Erika L. Pearce

Enhancing CD8 T-cell memory by modulating fatty acid metabolism

CD8 T cells, which have a crucial role in immunity to infection and cancer, are maintained in constant numbers, but on antigen stimulation undergo a developmental program characterized by distinct phases encompassing the expansion and then contraction of antigen-specific effector (TE) populations, followed by the persistence of long-lived memory (TM) cells. Although this predictable pattern of CD8 T-cell responses is well established, the underlying cellular mechanisms regulating the transition to TM cells remain undefined. Here we show that tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), an adaptor protein in the TNF-receptor and interleukin-1R/Toll-like receptor superfamily, regulates CD8 TM-cell development after infection by modulating fatty acid metabolism. We show that mice with a T-cell-specific deletion of TRAF6 mount robust CD8 TE-cell responses, but have a profound defect in their ability to generate TM cells that is characterized by the disappearance of antigen-specific cells in the weeks after primary immunization. Microarray analyses revealed that TRAF6-deficient CD8 T cells exhibit altered expression of genes that regulate fatty acid metabolism. Consistent with this, activated CD8 T cells lacking TRAF6 display defective AMP-activated kinase activation and mitochondrial fatty acid oxidation (FAO) in response to growth factor withdrawal. Administration of the anti-diabetic drug metformin restored FAO and CD8 TM-cell generation in the absence of TRAF6. This treatment also increased CD8 TM cells in wild-type mice, and consequently was able to considerably improve the efficacy of an experimental anti-cancer vaccine.

Checkpoint Blockade Cancer Immunotherapy Targets Tumour-Specific Mutant Antigens

The immune system influences the fate of developing cancers by not only functioning as a tumour promoter that facilitates cellular transformation, promotes tumour growth and sculpts tumour cell immunogenicity, but also as an extrinsic tumour suppressor that either destroys developing tumours or restrains their expansion. Yet, clinically apparent cancers still arise in immunocompetent individuals in part as a consequence of cancer-induced immunosuppression. In many individuals, immunosuppression is mediated by cytotoxic T-lymphocyte associated antigen-4 (CTLA-4) and programmed death-1 (PD-1), two immunomodulatory receptors expressed on T cells. Monoclonal-antibody-based therapies targeting CTLA-4 and/or PD-1 (checkpoint blockade) have yielded significant clinical benefits—including durable responses—to patients with different malignancies. However, little is known about the identity of the tumour antigens that function as the targets of T cells activated by checkpoint blockade immunotherapy and whether these antigens can be used to generate vaccines that are highly tumour-specific. Here we use genomics and bioinformatics approaches to identify tumour-specific mutant proteins as a major class of T-cell rejection antigens following anti-PD-1 and/or anti-CTLA-4 therapy of mice bearing progressively growing sarcomas, and we show that therapeutic synthetic long-peptide vaccines incorporating these mutant epitopes induce tumour rejection comparably to checkpoint blockade immunotherapy. Although mutant tumour-antigen-specific T cells are present in progressively growing tumours, they are reactivated following treatment with anti-PD-1 and/or anti-CTLA-4 and display some overlapping but mostly treatment-specific transcriptional profiles, rendering them capable of mediating tumour rejection. These results reveal that tumour-specific mutant antigens are not only important targets of checkpoint blockade therapy, but they can also be used to develop personalized cancer-specific vaccines and to probe the mechanistic underpinnings of different checkpoint blockade treatments.

Mitochondrial respiratory capacity is a critical regulator of CD8+ T cell memory development.

CD8(+) T cells undergo major metabolic changes upon activation, but how metabolism influences the establishment of long-lived memory T cells after infection remains a key question. We have shown here that CD8(+) memory T cells, but not CD8(+) T effector (Teff) cells, possessed substantial mitochondrial spare respiratory capacity (SRC). SRC is the extra capacity available in cells to produce energy in response to increased stress or work and as such is associated with cellular survival. We found that interleukin-15 (IL-15), a cytokine critical for CD8(+) memory T cells, regulated SRC and oxidative metabolism by promoting mitochondrial biogenesis and expression of carnitine palmitoyl transferase (CPT1a), a metabolic enzyme that controls the rate-limiting step to mitochondrial fatty acid oxidation (FAO). These results show how cytokines control the bioenergetic stability of memory T cells after infection by regulating mitochondrial metabolism.

