Conserved epigenetic hallmarks of T cell aging during immunity and malignancy.

Chronological aging correlates with epigenetic modifications at specific loci, calibrated to species lifespan. Such 'epigenetic clocks' appear conserved among mammals, but whether they are cell autonomous and restricted by maximal organismal lifespan remains unknown. We used a multilifetime murine model of repeat vaccination and memory T cell transplantation to test whether epigenetic aging tracks with cellular replication and if such clocks continue 'counting' beyond species lifespan. Here we found that memory T cell epigenetic clocks tick independently of host age and continue through four lifetimes. Instead of recording chronological time, T cells recorded proliferative experience through modification of cell cycle regulatory genes. Applying this epigenetic profile across a range of human T cell contexts, we found that naive T cells appeared 'young' regardless of organism age, while in pediatric patients, T cell acute lymphoblastic leukemia appeared to have epigenetically aged for up to 200 years. Thus, T cell epigenetic clocks measure replicative history and can continue to accumulate well-beyond organismal lifespan.

Cellular and molecular waypoints along the path of T cell exhaustion.

Thirty years of foundational research investigating molecular and cellular mechanisms promoting T cell exhaustion are now enabling rational design of T cell-based therapies for the treatment of chronic infections and cancer. Once described as a static cell fate, it is now well appreciated that the developmental path toward exhaustion is composed of a heterogeneous pool of cells with varying degrees of effector potential that ultimately converge on a terminally differentiated state. Recent description of the developmental stages along the differentiation trajectory of T cell exhaustion has provided insight into past immunotherapeutic success and future opportunities. Here, we discuss the hallmarks of distinct developmental stages occurring along the path to T cell dysfunction and the impact of these discrete CD8+ T cell fates on cancer immunotherapy.

Tonic-signaling chimeric antigen receptors drive human regulatory T cell exhaustion.

Regulatory T cell (Treg) therapy is a promising approach to improve outcomes in transplantation and autoimmunity. In conventional T cell therapy, chronic stimulation can result in poor in vivo function, a phenomenon termed exhaustion. Whether or not Tregs are also susceptible to exhaustion, and if so, if this would limit their therapeutic effect, was unknown. To "benchmark" exhaustion in human Tregs, we used a method known to induce exhaustion in conventional T cells: expression of a tonic-signaling chimeric antigen receptor (TS-CAR). We found that TS-CAR-expressing Tregs rapidly acquired a phenotype that resembled exhaustion and had major changes in their transcriptome, metabolism, and epigenome. Similar to conventional T cells, TS-CAR Tregs upregulated expression of inhibitory receptors and transcription factors such as PD-1, TIM3, TOX and BLIMP1, and displayed a global increase in chromatin accessibility-enriched AP-1 family transcription factor binding sites. However, they also displayed Treg-specific changes such as high expression of 4-1BB, LAP, and GARP. DNA methylation analysis and comparison to a CD8+ T cell-based multipotency index showed that Tregs naturally exist in a relatively differentiated state, with further TS-CAR-induced changes. Functionally, TS-CAR Tregs remained stable and suppressive in vitro but were nonfunctional in vivo, as tested in a model of xenogeneic graft-versus-host disease. These data are the first comprehensive investigation of exhaustion in Tregs and reveal key similarities and differences with exhausted conventional T cells. The finding that human Tregs are susceptible to chronic stimulation-driven dysfunction has important implications for the design of CAR Treg adoptive immunotherapy strategies.

T cell TET2 disruption cuts the breaks on antitumor CAR T cell therapy.

Functional persistence of chimeric antigen receptor (CAR) T cells is required for sustaining an antitumor response. Recently, Jain et al. revealed that disruption of TET2 in CAR T cells resulted in antigen-independent CAR T cell hyperproliferation that enhanced tumor control in mice, highlighting the potential of epigenetic strategies to improve T cell-based cancer immunotherapy.

Improving antitumor T cells.

Disrupting cell cycle regulators can overcome anticancer T cell dysfunction.

Mechanisms of T cell exhaustion guiding next-generation immunotherapy.

