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Systemic lupus erythematosus (SLE) is characterized by increased DNA demethylation in T cells, although it is unclear whether this occurs primarily in a subset of SLE T cells. The process driving the DNA demethylation and the consequences on overall gene expression are also poorly understood and whether this represents a secondary consequence of SLE or a primary contributing factor. Lupus-prone lpr mice accumulate large numbers of T cells with age because of a mutation in Fas (CD95). The accumulating T cells include an unusual population of CD4−CD8−TCR-αβ+ (DN) T cells that arise from CD8+ precursors and are also found in human SLE. We have previously observed that T cell accumulation in lpr mice is due to dysregulation of T cell homeostatic proliferation, which parallels an increased expression of numerous genes in the DN subset, including several proinflammatory molecules and checkpoint blockers. We thus determined the DNA methylome in lpr DN T cells compared with their CD8+ precursors. Our findings show that DN T cells manifest discrete sites of extensive demethylation throughout the genome, and these sites correspond to the location of a large proportion of the upregulated genes. Thus, dysregulated homeostatic proliferation in lpr mice and consequent epigenetic alterations may be a contributing factor to lupus pathogenesis.

SIGNIFICANCE: Numerous abnormalities in T cells have been described in patients with systemic lupus erythematosus (SLE), including lymphopenia, DNA demethylation, expression of endogenous retroviruses (ERVs), increased cell death, enlarged mitochondria, production of reactive oxygen species (ROS), and the appearance of unusual CD4(−)CD8(−) T cells. Our studies propose a model in which accelerated homeostatic proliferation of T cells promotes an epigenetic and metabolic program, leading to this cluster of abnormalities. RECENT ADVANCES: Growing knowledge of the innate immune disorders in SLE has included increased mitochondrial size and ROS production that induces oligomerization of the mitochondrial antiviral signaling (MAVS) protein and type I interferon production, as well as DNA demethylation, upregulation of inflammatory genes, and expression of certain ERVs in SLE peripheral blood mononuclear cells. All these events are part of the cellular program that occurs during homeostatic proliferation of T cells. Evidence from a murine model of SLE as well as in human SLE reveals that increased T cell homeostatic proliferation may be a driving factor in these processes. CRITICAL ISSUES: Despite extensive knowledge of the myriad autoantibodies in SLE and other immune abnormalities, a cogent model has been lacking to link the numerous and seemingly disparate immune aberrations. This may partly explain the general lack of new drugs specifically for SLE in over 50 years. A more coherent model of SLE would not only unify the variety of immune abnormalities is SLE but would also suggest new therapies. FUTURE DIRECTIONS: The model of augmented homeostatic proliferation leading to increased mitochondrial mass, ROS, DNA demethylation, and upregulation of inflammatory genes suggests strategic new targets for SLE, including antioxidants and certain inhibitors of metabolism. Antioxid. Redox Signal. 36, 410–422.

Interleukin 6 (IL-6) is known to regulate the CD4 T cell function by inducing gene expression of a number of cytokines through activation of Stat3 transcription factor. Here, we reveal that IL-6 strengthens the mechanics of CD4 T cells. The presence of IL-6 during activation of mouse and human CD4 T cells enhances their motility (random walk and exploratory spread), resulting in an increase in travel distance and higher velocity. This is an intrinsic effect of IL-6 on CD4 T-cell fitness that involves an increase in mitochondrial Ca2+. Although Stat3 transcriptional activity is dispensable for this process, IL-6 uses mitochondrial Stat3 to enhance mitochondrial Ca2+-mediated motility of CD4 T cells. Thus, through a noncanonical pathway, IL-6 can improve competitive fitness of CD4 T cells by facilitating cell motility. These results could lead to alternative therapeutic strategies for inflammatory diseases in which IL-6 plays a pathogenic role.

Interleukin 6 (IL-6) is known to regulate the CD4 T cell function by inducing gene expression of a number of cytokines through activation of Stat3 transcription factor. Here, we reveal that IL-6 strengthens the mechanics of CD4 T cells. The presence of IL-6 during activation of mouse and human CD4 T cells enhances their motility (random walk and exploratory spread), resulting in an increase in travel distance and higher velocity. This is an intrinsic effect of IL-6 on CD4 T-cell fitness that involves an increase in mitochondrial Ca(2+). Although Stat3 transcriptional activity is dispensable for this process, IL-6 uses mitochondrial Stat3 to enhance mitochondrial Ca(2+)-mediated motility of CD4 T cells. Thus, through a noncanonical pathway, IL-6 can improve competitive fitness of CD4 T cells by facilitating cell motility. These results could lead to alternative therapeutic strategies for inflammatory diseases in which IL-6 plays a pathogenic role.

Chemotherapy remains the standard of care for most cancers worldwide, however development of chemoresistance due to the presence of the drug-effluxing ATP binding cassette (ABC) transporters remains a significant problem. The development of safe and effective means to overcome chemoresistance is critical for achieving durable remissions in many cancer patients. We have investigated the energetic demands of ABC transporters in the context of the metabolic adaptations of chemoresistant cancer cells. Here we show that ABC transporters use mitochondrial-derived ATP as a source of energy to efflux drugs out of cancer cells. We further demonstrate that the loss of methylation-controlled J protein (MCJ) (also named DnaJC15), an endogenous negative regulator of mitochondrial respiration, in chemoresistant cancer cells boosts their ability to produce ATP from mitochondria and fuel ABC transporters. We have developed MCJ mimetics that can attenuate mitochondrial respiration and safely overcome chemoresistance in vitro and in vivo. Administration of MCJ mimetics in combination with standard chemotherapeutic drugs could therefore become an alternative strategy for treatment of multiple cancers., Drug efflux through ABC transporters is a common mechanism leading to chemoresistance in cancer. Here, the authors show that mitochondrial respiration provides ATP to allow ABC transporters activity so mitochondrial respiration inhibition overcomes chemoresistance in preclinical cancer models.

