Kathleen Yates

The epigenetic landscape of T cell exhaustion

Epigenetic profiling suggests that exhausted T cells are a distinct cell linage. The epigenetics of exhaustion During cancer or chronic infection, T cells become dysfunctional, eventually acquiring an “exhausted” phenotype. Immunotherapies aim to reverse this state. Using a mouse model of chronic infection, two studies now show that the epigenetic profile of exhausted T cells differs substantially from those of effector and memory T cells, suggesting that exhausted T cells are a distinct lineage (see the Perspective by Turner and Russ). Sen et al. defined specific functional modules of enhancers that are also conserved in exhausted human T cells. Pauken et al. examined the epigenetic profile of exhausted T cells after immunotherapy. Although there was transcriptional rewiring, the cells never acquired a memory T cell phenotype. Thus, epigenetic regulation may limit the success of immunotherapies. Science, this issue p. 1104, p. 1165; see also p. 1160 Exhausted T cells in cancer and chronic viral infection express distinctive patterns of genes, including sustained expression of programmed cell death protein 1 (PD-1). However, the regulation of gene expression in exhausted T cells is poorly understood. Here, we define the accessible chromatin landscape in exhausted CD8+ T cells and show that it is distinct from functional memory CD8+ T cells. Exhausted CD8+ T cells in humans and a mouse model of chronic viral infection acquire a state-specific epigenetic landscape organized into functional modules of enhancers. Genome editing shows that PD-1 expression is regulated in part by an exhaustion-specific enhancer that contains essential RAR, T-bet, and Sox3 motifs. Functional enhancer maps may offer targets for genome editing that alter gene expression preferentially in exhausted CD8+ T cells.

The transcription factor BATF operates as an essential differentiation checkpoint in early effector CD8 + T cells

The transcription factor BATF is required for the differentiation of interleukin 17 (IL-17)-producing helper T cells (TH17 cells) and follicular helper T cells (TFH 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.

In vivo CRISPR screening identifies Ptpn2 as a cancer immunotherapy target

Immunotherapy with PD-1 checkpoint blockade is effective in only a minority of patients with cancer, suggesting that additional treatment strategies are needed. Here we use a pooled in vivo genetic screening approach using CRISPR–Cas9 genome editing in transplantable tumours in mice treated with immunotherapy to discover previously undescribed immunotherapy targets. We tested 2,368 genes expressed by melanoma cells to identify those that synergize with or cause resistance to checkpoint blockade. We recovered the known immune evasion molecules PD-L1 and CD47, and confirmed that defects in interferon-γ signalling caused resistance to immunotherapy. Tumours were sensitized to immunotherapy by deletion of genes involved in several diverse pathways, including NF-κB signalling, antigen presentation and the unfolded protein response. In addition, deletion of the protein tyrosine phosphatase PTPN2 in tumour cells increased the efficacy of immunotherapy by enhancing interferon-γ-mediated effects on antigen presentation and growth suppression. In vivo genetic screens in tumour models can identify new immunotherapy targets in unanticipated pathways.

CD39 Expression Identifies Terminally Exhausted CD8+ T Cells.

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.

Vaccine-elicited CD4 T cells induce immunopathology after chronic LCMV infection.