The functional decline in T cells during their chronic stimulation, commonly referred to as T cell exhaustion, is a major limitation for current immunotherapy approaches. As modern medicine embraces therapeutic approaches that exploit the immuno-oncology interface, a primary question is how is T cell function maintained over time in scenarios of prolonged tumor burden. Deciphering the molecular mechanisms of T cell exhaustion is now enabling the field to begin using cardinal features of T cell differentiation to develop biomarkers that can delineate responders from nonresponders prior to treatment with T cell-based therapeutics. Furthermore, applying principles of basic T cell immunity toward the development of cancer treatments is laying a foundation for rational approaches to improve immunotherapy by redirecting T cells away from a dysfunctional developmental trajectory.

Defining the Molecular Hallmarks of T-Cell Memory.

The pool of memory CD8 T cells is comprised of highly specialized subpopulations of cells with both shared and distinct functions. The ongoing study of T-cell memory is focused on how these different subpopulations arise, how the cells are maintained over the life of the host, and how the cells protect a host against reinfection. As a field we have used the convenience of a narrow range of surface markers to define and study these memory T-cell subsets. However, as we learn more about these cells, it is becoming clear that these broad definitions are insufficient to capture the complexity of the CD8 memory T-cell pool, and an updated definition of these cellular states are needed. Here, we discuss data that have recently arisen that highlight the difficulty in using surface markers to functionally characterize CD8 T-cell populations, and the possibility of using the epigenetic state of cells to more clearly define the functional capacity of CD8 memory T-cell subsets.

CD19-CAR T cells undergo exhaustion DNA methylation programming in patients with acute lymphoblastic leukemia.

CD19-CAR T cell therapy has evolved into the standard of care for relapsed/refractory B cell acute lymphoblastic leukemia (ALL); however, limited persistence of the CAR T cells enables tumor relapse for many patients. To gain a deeper understanding of the molecular characteristics associated with CAR T cell differentiation, we performed longitudinal genome-wide DNA methylation profiling of CD8+ CD19-CAR T cells post-infusion in ALL patients. We report that CAR T cells undergo a rapid and broad erasure of repressive DNA methylation reprograms at effector-associated genes. The CAR T cell post-infusion changes are further characterized by repression of genes (e.g., TCF7 and LEF1) associated with memory potential and a DNA methylation signature (e.g., demethylation at CX3CR1, BATF, and TOX) demarcating a transition toward exhaustion-progenitor T cells. Thus, CD19-CAR T cells undergo exhaustion-associated DNA methylation programming, indicating that efforts to prevent this process may be an attractive approach to improve CAR T cell efficacy.

Deleting DNMT3A in CAR T cells prevents exhaustion and enhances antitumor activity.

Chimeric antigen receptor (CAR) T cell therapy is revolutionizing cancer immunotherapy for patients with B cell malignancies and is now being developed for solid tumors and chronic viral infections. Although clinical trials have demonstrated the curative potential of CAR T cell therapy, a substantial and well-established limitation is the heightened contraction and transient persistence of CAR T cells during prolonged antigen exposure. The underlying mechanism(s) for this dysfunctional state, often termed CAR T cell exhaustion, remains poorly defined. Here, we report that exhaustion of human CAR T cells occurs through an epigenetic repression of the T cell's multipotent developmental potential. Deletion of the de novo DNA methyltransferase 3 alpha (DNMT3A) in T cells expressing first- or second-generation CARs universally preserved the cells' ability to proliferate and mount an antitumor response during prolonged tumor exposure. The increased functionality of the exhaustion-resistant DNMT3A knockout CAR T cells was coupled to an up-regulation of interleukin-10, and genome-wide DNA methylation profiling defined an atlas of genes targeted for epigenetic silencing. This atlas provides a molecular definition of CAR T cell exhaustion, which includes many transcriptional regulators that limit the "stemness" of immune cells, including CD28, CCR7, TCF7, and LEF1. Last, we demonstrate that this epigenetically regulated multipotency program is firmly coupled to the clinical outcome of prior CAR T cell therapies. These data document the critical role epigenetic mechanisms play in limiting the fate potential of human T cells and provide a road map for leveraging this information for improving CAR T cell efficacy.

Proinflammatory cytokines promote TET2-mediated DNA demethylation during CD8 T cell effector differentiation.

