence that antibody cytokine complexes can effectively expand myeloid cells, and furthermore, that G-CSF/anti-G-CSF mAb complexes may provide an improved method for the administration of recombinant G-CSF.

After infection, many factors coordinate the population expansion and differentiation of CD8+ effector and memory T cells. Using data of unparalleled breadth from the Immunological Genome Project, we analyzed the CD8+ T cell transcriptome throughout infection to establish gene-expression signatures and identify putative transcriptional regulators. Notably, we found that the expression of key gene signatures can be used to predict the memory-precursor potential of CD8+ effector cells. Long-lived memory CD8+ cells ultimately expressed a small subset of genes shared by natural killer T and γδ T cells. Although distinct inflammatory milieu and T cell precursor frequencies influenced the differentiation of CD8+ effector and memory populations, core transcriptional signatures were regulated similarly, whether polyclonal or transgenic, and whether responding to bacterial or viral model pathogens. Our results provide insights into the transcriptional regulation that influence memory formation and CD8+ T cell immunity.

Using whole-genome microarray data sets of the Immunological Genome Project, we demonstrate a closer transcriptional relationship between NK cells and T cells than between any other leukocytes, distinguished by their shared expression of genes encoding molecules with similar signaling functions. Whereas resting NK cells are known to share expression of a few genes with cytotoxic CD8(+) T cells, our transcriptome-wide analysis demonstrates that the commonalities extend to hundreds of genes, many encoding molecules with unknown functions. Resting NK cells demonstrate a 'preprimed' state compared with naive T cells, which allows NK cells to respond more rapidly to viral infection. Collectively, our data provide a global context for known and previously unknown molecular aspects of NK cell identity and function by delineating the genome-wide repertoire of gene expression of NK cells in various states.