Background Fecal microbiota transplantation (FMT) is a promising new strategy in the treatment of Inflammatory Bowel Disease, but long-term delivery systems are lacking. This randomized study was designed as a safety and feasibility study of long-term FMT in subjects with mild to moderate UC using frozen, encapsulated oral FMT (cFMT). Methods Subjects were randomized 1:1 to receive FMT induction by colonoscopy, followed by 12 weeks of daily oral administration of frozen encapsulated cFMT or sham therpay. Subjects were followed for 36 weeks and longitudenal clinical assessments included multiple subjective and objective markers of disease severity. Ribosomal 16S bacterial sequencing was used to assess donor-induced changes in the gut microbiota. Changes in T regulatory (Treg) and mucosal associated invariant T (MAIT) cell populations were evaluated by flow cytometry as an exploratory endpoint. Results Twelve subjects with active UC were randomized: 6 subjects completed the full 12-week course of FMT plus cFMT, and 6 subjects received sham treatment by colonic installation and longitudinal oral placebo capules. Chronic administration of cFMT was found to be safe and well-tolerated but home storage concerns exist. Protocol adherence was high, and none of the study subjects experienced FMT-associated treatment emergent adverse events. Two subjects that received cFMT achieved clinical remission versus none in the placebo group (95% CI = 0.38-infinity, p = 0.45). cFMT was associated with sustained donor-induced shifts in fecal microbial composition. Changes in MAIT cell cytokine production were observed in cFMT recipients and correlated with treatment response. Conclusion These pilot data suggest that daily encapsulated cFMT may extend the durability of index FMT-induced changes in gut bacterial community structure and that an association between MAIT cell cytokine production and clinical response to FMT may exist in UC populations. Oral frozen encapsulated cFMT is a promising FMT delivery system and may be preferred for longterm treatment strategies in UC and other chronic diseases but further evaluations will have to address home storage concerns. Larger trials should be done to explore the benefits of cFMT and to determine its long-term impacts on the colonic microbiome. Trial registration: ClinicalTrials.gov (NCT02390726). Registered 17 March 2015, https://clinicaltrials.gov/ct2/show/NCT02390726?term=NCT02390726&draw=2&rank=1.

Nonalcoholic fatty liver disease (NAFLD) is considered the next major health epidemic with an estimated 25% worldwide prevalence. No drugs have yet been approved and NAFLD remains a major unmet need. Here, we identify MCJ (Methylation-Controlled J protein) as a target for non-alcoholic steatohepatitis (NASH), an advanced phase of NAFLD. MCJ is an endogenous negative regulator of the respiratory chain Complex I that acts to restrain mitochondrial respiration. We show that therapeutic targeting of MCJ in the liver with nanoparticle- and GalNAc-formulated siRNA efficiently reduces liver lipid accumulation and fibrosis in multiple NASH mouse models. Decreasing MCJ expression enhances the capacity of hepatocytes to mediate β-oxidation of fatty acids and minimizes lipid accumulation, which results in reduced hepatocyte damage and fibrosis. Moreover, MCJ levels in the liver of NAFLD patients are elevated relative to healthy subjects. Thus, inhibition of MCJ emerges as an alternative approach to treat NAFLD., Non-alcoholic fatty liver (NAFLD) disease causes degeneration of the liver, affects about 25% of people globally, and has no approved treatment. Here, the authors show that the therapeutic siRNA-driven silencing of MCJ in the liver is an effective and safe treatment for NAFLD in multiple mouse models.

ObjectivesRecent investigations in humans and mouse models with lupus have revealed evidence of mitochondrial dysfunction and production of mitochondrial reactive oxygen species (mROS) in T cells and neutrophils. This can provoke numerous cellular changes including oxidation of nucleic acids, proteins, lipids and even induction of cell death. We have previously observed that in T cells from patients with lupus, the increased mROS is capable of provoking oligomerisation of mitochondrial antiviral stimulator (MAVS) and production of type I interferon (IFN-I). mROS in SLE neutrophils also promotes the formation of neutrophil extracellular traps (NETs), which are increased in lupus and implicated in renal damage. As a result, in addition to traditional immunosuppression, more comprehensive treatments for lupus may also include non-immune therapy, such as antioxidants.MethodsLupus-prone MRL-lpr mice were treated from weaning for 11 weeks with the mitochondria-targeted antioxidant, MitoQ (200 µM) in drinking water. Mice were then assessed for ROS production in neutrophils, NET formation, MAVS oligomerisation, serum IFN-I, autoantibody production and renal function.ResultsMitoQ-treated mice manifested reduced neutrophil ROS and NET formation, decreased MAVS oligomerisation and serum IFN-I, and reduced immune complex formation in kidneys, despite no change in serum autoantibody .ConclusionsThese findings reveal the potential utility of targeting mROS in addition to traditional immunosuppressive therapy for lupus.

