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The T cell-associated CD28 molecule plays a key role in T cell co-stimulation. Its ligation induces the tyrosine phosphorylation of numerous proteins including CD28 itself as well as a restricted set of substrates of 97 and 62-68 kDa which are poorly phosphorylated by the tyrosine kinases induced by CD3-TCR triggering. In this study, we identify these substrates as the product of the vav proto-oncogene and as a 62 kDa protein that could correspond at least in part to p62dok the 62 kDa adaptor molecule associated to p120 Ras-GTPase activating protein. Both p97vav and p62 are tyrosine phosphorylated upon CD28 ligation by mAb or by its counter-receptor B7-1/CD80. Using CD28 mutants, we also show that Vav and p62 tyrosine phosphorylation is regulated by distinct domains within the CD28 cytoplasmic tail: residues 173-181 for Vav and residues 182-202 for P62. Finally, the phosphorylation of Vav and p62 does not require an intact binding site for Grb-2 or p85 SH2 domains. We thus demonstrate that the CD28 cytoplasmic domain contains at least three functionally independent regions involved in CD28-induced signal transduction, since in addition to the Grb-2 and p85 SH2 domain binding site (Tyr173), residues 173-181 and 182 202 are associated with Vav and p62 tyrosine phosphorylation respectively. [ABSTRACT FROM PUBLISHER]

Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications., Competing Interests: AB, LS, LD, AP, FS, EH, CE, HJ, HY, GF, DC No competing interests declared, EG Now works for Boehringer Ingelheim Pharma GmbH & Co KG, MP Board member of Tartan Cell Technologies Ltd, JS Board member of Tartan Cell Technologies Ltd and Glencoe Software Ltd, AL Board member of Tartan Cell Technologies Ltd and Platinum Informatics Ltd, (© 2024, Brenes et al.)

Background: Neutrophils are important in the pathophysiology of coronavirus disease 2019 (COVID-19), but the molecular changes contributing to altered neutrophil phenotypes following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection are not fully understood. We used quantitative mass spectrometry-based proteomics to explore neutrophil phenotypes immediately following acute SARS-CoV-2 infection and during recovery., Methods: Prospective observational study of hospitalised patients with PCR-confirmed SARS-CoV-2 infection (May to December 2020). Patients were enrolled within 96 h of admission, with longitudinal sampling up to 29 days. Control groups comprised non-COVID-19 acute lower respiratory tract infection (LRTI) and age-matched noninfected controls. Neutrophils were isolated from peripheral blood and analysed using mass spectrometry. COVID-19 severity and recovery were defined using the World Health Organization ordinal scale., Results: Neutrophil proteomes from 84 COVID-19 patients were compared to those from 91 LRTI and 42 control participants. 5800 neutrophil proteins were identified, with >1700 proteins significantly changed in neutrophils from COVID-19 patients compared to noninfected controls. Neutrophils from COVID-19 patients initially all demonstrated a strong interferon signature, but this signature rapidly declined in patients with severe disease. Severe disease was associated with increased abundance of proteins involved in metabolism, immunosuppression and pattern recognition, while delayed recovery from COVID-19 was associated with decreased granule components and reduced abundance of metabolic proteins, chemokine and leukotriene receptors, integrins and inhibitory receptors., Conclusions: SARS-CoV-2 infection results in the sustained presence of circulating neutrophils with distinct proteomes suggesting altered metabolic and immunosuppressive profiles and altered capacities to respond to migratory signals and cues from other immune cells, pathogens or cytokines., Competing Interests: Conflict of interest: H.R. Keir has received speaker fees from Insmed. A. Shoemark has received grant funding from AstraZeneca. A.J. Brenes has received support for attending meetings from the British Society for Proteome research. J.D. Chalmers has received research grants from AstraZeneca, Boehringer Ingelheim, GlaxoSmithKline, Gilead Sciences, Grifols, Novartis, Insmed and Trudell, and received consultancy or speaker fees from Antabio, AstraZeneca, Boehringer Ingelheim, Chiesi, GlaxoSmithKline, Insmed, Janssen, Novartis, Pfizer, Trudell and Zambon. All other authors declare no conflicts of interest., (Copyright ©The authors 2024.)

Rationale: Chronic obstructive pulmonary disease (COPD) is a disease characterized by persistent airway inflammation and disordered macrophage function. The extent to which alterations in macrophage bioenergetics contribute to impaired antioxidant responses and disease pathogenesis has yet to be fully delineated. Objectives: Through the study of COPD alveolar macrophages (AMs) and peripheral monocyte-derived macrophages (MDMs), we sought to establish if intrinsic defects in core metabolic processes drive macrophage dysfunction and redox imbalance. Methods: AMs and MDMs from donors with COPD and healthy donors underwent functional, metabolic, and transcriptional profiling. Measurements and Main Results: We observed that AMs and MDMs from donors with COPD display a critical depletion in glycolytic- and mitochondrial respiration-derived energy reserves and an overreliance on glycolysis as a source for ATP, resulting in reduced energy status. Defects in oxidative metabolism extend to an impaired redox balance associated with defective expression of the NADPH-generating enzyme, ME1 (malic enzyme 1), a known target of the antioxidant transcription factor NRF2 (nuclear factor erythroid 2-related factor 2). Consequently, selective activation of NRF2 resets the COPD transcriptome, resulting in increased generation of TCA cycle intermediaries, improved energetic status, favorable redox balance, and recovery of macrophage function. Conclusions: In COPD, an inherent loss of metabolic plasticity leads to metabolic exhaustion and reduced redox capacity, which can be rescued by activation of the NRF2 pathway. Targeting these defects, via NRF2 augmentation, may therefore present an attractive therapeutic strategy for the treatment of the aberrant airway inflammation described in COPD.