To gain insight into the signaling determinants of effector-associated DNA methylation programming among CD8 T cells, we explore the role of interleukin (IL)-12 in the imprinting of IFNg expression during CD8 T cell priming. We observe that anti-CD3/CD28-mediated stimulation of human naive CD8 T cells is not sufficient to induce substantial demethylation of the IFNg promoter. However, anti-CD3/CD28 stimulation in the presence of the inflammatory cytokine, IL-12, results in stable demethylation of the IFNg locus that is commensurate with IFNg expression. IL-12-associated demethylation of the IFNg locus is coupled to cell division through TET2-dependent demethylation in an ex vivo human chimeric antigen receptor T cell model system and an in vivo immunologically competent murine system. Collectively, these data illustrate that IL-12 signaling promotes TET2-mediated effector DNA demethylation programming in CD8 T cells and serve as proof of concept that cytokines can guide induction of epigenetically regulated traits for T cell-based immunotherapies.

CAR T cells need a pitstop to win the race.

Prolonged TCR-driven stimulation can induce a dysfunctional T cell state, broadly described as T cell exhaustion, limiting the clinical potential of chimeric antigen receptor (CAR) T cells. Recent findings in Science indicate that early cessation of CAR T cell tonic signaling can prevent stabilization of exhaustion-associated epigenetic programs, enabling a prolonged anti-tumor response.

Regnase-1 suppresses TCF-1+ precursor exhausted T-cell formation to limit CAR-T-cell responses against ALL.

Chimeric antigen receptor (CAR)-T-cell therapeutic efficacy is associated with long-term T-cell persistence and acquisition of memory. Memory-subset formation requires T-cell factor 1 (TCF-1), a master transcription factor for which few regulators have been identified. Here, we demonstrate using an immune-competent mouse model of B-cell acute lymphoblastic leukemia (ALL; B-ALL) that Regnase-1 deficiency promotes TCF-1 expression to enhance CAR-T-cell expansion and memory-like cell formation. This leads to improved CAR-T-mediated tumor clearance, sustained remissions, and protection against secondary tumor challenge. Phenotypic, transcriptional, and epigenetic profiling identified increased tumor-dependent programming of Regnase-1-deficient CAR-T cells into TCF-1+ precursor exhausted T cells (TPEX) characterized by upregulation of both memory and exhaustion markers. Regnase-1 directly targets Tcf7 messenger RNA (mRNA); its deficiency augments TCF-1 expression leading to the formation of TPEX that support long-term CAR-T-cell persistence and function. Regnase-1 deficiency also reduces exhaustion and enhances the activity of TCF-1- CAR-T cells. We further validate these findings in human CAR-T cells, where Regnase-1 deficiency mediates enhanced tumor clearance in a xenograft B-ALL model. This is associated with increased persistence and expansion of a TCF-1+ CAR-T-cell population. Our findings demonstrate the pivotal roles of TPEX, Regnase-1, and TCF-1 in mediating CAR-T-cell persistence and recall responses, and identify Regnase-1 as a modulator of human CAR-T-cell longevity and potency that may be manipulated for improved therapeutic efficacy.

Rewriting History: Epigenetic Reprogramming of CD8+ T Cell Differentiation to Enhance Immunotherapy.

The full potential of T cell-based immunotherapies remains limited by a variety of T cell extrinsic and intrinsic immunosuppressive mechanisms that can become imprinted to stably reduce the antitumor ability of T cells. Here, we discuss recent insights into memory CD8+ T cell differentiation and exhaustion and the association of these differentiation states with clinical outcomes during immune checkpoint blockade and chimeric antigen receptor (CAR) T cell therapeutic modalities. We consider the barriers limiting immunotherapy with a focus on epigenetic regulation impeding efficacy of adoptively transferred T cells and other approaches that augment T cell responses such as immune checkpoint blockade. Furthermore, we outline conceptual and technical breakthroughs that can be applied to existing therapeutic approaches and to the development of novel cutting-edge strategies.

Beta cell-specific CD8+ T cells maintain stem cell memory-associated epigenetic programs during type 1 diabetes.

The pool of beta cell-specific CD8+ T cells in type 1 diabetes (T1D) sustains an autoreactive potential despite having access to a constant source of antigen. To investigate the long-lived nature of these cells, we established a DNA methylation-based T cell 'multipotency index' and found that beta cell-specific CD8+ T cells retained a stem-like epigenetic multipotency score. Single-cell assay for transposase-accessible chromatin using sequencing confirmed the coexistence of naive and effector-associated epigenetic programs in individual beta cell-specific CD8+ T cells. Assessment of beta cell-specific CD8+ T cell anatomical distribution and the establishment of stem-associated epigenetic programs revealed that self-reactive CD8+ T cells isolated from murine lymphoid tissue retained developmentally plastic phenotypic and epigenetic profiles relative to the same cells isolated from the pancreas. Collectively, these data provide new insight into the longevity of beta cell-specific CD8+ T cell responses and document the use of this methylation-based multipotency index for investigating human and mouse CD8+ T cell differentiation.