T lymphocyte homeostatic proliferation, driven by the engagement of T cell antigen receptor with self-peptide/major histocompatibility complexes, and signaling through the common γ-chain-containing cytokine receptors, is critical for the maintenance of the T cell compartment and is regulated by the Fas death receptor (Fas, CD95). In the absence of Fas, Fas-deficient lymphoproliferation spontaneous mutation (Ipr) mice accumulate homeostatically expanded T cells. The functional consequences of sequential rounds of homeostatic expansion are not well defined. We thus examined the gene expression profiles of murine wild-type and Fas-deficient Ipr CD8+ T cell subsets that have undergone different amounts of homeostatic proliferation as defined by their level of CD44 expression, and the CD4−CD8−TCRαβ+ T cell subset that results from extensive homeostatic expansion of CD8+ T cells. Our studies show that recurrent T cell homeostatic proliferation results in global gene expression changes, including the progressive upregulation of both cytolytic proteins such as Fas-Ligand and granzyme B as well as inhibitory proteins such as programmed cell death protein 1 (PD-1) and lymphocyte activating 3 (Lag3). These findings provide an explanation for how augmented T cell homeostatic expansion could lead to the frequently observed clinical paradox of simultaneous autoinflammatory and immunodeficiency syndromes and provide further insight into the regulatory programs that control chronically stimulated T cells.

Background Systemic Lupus Erythematosus (SLE) is characterized by numerous seemingly unconnected abnormalities. These include: 1) a multisystem inflammatory syndrome, 2) a strong type I Interferon (IFN-I) gene signature in peripheral blood lymphocytes (PBL), 3) an unusual population of CD4-CD8- T-+cells, 4) SLE T cells containing enlarged mitochondria and reactive oxygen species (ROS), and 5) a polymorphism in Mitochondrial antiviral stimulator (MAVS C79F) associated with milder SLE. Our studies provide a unifying model for these abnormalities through augmented T cell homeostatic proliferation, which leads to two parallel cellular pathways: first, progressive upregulation of cytolytic inflammatory molecules, including high levels of Fas-Ligand (FasL), Granzyme B, and IFN-gamma, and second, generation of CD4-CD8- T cells from CD8 +precursors, which manifest disorganized enlarged mitochondria, elevated reactive oxygen species (ROS) that drives oligomerization of MAVS and IFN-I production. Methods PBL from active SLE patients and age- and sex-matched healthy controls were assessed for increased expression of the T cell homeostatic proliferation phenotype determined from our murine studies: CD45RO+PD-1+SLAMF7+IL-7R-alpha-. The same cells were analyzed for MAVS oligomerization. Young lupus-prone MRL-lpr mice were treated for 11 weeks with the mitochondrially targeted antioxidant MitoQ. Results Reactive oxygen species (ROS) is sufficient to drive MAVS oligomerization and IFN-I production in cell lines and primary human PBL. However, PBL from SLE patients manifested spontaneous MAVS oligomerization, which paralleled the levels of serum INF-I and SLEDAI score (figure 1). Furthermore, SLE patients had evidence of increased T cell homeostatic proliferation. In addition, SLE patients had evidence of increased I cell homeostatic proliferation, based on increased IL-7R- Ki67 +T cells. Very similar findings were observed in lupus prone MRL-lpr mice. Treatment of MRL-lpr mice over 11 weeks with the mitochondrially-targeted anti-oxidant MitoQ inhibited MAVS oligomerization, reduced serum IFN-I, improved dermatitis, and reduced kidney immune complexes. Conclusions Conclusions: Human and murine SLE have evidence of mitochondrial dysfunction, elevated ROS, spontaneous MAVS oligomerization and elevated IFN-I, as well as augmented T cell homeostatic proliferation. These abnormalities can be largely reversed with MitoQ, with alleviation of disease in murine SLE. Funding Source(s): NIH (NIAID, NIAMS, NIGMS)

An effective adaptive immune response requires rapid T cell proliferation, followed by equally robust cell death. These two processes are coordinately regulated to allow sufficient magnitude of response followed by its rapid resolution, while also providing the maintenance of T cell memory. Both aspects of this T cell response are characterized by profound changes in metabolism; glycolysis drives proliferation whereas oxidative phosphorylation supports the survival of memory T cells. While much is known about the separate aspects of T cell expansion and contraction, considerably less is understood regarding how these processes might be connected. We report a link between the induction of glycolysis in CD8+ T cells and upregulation of the inhibitor of complex I and oxidative phosphorylation, methylation-controlled J protein (MCJ). MCJ acts synergistically with glycolysis to promote caspase-3 activity. Effector CD8+ T cells from MCJ-deficient mice manifest reduced glycolysis and considerably less active caspase-3 compared to wild-type cells. Consistent with these observations, in non-glycolytic CD8+ T cells cultured in the presence of IL-15, MCJ expression is repressed by methylation, which parallels their reduced active caspase-3 and increased survival compared to glycolytic IL-2-cultured T cells. Elevated levels of MCJ are also observed in vivo in the highly proliferative and glycolytic subset of CD4-CD8- T cells in Fas-deficient lpr mice. This subset also manifests elevated levels of activated caspase-3 and rapid cell death. Collectively, these data demonstrate tight linkage of glycolysis, MCJ expression, and active caspase-3 that serves to prevent the accumulation and promote the timely death of highly proliferative CD8+ T cells.

γδ T cells function at the interface between innate and adaptive immunity and have well-demonstrated roles in response to infection, autoimmunity and tumors. A common characteristic of these seemingly disparate conditions may be cellular stress or death. However, the conditions under which ligands for γδ T cells are induced or exposed remain largely undefined. We observed that induction of necroptosis of murine or human dendritic cells (DC) by inhibition of caspase activity paradoxically augments their ability to activate γδ T cells. Furthermore, upregulation of the stabilizer of caspase-8 activity, c-FLIP, by IL-4, not only greatly reduced the susceptibility of DC to necroptosis, but also considerably decreased their ability to activate γδ T cells. Collectively, these findings suggest that the induction of necroptosis in DC upregulates or exposes the expression of γδ T cell ligands, and they support the view that γδ T cells function in the immune surveillance of cell stress.