Fueling Immunity: Insights into Metabolism and Lymphocyte Function

Background Naïve lymphocytes circulate in the body in a resting state, but upon recognition of foreign antigen and receipt of proper costimulatory signals, these cells become activated, undergo a rapid burst in proliferation, and assume effector functions aimed at controlling or killing the invader. There is a growing appreciation that changes in peripheral T cell function are not only supported by but are dependent on metabolic reprogramming and that specific effector functions cannot proceed without adopting the correct metabolism. However, the reasons underlying why T cells adopt specific metabolic programs and the impact that these programs have on T cell function and, ultimately, immunological outcome remain unclear. T cell function and fate are dependent on metabolic reprogramming. As T cells differentiate during an immune response, they move from what are presumably nutrient-replete lymphoid organs to sites of cancer or infection, where oxygen, nutrients, growth factors, and other signals may become limiting. These metabolically restrictive environments force T cells to metabolically adapt in order to survive and perform their necessary functions. Advances Research into the metabolism of tumor cells has provided valuable insight into the metabolic pathways important for cell proliferation and survival, as well as the influence of metabolites themselves on signal transduction and epigenetic programming. Many of these concepts have shaped how we view metabolism in T cells. However, it is important to note that, unlike tumors, T cells rapidly transition between resting catabolic states (naïve and memory T cells) to one of growth and proliferation (effector T cells) as part of a normal developmental program. In addition, as T cells differentiate during an immune response they also move from what are presumably nutrient-replete lymphoid organs to sites of cancer or infection, where oxygen, nutrients, and growth factors may become limiting. Thus, T cells must metabolically adapt to these changing conditions in order to perform their necessary functions. In this review, we highlight emerging areas in the metabolism of these dynamic cells and discuss the potential impact of metabolic control on T cell fate, plasticity, and effector function. Outlook It is becoming increasingly clear that T cell function is intimately linked to metabolic programs, and as such there is a considerable and growing interest in developing techniques that target metabolism for immunotherapy. Studying metabolism has often been difficult for the nonexpert, because many of the experimental approaches require specialized instrumentation that has not been widely available. Furthermore, acquiring sufficient cellular material for ex vivo analyses, coupled with the inherent difficulty of assessing cellular metabolism in vivo during an immune response, presents substantial challenges to scientists studying the metabolism of immune cells. Nevertheless, understanding how environmental cues and cellular metabolism influence the outcome of T cell–mediated immune responses will be critical for learning how to exploit metabolism to alter disease outcome. Overall, we are just beginning to understand the pathways that regulate metabolism in lymphocytes and how T cells adapt to changes in their microenvironment, particularly in vivo; this area of immunology is poised for substantial advances in the years to come. Lymphocyte Metabolism Lymphocytes are highly dynamic cells, undergoing extensive proliferation upon infection and then reducing in number upon pathogen clearance. Lymphocytes also circulate through many different tissue environments that vary in their nutrient and oxygen availability. Recent studies have revealed changes in metabolic programming that facilitate this dynamic behavior. How these changes occur and the specific effects that they have on lymphocyte function and on the ultimate outcome of an infection are not well understood. Pearce et al. (1242454) review recent progress in this area, suggest how parallels might be found in studying the metabolic changes seen in tumor cells, and propose challenges for the future. Lymphocytes face major metabolic challenges upon activation. They must meet the bioenergetic and biosynthetic demands of increased cell proliferation and also adapt to changing environmental conditions, in which nutrients and oxygen may be limiting. An emerging theme in immunology is that metabolic reprogramming and lymphocyte activation are intricately linked. However, why T cells adopt specific metabolic programs and the impact that these programs have on T cell function and, ultimately, immunological outcome remain unclear. Research on tumor cell metabolism has provided valuable insight into metabolic pathways important for cell proliferation and the influence of metabolites themselves on signal transduction and epigenetic programming. In this Review, we highlight emerging concepts regarding metabolic reprogramming in proliferating cells and discuss their potential impact on T cell fate and function.

Cell-intrinsic lysosomal lipolysis is essential for alternative activation of macrophages

Alternative (M2) activation of macrophages driven via the α-chain of the receptor for interleukin 4 (IL-4Rα) is important for immunity to parasites, wound healing, the prevention of atherosclerosis and metabolic homeostasis. M2 polarization is dependent on fatty acid oxidation (FAO), but the source of the fatty acids that support this metabolic program has not been clear. We found that the uptake of triacylglycerol substrates via the scavenger receptor CD36 and their subsequent lipolysis by lysosomal acid lipase (LAL) was important for the engagement of elevated oxidative phosphorylation, enhanced spare respiratory capacity (SRC), prolonged survival and expression of genes that together define M2 activation. Inhibition of lipolysis suppressed M2 activation during infection with a parasitic helminth and blocked protective responses to this pathogen. Our findings delineate a critical role for cell-intrinsic lysosomal lipolysis in M2 activation.

