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The protein LEM promotes CD8+ T cell immunity through effects on mitochondrial respiration
Abstract
Protective CD8(+) T cell-mediated immunity requires a massive expansion in cell number and the development of long-lived memory cells. Using forward genetics in mice, we identified an orphan protein named Lymphocyte Expansion Molecule (LEM) that promoted antigen-dependent CD8(+) T cell proliferation, effector function, and memory cell generation in response to infection with lymphocytic choriomeningitis virus. Generation of LEM-deficient mice confirmed these results. Through interaction with CR6 interacting factor (CRIF1), LEM controlled the levels of oxidative phosphorylation (OXPHOS) complexes and respiration resulting in the production of pro-proliferative mitochondrial Reactive Oxygen Species (mROS). LEM provides a link between immune activation and the expansion of protective CD8(+) T cells driven by OXPHOS and represents a pathway for the restoration of long-term protective immunity based on metabolically modified CTL.
Copyright © 2015, American Association for the Advancement of Science.
... Naive and resting T cells make use of fatty acid oxidation and the mitochondrial tricarboxylic acid (TCA) cycle, which provides reducing agents for energy production through oxidative phosphorylation (OXPHOS) (Pearce et al., 2009). Recently, it has been shown in murine models that mitochondrial activity is also needed for activating and maintaining antigen-specific responses (Okoye et al., 2015;Sena et al., 2013). Upon activation, CD8 T cells have been described to switch their metabolism to become heavily dependent on glycolysis, even in the presence of sufficient oxygen. ...
... Our findings indicate that, in some virus-specific effector T cells, mitochondrial respiration is used to supplement glycolysis in order to satisfy the demands of efficient cytokine production, in line with increased proliferation, survival, and anti-viral function in CD8 T cells with genetically enhanced OXPHOS (Okoye et al., 2015). The generation of reactive oxygen species (ROS) is vital in the activation of CD4 T cells (Sena et al., 2013), and an inability of HBV-specific T cells to use OXPHOS could partially be due to changes in ROS production. ...
... *p < 0.05; **p < 0.005. that a more protective T cell response requires mitochondrial respiration is consistent with findings from two recent papers emphasizing the importance of OXPHOS for antigen-specific T cell expansion and antiviral immunity in mice (Okoye et al., 2015;Sena et al., 2013). A better understanding of why this pathway fails in exhausted T cells may reveal mitochondrial targets for therapeutic boosting of antiviral immunity. ...
T cells undergo profound metabolic changes to meet the increased energy demands of maintaining an antiviral response. We postulated that differences in metabolic reprogramming would shape the efficacy of CD8 T cells mounted against persistent viral infections. We found that the poorly functional PD-1hi T cell response against HBV had upregulated the glucose transporter, Glut1, an effect recapitulated by oxygen deprivation to mimic the intrahepatic environment. Glut1hi HBV-specific T cells were dependent on glucose supplies, unlike the more functional CMV-specific T cells, that could utilise oxidative phosphorylation in the absence of glucose. The inability of HBV-specific T cells to switch to oxidative phosphorylation was accompanied by increased mitochondrial size and lower mitochondrial potential, indicative of mitochondrial dysfunction. IL-12, which recovers HBV-specific T cell effector function, increased their mitochondrial potential and reduced their dependence on glycolysis. Our findings suggest that mitochondrial defects limit the metabolic plasticity of exhausted HBV-specific T cells.
... Crif1 plays a key role in mitochondrial homeostasis of host cell and oxidative phosphorylation. Crif1 is involved in regulation of oxidative phosphorylation and respiration by lymphocyte expansion molecule (LEM) to promote antigen-dependent CD8(+) T cell proliferation [26] and by lymphocyte-specific protein tyrosine kinase (Lck) to cause blood malignancies [27]. The fundamental effect of Crif1 on mitochondrial function is not limited to the immune system [28]. ...
... This possibility is supported by the robust number of differentially expressed genes in the QTL region, by the allele-specific expression analysis, and by our discovery of several potential candidate genes. Second, the essential function maintaining mitochondrial homeostasis of host cell and oxidative phosphorylation [26,30,32] provides the possibility that Crif1 is merely a general host responsive factor coping with stress during inflammation. Third, S. aureus colonization can increase a patient's risk for subsequent S. aureus infection [58], and we did not limit control subjects to those who were colonized with S. aureus. ...
We previously showed that chromosome 8 of A/J mice was associated with susceptibility to S. aureus infection. However, the specific genes responsible for this susceptibility are unknown. Chromosome substitution strain 8 (CSS8) mice, which have chromosome 8 from A/J but an otherwise C57BL/6J genome, were used to identify the genetic determinants of susceptibility to S. aureus on chromosome 8. Quantitative trait loci (QTL) mapping of S. aureus-infected N2 backcross mice (F1 [C8A] × C57BL/6J) identified a locus 83180780–88103009 (GRCm38/mm10) on A/J chromosome 8 that was linked to S. aureus susceptibility. All genes on the QTL (n~ 102) were further analyzed by three different strategies: 1) different expression in susceptible (A/J) and resistant (C57BL/6J) mice only in response to S. aureus, 2) consistently different expression in both uninfected and infected states between the two strains, and 3) damaging non-synonymous SNPs in either strain. Eleven candidate genes from the QTL region were significantly differently expressed in patients with S. aureus infection vs healthy human subjects. Four of these 11 genes also exhibited significantly different expression in S. aureus-challenged human neutrophils: Ier2, Crif1, Cd97 and Lyl1. CD97 ligand binding was evaluated within peritoneal neutrophils from A/J and C57BL/6J. CD97 from A/J had stronger CD55 but weaker integrin α5β1 ligand binding as compared with C57BL/6J. Because CD55/CD97 binding regulates immune cell activation and cytokine production, and integrin α5β1 is a membrane receptor for fibronectin, which is also bound by S. aureus, strain-specific differences could contribute to susceptibility to S. aureus. Down-regulation of Crif1 with siRNA was associated with increased host cell apoptosis among both naïve and S. aureus-infected bone marrow-derived macrophages. Specific genes in A/J chromosome 8, including Cd97 and Crif1, may play important roles in host defense against S. aureus.
... Since it is known that the PI3K/AKT/mTOR pathway is required for metabolism, we analyzed the contribution of AKT and mTOR, both involved in signaling processes known to regulate cellular metabolism [6,32,33]. Inhibition of mTOR with rapamycin showed no effect on lactate production, while the inhibition of AKT completely abrogated lactate production. Additionaly, we show for the first time that activation of AKT upregulates the expression of LDH, the enzyme which converts pyruvate to lactate. ...
... The study by MacIntyre and coworkers [8] suggests that PDK1 is the master regulator of T cell metabolism by phosphorylating members of the AGC kinase family, like RSK, PKCs, and SGKs independent of the PI3K/AKT pathway. Recently a new molecule was identified called lymphocyte expansion molecule (LEM, [32]), which was shown to promote antigen specific CD8+ T cell expansion, effector function, and memory cell generation. LEM was observed to regulate the protein complexes of the oxidative phosphorylation pathway in the inner membrane of the mitochondria thereby upregulating the generation of reactive oxygen species which play a crucial role in T cell proliferation. ...
Background:
Antigenic stimulation of the T cell receptor (TCR) initiates a change from a resting state into an activated one, which ultimately results in proliferation and the acquisition of effector functions. To accomplish this task, T cells require dramatic changes in metabolism. Therefore, we investigated changes of metabolic intermediates indicating for crucial metabolic pathways reflecting the status of T cells. Moreover we analyzed possible regulatory molecules required for the initiation of the metabolic changes.
Results:
We found that proliferation inducing conditions result in an increase in key glycolytic metabolites, whereas the citric acid cycle remains unaffected. The upregulation of glycolysis led to a strong lactate production, which depends upon AKT/PKB, but not mTOR. The observed upregulation of lactate dehydrogenase results in increased lactate production, which we found to be dependent on IL-2 and to be required for proliferation. Additionally we observed upregulation of Glucose-transporter 1 (GLUT1) and glucose uptake upon stimulation, which were surprisingly not influenced by AKT inhibition.
Conclusions:
Our findings suggest that AKT plays a central role in upregulating glycolysis via induction of lactate dehydrogenase expression, but has no impact on glucose uptake of T cells. Furthermore, under apoptosis inducing conditions, T cells are not able to upregulate glycolysis and induce lactate production. In addition maintaining high glycolytic rates strongly depends on IL-2 production.
... This study aimed to investigate the role of Nrf2 signaling in influencing melanoma growth and lung metastasis using Nrf2-null mice. Melanoma is one of the most metastatic cancers, and B16-F10 melanoma cell line has been widely used to study melanoma growth and metastasis in animal models [15][16][17][18]. In this regard, intravenous inoculation of B16-F10 cells is a commonly used approach to creating lung metastasis in C57BL/6 mice [15][16][17][18]. ...
... Melanoma is one of the most metastatic cancers, and B16-F10 melanoma cell line has been widely used to study melanoma growth and metastasis in animal models [15][16][17][18]. In this regard, intravenous inoculation of B16-F10 cells is a commonly used approach to creating lung metastasis in C57BL/6 mice [15][16][17][18]. However, this procedure does not mimic the natural metastasis of melanoma from the skin to distant organs, including the lungs. ...
The role of Nrf2, a key regulator of antioxidant and cytoprotective genes, in tumorigenesis remains controversial. Here we showed that Nrf2 deficiency led to increased local tumor growth in mice following subcutaneous injection of B16-F10 melanoma cells, as indicated by increased proportion of animals with locally palpable tumor mass and time-dependent increases in tumor volume at the injection site. In vivo bioluminescence imaging also revealed increased growth of melanoma in Nrf2-null mice as compared with wild-type mice. By using a highly sensitive bioluminometric assay, we further found that Nrf2 deficiency resulted in a remarkable increase in lung metastasis of B16-F10 melanoma cells as compared with wild-type mice. Taken together, the results of this short communication for the first time demonstrated that Nrf2 deficiency promoted melanoma growth and lung metastasis following subcutaneous inoculation of B16-F10 cells in mice.
... In one study, LEM mutations were identified via high-throughput exome sequencing [24] in lymphocytic choriomeningitis virus clone13 (LCMV Cl13)-infected mice ('Retro' strain). Specific mutations enhanced the production of LCMV-specific cytotoxic CD8 + [ 1 0 _ T D $ D I F F ] T cells (CTLs) as well as long-lived memory T cell numbers [25]. Moreover, LEM in CTLs was shown to interact with CR6-interacting factor (CRIF1), a protein needed for the translation and insertion of OXPHOS peptides into the IMM [25,26]. ...
... Specific mutations enhanced the production of LCMV-specific cytotoxic CD8 + [ 1 0 _ T D $ D I F F ] T cells (CTLs) as well as long-lived memory T cell numbers [25]. Moreover, LEM in CTLs was shown to interact with CR6-interacting factor (CRIF1), a protein needed for the translation and insertion of OXPHOS peptides into the IMM [25,26]. Presumably, LEM interacts with the OXPHOS protein CRIF1 to increase the levels of mtROS ( Figure 1D). ...
The integration of the many roles of mitochondria in cellular function and the contribution of mitochondrial dysfunction to disease are major areas of research. Within this realm, the roles of mitochondria in immune defense, epigenetics, and stem cell (SC) development have recently come into the spotlight. With new understanding, mitochondria may bring together these seemingly unrelated fields, a crucial process in treatment and prevention for various diseases. In this review we describe novel findings in these three arenas, discussing the significance of the interplay between mitochondria and the cell nucleus in response to environmental cues. While we optimistically anticipate that further research in these areas can have a profound impact on disease management, we also bring forth some of the key questions and challenges that remain. Mitochondria play a pivotal role in the immune system by detecting foreign invaders through signaling pathways (e.g., inflammasomes) and generating immune responses. Modulation of this role might open up new therapeutic potential.Methylation by DNA methyltransferases contributes to the epigenetic modification of mitochondrial DNA. Dysregulation of the mitochondrial epigenome within cells has been implicated in various diseases.Mitochondria contribute to tissue regeneration and integrity, which are maintained by stem cell renewal and differentiation. Stem cells present exciting medical possibilities in regenerative medicine. Understanding specific mitochondrial biology in stem cells is vital.Novel techniques are allowing the study of mitochondria in much greater detail than before.Possible new therapeutic avenues are emerging with increased scientific knowledge linking mitochondria to immunity, epigenetics, and stem cell biology.
... Animal models are instrumental in the development of effective drugs for both preventive and therapeutic intervention of human cancer, a major cause of death worldwide [1,2]. In this context, inoculation of B16-F10 melanoma cell line in mice is among the most commonly used models for studying the molecular mechanisms of cancer metastasis and developing mechanistically based-chemotherapeutic modalities [3][4][5][6]. B16-F10 melanoma cells readily undergo metastasis to various organs, especially the lungs, following intravenous or subcutaneous inoculation, and as such, this cancer cell line has been widely used to study cancer metastasis to the lungs and its therapeutic intervention [3][4][5][6]. Traditionally, examination of the formation of melanoma cell foci on lung surface is employed to estimate cancer cell load in the lungs. ...