IL-6 promotes the differentiation of a subset of naïve CD8+ T cells into IL-21–producing B helper CD8+ T cells., IL-6 is known to contribute to the differentiation of CD4+ T cells into different subsets of effector T helper cells. Less is known about the potential of IL-6 in regulating CD8+ T cell effector function. Here, we identify IL-6 as a master regulator of IL-21 in effector CD8+ T cells. IL-6 promotes the differentiation of a subset of naive CD8+ T cells that express IL-6R into a unique population of effector CD8+ T cells characterized by the production of high levels of IL-21 and low levels of IFN-γ. Similar to CD4+ T follicular helper (Tfh) cells, IL-21–producing CD8+ T cells generated in the presence of IL-6 directly provide help to B cells to induce isotype switching. CD8+ T cell–derived IL-21 contributes to the production of protective virus-specific IgG antibodies during influenza virus infection. Thus, this study reveals the presence of a new mechanism by which IL-6 regulates antibody production during viral infection, and a novel function of effector CD8+ T cells in the protection against viruses.

Delivery of small interfering RNAs (siRNAs) into primary T cells is quite challenging because they are non-proliferating cells and are difficult to transfect with non-viral approaches. Because sonoporation is independent of the proliferation status of cells and siRNA acts in the cell cytoplasm, we investigated whether sonoporation could be used to deliver siRNA into mouse and human T cells. Cells mixed with Definity microbubbles and siRNA were sonicated with a non-focused transducer of center frequency 2.20 MHz producing ultrasound at a 10% duty cycle, pulse repetition frequency of 2.20 kHz and spatial average temporal average ultrasound intensity of 1.29 W/cm2 for 5 s and then examined for siRNA fluorescence by flow cytometry analysis. These sonoporation conditions resulted in high-efficiency transfection of siRNA in mouse and human T cells. Further, the efficacy of siRNA delivery by sonoporation was illustrated by the successful visualization of decreased methylation-controlled J protein expression in mouse and human CD8 T cells via Western blot analysis. The results provide the first evidence that sonoporation is a novel approach to delivery of siRNA into fresh isolated mouse and human T cells in vitro, and might be used for in vivo studies in the future.

Glioblastoma (GBM), the most common primary adult malignant brain tumor, is associated with a poor prognosis due, in part, to tumor recurrence mediated by chemotherapy and radiation resistant glioma stem-like cells (GSCs). The metabolic and epigenetic state of GSCs differs from their non-GSC counterparts, with GSCs exhibiting greater glycolytic metabolism and global hypoacetylation. However, little attention has been focused on the potential use of acetate supplementation as a therapeutic approach. N-acetyl-l-aspartate (NAA), the primary storage form of brain acetate, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis, are significantly reduced in GBM tumors. We recently demonstrated that NAA supplementation is not an appropriate therapeutic approach since it increases GSC proliferation and pursued an alternative acetate source. The FDA approved food additive Triacetin (glyceryl triacetate, GTA) has been safely used for acetate supplementation therapy in Canavan disease, a leukodystrophy due to ASPA mutation. This study characterized the effects of GTA on the proliferation and differentiation of six primary GBM-derived GSCs relative to established U87 and U251 GBM cell lines, normal human cerebral cortical astrocytes, and murine neural stem cells. GTA reduced proliferation of GSCs greater than established GBM lines. Moreover, GTA reduced growth of the more aggressive mesenchymal GSCs greater than proneural GSCs. Although sodium acetate induced a dose-dependent reduction of GSC growth, it also reduced cell viability. GTA-mediated growth inhibition was not associated with differentiation, but increased protein acetylation. These data suggest that GTA-mediated acetate supplementation is a novel therapeutic strategy to inhibit GSC growth.

Mitochondria are the main engine that generates ATP through oxidative phosphorylation within the respiratory chain. Mitochondrial respiration is regulated according to the metabolic needs of cells and can be modulated in response to metabolic changes. Little is known about the mechanisms that regulate this process. Here, we identify MCJ/DnaJC15 as a distinct cochaperone that localizes at the mitochondrial inner membrane, where it interacts preferentially with complex I of the electron transfer chain. We show that MCJ impairs the formation of supercomplexes and functions as a negative regulator of the respiratory chain. The loss of MCJ leads to increased complex I activity, mitochondrial membrane potential, and ATP production. Although MCJ is dispensable for mitochondrial function under normal physiological conditions, MCJ deficiency affects the pathophysiology resulting from metabolic alterations. Thus, enhanced mitochondrial respiration in the absence of MCJ prevents the pathological accumulation of lipids in the liver in response to both fasting and a high-cholesterol diet. Impaired expression or loss of MCJ expression may therefore result in a “rapid” metabolism that mitigates the consequences of metabolic disorders.

Summary Caspase-8 serves two paradoxical roles in T lymphocytes: it initiates apoptosis following death receptor engagement, and is also indispensible for proliferation following T-cell antigen receptor (TCR) signalling. These opposing processes appear to be controlled by both spatial and quantitative differences in caspase-8 activation. Given differences in the turnover of T-cell subsets, we compared caspase activity and susceptibility to cell death following TCR restimulation in murine CD4 + and CD8 + ab T cells and cd T cells. We observed a spectrum of caspase activity in non-dying effector T cells in which CD4 + T cells manifested the lowest levels of active caspases whereas cd T cells manifested the highest levels. Further analysis revealed that most of the difference in T-cell subsets was the result of high levels of active caspase-3 in non-dying effector cd T cells. Despite this, cd T cells manifested little spontaneous or CD3 restimulation-induced cell death as the result of confinement of active caspases to the cell membrane. By contrast, CD4 + T cells were highly sensitive to CD3-induced cell death, associated with the appearance of active caspases in the cytoplasm and cleavage of the caspase substrates Bid and ICAD. Hence, the location and amount of active caspases distinguishes effector T-cell subsets and profoundly influences the fate of the T-cell response.

