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The immune system of laboratory mice raised in an ultra-hygienic environment resembles that ofnewborn humans, but can be induced to resemble the immune system of adult humans or 'dirty' mice by co-housing with pet store-bought mice. Do 'dirty' mice make better immunological models? The laboratory mouse is by far the dominant model organism for in vivo immunological research which — particularly in the light of disappointing results obtained with some recent transfers of disease treatments from laboratory to clinic — raises the question of how accurately the model reflects the human immune system. These authors compare the immune status of laboratory mice with that of feral mice and with mice bought commercially as pets. They find that the immune system of the ubiquitous laboratory 'specific pathogen free' mouse approximates that of human neonates, rather than human adults. Co-housing laboratory mice with 'pet store' mice leads to maturation of the immune system, making it more similar to that of the human adult, and resulting in increased resistance in several models of infection. The use of such 'dirty' mice could supplement current models to either increase translational potential to human disease or to better inform the efficacy of preclinical prophylactic and therapeutic modalities. Our current understanding of immunology was largely defined in laboratory mice, partly because they are inbred and genetically homogeneous, can be genetically manipulated, allow kinetic tissue analyses to be carried out from the onset of disease, and permit the use of tractable disease models. Comparably reductionist experiments are neither technically nor ethically possible in humans. However, there is growing concern that laboratory mice do not reflect relevant aspects of the human immune system, which may account for failures to translate disease treatments from bench to bedside 1 , 2 , 3 , 4 , 5 , 6 , 7 , 8 . Laboratory mice live in abnormally hygienic specific pathogen free (SPF) barrier facilities. Here we show that standard laboratory mouse husbandry has profound effects on the immune system and that environmental changes produce mice with immune systems closer to those of adult humans. Laboratory mice—like newborn, but not adult, humans—lack effector-differentiated and mucosally distributed memory T cells. These cell populations were present in free-living barn populations of feral mice and pet store mice with diverse microbial experience, and were induced in laboratory mice after co-housing with pet store mice, suggesting that the environment is involved in the induction of these cells. Altering the living conditions of mice profoundly affected the cellular composition of the innate and adaptive immune systems, resulted in global changes in blood cell gene expression to patterns that more closely reflected the immune signatures of adult humans rather than neonates, altered resistance to infection, and influenced T-cell differentiation in response to a de novo viral infection. These data highlight the effects of environment on the basal immune state and response to infection and suggest that restoring physiological microbial exposure in laboratory mice could provide a relevant tool for modelling immunological events in free-living organisms, including humans.

T cell dysfunction is a hallmark of many cancers, but the basis for T cell dysfunction and the mechanisms by which antibody blockade of the inhibitory receptor PD-1 (anti-PD-1) reinvigorates T cells are not fully understood. Here we show that such therapy acts on a specific subpopulation of exhausted CD8 + tumor-infiltrating lymphocytes (TILs). Dysfunctional CD8 + TILs possess canonical epigenetic and transcriptional features of exhaustion that mirror those seen in chronic viral infection. Exhausted CD8 + TILs include a subpopulation of ‘progenitor exhausted’ cells that retain polyfunctionality, persist long term and differentiate into ‘terminally exhausted’ TILs. Consequently, progenitor exhausted CD8 + TILs are better able to control tumor growth than are terminally exhausted T cells. Progenitor exhausted TILs can respond to anti-PD-1 therapy, but terminally exhausted TILs cannot. Patients with melanoma who have a higher percentage of progenitor exhausted cells experience a longer duration of response to checkpoint-blockade therapy. Thus, approaches to expand the population of progenitor exhausted CD8 + T cells might be an important component of improving the response to checkpoint blockade. Exhausted cytotoxic T lymphocytes (CTLs) express the receptor PD-1 as a key signature. Haining and colleagues show that there are different ‘depths’ of exhaustion with a subset of exhausted CTLs that retain polyfunctionality and are responsive to PD-1 blockade.

CD8 + T cell exhaustion is a state of dysfunction acquired in chronic viral infection and cancer, characterized by the formation of Slamf6 + progenitor exhausted and Tim-3 + terminally exhausted subpopulations through unknown mechanisms. Here we establish the phosphatase PTPN2 as a new regulator of the differentiation of the terminally exhausted subpopulation that functions by attenuating type 1 interferon signaling. Deletion of Ptpn2 in CD8 + T cells increased the generation, proliferative capacity and cytotoxicity of Tim-3 + cells without altering Slamf6 + numbers during lymphocytic choriomeningitis virus clone 13 infection. Likewise , Ptpn2 deletion in CD8 + T cells enhanced Tim-3 + anti-tumor responses and improved tumor control. Deletion of Ptpn2 throughout the immune system resulted in MC38 tumor clearance and improved programmed cell death-1 checkpoint blockade responses to B16 tumors. Our results indicate that increasing the number of cytotoxic Tim-3 + CD8 + T cells can promote effective anti-tumor immunity and implicate PTPN2 in immune cells as an attractive cancer immunotherapy target. Exhaustion is an acquired state of T cell dysfunction. Sharpe and colleagues demonstrate that the phosphatase PTPN2 supports a T cell-intrinsic exhaustion program in both chronic infection and cancer models.