... In this context, inoculation of B16-F10 melanoma cell line in mice is among the most commonly used models for studying the molecular mechanisms of cancer metastasis and developing mechanistically based-chemotherapeutic modalities [3][4][5][6]. B16-F10 melanoma cells readily undergo metastasis to various organs, especially the lungs, following intravenous or subcutaneous inoculation, and as such, this cancer cell line has been widely used to study cancer metastasis to the lungs and its therapeutic intervention [3][4][5][6]. Traditionally, examination of the formation of melanoma cell foci on lung surface is employed to estimate cancer cell load in the lungs. However, different sizes of the surface foci make quantification of the melanoma cell load less accurate. ...
Animal models are essential for developing effective drugs for treating human cancer. Examination of the formation of lung surface foci of B16-F10 melanoma cells is a widely used animal model for studying cancer metastasis and drug intervention. This model, however, suffers from several drawbacks, including its non-quantitative nature and inability to yield information on cancer cell load inside the target organ. Here we report the development of a highly sensitive, bioluminescence-based method for quantifying melanoma cell load in mouse lungs following intravenous injection of luciferase-expressing B16-F10 melanoma cells. This method could readily detect as few as 1–10 cells in the samples and enable quantification of cancer cell load before the formation of surface foci in mouse lungs following metastasis of intravenously inoculated B16-F10 melanoma cells. This innovative bioluminometry-based method has important implications for studying anticancer drugs, including naturally occurring redox-active quinones that generate reactive oxygen species to kill cancer cells.
... group identified mice with enhanced CD8 T cell responses to viral and tumor challenge (Okoye et al., 2015). The source of the heightened immunity gained after germline mutagenesis was the increased expression of an orphan protein, identified as lymphocyte expansion molecule (LEM). ...
... Interestingly, augmented OXPHOS and mitochondrial ROS levels were detected in CD8 T cells isolated from LEM-deficient mice after infection, whereas heterozygous LEM-deficient CD8 T cells had reduced OXPHOS and mitochondrial ROS levels. LEM helps stabilize a protein involved in inserting ETC complex proteins in the mitochondrial membrane, which may account for the increased ROS and enhanced proliferation evident in CD8 T cells from these mice (Okoye et al., 2015). ...
Lymphocytes must adapt to a wide array of environmental stressors as part of their normal development, during which they undergo a dramatic metabolic remodeling process. Research in this area has yielded surprising findings on the roles of diverse metabolic pathways and metabolites, which have been found to regulate lymphocyte signaling and influence differentiation, function and fate. In this review, we integrate the latest findings in the field to provide an up-to-date resource on lymphocyte metabolism.
© 2015 Buck et al.
... However, in chronic infections signaling of inhibitory receptors like PD-1 and CTLA4, inhibit glycolysis which results in a metabolic switch to fatty acid dependent oxidative phosphorylation, which will not su ce in energy demands resulting in poor CD8 T cell functionality 32,33 . In addition, mitochondrial dysfunction has been observed in exhausted T cells which prevents e cient oxidative phosphorylation 34,35 . ...
Antiretroviral treatment of HIV infected individuals cannot eliminate the HIV reservoir and immune control of HIV is rarely seen upon treatment interruption. In long-term non-progressors (LTNP), an effective CD8 T cell response is thought to contribute to be immune control of HIV. Here we studied the transcriptional profile of virus specific CD8 T cells to gain molecular insights in CD8 T cell functionality in HIV progressors and LTNP. Principal component analysis revealed distinct overall transcriptional profiles between the groups. The transcription profile of HIV-specific CD8 T cells of LTNP groups was associated with increased protein/RNA metabolism pathways, indicating an increased CD8 T cell functionality. In contrast, CMV-specific CD8 T cells from progressors showed increased expression of genes related to effector functions and suggests recent antigen exposure. Our data shows that changes in the energy demanding RNA and protein metabolism may drive dysfunctionality of HIV-specific T cells changes during chronic HIV infection.
... Current findings also indicate that OXPHOS is required to complement glycolysis because it is capable of fulfilling cytokine production demand, sustaining proliferation and antiviral function in CD8 + T cells [86,87]. These findings collectively denote that mitochondrial OXPHOS is still engaged in effector activity, although glycolysis is regarded as the predominant pathway. ...
The progressive decline of CD8+ cytotoxic T cells in human immunodeficiency virus (HIV)-infected patients due to infection-triggered cell exhaustion and cell death is significantly correlated with disease severity and progression into the life-threatening acquired immunodeficiency syndrome (AIDS) stage. T cell exhaustion is a condition of cell dysfunction despite antigen engagement, characterized by augmented surface expression of immune checkpoint molecules such as programmed cell death protein 1 (PD-1), which suppress T cell receptor (TCR) signaling and negatively impact the proliferative and effector activities of T cells. T cell function is tightly modulated by cellular glucose metabolism, which produces adequate energy to support a robust reaction when battling pathogen infection. The transition of the T cells from an active to an exhausted state following pathogen persistence involves a drastic change in metabolic activity. This review highlights the interplay between immune checkpoint molecules and glucose metabolism that contributes to T cell exhaustion in the context of chronic HIV infection, which could deliver an insight into the rational design of a novel therapeutic strategy.
... Митохондрии регулируют процессинг и презентацию антигена и локализуются в иммунных синапсах во время активации Т-клеток [17]. Митохондриальный метаболизм также поддерживает фенотип Т-клеток [18,19]. Незаменимыми факторами для активации Т-клеток, их эффекторных функций и дифференциации могут являться мтАФК [13,20,21]. ...
Introduction. Chronic respiratory diseases are one of the most common types of non-communicable diseases and are an important problem of our time. The induction of oxidative stress, chronic inflammation and hypoxia, which underlie the pathogenesis of chronic diseases of the bronchopulmonary system, can be determined at the cellular and molecular level by impaired mitochondrial functioning.
Aim. This review is devoted to the prospects for assessing the functional state of mitochondria as a fine indicator of the course of chronic respiratory diseases.
Results. The data of domestic and foreign sources on the most important parameters of mitochondrial functioning in normal and chronic bronchopulmonary pathology were analyzed. It has been shown that mitochondria are highly sensitive to changes in both exogenous and endogenous homeostasis. Functional parameters of mitochondria, the level of mitochondrial reactive oxygen species, mitochondrial membrane potential, and fatty acid composition of mitochondrial membranes can be used as diagnostic and prognostic criteria for chronic respiratory diseases. The data presented in the review indicate the need for further studies of the functional state of mitochondria in chronic bronchopulmonary pathology.
... Once the target cells are eliminated, antigen (Ag)-specific CD8 + T cells undergo contraction and a smaller pool of these Ag-specific CD8 + T cells live on as memory cells primed for immediate immune response to re-challenge (Hosking et al, 2014). Recent work has highlighted that metabolic programming itself influences CD8 + T-cell proliferation, differentiation, effector function, persistence, and survival into memory Chang et al, 2013;Okoye et al, 2015;Bengsch et al, 2016;Schurich et al, 2016). Yet, the widespread presence of chronic viral infection and cancer indicate that CD8 + T-cell-mediated immune response fails quite often. ...
The mechanisms inducing exhaustion of HIV-specific CD8 ⁺ T cells are not fully understood. Metabolic programming directly influences T-cell differentiation, effector function, and memory. We evaluated metabolic profiles of ex vivo CD8 ⁺ T cells in HIV-infected individuals. The baseline oxygen consumption rate of CD8 ⁺ T cells was elevated in all infected individuals and CD8 ⁺ T cells were working at maximal respiratory capacity. The baseline glycolysis rate was enhanced only during early untreated HIV and in viral controllers, but glycolytic capacity was conserved at all stages of infection. CD8 ⁺ T-cell mTOR activity was found to be reduced. Enhanced glycolysis was crucial for HIV-specific killing of CD8 ⁺ T cells. CD8 ⁺ T-cell cytoplasmic GAPDH content was reduced in HIV, but less in early infection and viral controllers. Thus, CD8 ⁺ T-cell exhaustion in HIV is characterized by reduced glycolytic activity, enhanced OXPHOS demands, dysregulated mTOR, and reduced cytoplasmic GAPDH. These data provide potential metabolic strategies to reverse CD8 ⁺ T-cell dysfunction in HIV.
... The more I tried to cover new perspectives that are rooted in the history of mitochondrial research, the more I realized that my selective account was inevitably leaving out a number exciting developments. I will mention the relationships of autophagy with mitochondrial fission-fusion events (Twig et al., 2008;Lazarou et al., 2015;Dorn, 2016); the role of mitochondria in the antiviral response (Kozaki et al., 2017), in the growth of intracellular parasites (Pernas et al., 2018), in antigen presentation (Matheoud et al., 2016), in T cell function (Okoye et al., 2015;Weinberg et al., 2019) and dysfunction (Desdin-Mico et al., 2020), in metabolic reprogramming of macrophages (Mills et al., 2016;Acín-Pérez et al., 2020), in angiogenesis (Herkenne et al., 2020), in systemic stress response mediated by FGF21 (Forsstrom et al., 2019), in non-alcoholic steatohepatitis, where downregulation of mitochondrial circular RNA prevents inhibition of the permeability transition pore by the SCAR protein (Zhao et al., 2020); recent advances on the mechanism of germline selection of human mtDNA (Wei et al., 2019); and the most unexpected finding that the protein product of the ARHGAP11B gene, which plays an essential role in development of the human neocortex (Heide et al., 2020), localizes to mitochondria to inhibit the permeability transition pore (Namba et al., 2020). It is reassuring, indeed, that not all could be predicted. ...
... Consistently, as well as increasing expression of glutamine transporters, activated T cells also upregulate glutaminolytic enzymes that metabolize glutamine to α-ketoglutarate ( Wang et al., 2011; Figure 1). Mechanistically, increased mitochondrial OXPHOS of these substrates is linked to T cell proliferation and function, not only through generation of required ATP, but also of ROS that stabilize transcription factors, promoting effector molecule expression ( Sena et al., 2013;Okoye et al., 2015) and supporting the activity of metabolic pathways that maintain redox homeostasis, such as the malate-aspartate shuttle ( Bailis et al., 2019). Furthermore, TCA cycle intermediates are used for anabolic processes, as well as post-translational modifications of proteins-notably the acetylation of histones, permitting gene transcription ( Peng et al., 2016;Bailis et al., 2019;Qiu et al., 2019) and enzymes such as GAPDH, thereby augmenting their function ( Balmer et al., 2016). ...
T lymphocytes are a critical component of the adaptive immune system, with key roles in the immune response to infection and cancer. Their activity is fundamentally underpinned by dynamic, regulated changes in their metabolism. This ensures adequate availability of energy and biosynthetic precursors for clonal expansion and effector function, and also directly regulates cell signaling, gene transcription, and protein translation. In health, distinct T cells subtypes demonstrate differences in intrinsic metabolic capacity which correlate with their specialized immune functions. In disease, T cells with impaired immune function appear to be likewise metabolically impaired. Furthermore, diseased tissue environments—through inadequate provision of nutrients and oxygen, or accumulation of metabolic intermediates, end-products, and cytokines- can impose metabolic insufficiency upon these cells, and further compound intrinsic impairments. These intrinsic and extrinsic determinants of T cell metabolism and their potential compound effects, together with the mechanisms involved form the subject of this review. We will also discuss how dysfunctional metabolic pathways may be therapeutically targeted to restore normal T cell function in disease.
... In line with this, T cells from Uqcrfs-deficient mice (complex III subunit 5) show decreased activation and diminished clonal expansion upon in vivo antigen encounter (28). Furthermore, increased expression of lymphocyte expansion molecule (LEM), which positively regulates the expression of the mitochondrial electron transport chain complex, controlling the activity of OXPHOS proteins and mROS production, resulted in increased CD8 T cell proliferation and function (29). The removal of LEM reduced CD8 T cell proliferation and reduced levels of mROS. ...
In the past years, there have been significant advances in the understanding of how environmental conditions alone or in conjunction with pathogen invasion affect the metabolism of T cells, thereby influencing their activation, differentiation, and longevity. Detailed insights of the interlinked processes of activation and metabolism can contribute to major advances in immunotherapies. Naive and memory T cells circulate the body. In a quiescent state with low metabolic demands, they predominantly use oxidative phosphorylation for their energy needs. Recognition of cognate antigen combined with costimulatory signals results in a proliferative burst and effector molecule production, requiring rapid release of energy, achieved via dynamically reprogramming metabolic pathways. After activation, most T cells succumb to activation induced cell death, but few differentiate into memory T cells. Of note, some memory T cells permanently occupy tissues without circulating. These, tissue resident T cells are predominantly CD8 T cells, maintained in a metabolic state distinct from naïve and circulating memory CD8 T cells with elements similar to effector CD8 T cells but without undergoing proliferative burst or secreting immune mediators. They continually interact with tissue cells as part of an immune surveillance network, are well-adapted to the tissues they have made their home and where they may encounter different metabolic environments. In this review, we will discuss recent insights in metabolic characteristics of CD8 T cell biology, with emphasis on tissue resident CD8 T cells at the epithelial barriers.