For vaccines, CD4+ T cells can spell trouble The ideal vaccine elicits immune memory—either antibodies or memory T cells—to protect the host from subsequent infections. T cell–mediated immunity requires both helper CD4+ T cells and cytotoxic CD8+ T cells to kill virus-infected cells. But what happens when a vaccine only elicits CD4+ memory T cells? Penaloza-MacMaster et al. probed this question by giving mice a vaccine that generated only memory CD4+ T cells against lymphocytic choriomeningitis virus (LCMV). Instead of protecting mice against chronic LCMV, vaccinated mice developed massive inflammation and died. Virus-specific CD8+ T cells or antibodies protected mice from the pathology. These results may have implications for vaccines against chronic viruses such as HIV. Science, this issue p. 278 Severe immunopathology kills virally infected mice that received vaccines targeting only CD4+ T cells. CD4 T cells promote innate and adaptive immune responses, but how vaccine-elicited CD4 T cells contribute to immune protection remains unclear. We evaluated whether induction of virus-specific CD4 T cells by vaccination would protect mice against infection with chronic lymphocytic choriomeningitis virus (LCMV). Immunization with vaccines that selectively induced CD4 T cell responses resulted in catastrophic inflammation and mortality after challenge with a persistent strain of LCMV. Immunopathology required antigen-specific CD4 T cells and was associated with a cytokine storm, generalized inflammation, and multi-organ system failure. Virus-specific CD8 T cells or antibodies abrogated the pathology. These data demonstrate that vaccine-elicited CD4 T cells in the absence of effective antiviral immune responses can trigger lethal immunopathology.

Targeted reconstruction of T cell receptor sequence from single cell RNA-seq links CDR3 length to T cell differentiation state

Abstract The T cell compartment must contain diversity in both T cell receptor (TCR) repertoire and cell state to provide effective immunity against pathogens. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current methods for TCR inference from scRNA-seq are limited in their sensitivity and require long sequencing reads, thus increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present TRAPeS, a publicly available tool that can efficiently extract TCR sequence information from short-read scRNA-seq libraries. We apply it to investigate heterogeneity in the CD8+ T cell response in humans and mice, and show that it is accurate and more sensitive than existing approaches. Coupling TRAPeS with transcriptome analysis of CD8+ T cells specific for a single epitope from Yellow Fever Virus (YFV), we show that the recently described ‘naive-like’ memory population have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype cells. This suggests that TCR usage is associated with the differentiation state of the CD8+ T cell response to YFV.

CDK4/6 Inhibition Augments Antitumor Immunity by Enhancing T-cell Activation

Immune checkpoint blockade, exemplified by antibodies targeting the PD-1 receptor, can induce durable tumor regressions in some patients. To enhance the efficacy of existing immunotherapies, we screened for small molecules capable of increasing the activity of T cells suppressed by PD-1. Here, we show that short-term exposure to small-molecule inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6) significantly enhances T-cell activation, contributing to antitumor effects in vivo, due in part to the derepression of NFAT family proteins and their target genes, critical regulators of T-cell function. Although CDK4/6 inhibitors decrease T-cell proliferation, they increase tumor infiltration and activation of effector T cells. Moreover, CDK4/6 inhibition augments the response to PD-1 blockade in a novel ex vivo organotypic tumor spheroid culture system and in multiple in vivo murine syngeneic models, thereby providing a rationale for combining CDK4/6 inhibitors and immunotherapies.Significance: Our results define previously unrecognized immunomodulatory functions of CDK4/6 and suggest that combining CDK4/6 inhibitors with immune checkpoint blockade may increase treatment efficacy in patients. Furthermore, our study highlights the critical importance of identifying complementary strategies to improve the efficacy of immunotherapy for patients with cancer. Cancer Discov; 8(2); 216-33. ©2017 AACR.See related commentary by Balko and Sosman, p. 143See related article by Jenkins et al., p. 196This article is highlighted in the In This Issue feature, p. 127.

Immediate Dysfunction of Vaccine-Elicited CD8+ T Cells Primed in the Absence of CD4+ T Cells