T cell exhaustion is an induced state of dysfunction that arises in response to chronic infection and cancer. Exhausted CD8 + T cells acquire a distinct epigenetic state, but it is not known whether that chromatin landscape is fixed or plastic following the resolution of a chronic infection. Here we show that the epigenetic state of exhaustion is largely irreversible, even after curative therapy. Analysis of chromatin accessibility in HCV- and HIV-specific responses identifies a core epigenetic program of exhaustion in CD8 + T cells, which undergoes only limited remodeling before and after resolution of infection. Moreover, canonical features of exhaustion, including super-enhancers near the genes TOX and HIF1A , remain ‘epigenetically scarred.’ T cell exhaustion is therefore a conserved epigenetic state that becomes fixed and persists independent of chronic antigen stimulation and inflammation. Therapeutic efforts to reverse T cell exhaustion may require new approaches that increase the epigenetic plasticity of exhausted T cells. The degree of plasticity in the epigenetic landscape of exhausted T cells has been unclear. Sen and colleagues find that exhausted CD8 + T cells demonstrate a stable core epigenetic exhaustion signature that persists independent of inflammation or viral antigen.

The cyclin D3–CDK6 kinase complex, which is overactive in some cancers, inhibits two key glycolysis enzymes and thereby enhances the levels of antioxidants in cells, promoting tumour cell survival. Cancer cell survival by cyclin D3–CDK6 metabolism Cyclin–CDK complexes are commonly amplified in cancer and promote cell cycle progression. Inhibitors for CDK4/6 are being tested in clinical trials and are thought to work in patients that retain expression of the CDK substrate RB1. Here, the authors describe an additional pro-survival role of one cyclin–CDK complex, D3–CDK6, which controls cellular metabolism. When hyperactivated in cancer cells, the complex phosphorylates and inactivates two glycolysis enzymes. This redirects glycolytic intermediates to the pentose phosphate and serine pathways, providing enhanced antioxidant capacity. CDK4/6 inhibitors can induce apoptosis by increasing the oxidative stress in tumour cells expressing high levels of D3–CDK6 complexes. The findings suggest that, in addition to RB1, markers such as levels of D3–CDK6 complexes could be useful for identifying patients likely to respond to CDK4/6 inhibitors. D-type cyclins (D1, D2 and D3) and their associated cyclin-dependent kinases (CDK4 and CDK6) are components of the core cell cycle machinery that drives cell proliferation 1 , 2 . Inhibitors of CDK4 and CDK6 are currently being tested in clinical trials for patients with several cancer types, with promising results 2 . Here, using human cancer cells and patient-derived xenografts in mice, we show that the cyclin D3–CDK6 kinase phosphorylates and inhibits the catalytic activity of two key enzymes in the glycolytic pathway, 6-phosphofructokinase and pyruvate kinase M2. This re-directs the glycolytic intermediates into the pentose phosphate (PPP) and serine pathways. Inhibition of cyclin D3–CDK6 in tumour cells reduces flow through the PPP and serine pathways, thereby depleting the antioxidants NADPH and glutathione. This, in turn, increases the levels of reactive oxygen species and causes apoptosis of tumour cells. The pro-survival function of cyclin D-associated kinase operates in tumours expressing high levels of cyclin D3–CDK6 complexes. We propose that measuring the levels of cyclin D3–CDK6 in human cancers might help to identify tumour subsets that undergo cell death and tumour regression upon inhibition of CDK4 and CDK6. Cyclin D3–CDK6, through its ability to link cell cycle and cell metabolism, represents a particularly powerful oncoprotein that affects cancer cells at several levels, and this property can be exploited for anti-cancer therapy.

Therapies that target the function of immune cells have significant clinical efficacy in diseases such as cancer and autoimmunity. Although functional genomics has accelerated therapeutic target discovery in cancer, its use in primary immune cells is limited because vector delivery is inefficient and can perturb cell states. Here we describe CHIME: CHimeric IMmune Editing, a CRISPR-Cas9 bone marrow delivery system to rapidly evaluate gene function in innate and adaptive immune cells in vivo without ex vivo manipulation of these mature lineages. This approach enables efficient deletion of genes of interest in major immune lineages without altering their development or function. We use this approach to perform an in vivo pooled genetic screen and identify Ptpn2 as a negative regulator of CD8 + T cell-mediated responses to LCMV Clone 13 viral infection. These findings indicate that this genetic platform can enable rapid target discovery through pooled screening in immune cells in vivo. The use of functional genomics in primary immune cells has been limited by inefficient vector delivery and risk of perturbing cell states. Here the authors present CHimeric IMmune Editing (CHIME) for in vivo evaluation of gene function and pooled screening approaches.