TLR-driven early glycolytic reprogramming via the kinases TBK1-IKKɛ supports the anabolic demands of dendritic cell activation

The ligation of Toll-like receptors (TLRs) leads to rapid activation of dendritic cells (DCs). However, the metabolic requirements that support this process remain poorly defined. We found that DC glycolytic flux increased within minutes of exposure to TLR agonists and that this served an essential role in supporting the de novo synthesis of fatty acids for the expansion of the endoplasmic reticulum and Golgi required for the production and secretion of proteins that are integral to DC activation. Signaling via the kinases TBK1, IKKɛ and Akt was essential for the TLR-induced increase in glycolysis by promoting the association of the glycolytic enzyme HK-II with mitochondria. In summary, we identified the rapid induction of glycolysis as an integral component of TLR signaling that is essential for the anabolic demands of the activation and function of DCs.

Memory CD8+ T Cells Use Cell-Intrinsic Lipolysis to Support the Metabolic Programming Necessary for Development

Generation of CD8(+) memory T cells requires metabolic reprogramming that is characterized by enhanced mitochondrial fatty-acid oxidation (FAO). However, where the fatty acids (FA) that fuel this process come from remains unclear. While CD8(+) memory T cells engage FAO to a greater extent, we found that they acquired substantially fewer long-chain FA from their external environment than CD8(+) effector T (Teff) cells. Rather than using extracellular FA directly, memory T cells used extracellular glucose to support FAO and oxidative phosphorylation (OXPHOS), suggesting that lipids must be synthesized to generate the substrates needed for FAO. We have demonstrated that memory T cells rely on cell intrinsic expression of the lysosomal hydrolase LAL (lysosomal acid lipase) to mobilize FA for FAO and memory T cell development. Our observations link LAL to metabolic reprogramming in lymphocytes and show that cell intrinsic lipolysis is deterministic for memory T cell fate.

Commitment to glycolysis sustains survival of NO-producing inflammatory dendritic cells

TLR agonists initiate a rapid activation program in dendritic cells (DCs) that requires support from metabolic and bioenergetic resources. We found previously that TLR signaling promotes aerobic glycolysis and a decline in oxidative phosphorylation (OXHPOS) and that glucose restriction prevents activation and leads to premature cell death. However, it remained unclear why the decrease in OXPHOS occurs under these circumstances. Using real-time metabolic flux analysis, in the present study, we show that mitochondrial activity is lost progressively after activation by TLR agonists in inflammatory blood monocyte-derived DCs that express inducible NO synthase. We found that this is because of inhibition of OXPHOS by NO and that the switch to glycolysis is a survival response that serves to maintain ATP levels when OXPHOS is inhibited. Our data identify NO as a profound metabolic regulator in inflammatory monocyte-derived DCs.

CD8 memory T cells have a bioenergetic advantage that underlies their rapid recall ability

A characteristic of memory T (TM) cells is their ability to mount faster and stronger responses to reinfection than naïve T (TN) cells do in response to an initial infection. However, the mechanisms that allow this rapid recall are not completely understood. We found that CD8 TM cells have more mitochondrial mass than CD8 TN cells and, that upon activation, the resulting secondary effector T (TE) cells proliferate more quickly, produce more cytokines, and maintain greater ATP levels than primary effector T cells. We also found that after activation, TM cells increase oxidative phosphorylation and aerobic glycolysis and sustain this increase to a greater extent than TN cells, suggesting that greater mitochondrial mass in TM cells not only promotes oxidative capacity, but also glycolytic capacity. We show that mitochondrial ATP is essential for the rapid induction of glycolysis in response to activation and the initiation of proliferation of both TN and TM cells. We also found that fatty acid oxidation is needed for TM cells to rapidly respond upon restimulation. Finally, we show that dissociation of the glycolysis enzyme hexokinase from mitochondria impairs proliferation and blocks the rapid induction of glycolysis upon T-cell receptor stimulation in TM cells. Our results demonstrate that greater mitochondrial mass endows TM cells with a bioenergetic advantage that underlies their ability to rapidly recall in response to reinfection.

The Energy Sensor AMPK Regulates T Cell Metabolic Adaptation and Effector Responses In Vivo

Naive T cells undergo metabolic reprogramming to support the increased energetic and biosynthetic demands of effector T cell function. However, how nutrient availability influences T cell metabolism and function remains poorly understood. Here we report plasticity in effector T cell metabolism in response to changing nutrient availability. Activated T cells were found to possess a glucose-sensitive metabolic checkpoint controlled by the energy sensor AMP-activated protein kinase (AMPK) that regulated mRNA translation and glutamine-dependent mitochondrial metabolism to maintain T cell bioenergetics and viability. T cells lacking AMPKα1 displayed reduced mitochondrial bioenergetics and cellular ATP in response to glucose limitation in vitro or pathogenic challenge in vivo. Finally, we demonstrated that AMPKα1 is essential for T helper 1 (Th1) and Th17 cell development and primary T cell responses to viral and bacterial infections in vivo. Our data highlight AMPK-dependent regulation of metabolic homeostasis as a key regulator of T cell-mediated adaptive immunity.