Fas-deficient mice (Fas(lpr/lpr)) and humans have profoundly dysregulated T lymphocyte homeostasis, which manifests as an accumulation of CD4(+) and CD8(+) T cells as well as an unusual population of CD4(-)CD8(-)TCRαβ(+) T cells. To date, no unifying model has explained both the increased T-cell numbers and the origin of the CD4(-)CD8(-)TCRαβ(+) T cells. As Fas(lpr/lpr) mice raised in a germ-free environment still manifest lymphadenopathy, we considered that this process is primarily driven by recurrent low-avidity TCR signaling in response to self-peptide/MHC as occurs during homeostatic proliferation. In these studies, we developed two independent systems to decrease the number of self-peptide/MHC contacts. First, expression of MHC class I was reduced in OT-I TCR transgenic mice. Although OT-I Fas(lpr/lpr) mice did not develop lymphadenopathy characteristic of Fas(lpr/lpr) mice, in the absence of MHC class I, OT-I Fas(lpr/lpr) T cells accumulated as both CD8(+) and CD4(-)CD8(-) T cells. In the second system, re-expression of β(2)m limited to thymic cortical epithelial cells of Fas(lpr/lpr) β(2)m-deficient mice yielded a model in which polyclonal CD8(+) thymocytes entered a peripheral environment devoid of MHC class I. These mice accumulated significantly greater numbers of CD4(-)CD8(-)TCRαβ(+) T cells than conventional Fas(lpr/lpr) mice. Thus, Fas shapes the peripheral T-cell repertoire by regulating the survival of a subset of T cells proliferating in response to limited self-peptide/MHC contacts.

Evolving models of immune tolerance have challenged the view that the response of the maternal immune system to environmental or fetal antigens must be suppressed or deviated. CD8 T cells play a central role in the immune response to viruses and intracellular pathogens so the maintenance of both the number and function of these cells is critical to protect both the mother and fetus. We show that the numbers of maternal CD8 T cells in both the spleen and the uterine draining lymph nodes are transiently increased at mid-gestation and this correlates with enhanced CD8 T-cell proliferation and an increased relative expression of both pro-survival and pro-apoptotic molecules. In transgenic mice bearing T-cell antigen receptors specific for the male HY or allo-antigens, the transgenic CD8 T cells retain the ability to proliferate and function during pregnancy. Moreover, anti-HY T-cell receptor transgenic mice have normal numbers of male pups despite the presence of CD8 T cells at the maternal–fetal interface. These data suggest that pregnancy is a dynamic state in which CD8 T-cell turnover is increased while the function and ending size of the CD8 T-cell compartment are maintained.

Apoptotic death of T lymphocytes is critical for shutdown of immune responses and hemopoietic cell homeostasis. Both death receptor (Fas) activation and mitochondrial apoptosis triggered by the BH3-only protein Bim have been implicated in the killing of antigen-stimulated T cells. We examined mice lacking the gene encoding Bim (Bcl2l11) and with the inactivating lpr mutation in the gene encoding Fas (Fas), designated Bcl2l11(-/-)Fas(lpr/lpr) mice. Shutdown of an acute T cell response to herpes simplex virus involved only Bim with no contribution by Fas, whereas both pathways synergized in killing antigen-stimulated T cells in chronic infection with murine gamma-herpesvirus. Bcl2l11(-/-)Fas(lpr/lpr) mice developed remarkably enhanced and accelerated fatal lymphadenopathy and autoimmunity compared to mice lacking only one of these apoptosis inducers. These results identify critical overlapping roles for Fas and Bim in T cell death in immune response shutdown and prevention of immunopathology and thereby resolve a long-standing controversy.

γδ T cells function at the interface between innate and adaptive immunity and have well-demonstrated roles in response to infection, autoimmunity, and tumors. A common characteristic of these seemingly disparate conditions may be cellular stress or death. Yet, the conditions under which ligands for γδ T cells are induced or exposed remain largely undefined. We now observe that induction of necroptosis of murine or human dendritic cells by inhibition of caspase activity paradoxically augments their ability to activate γδ T cells. Furthermore, upregulation of the stabilizer of caspase-8 activity, c-FLIP, by IL-4 not only greatly reduced the susceptibility of dendritic cells to necroptosis, but also considerably decreased their ability to activate γδ T cells. Collectively these findings suggest that induction of necroptosis in dendritic cells upregulates or exposes expression of γδ T cell ligands, and supports the view that γδ T cells function in the immune surveillance of cell stress.

Summary 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.

Background Evidence for association between asthma and the unfolded protein response is emerging. Endoplasmic reticulum resident protein 57 (ERp57) is an endoplasmic reticulum–localized redox chaperone involved in folding and secretion of glycoproteins. We have previously demonstrated that ERp57 is upregulated in allergen-challenged human and murine lung epithelial cells. However, the role of ERp57 in asthma pathophysiology is unknown. Objectives Here we sought to examine the contribution of airway epithelium–specific ERp57 in the pathogenesis of allergic asthma. Methods We examined the expression of ERp57 in human asthmatic airway epithelium and used murine models of allergic asthma to evaluate the relevance of epithelium-specific ERp57. Results Lung biopsy specimens from asthmatic and nonasthmatic patients revealed a predominant increase in ERp57 levels in epithelium of asthmatic patients. Deletion of ERp57 resulted in a significant decrease in inflammatory cell counts and airways resistance in a murine model of allergic asthma. Furthermore, we observed that disulfide bridges in eotaxin, epidermal growth factor, and periostin were also decreased in the lungs of house dust mite–challenged ERp57-deleted mice. Fibrotic markers, such as collagen and α smooth muscle actin, were also significantly decreased in the lungs of ERp57-deleted mice. Furthermore, adaptive immune responses were dispensable for house dust mite–induced endoplasmic reticulum stress and airways fibrosis. Conclusions Here we show that ERp57 levels are increased in the airway epithelium of asthmatic patients and in mice with allergic airways disease. The ERp57 level increase is associated with redox modification of proinflammatory, apoptotic, and fibrotic mediators and contributes to airways hyperresponsiveness. The strategies to inhibit ERp57 specifically within the airways epithelium might provide an opportunity to alleviate the allergic asthma phenotype.