... I mmunological memory is a hallmark of adaptive immunity [1][2][3][4][5][6] , and the quality of adaptive immune responses depends highly on the size of the antigen-specific memory T cell pool [7][8][9][10] . In recent years, mounting evidence has shown the importance of cellular metabolism in many aspects of T cell biology [11][12][13][14][15][16][17][18] . Inhibition of mTOR, a critical kinase that regulates cellular metabolism via glycolysis, glutaminolysis and fatty acid biosynthesis, enhances the generation of memory CD8 + T cells 19 . ...
Immunological memory is central to adaptive immunity and protection from disease. Changing metabolic demands as antigen-specific T cells transition from effector to memory cells have been well documented, but the cell-specific pathways and molecules that govern this transition are poorly defined. Here we show that genetic deletion of ACC1, a rate-limiting enzyme in fatty acid biosynthesis, enhances the formation of CD4+ T memory cells. ACC1-deficient effector helper T (Th) cells have similar metabolic signatures to wild-type memory Th cells, and expression of the gene encoding ACC1, Acaca, was inversely correlated with a memory gene signature in individual cells. Inhibition of ACC1 function enhances memory T cell formation during parasite infection in mice. Using single-cell analyses we identify a memory precursor-enriched population (CCR7hiCD137lo) present during early differentiation of effector CD4+ T cells. Our data indicate that fatty acid metabolism directs cell fate determination during the generation of memory CD4+ T cells.
... Naive and resting T cells make use of fatty acid oxidation (FAO) and the mitochondrial tricarboxylic acid (TCA) cycle to generate large amounts of ATP through oxidative phosphorylation (OXPHOS) (121,122). Recent studies in a murine model revealed that mitochondrial activity was one of the requirements for the activation and sustenance of antigen- specific responses (123,124). Upon activation, T cells switch their metabolism to high rates of glycolysis even in the presence of sufficient oxygen and this support proliferation and effector function via providing fast energy and metabolites (125). ...
T-cell exhaustion is a phenomenon of dysfunction or physical elimination of antigen-specific T cells reported in human immunodeficiency virus (HIV), hepatitis B virus (HBV), and hepatitis C virus (HCV) infections as well as cancer. Exhaustion appears to be often restricted to CD8+ T cells responses in the literature, although CD4+ T cells have also been reported to be functionally exhausted in certain chronic infections. Although our understanding of the molecular mechanisms associated with the transcriptional regulation of T-cell exhaustion is advancing, it is imperative to also explore the central mechanisms that control the altered expression patterns. Targeting metabolic dysfunctions with mitochondrion-targeted antioxidants are also expected to improve the antiviral functions of exhausted virus-specific CD8+ T cells. In addition, it is crucial to consider the contributions of mitochondrial biogenesis on T-cell exhaustion and how mitochondrial metabolism of T cells could be targeted whilst treating chronic viral infections. Here, we review the current understanding of cardinal features of T-cell exhaustion in chronic infections, and have attempted to focus on recent discoveries, potential strategies to reverse exhaustion and reinvigorate optimal protective immune responses in the host.
... The effects of ROS on CD8+ T-cell function are mediated by lymphocyte expansion molecule (LEM), which regulates the expression of OXPHOS proteins (such as NADH ubiquinone oxidoreductase chain 1) and, accordingly, ROS production. LEM is necessary for cytotoxic T-cell expansion and memory T-cell development (Okoye et al., 2015). Therefore, basal ROS levels are required for proper T-cell signaling. ...
The scientific knowledge about tumor metabolism has grown at a fascinating rate in recent decades. We now know that tumors are highly active both in their metabolism of available nutrients and in the secretion of metabolic by-products. However, cancer cells can modulate metabolic pathways and thus adapt to specific nutrients. Unlike tumor cells, immune cells are not subject to a 'micro-evolution' that would allow them to adapt to progressing tumors that continuously develop new mechanisms of immune escape. Consequently, immune cells are often irreversibly affected and may allow or even support cancer progression. The mechanisms of how tumors change immune cell function are not sufficiently explored. It is, however, clear that commonly shared features of tumor metabolism, such as local nutrient depletion or production of metabolic 'waste' can broadly affect immune cells and contribute to immune evasion. Moreover, immune cells utilize different metabolic programs based on their subtype and function, and these immunometabolic pathways can be modified in the tumor microenvironment. In this review and accompanying poster, we identify and describe the common mechanisms by which tumors metabolically affect the tumor-infiltrating cells of native and adaptive immunity, and discuss how these mechanisms may lead to novel therapeutic opportunities.
... The modulation of TCR signalling has been exploited in adoptive cell therapy [11]. While much focus has been on how to increase TCR stimulation and hence improve the cytotoxic effect [12][13][14][15][16][17], the possibility of non-specific binding or cross reactivity still represents a severe complication [18]. Various safeguard methods have been proposed to control and block the unexpected side effects [19,20], but novel methods for pre-clinical or early clinical validation of TCR selectivity and specificity still represent an important innovation, since safeguard kinetics might be too slow to rescue a patient if T-cell activation occurs against an unpredicted target or at an unpredicted site. ...
Adoptive cell therapy with T-cell receptor (TCR)-engineered T cells represents a powerful method to redirect the immune system against tumours. However, although TCR recognition is restricted to a specific peptide–MHC (pMHC) complex, increasing numbers of reports have shown cross-reactivity and off-target effects with severe consequences for the patients. This demands further development of strategies to validate TCR safety prior to clinical use. We reasoned that the desired TCR signalling depends on correct pMHC recognition on the outside and a restricted clustering on the inside of the cell. Since the majority of the adverse events are due to TCR recognition of the wrong target, we tested if blocking the signalling would affect the binding. By over-expressing the c-SRC kinase (CSK), a negative regulator of LCK, in redirected T cells, we showed that peripheral blood T cells inhibited anti-CD3/anti-CD28-induced phosphorylation of ERK, whereas TCR proximal signalling was not affected. Similarly, overexpression of CSK together with a therapeutic TCR prevented pMHC-induced ERK phosphorylation. Downstream effector functions were also almost completely blocked, including pMHC-induced IL-2 release, degranulation and, most importantly, target cell killing. The lack of effector functions contrasted with the unaffected TCR expression, pMHC recognition, and membrane exchange activity (trogocytosis). Therefore, co-expression of CSK with a therapeutic TCR did not compromise target recognition and binding, but rendered T cells incapable of executing their effector functions. Consequently, we named these redirected T cells “dummy T cells” and propose to use them for safety validation of new TCRs prior to therapy.
... LEM regulates OXPHOS through interaction with CR6interacting factor (CRIF1) to promote the production of mitochondrial reactive oxygen species (mROS). Thus, it appears that LEM controls CTL expansion and memory cell generation by increasing mROS production after activation [ (45).] ...
T cells play a critical role to defend against tumor and maintain immune homeostasis. The diverse functions of T cells require precise regulation of metabolic pathways. Recent studies reveal that metabolic changes are tightly linked to the activation and function of T cells. Given the importance of these cells in tumor progression, it is important to understand how the tumor microenvironment regulates metabolism of T cells and how the metabolic reprogramming of T cells affects tumor growth. Here, we review new findings and discuss how metabolic reprogramming of different types of T cells affects the immune response in tumors.
... The important involvement of ROS in T cell metabolic fate and function was recently shown by the identification of lymphocyte expansion molecule (LEM). LEM controls the levels of OXPHOS complexes and respiration, resulting in the production of pro-proliferative mitochondrial ROS, which is critical for promoting antigendependent CD8 + T cell proliferation, effector function, and long-term protective memory cells in response to infection with lymphocytic choriomeningitis virus [79]. ...
T lymphocytes undergo extensive changes in their metabolic properties during their transition through various differentiation states, from naïve to effector to memory or regulatory roles. The cause and effect relationship between metabolism and differentiation is a field of intense investigation. Many recent studies demonstrate the dependency of T cell functional outcomes on metabolic pathways and the possibility of metabolic intervention to modify these functions. In this review, we describe the basic metabolic features of T cells and new findings on how these correlate with various differentiation fates and functions. We also highlight the latest information regarding the main factors that affect T cell metabolic reprogramming.
... Intriguingly, a novel link between OXPHOS and immune response through development of long-lived T-memory cells has been demonstrated, due to a mechanism of immune activation guided by the newly discovered protein LEM (Okoye et al., 2015). Through interaction with CR6 interacting factor, LEM controlled the levels of OXPHOS complexes and respiration, resulting in the production of proproliferative mitochondrial ROS. ...
Metabolic reprogramming is one of the most frequent stress-adaptive response of cancer cells to survive environmental changes and meet increasing nutrient requirements during their growth. These modifications involve cellular bioenergetics and cross talk with surrounding microenvironment, in a dynamic network that connect different molecular processes, such as energy production, inflammatory response, and drug resistance. Even though the Warburg effect has long been considered the main metabolic feature of cancer cells, recent reports identify mitochondrial oxidative metabolism as a driving force for tumor growth in an increasing number of cellular contexts. In recent years, oxidative phosphorylation has been linked to a remodeling of inflammatory response due to autocrine or paracrine secretion of interleukines that, in turn, induces a regulation of gene expression involving, among others, molecules responsible for the onset of drug resistance. This process is especially relevant in ovarian cancer, characterized by low survival, high frequency of disease relapse and chemoresistance. Recently, the molecular chaperone TRAP1 (tumor necrosis factor-associated protein 1) has been identified as a key junction molecule in these processes in ovarian cancer: in fact, TRAP1 mediates a metabolic switch toward oxidative phosphorylation that, in turn, triggers cytokines secretion, with consequent gene expression remodeling, finally leading to cisplatin resistance and epithelial-to-mesenchymal transition in ovarian cancer models. This review summarizes how metabolism, chemoresistance, inflammation, and epithelial-to-mesenchymal transition are strictly interconnected, and how TRAP1 stays at the crossroads of these processes, thus shedding new lights on molecular networks at the basis of ovarian cancer.
... Mitochondria-derived ROS have recently been demonstrated to play important roles in adaptive immunity, including regulation of T cell activation and CD8 + memory T cell formation, as well as B cell fate determination upon activation [42][43][44]. Although the exact ROS involved in the above processes remain unclear, H 2 O 2 appears to be the most likely ROS that acts as a signaling molecule to regulate adaptive immunity [45]. ...
Hydrogen peroxide (H 2 O 2 ) is one of the most extensively studied reactive oxygen species (ROS) in biology and medicine. It is generated constitutively from various cellular processes either directly via two-electron reduction of molecular oxygen indirectly via dismutation of superoxide. The notable direct cellular sources for H 2 O 2 include xanthine oxidoreductase, monoamine oxidase, endoplasmic reticulum oxicireductin 1, oxidases in peroxisomes, and possibly certain members of the NOX/DUOX family. Because of the high activation energy, H 2 O 2 reacts poorly with most cellular constituents. However, it may oxidize the thiol groups in certain proteins and enzymes, including these involved in cell signaling transduction. The potential of H 2 O 2 to cause oxidative stress and tissue injury primarily results from its reactions with other molecules to form secondary reactive species, including hydroxyl radical and hypochlorous acid. While the tightly controlled production of H 2 O 2 plays important roles in various physiological responses, overproduction of this ROS contributes to the pathophysiology of a variety of disease processes and related conditions, including cardiovascular diseases, diabetes, neurodegeneration, cancer, and aging, among many others.
... 66 In murine CD8 + T cells, the lymphocyte expansion molecule assists in the formation of the mitochondrial electron transport chain, increases ROS, improves memory formation, and enhances effector functions including IFN-γ production. 86 However, direct regulation of IFN-γ production by ROS has not been shown. ...
Metabolism is critical for a host of cellular functions and provides a source of intracellular energy. It has been recognized recently that metabolism also regulates differentiation and effector functions of immune cells. Although initial work in this field has focused largely on T lymphocytes, recent studies have demonstrated metabolic control of innate immune cells, including natural killer (NK) cells. Here, we review what is known regarding the metabolic requirements for NK cell activation, focusing on NK cell production of interferon-gamma (IFN-γ). NK cells are innate immune lymphocytes that are poised for rapid activation during the early immune response. Although their basal metabolic rates do not change with short-term activation, they exhibit specific metabolic requirements for activation depending upon the stimulus received. These metabolic requirements for NK cell activation are altered by culturing NK cells with interleukin-15, which increases NK cell metabolic rates at baseline and shifts them toward aerobic glycolysis. We discuss the metabolic pathways important for NK cell production of IFN-γ protein and potential mechanisms whereby metabolism regulates NK cell function.