The transcription factor BATF is required for differentiation of certain helper T cell subsets. Haining and colleagues show that BATF crucially regulates CD8 + effector cells by coordinating a transcription factor network. The transcription factor BATF is required for the differentiation of interleukin 17 (IL-17)-producing helper T cells (T H 17 cells) and follicular helper T cells (T FH cells). Here we identified a fundamental role for BATF in regulating the differentiation of effector of CD8 + T cells. BATF-deficient CD8 + T cells showed profound defects in effector population expansion and underwent proliferative and metabolic catastrophe early after encountering antigen. BATF, together with the transcription factors IRF4 and Jun proteins, bound to and promoted early expression of genes encoding lineage-specific transcription-factors (T-bet and Blimp-1) and cytokine receptors while paradoxically repressing genes encoding effector molecules (IFN-γ and granzyme B). Thus, BATF amplifies T cell antigen receptor (TCR)-dependent expression of transcription factors and augments the propagation of inflammatory signals but restrains the expression of genes encoding effector molecules. This checkpoint prevents irreversible commitment to an effector fate until a critical threshold of downstream transcriptional activity has been achieved.

Exhausted T cells express multiple co-inhibitory molecules that impair their function and limit immunity to chronic viral infection. Defining novel markers of exhaustion is important both for identifying and potentially reversing T cell exhaustion. Herein, we show that the ectonucleotidse CD39 is a marker of exhausted CD8+ T cells. CD8+ T cells specific for HCV or HIV express high levels of CD39, but those specific for EBV and CMV do not. CD39 expressed by CD8+ T cells in chronic infection is enzymatically active, co-expressed with PD-1, marks cells with a transcriptional signature of T cell exhaustion and correlates with viral load in HIV and HCV. In the mouse model of chronic Lymphocytic Choriomeningitis Virus infection, virus-specific CD8+ T cells contain a population of CD39high CD8+ T cells that is absent in functional memory cells elicited by acute infection. This CD39high CD8+ T cell population is enriched for cells with the phenotypic and functional profile of terminal exhaustion. These findings provide a new marker of T cell exhaustion, and implicate the purinergic pathway in the regulation of T cell exhaustion.

T cell exhaustion is associated with failure to clear chronic infections and malignant cells. Defining the molecular mechanisms of T cell exhaustion and reinvigoration is essential to improving immunotherapeutic modalities. Here we confirmed pervasive phenotypic, functional and transcriptional differences between memory and exhausted antigen-specific CD8 + T cells in human hepatitis C virus (HCV) infection before and after treatment. After viral cure, phenotypic changes in clonally stable exhausted T cell populations suggested differentiation toward a memory-like profile. However, functionally, the cells showed little improvement, and critical transcriptional regulators remained in the exhaustion state. Notably, T cells from chronic HCV infection that were exposed to antigen for less time because of viral escape mutations were functionally and transcriptionally more similar to memory T cells from spontaneously resolved HCV infection. Thus, the duration of T cell stimulation impacts exhaustion recovery, with antigen removal after long-term exhaustion being insufficient for the development of functional T cell memory. Lauer and colleagues examine CD8 + T cells following cure of human hepatitis C virus (HCV) infection. CD8 + T cells exposed to chronic HCV-specific activation show durable functional, phenotypic and transcriptional exhaustion that is maintained even after antigen stimulus is removed.

PD-1 is a key negative regulator of CD8 + T cell activation and is highly expressed by exhausted T cells in cancer and chronic viral infection. Although PD-1 blockade can improve viral and tumor control, physiological PD-1 expression prevents immunopathology and improves memory formation. The mechanisms driving high PD-1 expression in exhaustion are not well understood and could be critical to disentangling its beneficial and detrimental effects. Here, we functionally interrogated the epigenetic regulation of PD-1 using a mouse model with deletion of an exhaustion-specific PD-1 enhancer. Enhancer deletion exclusively alters PD-1 expression in CD8 + T cells in chronic infection, creating a ‘sweet spot’ of intermediate expression where T cell function is optimized compared to wild-type and Pdcd1 -knockout cells. This permits improved control of chronic infection without additional immunopathology. Together, these results demonstrate that tuning PD-1 via epigenetic editing can reduce CD8 + T cell dysfunction while avoiding excess immunopathology. PD-1 is a critical modulator of CD8 + T cell activation and exhaustion. Sen and colleagues show that a cell-state-specific enhancer tunes PD-1 expression exclusively in exhaustion and that deletion of this enhancer improves CD8 + T cell function.