Caspase activity is required not only for the death of T cells, but also for their activation. A delicate balance of caspase activity is thus required during T cell activation at a level that will not drive cell death. How caspase activity is initiated and regulated during T cell activation is not known. One logical candidate for this process is cellular FLIP long form (c-FLIPL), because it can block caspase-8 recruitment after Fas (CD95) ligation as well as directly heterodimerize with and activate caspase-8. The current findings demonstrate that after T cell activation, caspase-8 and c-FLIPL associate in a complex enriched for active caspases. This occurs coincidently with the cleavage of two known caspase-8 substrates, c-FLIPL and receptor interacting protein 1. Caspase activity is higher in wild-type CD8+ than CD4+ effector T cells. Increased expression of c-FLIPL results in augmented caspase activity in resting and effector T cells to levels that provoke cell death, especially of the CD8 subset. c-FLIPL is thus not only an inhibitor of cell death by Fas, it can also act as a principal activator of caspases independently of Fas.

Cellular FLIP long form (c-FLIPL) is a caspase-defective homologue of caspase-8 that blocks apoptosis by death receptors. The expression of c-FLIPL in T cells can also augment extracellular signal-regulated kinase phosphorylation after TCR ligation via the association of c-FLIPL with Raf-1. This contributes to the hyperproliferative capacity of T cells from c-FLIPL-transgenic mice. In this study we show that activated CD4+ T cells from c-FLIPL-transgenic mice produce increased amounts of Th2 cytokines and decreased amounts of Th1 cytokines. This correlates with increased serum concentrations of the Th2-dependent IgG1 and IgE. The Th2 bias of c-FLIPL-transgenic CD4+ T cells parallels impaired NF-κB activity and increased levels of GATA-3, which contribute, respectively, to decreased IFN-γ and increased Th2 cytokines. The Th2 bias of c-FLIPL-transgenic mice extends to an enhanced sensitivity to OVA-induced asthma. Taken together, these results show that c-FLIPL can influence cytokine gene expression to promote Th2-driven allergic reaction, in addition to its traditional role of blocking caspase activation induced by death receptors.

Glioblastoma (GBM), the most common primary adult malignant brain tumor, is associated with a poor prognosis due, in part, to tumor recurrence mediated by chemotherapy and radiation resistant glioma stem-like cells (GSCs). The metabolic and epigenetic state of GSCs differs from their non-GSC counterparts, with GSCs exhibiting greater glycolytic metabolism and global hypoacetylation. However, little attention has been focused on the potential use of acetate supplementation as a therapeutic approach. N-acetyl-L-aspartate (NAA), the primary storage form of brain acetate, and aspartoacylase (ASPA), the enzyme responsible for NAA catalysis, are significantly reduced in GBM tumors. We recently demonstrated that NAA supplementation is not an appropriate therapeutic approach since it increases GSC proliferation and pursued an alternative acetate source. The FDA approved food additive Triacetin (glyceryl triacetate, GTA) has been safely used for acetate supplementation therapy in Canavan disease, a leukodystrophy due to ASPA mutation. This study characterized the effects of GTA on the proliferation and differentiation of six primary GBM-derived GSCs relative to established U87 and U251 GBM cell lines, normal human cerebral cortical astrocytes, and murine neural stem cells. GTA reduced proliferation of GSCs greater than established GBM lines. Moreover, GTA reduced growth of the more aggressive mesenchymal GSCs greater than proneural GSCs. Although sodium acetate induced a dose-dependent reduction of GSC growth, it also reduced cell viability. GTA-mediated growth inhibition was not associated with differentiation, but increased protein acetylation. These data suggest that GTA-mediated acetate supplementation is a novel therapeutic strategy to inhibit GSC growth.

Caspase activity is critical for both T-cell survival and death. However, little is known regarding what determines caspase activity in cycling T cells. Interleukin (IL)-2 and IL-15 confer very different susceptibilities to T-cell death. We therefore considered that IL-2 and IL-15 differentially regulate caspase activity to influence T-cell survival. We observed that IL-2-cultured primary murine effector T cells manifested elevated levels of caspase-3 activity compared with IL-15-cultured T cells. T cell receptor (TCR) restimulation further increased caspase activity and induced considerable cell death in IL-2-cultured T cells, but provoked only a minimal increase of caspase activity and cell death in IL-15-cultured T cells. IL-2 sensitization to cell death was caspase-3 mediated. Interestingly, increased active caspase-3 levels with IL-2 were independent of active initiator caspase-8 and caspase-9 that were similar with IL-2 and IL-15. Rather, caspase-3 activity was inhibited by posttranslational S-nitrosylation in IL-15-cultured T cells, but not in the presence of IL-2. This paralleled increased reactive nitrogen and oxygen species with IL-15 and reduced glycolysis. Taken together, these data suggest that the metabolic state conferred by IL-15 inhibits T-cell apoptosis in part by maintaining low levels of active caspase-3 via S-nitrosylation.