... Recent studies have demonstrated that skewing of metabolic pathways in CD8 + T cells correlates with formation of memory CD8 + T cells and pharmacological inhibition of critical metabolic sensors can perturb CD8 + T cell differentiation (Araki et al., 2009;Pearce et al., 2009;van der Windt et al., 2012). Furthermore, deletion of molecules critical for various metabolic processes can have both deleterious or beneficial effects on effector and memory cell generation and survival (Chaoul et al., 2015;Cui et al., 2015;O'Sullivan et al., 2014;Okoye et al., 2015;Rao et al., 2010;Rolf et al., 2013;Sena et al., 2013;Shrestha et al., 2014). These data support a role for cellular metabolism in CD8 + T cell differentiation and function; however, the critical question of whether metabolic pathway choice is the driving force behind memory cell differentiation is not yet resolved. ...
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.
... To see this illustration in color, the reader is referred to the web version of this article at www.liebertpub.com/ars ‰ OXPHOS IN CANCER AND TUMOR MICROENVIRONMENT proliferation, another study confirmed that mitochondrial ROS affect respiratory chain assembly into the inner mitochondrial membrane and CD8 + T-cell expansion, thus demonstrating that long-term protective immunity can be OXPHOS driven (151). Antineoplastic chemotherapies stimulate autophagy, which appears as an adaptive metabolic mechanism of resistance to treatment (113). ...
The worldwide epidemic of obesity is a major public health concern. Obesity is a major risk factor for non-communicable diseases such as Type 2 diabetes and cardiovascular diseases, clustered in the so-called metabolic syndrome (MS). Other main chronic illnesses are promoted by excessive body weight, including cancer and neurodegenerative pathologies, both affecting a great number of people worldwide. In the recent years, the primary role of an excess of reactive oxygen species (oxidative stress) resulting from altered redox control in the etiology of all of these pathologies has been unveiled. Interestingly, it appears that oxidative stress is both the cause and the consequence of obesity and associated disorders. This Forum features reviews which recapitulate the current knowledge on the link between oxidative stress and MS in the physio-pathology of different biological systems.
... The important involvement of ROS to T cell metabolic fate and function was highlighted by the recent identification of lymphocyte expansion molecule (LEM). LEM has no effects on glycolysis but controls the levels of OXPHOS complexes and respiration, resulting in the production of pro-proliferative mitochondrial ROS, which is critical for promoting antigen-dependent CD8 + T cell proliferation, effector function, and longterm protective memory cells in response to infection with lymphocytic choriomeningitis virus [227]. ...
Conversion of normal cells to cancer is accompanied with changes in their metabolism. During this conversion, cell metabolism undergoes a shift from oxidative phosphorylation to aerobic glycolysis, also known as Warburg effect, which is a hallmark for cancer cell metabolism. In cancer cells, glycolysis functions in parallel with the TCA cycle and other metabolic pathways to enhance biosynthetic processes and thus support proliferation and growth. Similar metabolic features are observed in T cells during activation but, in contrast to cancer, metabolic transitions in T cells are part of a physiological process. Currently, there is intense interest in understanding the cause and effect relationship between metabolic reprogramming and T cell differentiation. After the recent success of cancer immunotherapy, the crosstalk between immune system and cancer has come to the forefront of clinical and basic research. One of the key goals is to delineate how metabolic alterations of cancer influence metabolism-regulated function and differentiation of tumor resident T cells and how such effects might be altered by immunotherapy. Here, we review the unique metabolic features of cancer, the implications of cancer metabolism on T cell metabolic reprogramming during antigen encounters, and the translational prospective of harnessing metabolism in cancer and T cells for cancer therapy.
... In this model, restriction of mTOR signaling in early Tex cells by rapamycin might help adapt the nutrient demand to availability, preventing proton gradient breakdown and ROS production. In support of this notion, improving mitochondrial OXPHOS by overexpression or gain-of-function of lymphocyte expansion molecule (lem) increased virus-specific T cell function and viral control early during clone 13 infection (Okoye et al., 2015). Despite the advantages of rapamycin treatment for the metabolic phenotype of early Tex cells, its use is unlikely to be of overall benefit in a therapeutic setting, given its immunosuppressive effects (Powell et al., 2012). ...
Dynamic reprogramming of metabolism is essential for T cell effector function and memory formation. However, the regulation of metabolism in exhausted CD8+ T (Tex) cells is poorly understood. We found that during the first week of chronic lymphocytic choriomeningitis virus (LCMV) infection, before severe dysfunction develops, virus-specific CD8+ T cells were already unable to match the bioenergetics of effector T cells generated during acute infection. Suppression of T cell bioenergetics involved restricted glucose uptake and use, despite persisting mechanistic target of rapamycin (mTOR) signaling and upregulation of many anabolic pathways. PD-1 regulated early glycolytic and mitochondrial alterations and repressed transcriptional coactivator PGC-1α. Improving bioenergetics by overexpression of PGC-1α enhanced function in developing Tex cells. Therapeutic reinvigoration by anti-PD-L1 reprogrammed metabolism in a subset of Tex cells. These data highlight a key metabolic control event early in exhaustion and suggest that manipulating glycolytic and mitochondrial metabolism might enhance checkpoint blockade outcomes.
... 30 The importance of mROS for CD8 + T-cell function was further confirmed by identification of the protein lymphocyte expansion molecule (LEM), which regulates expression of the mitochondrial electron transport chain complex, and consequent mROS production, and is crucial for CD8 + T-cell proliferation, cytotoxicity, memory generation and control of lymphocytic choriomeningitis virus infection in vivo. 40 In addition to increased function, there is also evidence for mitochondrial biogenesis following T-cell activation, reflected by increases in mitochondrial mass and mitochondrial DNA abundance. 41 How this process is regulated in T cells is largely unknown and warrants further investigation. ...
T lymphocytes are a critical component of the adaptive immune system mediating protection against infection and malignancy, but also implicated in many immune pathologies. Upon recognition of specific antigens T cells clonally expand, traffic to inflamed sites and acquire effector functions, such as the capacity to kill infected and malignantly transformed cells and secrete cytokines to coordinate the immune response. These processes have significant bioenergetic and biosynthetic demands, which are met by dynamic changes in T cell metabolism, specifically increases in glucose uptake and metabolism; mitochondrial function; amino acid uptake, and cholesterol and lipid synthesis. These metabolic changes are co-ordinated by key cellular kinases and transcription factors. Dysregulated T cell metabolism is associated with impaired immunity in chronic infection and cancer and conversely with excessive T cell activity in autoimmune and inflammatory pathologies. Here we review the key aspects of T cell metabolism relevant to their immune function, and discuss evidence for the potential to therapeutically modulate T cell metabolism in disease. This article is protected by copyright. All rights reserved.
... However, whether this is indeed the mechanistic explanation for the decreased fitness of autophagy-deficient CD4 + T cells in vivo remains unclear, as other studies did not observe any increase in mitochondrial mass or ROS production (310,319), and mitochondria were shown to be excluded from autophagosome degradation in activated wild-type CD4 + T cells (317). Further complication arises from the findings that although increased ROS can be detrimental for T cells (320), ROS production is increased after TCR triggering and is, in fact, required for T cell proliferation, particularly in CD8 + T cells (321)(322)(323)(324). It was also proposed that imbalanced expression or accumulation of apoptosisrelated proteins might contribute to the defective proliferation and survival of autophagy-deficient T cells. ...
The gastrointestinal tract presents a unique challenge to the mucosal immune system, which has to constantly monitor the vast surface for the presence of pathogens, while at the same time maintaining tolerance to beneficial or innocuous antigens. In the intestinal mucosa, specialized innate and adaptive immune components participate in directing appropriate immune responses toward these diverse challenges. Recent studies provide compelling evidence that the process of autophagy influences several aspects of mucosal immune responses. Initially described as a “self-eating” survival pathway that enables nutrient recycling during starvation, autophagy has now been connected to multiple cellular responses, including several aspects of immunity. Initial links between autophagy and host immunity came from the observations that autophagy can target intracellular bacteria for degradation. However, subsequent studies indicated that autophagy plays a much broader role in immune responses, as it can impact antigen processing, thymic selection, lymphocyte homeostasis, and the regulation of immunoglobulin and cytokine secretion. In this review, we provide a comprehensive overview of mucosal immune cells and discuss how autophagy influences many aspects of their physiology and function. We focus on cell type-specific roles of autophagy in the gut, with a particular emphasis on the effects of autophagy on the intestinal T cell compartment. We also provide a perspective on how manipulation of autophagy may potentially be used to treat mucosal inflammatory disorders.
... We found that lungs of IKKε-deficient mice harbored 4-fold as many CD8 + T cells as those of WT mice ( Figure 6D). A high level of CD44 expression serves as a marker for activated antitumor T cells (Okoye et al., 2015). There were 0.3% and 2% of CD44 high T cells in the lungs of WT and IKKε-deficient mice, respectively ( Figure 6E). ...
Activation of nuclear factor of activated T cells (NFAT) is crucial for immune responses. IKKε is an IκB kinase (IKK)-related kinase, and the function of IKKε remains obscure in T cells, despite its abundant expression. We report that IKKε inhibits NFAT activation and T cell responses by promoting NFATc1 phosphorylation. During T cell activation, IKKε was transiently activated to phosphorylate NFATc1. Loss of IKKε elevated T cell antitumor and antiviral immunity and, therefore, reduced tumor development and persistent viral infection. IKKε was activated in CD8+ T cells of mice bearing melanoma or persistently infected with a model herpesvirus. These results collectively show that IKKε promotes NFATc1 phosphorylation and inhibits T cell responses, identifying IKKε as a crucial negative regulator of T cell activation and a potential target for immunotherapy.
... Oxidative metabolism is also important for effector T cell responses; inhibition of OxPhos or glutamine deprivation suppresses T cell clonal expansion and effector function (24,91,92). Indeed, inhibition of mitochondrial ATP synthesis was sufficient to inhibit alloreactive T cells in graft-versus-host disease models (93). ...
Immune cell metabolism is dynamically regulated in parallel with the substantial changes in cellular function that accompany immune cell activation. While these changes in metabolism are important for facilitating the increased energetic and biosynthetic demands of activated cells, immune cell metabolism also has direct roles in controlling the functions of immune cells and shaping the immune response. A theme is emerging wherein nutrients, metabolic enzymes, and metabolites can act as an extension of the established immune signal transduction pathways, thereby adding an extra layer of complexity to the regulation of immunity. This Review will outline the metabolic configurations adopted by different immune cell subsets, describe the emerging roles for metabolic enzymes and metabolites in the control of immune cell function, and discuss the therapeutic implications of this emerging immune regulatory axis.
... proliferation, another study confirmed that mitochondrial ROS affect respiratory chain assembly into the inner mitochondrial membrane and CD8 + T-cell expansion, thus demonstrating that long-term protective immunity can be OXPHOS driven (151). Antineoplastic chemotherapies stimulate autophagy, which appears as an adaptive metabolic mechanism of resistance to treatment (113). ...
Significance:
In the last years, metabolic reprogramming, fluctuations in bioenergetic fuels and modulation of oxidative stress became new key hallmarks of tumor development. In cancer, elevated glucose uptake and high glycolytic rate, as source of ATP, constitute a growth advantage for tumors. This represents the universally known "Warburg effect", which gave rise to one major clinical application for detecting cancer cells using glucose analogues: the PET-scan imaging.
Recent advances:
Glucose utilization and carbon sources in tumors are much more heterogeneous than initially thought. Indeed, new studies emerged and revealed a dual capacity of tumor cells for glycolytic and oxidative phosphorylation (OXPHOS) metabolism. OXPHOS metabolism, which relies predominantly on mitochondrial respiration, exhibits fine-tuned regulation of respiratory chain complexes and enhanced antioxidant response or detoxification capacity.
Critical issues:
OXPHOS-dependent cancer cells use alternative oxidizable substrates, such as glutamine and fatty acids. The diversity of carbon substrates fuelling neoplastic cells is indicative of metabolic heterogeneity, even within tumors sharing the same clinical diagnosis. Metabolic switch supports cancer cell stemness and their bioenergy-consuming functions, such as proliferation, survival, migration and invasion. Moreover, ROS-induced mitochondrial metabolism and nutrient availability are important for interaction with tumor microenvironment components. Carcinoma-associated fibroblasts and immune cells participate in the metabolic interplay with neoplastic cells. They collectively adapt in a dynamic manner to the metabolic needs of cancer cells, thus participating to tumorigenesis and resistance to treatments.
Future directions:
Characterizing the reciprocal metabolic interplay between stromal, immune and neoplastic cells will provide a better understanding of treatment resistance.