Developmental plasticity allows outside-in immune responses by resident memory T cells.

Central memory T (TCM) cells patrol lymph nodes and perform conventional memory responses on restimulation: proliferation, migration and differentiation into diverse T cell subsets while also self-renewing. Resident memory T (TRM) cells are parked within single organs, share properties with terminal effectors and contribute to rapid host protection. We observed that reactivated TRM cells rejoined the circulating pool. Epigenetic analyses revealed that TRM cells align closely with conventional memory T cell populations, bearing little resemblance to recently activated effectors. Fully differentiated TRM cells isolated from small intestine epithelium exhibited the potential to differentiate into TCM cells, effector memory T cells and TRM cells on recall. Ex-TRM cells, former intestinal TRM cells that rejoined the circulating pool, heritably maintained a predilection for homing back to their tissue of origin on subsequent reactivation and a heightened capacity to redifferentiate into TRM cells. Thus, TRM cells can rejoin the circulation but are advantaged to re-form local TRM when called on.

Epigenetic Maintenance of Acquired Gene Expression Programs during Memory CD8 T Cell Homeostasis.

Memory CD8 T cells have a unique ability to provide lifelong immunity against pathogens containing their cognate epitope. Because of their ability to provide lifelong protection, the generation of memory T cells is now a major focus for current vaccination or adoptive cell therapy approaches to treat chronic viral infections and cancer. It is now clear that maintenance of memory CD8 T cells occurs through a process of antigen-independent homeostatic proliferation, which is regulated in part by the gamma chain cytokines IL-7 and IL-15. Here, we will describe the role of these cytokines in the survival and self-renewal of memory CD8 T cells. Further, we will describe the role of epigenetics in the maintenance of acquired functions among memory CD8 T cells during homeostatic proliferation.

In vitro Homeostatic Proliferation of Human CD8 T Cells.

Long-lived T-cell-mediated immunity requires persistence of memory T cells in an antigen-free environment while also maintaining a heightened capacity to recall effector functions. Such antigen-independent homeostatic proliferation is mediated in part by the common gamma-chain cytokines IL-7 and IL-15. To further explore the mechanisms governing maintenance of effector functions in long-lived memory T cells during antigen-independent proliferation, human naïve and memory CD8 T cells can be sorted from peripheral blood mononuclear cells (PBMCs), labeled with the proliferation-tracking dye carboxyfluorescein succinimidyl ester (CFSE), and then purified based on their levels of cell division. This allows investigators to assess differences in the desired molecular target in cells that have undergone cytokine-driven proliferation. We provide here a protocol for assessing epigenetic programs in divided and undivided human naïve and memory CD8 T cells following 7 days in culture with IL-7 and IL-15 to illustrate how this approach can shed light on the mechanism(s) that governs the preservation of effector functions during homeostasis of long-lived memory CD8 T cells.

Human memory CD8 T cell effector potential is epigenetically preserved during in vivo homeostasis.

Antigen-independent homeostasis of memory CD8 T cells is vital for sustaining long-lived T cell-mediated immunity. In this study, we report that maintenance of human memory CD8 T cell effector potential during in vitro and in vivo homeostatic proliferation is coupled to preservation of acquired DNA methylation programs. Whole-genome bisulfite sequencing of primary human naive, short-lived effector memory (TEM), and longer-lived central memory (TCM) and stem cell memory (TSCM) CD8 T cells identified effector molecules with demethylated promoters and poised for expression. Effector-loci demethylation was heritably preserved during IL-7- and IL-15-mediated in vitro cell proliferation. Conversely, cytokine-driven proliferation of TCM and TSCM memory cells resulted in phenotypic conversion into TEM cells and was coupled to increased methylation of the CCR7 and Tcf7 loci. Furthermore, haploidentical donor memory CD8 T cells undergoing in vivo proliferation in lymphodepleted recipients also maintained their effector-associated demethylated status but acquired TEM-associated programs. These data demonstrate that effector-associated epigenetic programs are preserved during cytokine-driven subset interconversion of human memory CD8 T cells.