CD4+ T cell help is critical for optimal CD8+ T cell memory differentiation and maintenance in many experimental systems. In addition, many reports have identified reduced primary CD8+ T cell responses in the absence of CD4+ T cell help, which often coincides with reduced Ag or pathogen clearance. In this study, we demonstrate that absence of CD4+ T cells at the time of adenovirus vector immunization of mice led to immediate impairments in early CD8+ T cell functionality and differentiation. Unhelped CD8+ T cells exhibited a reduced effector phenotype, decreased ex vivo cytotoxicity, and decreased capacity to produce cytokines. This dysfunctional state was imprinted within 3 d of immunization. Unhelped CD8+ T cells expressed elevated levels of inhibitory receptors and exhibited transcriptomic exhaustion and anergy profiles by gene set enrichment analysis. Dysfunctional, impaired effector differentiation also occurred following immunization of CD4+ T cell–deficient mice with a poxvirus vector. This study demonstrates that following priming with viral vectors, CD4+ T cell help is required to promote both the expansion and acquisition of effector functions by CD8+ T cells, which is accomplished by preventing immediate dysfunction.

Isolation of RNA and the synthesis and amplification of cDNA from antigen-specific T cells for genome-wide expression analysis.

Genome-wide gene expression analysis has become a very powerful routine tool for the study of distinct differentiation states. However, the examination of total populations of cells that contain high levels of heterogeneity, such as the total CD8(+) T cell population during an immune response, is limited because that complexity hampers accurate interpretation. The gene expression signatures from populations represent the average of all cells within the populations, which will smooth out large expression changes within small subpopulations and virtually eliminate any small changes. However, small expression changes within a minor subpopulation, such as antigen-specific CD8(+) T cells responding to an infection, can have relevant biological consequences. Although very limited amounts of RNA can be isolated from small subpopulations of cells, there are now methods to synthesize and amplify cDNA from this limited RNA in sufficient quantities needed for microarray analysis. Here, we describe a complete protocol to extract RNA from small numbers of cells, synthesize cDNA from that RNA, and amplify that cDNA in an unbiased method. This protocol is a useful tool for the study of genome-wide expression signatures from many of the subpopulations that are numerically small but important in immune responses and homeostasis.

Comparative transcriptome analysis reveals distinct genetic modules associated with Helios expression in intratumoral regulatory T cells

Significance Regulatory T cells (Tregs) play a critical role in inflammatory, autoimmune, and antitumor immune responses. Increased expression of transcription factor Helios by tumor-infiltrating Tregs can enhance immune-suppressive activity, while deletion of Helios promotes an effector T helper (Th) cell phenotype that can contribute to the host antitumor immune response. We report that chronic inflammatory conditions of tumors induce Helios-deficient Tregs to express increased levels of genes associated with T cell activation and Th cell differentiation. Helios-dependent changes in gene expression are restricted to tumor sites and not observed in peripheral lymphoid tissues. We suggest that Helios-deficient Tregs that recognize tumor-associated self-antigens may become unstable in the tumor microenvironment and undergo reprogramming into effector T cells that can inhibit tumor growth. Regulatory T cells (Tregs) are key modulators of immune tolerance, capable of suppressing inflammatory immune responses and promoting nonlymphoid tissue homeostasis. Helios, a transcription factor (TF) that is selectively expressed by Tregs, has been shown to be essential for the maintenance of Treg lineage stability in the face of inflammatory conditions that include autoimmune disease and cancer. Helios-deficient Tregs within tumors acquire effector T cell function and contribute to immune responses against cancer. However, the underlying genetic basis of this Treg reprogramming is not well understood. Here, we report that Helios-deficient Tregs within the chronic inflammatory tumor microenvironment (TME) derepress genetic programs associated with T helper (Th) cell differentiation by up-regulating Th cell-associated TFs and effector cytokines. These genetic changes of Helios-deficient Tregs are most apparent in a Treg subpopulation with high affinity for self-antigens, as detected by both increased GITR/PD-1 expression and increased responsiveness to self-antigens. Their combined effects may promote a phenotype conversion of Tregs into effector T cells within the TME, where TCR engagement and costimulatory receptor expression by Tregs are increased. These data provide a genetic basis for the unstable phenotype of Helios-deficient Tregs within the inflammatory environment of tumors and suggest that immune milieu-dependent alterations in gene expression are a central feature of Treg conversion.