... Pathways leading to increased mitochondrial respiration are associated with a superior memory CD8 response. A recent study has identified Lymphocyte Expansion Molecule (LEM) as a novel protein that promotes memory CD8 response by increasing OXPHOS indirectly by boosting the translation of proteins synthesized in the mitochondria (Okoye et al., 2015). In our study, we identify MCJ as an endogenous negative regulator of OXPHOS in CD8 cells. ...
Mitochondrial respiration is regulated in CD8+ T cells during the transition from naive to effector and memory cells, but mechanisms controlling this process have not been defined. Here we show that MCJ (methylation-controlled J protein) acted as an endogenous break for mitochondrial respiration in CD8+ T cells by interfering with the formation of electron transport chain respiratory supercomplexes. Metabolic profiling revealed enhanced mitochondrial metabolism in MCJ-deficient CD8+ T cells. Increased oxidative phosphorylation and subcellular ATP accumulation caused by MCJ deficiency selectively increased the secretion, but not expression, of interferon-γ. MCJ also adapted effector CD8+ T cell metabolism during the contraction phase. Consequently, memory CD8+ T cells lacking MCJ provided superior protection against influenza virus infection. Thus, MCJ offers a mechanism for fine-tuning CD8+ T cell mitochondrial metabolism as an alternative to modulating mitochondrial mass, an energetically expensive process. MCJ could be a therapeutic target to enhance CD8+ T cell responses.
... It has been established that mitochondrial ROS play important roles under both physiological and pathophysiological conditions [20]. In this context, multiple recent studies have demonstrated that the tightly regulated formation of mitochondrial ROS serves as an important signaling mechanism for T cell immunity as well as innate immunity [21][22][23], and as such, inhibition of this regulated formation of mitochondrial ROS by antioxidant compounds would compromise T cell immunity, leading to detrimental effects. On the other hand, dysregulated induction of mitochondrial ROS, as suggested by the study of Ma et al. [17] in NAFLD, may cause selective CD4 + T cell depletion, leading to compromised immunosurveillance and consequent promotion of hepatocarcinogenesis. ...
... Specifically, mitochondria regulate antigen processing and presentation and localize to the immune synapse during T cell activation (121). Mitochondrial metabolism also maintains the memory T cell phenotype (122) and dictates the different inflammatory and suppressive CD4 + Th cells (109). A balanced Th1 and Th2 response is suited to the immune challenge, and a dysregulated response is linked to a variety of chronic inflammatory lung conditions such as asthma and chronic bronchitis (123). ...
Mitochondria are a distinguishing feature of eukaryotic cells. Best known for their critical function in energy production via oxidative phosphorylation (OXPHOS), mitochondria are essential for nutrient and oxygen sensing and for the regulation of critical cellular processes, including cell death and inflammation. Such diverse functional roles for organelles that were once thought to be simple may be attributed to their distinct heteroplasmic genome, exclusive maternal lineage of inheritance, and ability to generate signals to communicate with other cellular organelles. Mitochondria are now thought of as one of the cell's most sophisticated and dynamic responsive sensing systems. Specific signatures of-mitochondrial dysfunction that are associated with disease pathogenesis and/or progression are becoming increasingly important. In particular, the centrality of mitochondria in the pathological processes and clinical phenotypes associated with a range of lung diseases is emerging. Understanding the molecular mechanisms regulating the mitochondrial processes of lung cells will help to better define phenotypes and clinical manifestations associated with respiratory disease and to identify potential diagnostic and therapeutic targets.
... Future mechanistic studies that manipulate components of this pathway, including COXVIIb, using a systematic knockdown or gene expressionmodulation approach may provide further insights into the individual contributions of metabolism-related proteins in mediating aSR1 expansion effects. Currently, we propose that aSR1-mediated metabolic changes may provide a cellular context that promotes AML expansion ex vivo in a manner similar to OXPHOS-driven expansion in CD8 + T cells [27]. ...
Small molecule-based antagonism of the aryl hydrocarbon receptor (AHR) by StemRegenin1 (SR1) promotes ex vivo expansion and maintenance of primary human hematopoietic stem cells (HSCs) as well as acute myeloid leukemia (AML) cells. However, basis and nature of SR1 induced expansion of human AML remains unknown. Here, global expression profiling on 7 clinically diverse human AML patient samples treated ex vivo with a synthesized analog of SR1 (aSR1) uncovered that only as few as 750 genes were differentially regulated. Uniquely, aSR1 treatment did not modulate self-renewal associated pathways including Hedgehog, Notch or Wnt across patient samples, but instead resulted in overall upregulation of the oxidative phosphorylation metabolic pathway. Higher oxygen consumption rates, along with increased sensitivity to the chemotherapeutic agent cytarabine (AraC) validated that aSR1-induced transcriptional profiles lead to functional enhancement of oxidative phosphorylation. Our study reveals that aSR1 induces minor alterations to the leukemic transcriptional profile leading to a shift in cellular metabolism. This finding should further instruct use of SR1-mediated expansion for mechanistic studies of leukemic self-renewal and the development of drug screening platforms using patient specific AML samples.
... Mitochondrial respiration through reactive oxygen species signaling and the mTOR pathway appear critical for antigenspecific T-cell activation and memory development (Araki et al., 2009; Sena et al., 2013; van der Windt et al., 2012 ). Manipulation of these pathways might promote persistence and effector function of adoptively transferred T cells (Okoye et al., 2015; Pearce et al., 2009). ...
T lymphocytes can be redirected to recognize a tumor target and harnessed to combat cancer by genetic introduction of T-cell receptors of a defined specificity. This approach has recently mediated encouraging clinical responses in patients with cancers previously regarded as incurable. However, despite the great promise, T-cell receptor gene therapy still faces a multitude of obstacles. Identification of epitopes that enable effective targeting of all the cells in a heterogeneous tumor while sparing normal tissues remains perhaps the most demanding challenge. Experience from clinical trials has revealed the dangers associated with T-cell receptor gene therapy and highlighted the need for reliable preclinical methods to identify potentially hazardous recognition of both intended and unintended epitopes in healthy tissues. Procedures for manufacturing large and highly potent T-cell populations can be optimized to enhance their antitumor efficacy. Here, we review the current knowledge gained from preclinical models and clinical trials using adoptive transfer of T-cell receptor-engineered T lymphocytes, discuss the major challenges involved and highlight potential strategies to increase the safety and efficacy to make T-cell receptor gene therapy a standard-of-care for large patient groups.
... Consistent with this idea, a recent study has shown that the enhancement of mitochondrial oxidative phosphorylation could improve long-term protective immunity through the promotion of CD8+ T effector and memory cell proliferation and survival in the context of viral and tumor clearance. Some of the biologic outcomes of T cells are likely attributable to the production of pro-proliferative mitochondrial reactive oxygen species (mROS) [41]. Taken together, T cell activation and differentiation are tightly coupled with metabolic reprogramming. ...
Upon antigen stimulation, small and quiescent naïve T cells undergo an approximately 24h growth phase followed by rapid proliferation. Depending on the nature of the antigen and cytokine milieu, these proliferating T cells differentiate into distinctive functional subgroups that are essential for appropriate immune defense and regulation. T cells undergo a characteristic metabolic rewiring that fulfills the dramatically increased bioenergetic and biosynthetic demands during the transition between resting, activation and differentiation. Beyond this, T cells are distributed throughout the body and are able to function in a wide range of physio-pathological environments, including some with a dramatic metabolic derangement. As such, T cells must quickly respond to and adapt to fluctuations in environmental nutrient levels. We consider such responsiveness and adaptation in terms of metabolic plasticity, that is, an evolutionarilly selected process which allows T cells to illicit robust immune functions in response to either a continuous or disrupted nutrient supply. In this review, we illustrate the relevant metabolic pathways in T cells and discuss the ability of T cells to change their metabolic substrates in response to changes in the environment.
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Sustaining proliferative signaling and enabling replicative immortality are two important hallmarks of cancer. The complex of cyclin-dependent kinase (CDK) and its cyclin plays a decisive role in the transformation of the cell cycle and is also critical in the initiation and progression of cancer. CRIF1, a multifunctional factor, plays a pivotal role in a series of cell biological progresses such as cell cycle, cell proliferation, and energy metabolism. CRIF1 is best known as a negative regulator of the cell cycle, on account of directly binding to Gadd45 family proteins or CDK2. In addition, CRIF1 acts as a regulator of several transcription factors such as Nur77 and STAT3 and partly determines the proliferation of cancer cells. Many studies showed that the expression of CRIF1 is significantly altered in cancers and potentially regarded as a tumor suppressor. This suggests that targeting CRIF1 would enhance the selectivity and sensitivity of cancer treatment. Moreover, CRIF1 might be an indispensable part of mitoribosome and is involved in the regulation of OXPHOS capacity. Further, CRIF1 is thought to be a novel target for the underlying mechanism of diseases with mitochondrial dysfunctions. In summary, this review would conclude the latest aspects of studies about CRIF1 in cancers and mitochondria-related diseases, shed new light on targeted therapy, and provide a more comprehensive holistic view.
CD8 T cell exhaustion is a hallmark of HIV-1 infection, characterized by phenotypic and functional CD8 T cell abnormalities that persist despite years of effective antiretroviral treatment (ART). More recently, the importance of cellular metabolism in shaping T cell antiviral function has emerged as a crucial aspect of immunotherapeutics aimed at re-invigorating exhausted CD8 T cells but remains under-investigated in HIV-1 infection. To gain a better insight into this process and identify new targets for effective CD8 T cell restoration we examined the metabolic profile of exhausted CD8 T cells in HIV-1 infection. We show that relative to HIV-1 elite controllers (EC) and HIV-1 seronegative donors, CD8 T cells from HIV-1 viraemic individuals are skewed toward a PD-1hiEOMEShiT-betlowTIGIT+ phenotype that is maintained during ART. This exhausted signature is enriched in HIV-specific CD8 T cells, compared to CMV-specific CD8 T cell populations, and further delineated by higher expression of the glucose transporter, Glut-1, impaired mitochondrial function and biogenesis, reflecting underlying metabolic defects. A notable improvement in antiviral HIV-specific CD8 T cell function was elicited via mitochondrial antioxidant treatment in combination with pharmacological modulation of mitochondrial dynamics and IL-15 treatment. These findings identify mitochondria as promising targets for combined reconstitution therapies in HIV-1 infection.
Data is the cornerstone of the modern academic industry, like a constant production line of consumable goods packaged with a veneer of statistical techniques. Biomedical and psychological sciences invert the traditional logic of the physical sciences where hypotheses were tested against data rather than data against hypotheses. This is likely to reflect the immaturity of the new paradigms that barely cope with the data output of a precocious enterprise. However, the current state leads to distortions of the practice of science and entrenchment of its dysfunctional politics reflected in the science itself. With the debate now on the reproduction of data and statistical sleights of hand accompanying many if not most studies, what is lost in the debate is the preeminent role of good theoretical discovery. This is partly as a result of the structure of funding, the nature of reporting in biomedical fields based on the symbiotic relationship of high ranking institutions and journals. Open access could fill a gap in traditional publishing literature which has entrenched a culture of highly restrictive practices at a time when revolutionary science is required. OA is not in danger of lowering the standards of science as its critics claim, but because it is ‘open’ to new and radical ideas that would never see the light of day otherwise, it may well provide a rejuvenating energy. OA is a self-regulatory response to the consequences of the distorted and inhibitory contemporary practice of science.
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.
Purpose of review
An increasing body of evidence indicates that bio-energetic metabolism of activated T cells is a potential target to control the autoimmune response in type 1 diabetes (T1D).
Recent findings
T-cell activation and proliferation is linked to the cell capacity to provide sufficient energy and biosynthesis molecules to support T-cell growth and division. This makes T cells susceptible to metabolic inhibition for the control of the T-cell response. There is a wide therapeutic arsenal of metabolic inhibitors, including novel classes of drugs that have become recently available.
Summary
With the current knowledge and availability of metabolic inhibitors, we are now in the position to design a metabolic inhibition strategy to determine whether targeting of autoreactive T cells is an effective strategy to control the process of ?-cell destruction in T1D.
Data is the cornerstone of the modern academic industry, like a constant production line of consumable goods packaged with a veneer of statistical techniques. Biomedical and psychological sciences invert the traditional logic of the physical sciences where hypotheses were tested against data rather than data against hypotheses. This is likely to reflect the immaturity of the new paradigms that barely cope with the data output of a precocious enterprise. However, the current state leads to distortions of the practice of science and entrenchment of its dysfunctional politics reflected in the science itself. With the debate now on the reproduction of data and statistical sleights of hand accompanying many if not most studies, what is lost in the debate is the preeminent role of good theoretical discovery. This is partly as a result of the structure of funding, the nature of reporting in biomedical fields based on the symbiotic relationship of high ranking institutions and journals. Open access could fill a gap in traditional publishing literature which has entrenched a culture of highly restrictive practices at a time when revolutionary science is required. OA is not in danger of lowering the standards of science as its critics claim as the studies on non replication show that they do not discriminate against OA papers specifically. Because it is ?open? to new and radical ideas that would never see the light of day otherwise, it may well provide a rejuvenating energy. OA is a self-regulatory response to the consequences of the distorted and inhibitory contemporary practice of science as the popularity of OA journals testify to.