Mitogen-activated protein (MAP) kinases, which include the extracellular response kinases, p38 and c-Jun amino terminal kinases (JNK), play a significant role in mediating signals triggered by cytokines, growth factors and environmental stress. The JNK and p38 MAP kinases have been involved in growth, differentiation and cell death in different cell types. In the present paper, we describe how the JNK and p38 MAP kinase signalling pathways are regulated and their role during thymocyte development and the activation and differentiation of T cells in the peripheral immune system. The results from these studies demonstrate that the JNK and p38 MAP kinase signalling pathways regulate different aspects of T-cell mediated immune responses.

The development of T cells in the thymus is coordinated by cell-specific gene expression programs that involve multiple transcription factors and signaling pathways. Here, we show that the p38 mitogen-activated protein (MAP) kinase signaling pathway is strictly regulated during the differentiation of CD4−CD8− thymocytes. Persistent activation of p38 MAP kinase blocks fetal thymocyte development at the CD25+CD44− stage in vivo, and results in the lack of T cells in the peripheral immune system of adult mice. Inactivation of p38 MAP kinase is required for further differentiation of these cells into CD4+CD8+ thymocytes. The arrest of cell cycle in mitosis is partially responsible for the blockade of differentiation. Therefore, the p38 MAP kinase pathway is a critical regulatory element of differentiation and proliferation during the early stages of in vivo thymocyte development.

CD2 is a cell surface glycoprotein expressed on most T lymphocytes that is generally viewed as a cell adhesion molecule and, in this capacity, contributes to T cell receptor (TCR) signaling. CD2 has a relatively long cytoplasmic tail which associates with the src family tyrosine kinases, p56(lck) and p59(fyn), and could potentially signal directly. Down-modulation of CD2 on T cells has been shown to result in diminished proliferative capacity and interleukin (IL)-2 production. Furthermore, re-expression of CD2 can result in the restoration of these functions. This suggests that CD2 can influence the intensity of TCR signaling. As TCR signal intensity is pivotal to the induction of T cell apoptosis, we considered the hypothesis that the level of CD2 on the T cell surface may influence its propensity toward apoptosis. Using an anti-CD2 antibody, CD2 was down-modulated in vivo on mouse T lymphocytes without affecting the levels of surface CD3, TCR alphabeta, CD4 or CD8. Deletion of superantigen-responsive T cells was delayed in mice with down-modulated CD2 following the administration of staphylococcal enterotoxin B (SEB). This was paralleled by diminished apoptosis of SEB-responsive cells. The findings suggest a model whereby the level of CD2 expression influences the intensity of TCR signaling and the ability to undergo apoptosis.

Caspase-8 is now appreciated to govern both apoptosis following death receptor ligation and cell survival and growth via inhibition of the Ripoptosome. Cells must therefore carefully regulate the high level of caspase-8 activity during apoptosis versus the modest levels observed during cell growth. The caspase-8 paralogue c-FLIP is a good candidate for a molecular rheostat of caspase-8 activity. c-FLIP can inhibit death receptor-mediated apoptosis by competing with caspase-8 for recruitment to FADD. However, full-length c-FLIPL can also heterodimerize with caspase-8 independent of death receptor ligation and activate caspase-8 via an activation loop in the C terminus of c-FLIPL. This triggers cleavage of c-FLIPL at Asp-376 by caspase-8 to produce p43FLIP. The continued function of p43FLIP has, however, not been determined. We demonstrate that acute deletion of endogenous c-FLIP in murine effector T cells results in loss of caspase-8 activity and cell death. The lethality and caspase-8 activity can both be rescued by the transgenic expression of p43FLIP. Furthermore, p43FLIP associates with Raf1, TRAF2, and RIPK1, which augments ERK and NF-κB activation, IL-2 production, and T cell proliferation. Thus, not only is c-FLIP the initiator of caspase-8 activity during T cell activation, it is also an initial caspase-8 substrate, with cleaved p43FLIP serving to both stabilize caspase-8 activity and promote activation of pathways involved with T cell growth.

Fas is a cell surface molecule that is expressed on a wide array of cell types and triggers apoptosis. While in most situations Fas ligation activates programmed cell death, on resting T lymphocytes it can co-stimulate proliferation with the T cell receptor (TCR)/CD3 complex. This incongruity suggests that Fas may elicit signaling events that overlap with those used by proliferation cues. We observe that in the human T cell line Jurkat and in human peripheral blood lymphocytes, Fas stimulation does not signal by the Ras/Raf-1/mitogen-activated protein kinase (MAPK) pathway or by increased intracellular calcium. Rather, Fas ligation strongly activates Jun kinase (JNK). This activity, as well as Fas-induced apoptosis, is blocked by increased levels of cAMP. The balance between proliferation and apoptosis by Fas triggering of T lymphocytes may therefore reflect a signaling ratio between TCR activation of the Ras/Raf-1/MAPK pathway versus JNK activation by Fas.

Rab3A is a small, Ras-like GTPase expressed in neuroendocrine cells, in which it is associated with secretory vesicle membranes and regulates exocytosis. Using the yeast two-hybrid system, we have identified a rat brain cDNA encoding a novel 50-kDa protein, which we have named Rabin3, that interacts with Rab3A and Rab3D but not with other small GTPases (Rab3C, Rab2, Ran, or Ras). Several independent point mutations in the effector domain of Rab3A (F51L, V55E, and G56D) which do not alter nucleotide binding by the GTPase abolish the interaction with Rabin3, while another mutation (V52A) appears to increase the interaction. These results demonstrate that the interaction is highly specific. However, a glutathione S-transferase-Rabin3 fusion protein associates only weakly in vitro with recombinant Rab3A and possesses no detectable GTPase-activating protein or nucleotide exchange activity, and Rabin3 overexpressed in adrenal chromaffin cells has no observable effect on secretion. The protein possess a sequence characteristic of coiled-coil domains and a second small region with sequence similarity to a Saccharomyces cerevisiae protein, Sec2p, Sec2p is essential for constitutive secretion in yeast cells and interacts with Sec4p, a close relative of the Rab3A GTPase. Rabin3 mRNA and protein are widely expressed but are particularly abundant in testes.