Data is the cornerstone of the modern academic industry, like a constant production line of consumable goods packaged with a veneer of statistical techniques. Biomedical and psychological sciences invert the traditional logic of the physical sciences where hypotheses were tested against data rather than data against hypotheses. This is likely to reflect the immaturity of the new paradigms that barely cope with the data output of a precocious enterprise. However, the current state leads to distortions of the practice of science and entrenchment of its dysfunctional politics reflected in the science itself. With the debate now on the reproduction of data and statistical sleights of hand accompanying many if not most studies, what is lost in the debate is the preeminent role of good theoretical discovery. This is partly as a result of the structure of funding, the nature of reporting in biomedical fields based on the symbiotic relationship of high ranking institutions and journals. Open access could fill a gap in traditional publishing literature which has entrenched a culture of highly restrictive practices at a time when revolutionary science is required. OA is not in danger of lowering the standards of science as its critics claim as the studies on non replication show that they do not discriminate against OA papers specifically. Because it is ?open? to new and radical ideas that would never see the light of day otherwise, it may well provide a rejuvenating energy. OA is a self-regulatory response to the consequences of the distorted and inhibitory contemporary practice of science as the popularity of OA journals testify to.
The development and function of cytotoxic CD8 T cells (CTLs), which provide immunity to viral infections, are regulated by changes in mitochondrial respiration. Champagne et al. (2016) describe a new mechanism through which mitochondrial metabolism controls production of ATP required for the secretion of critical anti-viral molecules by CTLs.
The question whether low doses and low dose-rates of ionizing radiation pose a health risk to people is of public, scientific and regulatory concern. It is a subject of intense debate and causes much fear. The controversy is to what extent low-dose effects, if any, cause or protect against damage such as cancer. Even if immediate molecular damage in exposed biological systems rises linearly with the number of energy deposition events (i.e., with absorbed dose), the response of the whole biological system to that damage is not linear. To understand how initial molecular damage affects a complex living system is the current challenge.
Immune cells play a key role in host defense against infection and cancer. Upon encountering danger signals, these cells undergo activation leading to a modulation in their immune functions. However, recent studies reveal that immune cells upon activation also show distinct metabolic changes that impact their immune functions. Such metabolic reprogramming and its functional effects are well known for cancer cells. Given that immune cells have emerged as crucial players in cancer progression, it is important to understand whether immune cells also undergo metabolic reprogramming in tumors and how this might affect their contribution in cancer progression. This emerging aspect of tumor-associated immune cells is reviewed here, discussing metabolic reprogramming of different immune cell types, the key pathways involved, and its impact on tumor progression.
Understanding the mechanistic processes that govern T cell responses is crucial to enhancing immunotherapies against human disease. Naive CD8 T cells extensively proliferate in response to antigen, creating a large pool of effector T cells that clear tumors or infected cells. During this process, T cells metabolically reprogram to meet energy demands and supply biosynthetic precursors necessary for proliferation ( 1 ). On page 995 of this issue, Okoye et al. ( 2 ) identify lymphocyte expansion molecule (LEM), a protein that targets T cell metabolism and enhances proliferation. LEM improved CD8 T cell–mediated viral and tumor clearance and boosted memory T cell numbers. These findings suggest that modulating T cell metabolism by targeting LEM could alter the course of cancer, autoimmunity, or infection.
The inhibitory programmed death 1 (PD-1)-programmed death ligand 1 (PD-L1) pathway contributes to the functional down-regulation of T cell responses during persistent systemic and local virus infections. The blockade of PD-1-PD-L1-mediated inhibition is considered as a therapeutic approach to reinvigorate antiviral T cell responses. Yet previous studies reported that PD-L1-deficient mice develop fatal pathology during early systemic lymphocytic choriomeningitis virus (LCMV) infection, suggesting a host protective role of T cell down-regulation. As the exact mechanisms of pathology development remained unclear, we set out to delineate in detail the underlying pathogenesis. Mice deficient in PD-1-PD-L1 signaling or lacking PD-1 signaling in CD8 T cells succumbed to fatal CD8 T cell-mediated immunopathology early after systemic LCMV infection. In the absence of regulation via PD-1, CD8 T cells killed infected vascular endothelial cells via perforin-mediated cytolysis, thereby severely compromising vascular integrity. This resulted in systemic vascular leakage and a consequential collapse of the circulatory system. Our results indicate that the PD-1-PD-L1 pathway protects the vascular system from severe CD8 T cell-mediated damage during early systemic LCMV infection, highlighting a pivotal physiological role of T cell down-regulation and suggesting the potential development of immunopathological side effects when interfering with the PD-1-PD-L1 pathway during systemic virus infections.
Although substantial progress has been made in understanding the mechanisms underlying the expression of mtDNA-encoded polypeptides, the regulatory factors involved in mitoribosome-mediated synthesis and simultaneous insertion of mitochondrial oxidative phosphorylation (OXPHOS) polypeptides into the inner membrane of mitochondria are still unclear. In the present study, disruption of the mouse Crif1 gene, which encodes a mitochondrial protein, resulted in a profound deficiency in OXPHOS caused by the disappearance of OXPHOS subunits and complexes in vivo. CRIF1 was associated with large mitoribosomal subunits that were located close to the polypeptide exit tunnel, and the elimination of CRIF1 led to both aberrant synthesis and defective insertion of mtDNA-encoded nascent OXPHOS polypeptides into the inner membrane. CRIF1 interacted with nascent OXPHOS polypeptides and molecular chaperones, e.g., Tid1. Taken together, these results suggest that CRIF1 plays a critical role in the integration of OXPHOS polypeptides into the mitochondrial membrane in mammals.
The persistence of memory T lymphocytes confers lifelong protection from pathogens. Memory T cells survive and undergo homeostatic proliferation (HSP) in the absence of Ag, although the cell-intrinsic mechanisms by which cytokines drive the HSP of memory T cells are not well understood. In this study we report that lysosome stability limits the long-term maintenance of memory CD8(+) T cell populations. Serine protease inhibitor (Spi) 2A, an anti-apoptotic cytosolic cathepsin inhibitor, is induced by both IL-15 and IL-7. Mice deficient in Spi2A developed fewer memory phenotype CD44(hi)CD8(+) T cells with age, which underwent reduced HSP in the bone marrow. Spi2A was also required for the maintenance of central memory CD8(+) T cell populations after acute infection with lymphocytic choriomeningitis virus. Spi2A-deficient Ag-specific CD8(+) T cell populations declined more than wild-type competitors after viral infection, and they were eroded further after successive infections. Spi2A protected memory cells from lysosomal breakdown by inhibiting cathepsin B. The impaired maintenance of Spi2A-deficient memory CD8(+) T cells was rescued by concomitant cathepsin B deficiency, demonstrating that cathepsin B was a physiological target of Spi2A in memory CD8(+) T cell survival. Our findings support a model in which protection from lysosomal rupture through cytokine-induced expression of Spi2A determines the long-term persistence of memory CD8(+) T cells.
Blockade of programmed death 1 (PD-1), an inhibitory receptor expressed by T cells, can overcome immune resistance. We assessed the antitumor activity and safety of BMS-936558, an antibody that specifically blocks PD-1.
We enrolled patients with advanced melanoma, non-small-cell lung cancer, castration-resistant prostate cancer, or renal-cell or colorectal cancer to receive anti-PD-1 antibody at a dose of 0.1 to 10.0 mg per kilogram of body weight every 2 weeks. Response was assessed after each 8-week treatment cycle. Patients received up to 12 cycles until disease progression or a complete response occurred.
A total of 296 patients received treatment through February 24, 2012. Grade 3 or 4 drug-related adverse events occurred in 14% of patients; there were three deaths from pulmonary toxicity. No maximum tolerated dose was defined. Adverse events consistent with immune-related causes were observed. Among 236 patients in whom response could be evaluated, objective responses (complete or partial responses) were observed in those with non-small-cell lung cancer, melanoma, or renal-cell cancer. Cumulative response rates (all doses) were 18% among patients with non-small-cell lung cancer (14 of 76 patients), 28% among patients with melanoma (26 of 94 patients), and 27% among patients with renal-cell cancer (9 of 33 patients). Responses were durable; 20 of 31 responses lasted 1 year or more in patients with 1 year or more of follow-up. To assess the role of intratumoral PD-1 ligand (PD-L1) expression in the modulation of the PD-1-PD-L1 pathway, immunohistochemical analysis was performed on pretreatment tumor specimens obtained from 42 patients. Of 17 patients with PD-L1-negative tumors, none had an objective response; 9 of 25 patients (36%) with PD-L1-positive tumors had an objective response (P=0.006).
Anti-PD-1 antibody produced objective responses in approximately one in four to one in five patients with non-small-cell lung cancer, melanoma, or renal-cell cancer; the adverse-event profile does not appear to preclude its use. Preliminary data suggest a relationship between PD-L1 expression on tumor cells and objective response. (Funded by Bristol-Myers Squibb and others; ClinicalTrials.gov number, NCT00730639.).
The homeostasis of the immune system is tightly controlled by both cell-extrinsic and -intrinsic mechanisms. These regulators, not all known to date, drive cells in and out of quiescence when and where required to allow the immune system to function. In this article, we describe a deficiency in deoxycytidine kinase (DCK), one of the major enzymes of the nucleoside salvage pathway, which affects peripheral T cell homeostatic proliferation and survival. As a result of an N-ethyl-N-nitrosourea-induced mutation in the last α helix of DCK, a functionally null protein has been generated in the mouse and affects the composition of the hematopoietic system. Both B and T lymphocyte development is impaired, leading to a state of chronic lymphopenia and to a significant increase in the number of myeloid cells and erythrocytes. In the periphery, we found that mutant lymphocytes adopt a CD44(high)CD62L(low) memory phenotype, with high levels of proliferation and apoptosis. These phenotypes are notably the result of a cell-extrinsic-driven lymphopenia-induced proliferation as wild-type cells transferred into DCK-deficient recipients adopt the same profile. In addition, DCK also regulates lymphocyte quiescence in a cell-intrinsic manner. These data establish dCK as a new regulator of hematopoietic integrity and lymphocyte quiescence and survival.
We report the development and optimization of reagents for in-solution, hybridization-based capture of the mouse exome. By validating this approach in a multiple inbred strains and in novel mutant strains, we show that whole exome sequencing is a robust approach for discovery of putative mutations, irrespective of strain background. We found strong candidate mutations for the majority of mutant exomes sequenced, including new models of orofacial clefting, urogenital dysmorphology, kyphosis and autoimmune hepatitis.
Assessing mitochondrial dysfunction requires definition of the dysfunction to be investigated. Usually, it is the ability of the mitochondria to make ATP appropriately in response to energy demands. Where other functions are of interest, tailored solutions are required. Dysfunction can be assessed in isolated mitochondria, in cells or in vivo, with different balances between precise experimental control and physiological relevance. There are many methods to measure mitochondrial function and dysfunction in these systems. Generally, measurements of fluxes give more information about the ability to make ATP than do measurements of intermediates and potentials. For isolated mitochondria, the best assay is mitochondrial respiratory control: the increase in respiration rate in response to ADP. For intact cells, the best assay is the equivalent measurement of cell respiratory control, which reports the rate of ATP production, the proton leak rate, the coupling efficiency, the maximum respiratory rate, the respiratory control ratio and the spare respiratory capacity. Measurements of membrane potential provide useful additional information. Measurement of both respiration and potential during appropriate titrations enables the identification of the primary sites of effectors and the distribution of control, allowing deeper quantitative analyses. Many other measurements in current use can be more problematic, as discussed in the present review.
Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.
Virus-specific T cells capable of controlling HBV and eliminating hepatocellular carcinoma (HCC) expressing HBV antigens are deleted or dysfunctional in patients with chronic HBV or HBV-related HCC. The goal of this study was to determine if T cell receptor (TCR) gene transfer can reconstitute HBV-specific T cell immunity in lymphocytes of chronic HBV patients and investigate whether HCC cells with natural HBV-DNA integration can be recognized by genetically modified T cells.
We used vector-mediated gene transfer to introduce HLA-A2-restricted, HBV-specific TCRs into T cells of chronic HBV as well as HBV-related HCC patients.
The introduced TCRs were expressed on the cell surface, evidenced by Vβ and pentamer staining. TCR transduced T cells produced IFN-γ, TNF-α, IL-2, and lysed HBV infected hepatocyte-like cell lines. Furthermore, HCC cell lines with natural HBV-DNA integration could be recognized by HBV-specific TCR-re-directed T cells.
TCR re-directed HBV-specific T cells generated from PBMC of chronic HBV and HBV-related HCC patients were multifunctional and capable of recognizing HBV-infected cells and HCC tumor cells expressing viral antigens from naturally integrated HBV DNA. These genetically modified T cells could be used to reconstitute virus-specific T cell immunity in chronic HBV patients and target tumors in HBV-related HCC.
Vaccination with irradiated B16 melanoma cells expressing either GM-CSF (Gvax) or Flt3-ligand (Fvax) combined with antibody blockade of the negative T-cell costimulatory receptor cytotoxic T-lymphocyte antigen-4 (CTLA-4) promotes rejection of preimplanted tumors. Despite CTLA-4 blockade, T-cell proliferation and cytokine production can be inhibited by the interaction of programmed death-1 (PD-1) with its ligands PD-L1 and PD-L2 or by the interaction of PD-L1 with B7-1. Here, we show that the combination of CTLA-4 and PD-1 blockade is more than twice as effective as either alone in promoting the rejection of B16 melanomas in conjunction with Fvax. Adding alphaPD-L1 to this regimen results in rejection of 65% of preimplanted tumors vs. 10% with CTLA-4 blockade alone. Combination PD-1 and CTLA-4 blockade increases effector T-cell (Teff) infiltration, resulting in highly advantageous Teff-to-regulatory T-cell ratios with the tumor. The fraction of tumor-infiltrating Teffs expressing CTLA-4 and PD-1 increases, reflecting the proliferation and accumulation of cells that would otherwise be anergized. Combination blockade also synergistically increases Teff-to-myeloid-derived suppressor cell ratios within B16 melanomas. IFN-gamma production increases in both the tumor and vaccine draining lymph nodes, as does the frequency of IFN-gamma/TNF-alpha double-producing CD8(+) T cells within the tumor. These results suggest that combination blockade of the PD-1/PD-L1- and CTLA-4-negative costimulatory pathways allows tumor-specific T cells that would otherwise be inactivated to continue to expand and carry out effector functions, thereby shifting the tumor microenvironment from suppressive to inflammatory.
Memory CD8 T cells are a critical component of protective immunity, and inducing effective memory T-cell responses is a major goal of vaccines against chronic infections and tumours. Considerable effort has gone into designing vaccine regimens that will increase the magnitude of the memory response, but there has been minimal emphasis on developing strategies to improve the functional qualities of memory T cells. Here we show that mTOR (mammalian target of rapamycin, also known as FRAP1) is a major regulator of memory CD8 T-cell differentiation, and in contrast to what we expected, the immunosuppressive drug rapamycin has immunostimulatory effects on the generation of memory CD8 T cells. Treatment of mice with rapamycin following acute lymphocytic choriomeningitis virus infection enhanced not only the quantity but also the quality of virus-specific CD8 T cells. Similar effects were seen after immunization of mice with a vaccine based on non-replicating virus-like particles. In addition, rapamycin treatment also enhanced memory T-cell responses in non-human primates following vaccination with modified vaccinia virus Ankara. Rapamycin was effective during both the expansion and contraction phases of the T-cell response; during the expansion phase it increased the number of memory precursors, and during the contraction phase (effector to memory transition) it accelerated the memory T-cell differentiation program. Experiments using RNA interference to inhibit expression of mTOR, raptor (also known as 4932417H02Rik) or FKBP12 (also known as FKBP1A) in antigen-specific CD8 T cells showed that mTOR acts intrinsically through the mTORC1 (mTOR complex 1) pathway to regulate memory T-cell differentiation. Thus these studies identify a molecular pathway regulating memory formation and provide an effective strategy for improving the functional qualities of vaccine- or infection-induced memory T cells.
We report the characterization of a highly germline competent C57BL/6N mouse embryonic stem cell line, JM8. To simplify breeding schemes, the dominant agouti coat color gene was restored in JM8 cells by targeted repair of the C57BL/6 nonagouti mutation. These cells provide a robust foundation for large-scale mouse knockout programs that aim to provide a public resource of targeted mutations in the C57BL/6 genetic background.
Two-hybrid methods have augmented the classical genetic techniques biologists use to assign function to genes. Here, we describe construction of a two-bait interaction trap that uses yeast cells to register more complex protein relationships than those detected in existing two-hybrid systems. We show that such cells can identify bridge or connecting proteins and peptide aptamers that discriminate between closely related allelic variants. The protein relationships detected by these cells are analogous to classical genetic relationships, but lend themselves to systematic application to the products of entire genomes and combinatorial libraries. We show that, by performing logical operations on the phenotypic outputs of these complex cells and existing two-hybrid cells, we can make inferences about the topology and order of protein interactions. Finally, we show that cells that register such relationships can perform logical operations on protein inputs. Thus these cells will be useful for analysis of gene and allele function, and may also define a path for construction of biological computational devices.
During acute lymphocytic choriomeningitis virus (LCMV) infection, CD8 T cells rapidly expand and differentiate into effectors that are required for viral clearance. The accumulation of activated T cells is greatly reduced in mice lacking the adaptor molecule MyD88. Although MyD88 has generally been considered to indirectly regulate adaptive immune responses by controlling inflammatory cytokine production and Ag presentation in innate immune cells, in this study, we identify an unappreciated cell-intrinsic role for MyD88 in LCMV-specific CD8 T cells. Using reciprocal adoptive transfer models and bone marrow chimeras, we show that Myd88(-/-) CD8 T cells are defective in their clonal expansion in response to LCMV infection, independent of their environment. Furthermore, we show that while MyD88 is dispensable for initial activation and division of LCMV-specific CD8 T cells during the early stages of viral infection, MyD88-dependent signals are critical for supporting their survival and sustained accumulation.
A mAb J43 has been produced against the product of the mouse PD-1 gene, a member of the Ig gene superfamily, which was previously isolated from an apoptosis-induced T cell hybridoma (2B4.11) by using subtractive hybridization. Analyses by flow cytometry and immunoprecipitation using the J43 mAb revealed that the PD-1 gene product is a 50-55 kDa membrane protein expressed on the cell surface of several PD-1 cDNA transfectants and 2B4.11 cells. Since the molecular weight calculated from the amino acid sequence is 29, 310, the PD-1 protein appears to be heavily glycosylated. Normal murine lymphoid tissues such as thymus, spleen, lymph node and bone marrow contained very small numbers of PD-1(+) cells. However, a significant PD-1(+) population appeared in the thymocytes as well as T cells in spleen and lymph nodes by the in vivo anti-CD3 mAb treatment. Furthermore, the PD-1 antigen expression was strongly induced in distinct subsets of thymocytes and spleen T cells by in vitro stimulation with either anti-CD3 mAb or concanavalin A (Con A) which could lead T cells to both activation and cell death. Similarly, PD-1 expression was induced on spleen B cells by in vitro stimulation with anti-IgM antibody. By contrast, PD-1 was not significantly expressed on lymphocytes by treatment with growth factor deprivation, dexamethasone or lipopolysaccharide. These results suggest that the expression of the PD-1 antigen is tightly regulated and induced by signal transduction through the antigen receptor and do not exclude the possibility that the PD-1 antigen may play a role in clonal selection of lymphocytes although PD-1 expression is not required for the common pathway of apoptosis.
A central question in immunology is the origin of long-lived T cell memory that confers protection against recurrent infection.
The differentiation of naı̈ve T cell receptor transgenic CD8+ cells into effector cytotoxic T lymphocytes (CTLs) and memory CD8+ cells was studied. Memory CD8+cells that were generated after strong antigenic stimulation were the progeny of cytotoxic effectors and retained antigen-specific
cytolytic activity 10 weeks after adoptive transfer to antigen-free recipient mice. Thus, potential vaccines based on CTL
memory will require the differentiation of naı̈ve cells into post-effector memory T cells.
During many viral infections, antigen-specific CD8+ T cells undergo large-scale expansion. After viral clearance, the vast majority of effector CD8+ T cells undergo apoptosis. Previous studies have implicated reactive oxygen intermediates (ROI) in lymphocyte apoptosis.
The purpose of the experiments presented here was to determine the role of ROI in the expansion and contraction of CD8+ T cells in vivo during a physiological response such as viral infection. Mice were infected with lymphocytic choriomeningitis
virus (LCMV) and treated with Mn(III)tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP), a metalloporphyrin-mimetic compound
with superoxide dismutase activity, from days 0 to 8 postinfection. At the peak of CD8+-T-cell response, on day 8 postinfection, the numbers of antigen-specific cells were 10-fold lower in MnTBAP-treated mice
than in control mice. From days 8 to 30, a contraction phase ensued where the numbers of antigen-specific CD8+ T cells declined 25-fold in vehicle-treated mice compared to a 3.5-fold decrease in MnTBAP-treated mice. Differences in contraction
appeared to be due to greater proliferation in drug-treated mice. By day 38, the numbers of antigen-specific CD8+ memory T cells were equivalent for the two groups. The administration of MnTBAP during secondary viral infection had no effect
on the expansion of antigen-specific CD8+ secondary effector T cells. These data suggest that ROI production is critical for the massive expansion and contraction
of antigen-specific CD8+ T cells during primary, but not secondary, viral infection.
Many gene sequences in eukaryotic genomes encode entire proteins or large segments of proteins that lack a well-structured three-dimensional fold. Disordered regions can be highly conserved between species in both composition and sequence and, contrary to the traditional view that protein function equates with a stable three-dimensional structure, disordered regions are often functional, in ways that we are only beginning to discover. Many disordered segments fold on binding to their biological targets (coupled folding and binding), whereas others constitute flexible linkers that have a role in the assembly of macromolecular arrays.
Functional impairment of antigen-specific T cells is a defining characteristic of many chronic infections, but the underlying mechanisms of T-cell dysfunction are not well understood. To address this question, we analysed genes expressed in functionally impaired virus-specific CD8 T cells present in mice chronically infected with lymphocytic choriomeningitis virus (LCMV), and compared these with the gene profile of functional memory CD8 T cells. Here we report that PD-1 (programmed death 1; also known as Pdcd1) was selectively upregulated by the exhausted T cells, and that in vivo administration of antibodies that blocked the interaction of this inhibitory receptor with its ligand, PD-L1 (also known as B7-H1), enhanced T-cell responses. Notably, we found that even in persistently infected mice that were lacking CD4 T-cell help, blockade of the PD-1/PD-L1 inhibitory pathway had a beneficial effect on the 'helpless' CD8 T cells, restoring their ability to undergo proliferation, secrete cytokines, kill infected cells and decrease viral load. Blockade of the CTLA-4 (cytotoxic T-lymphocyte-associated protein 4) inhibitory pathway had no effect on either T-cell function or viral control. These studies identify a specific mechanism of T-cell exhaustion and define a potentially effective immunological strategy for the treatment of chronic viral infections.
Inhibition of the nonsense-mediated decay (NMD) mechanism in cells results in stabilization of transcripts carrying premature translation termination codons. A strategy referred to as gene identification by NMD inhibition (GINI) has been proposed to identify genes carrying nonsense mutations. Genes containing frameshift mutations in colon cancer cell line have been identified using a modified version of GINI. To increase the efficiency of identifying mutant genes using GINI, we have now further improved the strategy. In this approach, inhibition of NMD with emetine is complemented with inhibiting NMD by blocking the phosphorylation of the hUpf1 protein with caffeine. In addition, to enhance the GINI strategy, comparing mRNA level alterations produced by inhibiting transcription alone or inhibiting transcription together with NMD following caffeine pretreatment were used for the efficient identification of false positives produced as a result of stress response to NMD inhibition. To demonstrate the improved efficiency of this approach, we analysed colon cancer cell lines showing microsatellite instability. Bi-allelic inactivating mutations were found in the FXR1, SEC31L1, NCOR1, BAT3, PHF14, ZNF294, C19ORF5 genes as well as genes coding for proteins with yet unknown functions.
What governs the increased apoptosis sensitivity of HIV-specific CD8(+) T cells is poorly understood. Here, we examined the involvement of mitochondria in this apoptosis. Remarkably higher mitochondrial mass (MM) was found in HIV-specific compared with CMV-specific CD8(+) T cells from HIV(+) patients and this could not be attributed to their different differentiation status. MM(High) phenotype characterized those CD8(+) T cells from HIV(+) patients that are sensitive to spontaneous and CD95/Fas-induced apoptosis. CD38 expression did not correlate with high MM, whereas Bcl-2 levels were significantly reduced in both CD38(+) and CD38(-) HIV-specific CD8(+) T cells. Although CD38(+) HIV-specific CD8(+) T cells were more susceptible to apoptosis, CD38 expression does not explain on its own the selective apoptosis sensitivity of HIV-specific CD8(+) T cells, as CD38(-) HIV-specific CD8(+) T cells were more apoptotic than CD38(+) CMV-specific ones. Proapoptotic HIV-specific CD8(+) T cells were CD38(+)Bcl-2(Low)MM(High). Copolarization of mitochondria with CD95/Fas capping, very early in CD95/Fas-induced apoptosis of HIV-specific CD8(+) T cells, suggests that mitochondria act as an amplification step for this apoptosis. Thus, an extensive mitochondrial network contributes to apoptosis sensitivity of CD8(+) T cells and, when this occurs together with reduced levels of Bcl-2 and chronic activation, determines the proapoptotic state of HIV-specific CD8(+) T cells.