Little is known regarding the function of γδ T cells, although they accumulate at sites of inflammation in infections and autoimmune disorders. We previously observed that γδ T cells in vitro are activated by Borrelia burgdorferi in a TLR2-dependent manner. We now observe that the activated γδ T cells can in turn stimulate dendritic cells in vitro to produce cytokines and chemokines that are important for the adaptive immune response. This suggested that in vivo γδ T cells may assist in activating the adaptive immune response. We examined this possibility in vivo and observed that γδ T cells are activated and expand in number during Borrelia infection, and this was reduced in the absence of TLR2. Furthermore, in the absence of γδ T cells, there was a significantly blunted response of adaptive immunity, as reflected in reduced expansion of T and B cells and reduced serum levels of anti- Borrelia antibodies, cytokines, and chemokines. This paralleled a greater Borrelia burden in γδ-deficient mice as well as more cardiac inflammation. These findings are consistent with a model of γδ T cells functioning to promote the adaptive immune response during infection.

γδ T cells function between the innate and adaptive immune responses, promoting antigen-presenting cell function and manifesting cytolytic activity. Their numbers often increase during infections, such as human immunodeficiency virus, and at sites of chronic inflammation. However, the turnover dynamics of human γδ T cells are poorly understood. Here we observed that despite more rapid proliferation in vitro by human Lyme arthritis synovial γδ T cells of the Vδ1 subset, they have reduced surviving cell numbers compared with αβ T cells because of increased cell death by the γδ T cells. Because caspases are involved in cell proliferation and death, and because signaling is more efficient through T cell receptor (TCR)-γδ than through TCR-αβ, we examined the levels of active caspases during cell cycling and following TCR restimulation. We observed higher overall caspase activity in Borrelia-reactive γδ T cells than in comparable αβ T cells. This was paralleled by greater spontaneous cell death and TCR restimulation-induced cell death of the γδ T cells, which was caspase dependent. Our current findings thus are consistent with a model in which human γδ T cells evolved to function quickly and transiently in an innate fashion.

Evolving models of immune tolerance have challenged the view that the response of the maternal immune system to environmental or fetal antigens must be suppressed or deviated. CD8 T cells play a central role in the immune response to viruses and intracellular pathogens so the maintenance of both the number and function of these cells is critical to protect both the mother and fetus. We show that the numbers of maternal CD8 T cells in both the spleen and the uterine draining lymph nodes are transiently increased at mid-gestation and this correlates with enhanced CD8 T-cell proliferation and an increased relative expression of both pro-survival and pro-apoptotic molecules. In transgenic mice bearing T-cell antigen receptors specific for the male HY or allo-antigens, the transgenic CD8 T cells retain the ability to proliferate and function during pregnancy. Moreover, anti-HY T-cell receptor transgenic mice have normal numbers of male pups despite the presence of CD8 T cells at the maternal–fetal interface. These data suggest that pregnancy is a dynamic state in which CD8 T-cell turnover is increased while the function and ending size of the CD8 T-cell compartment are maintained.

Pregnancy induces dynamic changes in the maternal environment that include reversible modifications in response to systemic mediators and local signals. The spleen can be used to determine the effects of pregnancy on multiple cellular populations, including those of the erythroid lineage and the immune system. Current evidence suggests that the transient increase in the size of the spleen during pregnancy is due to the expansion of erythroid precursors. However, it is unclear what factors contribute to this increase. Moreover, the additional erythroid cells may compete with neighboring leukocytes for growth factors or space, and this may in turn alter the function of these populations. Therefore, we assessed proliferation and apoptosis throughout gestation using in vivo bromodeoxyuridine incorporation and the TUNEL assay, respectively. Here, we show that erythroid-lineage TER-119(+) cells expanded significantly in midgestation because of enhanced proliferation and diminished apoptosis. This correlated with increased expression of the erythropoietin receptor (Epor) and decreased expression of the death receptor Fas, respectively. Leukocytes demonstrated population-specific responses. Natural killer cells proliferated in early pregnancy. Both lymphocytes and CD11B(+) cells underwent enhanced proliferation during midgestation. In contrast, neutrophils exhibited augmented proliferation throughout pregnancy. These subset-specific alterations in proliferation and death in the spleen suggest that complex regulation of population dynamics exists during pregnancy.

Activation of the innate immune system typically precedes engagement of adaptive immunity. Cells at the interface between these two arms of the immune response are thus critical to provide full engagement of host defense. Among the innate T cells at this interface are γδ T cells. γδ T cells contribute to the defense from a variety of infectious organisms, yet little is understood regarding how they are activated. We have previously observed that human γδ T cells of the Vδ1 subset accumulate in inflamed joints in Lyme arthritis and proliferate in response to stimulation with the causative spirochete, Borrelia burgdorferi. We now observe that murine γδ T cells are also activated by B. burgdorferi and that in both cases the activation is indirect via TLR stimulation on dendritic cells or monocytes. Furthermore, B. burgdorferi stimulation of monocytes via TLR, and secondary activation of γδ T cells, are both caspase-dependent.