As acute infections resolve, effector CD8(+) T cells differentiate into interleukin-7 receptor(lo) (IL-7R(lo)) short-lived effector cells (SLECs) and IL-7R(hi) memory precursor effector cells (MPECs) capable of generating long-lived memory CD8(+) T cells. By using another SLEC marker, KLRG1, we found that KLRG1(hi) effector cells began appearing early during infection and were committed to downregulating IL-7R. Unlike IL-7R(hi) MPECs, KLRG1(hi) IL-7R(lo) SLECs relied on IL-15, but IL-15 could not sustain their long-term maintenance or homeostatic turnover. The decision between SLEC and MPEC fates was regulated by the amount of inflammatory cytokines (i.e., IL-12) present during T cell priming. According to the amount of inflammation, a gradient of T-bet was created in which high T-bet expression induced SLECs and low expression promoted MPECs. These results elucidate a mechanism by which the innate immune system sets the relative amounts of a lineage-determining transcription factor in activated CD8(+) T cells and, correspondingly, regulates their memory cell potential.
Quantitative PCR (QPCR, or real time PCR (rtPCR)) has emerged as a powerful virologic technique for measuring viral replication and viral loads in humans and animal models. We have developed a QPCR assay to accurately quantify lymphocytic choriomeningitis virus (LCMV) in infected mice. We first validated this assay using plasmid DNA and LCMV viral stocks. We then demonstrated that the LCMV QPCR assay can detect LCMV in serum and tissues of chronically infected mice (LCMV-clone 13), with greater sensitivity than conventional plaque assay. Subsequently, we demonstrated that the QPCR assay can detect LCMV in tissues of CD40L(-/-) mice during a low grade chronic infection with LCMV Armstrong. Finally, we improved the assay further such that it was approximate 1000-fold more sensitive than plaque assay for detection of the presence of LCMV in tissue.
Signal transducer and activator of transcription 3 (STAT3) is a transcriptional factor that performs a broad spectrum of biological functions in response to various stimuli. However, no specific coactivator that regulates the transcriptional activity of STAT3 has been identified. Here we report that CR6-interacting factor 1 (Crif1) is a specific transcriptional coactivator of STAT3, but not of STAT1 or STAT5a. Crif1 interacts with STAT3 and positively regulates its transcriptional activity. Crif1-/- embryos were lethal around embryonic day 6.5, and manifested developmental arrest accompanied with defective proliferation and massive apoptosis. The expression of STAT3 target genes was markedly reduced in a Crif1-/- blastocyst culture and in Oncostatin M-stimulated Crif1-deficient MEFs. Importantly, the key activities of constitutively active STAT3-C, such as transcription, DNA binding, and cellular transformation, were abolished in the Crif1-null MEFs, suggesting the essential role of Crif1 in the transcriptional activity of STAT3. Our results reveal that Crif1 is a novel and essential transcriptional coactivator of STAT3 that modulates its DNA binding ability, and shed light on the regulation of oncogenic STAT3.
It is widely appreciated that T cells increase glycolytic flux during activation, but the role of mitochondrial flux is unclear. Here, we have shown that mitochondrial metabolism in the absence of glucose metabolism is sufficient to support interleukin-2 (IL-2) induction. Furthermore, we used mice with reduced mitochondrial reactive oxygen species (mROS) production in T cells (T-Uqcrfs(-/-) mice) to show that mitochondria are required for T cell activation to produce mROS for activation of nuclear factor of activated T cells (NFAT) and subsequent IL-2 induction. These mice could not induce antigen-specific expansion of T cells in vivo, but Uqcrfs1(-/-) T cells retained the ability to proliferate in vivo under lymphopenic conditions. This suggests that Uqcrfs1(-/-) T cells were not lacking bioenergetically but rather lacked specific ROS-dependent signaling events needed for antigen-specific expansion. Thus, mitochondrial metabolism is a critical component of T cell activation through the production of complex III ROS.
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.
Many persistent viral infections induce dysfunctional T cell responses. Although a negative correlation exists between viral load and T cell responses during chronic infection, it is not known whether high antigen levels are the cause or just the consequence of T cell exhaustion. Furthermore, it is unclear what role antigen presentation by bone-marrow (BM) derived versus infected parenchymal cells has on T cell exhaustion. To address these issues, we examined the influence of antigen presentation by different cell types on CD8(+) T cell responses during persistent infection of mice with lymphocytic choriomeningitis virus (LCMV) clone 13. We generated BM chimeric mice, in which non-BM derived cells were MHC class I deficient. Virus-specific CD8(+) T cells in lymphoid and nonlymphoid tissues were increased in both number and ability to produce cytokines in these mice soon after infection. However, viral clearance from infected MHC I(-/-) parenchyma was significantly impaired, despite increased populations of cytokine producing CTL. The CD8(+) T cell response was overwhelmed by sustained antigen persistence, becoming increasingly exhausted within 4-6 weeks. Thus, we find that (i) sustained antigen presentation directly drives T cell exhaustion during a chronic viral infection, (ii) CTL require direct antigen-MHC interactions to clear virus-infected cells, and (iii) persistent interactions with antigen presented on both hematopoietic and nonhematopoietic cells negatively impacts virus-specific T cell responses during chronic infection.
Memory is a hallmark of the immune system and ever since its recognition there has been considerable interest in understanding how immunity is maintained. The current model is that long-term memory is dependent on persistent antigenic stimulation. We report here results that challenge this view and provide evidence that antigen is not essential for the maintenance of CD8+ T-cell memory. We show that memory CD8+ cytotoxic T lymphocytes persist indefinitely in the absence of priming antigen, retain the memory phenotype (CD44hi), and provide protection against virus challenge. These findings suggest a re-evaluation of our current thinking on mechanisms involved in maintaining immunity and have implications towards designing effective vaccination strategies.
Virtually all of the measurable cell-mediated cytotoxicity delivered by cytotoxic T lymphocytes and natural killer cells comes from either the granule exocytosis pathway or the Fas pathway. The granule exocytosis pathway utilizes perforin to traffic the granzymes to appropriate locations in target cells, where they cleave critical substrates that initiate DNA fragmentation and apoptosis; granzymes A and B induce death via alternate, nonoverlapping pathways. The Fas/FasL system is responsible for activation-induced cell death but also plays an important role in lymphocyte-mediated killing under certain circumstances. The interplay between these two cytotoxic systems provides opportunities for therapeutic interventions to control autoimmune diseases and graft vs. host disease, but oversuppression of these pathways may also lead to increased viral susceptibility and/or decreased tumor cell killing.
Ever since Paul Ehrlich introduced the term “magic bullet,” the exquisite capacity for specificity afforded by the immune system has always underpinned its appeal as a therapeutic weapon against cancer. The first clinical validation of this principle came in the form of mAb administration, which, after a decade of skepticism, produced therapeutic successes in breast cancer and B cell lymphomas. T cell-based immunotherapy offers an even broader therapeutic potential, owing to the ability of T cells to recognize peptides derived from proteins in any cellular compartment. These peptides are produced when proteins are processed by specialized machinery; they then combine with MHC molecules, which transport them to the cell surface where the peptide–MHC complexes can be recognized by T cells. Although hundreds of experiments in rodent tumor models support the notion that tumor-specific T cells can be activated to inhibit tumor growth, direct evidence for therapeutic capacity in human cancer has been marginal. Now, two clinical studies using adoptive transfer of melanoma-specific T cells provide clear evidence for the ability of T cells to mediate antitumor activity and provide important general principles for T cell immunotherapy (1, 2).
Therapeutic vaccines aim to prevent severe complications of a chronic infection by reinforcing host defenses when some immune
control, albeit insufficient, can already be demonstrated and when a conventional antimicrobial therapy either is not available
or has limited efficacy. We focus on the rationale and challenges behind this still controversial strategy and provide examples
from three major chronic infectious diseases— human immunodeficiency virus, hepatitis B virus, and human papillomavirus—for
which the efficacy of therapeutic vaccines is currently being evaluated.
An essential event in the development of memory CD8(+) T lymphocytes is the escape of progenitors from programmed cell death, but how this is mediated is unclear. Here we report that the gene encoding serine protease inhibitor 2A (Spi2A), an inhibitor of lysosomal executioner proteases dependent on transcription factor NF-kappaB, is upregulated in memory cell precursors. Spi2A upregulation protected lymphocytic choriomeningitis virus-specific memory progenitors from programmed cell death. Thus, Spi2A promotes the survival of cytotoxic T lymphocytes, allowing them to differentiate into memory CD8 T cells. These findings suggest a model in which commitment to the memory lineage is facilitated by the upregulation of protective genes.
How cytotoxic T lymphocytes (CTLs) kill intracellular pathogens without killing themselves has been a recurring question ever since their discovery. By using mice deficient in Serine Protease Inhibitor 6 (Spi6), we show that by inhibiting granzyme B (GrB), Spi6 protects CTLs from self-inflicted injury. Infection with either Lymphocytic Choriomeningitis virus (LCMV) or Listeria monocytogenes (LM) revealed increased apoptosis and diminished survival of Spi6 knockout (KO) CTLs, which was cell autonomous and could be corrected by GrB deficiency. Spi6 KO mice in turn were impaired in their ability to clear LCMV infection. Spi6 KO CTLs revealed a breakdown in the integrity of cytotoxic granules, increased cytoplasmic GrB, and ensuing apoptosis. We conclude that Spi6 protects CTLs from suicide caused by GrB-mediated breakdown of cytotoxic granules.
Functional impairment of T cells is characteristic of many chronic mouse and human viral infections. The inhibitory receptor programmed death 1 (PD-1; also known as PDCD1), a negative regulator of activated T cells, is markedly upregulated on the surface of exhausted virus-specific CD8 T cells in mice. Blockade of this pathway using antibodies against the PD ligand 1 (PD-L1, also known as CD274) restores CD8 T-cell function and reduces viral load. To investigate the role of PD-1 in a chronic human viral infection, we examined PD-1 expression on human immunodeficiency virus (HIV)-specific CD8 T cells in 71 clade-C-infected people who were naive to anti-HIV treatments, using ten major histocompatibility complex (MHC) class I tetramers specific for frequently targeted epitopes. Here we report that PD-1 is significantly upregulated on these cells, and expression correlates with impaired HIV-specific CD8 T-cell function as well as predictors of disease progression: positively with plasma viral load and inversely with CD4 T-cell count. PD-1 expression on CD4 T cells likewise showed a positive correlation with viral load and an inverse correlation with CD4 T-cell count, and blockade of the pathway augmented HIV-specific CD4 and CD8 T-cell function. These data indicate that the immunoregulatory PD-1/PD-L1 pathway is operative during a persistent viral infection in humans, and define a reversible defect in HIV-specific T-cell function. Moreover, this pathway of reversible T-cell impairment provides a potential target for enhancing the function of exhausted T cells in chronic HIV infection.
Technological advances in recent years have allowed for an ever-expanding ability to analyze and quantify in vivo immune responses. MHC tetramers, intracellular cytokine staining, an increasing repertoire of transgenic and "knockout" mice, and the detailed characterization of a variety of infectious models have all facilitated more precise and definitive analyses of the generation and function of cytotoxic T lymphocytes (CTL). Understanding the mechanisms behind the differentiation of effector and memory CTL is of increasing importance to develop vaccination strategies against a variety of established and emerging infectious diseases. This review focuses on recent advances in our understanding of how effector and memory CTL differentiate and survive in vivo in response to viral or bacterial infection.
In mammalian cells, nonsense-mediated mRNA decay (NMD) is a consequence of nonsense codon recognition during a pioneer round of translation. This round can occur largely before or largely after the release of newly synthesized mRNA from nuclei, depending on the mRNA, and likely utilizes cytoplasmic ribosomes. We show that increasing the cellular concentration of the splicing factor SF2/ASF augments the efficiency of NMD and ultimately shifts NMD that takes place after mRNA export to the cytoplasm to NMD that occurs before mRNA release from nuclei. These changes are accompanied by an increased association of pioneer translation initiation complexes with SF2/ASF, translationally active ribosomes, and the translational activator TAP. Increased TAP binding correlates with increased SF2/ASF binding, but not increased REF/Aly or Y14 binding. Our results uncover an additional role for SF2/ASF and indicate that the efficiency of the pioneer round of translation influences the efficiency of subsequent rounds of translation.
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