Anticancer T cells acquire a dysfunctional state characterized by poor effector function and expression of inhibitory receptors, such as PD-1. Blockade of PD-1 leads to T cell reinvigoration and is increasingly applied as an effective anticancer treatment. Recent work challenged the commonly held view that the phosphatase PTPN11 (known as SHP-2) is essential for PD-1 signaling in T cells, suggesting functional redundancy with the homologous phosphatase PTPN6 (SHP-1). Therefore, we investigated the effect of concomitant Ptpn6 and Ptpn11 deletion in T cells on their ability to mount antitumour responses. In vivo data show that neither sustained nor acute Ptpn6/11 deletion improves T cell-mediated tumor control. Sustained loss of Ptpn6/11 also impairs the therapeutic effects of anti-PD1 treatment. In vitro results show that Ptpn6/11-deleted CD8 + T cells exhibit impaired expansion due to a survival defect and proteomics analyses reveal substantial alterations, including in apoptosis-related pathways. These data indicate that concomitant ablation of Ptpn6/11 in polyclonal T cells fails to improve their anticancer properties, implying that caution shall be taken when considering their inhibition for immunotherapeutic approaches., (© 2022 The Authors. Published under the terms of the CC BY 4.0 license.)

Bach2 codes for a transcriptional regulator exerting major influences on T cell-mediated immune regulation. Effector CTLs derived from in vitro activation of murine CD8 + T cells showed increased proliferative and cytolytic capacity in the absence of BACH2. Before activation, BACH2-deficient splenic CD8 + T cells had a higher abundance of memory and reduced abundance of naïve cells compared to wild-type. CTLs derived from central memory T cells were more potently cytotoxic than those derived from naïve T cells, but even within separated subsets, BACH2-deficiency conferred a cytotoxic advantage. Immunofluorescence and electron microscopy revealed larger granules in BACH2-deficient compared to wild-type CTLs, and proteomic analysis showed an increase in granule content, including perforin and granzymes. Thus, the enhanced cytotoxicity observed in effector CTLs lacking BACH2 arises not only from differences in their initial differentiation state but also inherent production of enlarged cytolytic granules. These results demonstrate how a single gene deletion can produce a CTL super-killer., (© 2022 The Authors. European Journal of Immunology published by Wiley-VCH GmbH.)

T cell activation, proliferation, and differentiation into effector and memory states involve massive remodeling of T cell size and molecular content and create a massive increase in demand for energy and amino acids. Protein synthesis is an energy- and resource-demanding process; as such, changes in T cell energy production are intrinsically linked to proteome remodeling. In this review, we discuss how protein synthesis and degradation change over the course of a T cell immune response and the crosstalk between these processes and T cell energy metabolism. We highlight how the use of high-resolution mass spectrometry to analyze T cell proteomes can improve our understanding of how these processes are regulated., (© 2021. The Author(s).)

To overcome oxidative, inflammatory, and metabolic stress, cells have evolved cytoprotective protein networks controlled by nuclear factor-erythroid 2 p45-related factor 2 (Nrf2) and its negative regulator, Kelch-like ECH associated protein 1 (Keap1). Here, using high-resolution mass spectrometry we characterize the proteomes of macrophages with altered Nrf2 status revealing significant differences among the genotypes in metabolism and redox homeostasis, which were validated with respirometry and metabolomics. Nrf2 affected the proteome following lipopolysaccharide (LPS) stimulation, with alterations in redox, carbohydrate and lipid metabolism, and innate immunity. Notably, Nrf2 activation promoted mitochondrial fusion. The Keap1 inhibitor, 4-octyl itaconate remodeled the inflammatory macrophage proteome, increasing redox and suppressing type I interferon (IFN) response. Similarly, pharmacologic or genetic Nrf2 activation inhibited the transcription of IFN-β and its downstream effector IFIT2 during LPS stimulation. These data suggest that Nrf2 activation facilitates metabolic reprogramming and mitochondrial adaptation, and finetunes the innate immune response in macrophages., Competing Interests: A.T.D.K. is a member of the scientific advisory board of Evgen Pharma., (© 2022 The Author(s).)

T cell receptor activation of naïve CD8 + T lymphocytes initiates their maturation into effector cytotoxic T lymphocytes (CTLs), which can kill cancer and virally infected cells. Although CTLs show an increased reliance on glycolysis upon acquisition of effector function, we found an essential requirement for mitochondria in target cell–killing. Acute mitochondrial depletion in USP30 (ubiquitin carboxyl-terminal hydrolase 30)–deficient CTLs markedly diminished killing capacity, although motility, signaling, and secretion were all intact. Unexpectedly, the mitochondrial requirement was linked to mitochondrial translation, inhibition of which impaired CTL killing. Impaired mitochondrial translation triggered attenuated cytosolic translation, precluded replenishment of secreted killing effectors, and reduced the capacity of CTLs to carry out sustained killing. Thus, mitochondria emerge as a previously unappreciated homeostatic regulator of protein translation required for serial CTL killing.

Metabolic and nutrient-sensing pathways play an important role in controlling the efficacy of effector T cells. Oxygen is a critical regulator of cellular metabolism. However, during immune responses T cells must function in oxygen-deficient, or hypoxic, environments. Here, we used high resolution mass spectrometry to investigate how the proteome of primary murine CD8 + cytotoxic T lymphocytes (CTLs) is reconfigured in response to hypoxia in vitro . We identified and quantified over 7,600 proteins and discovered that hypoxia increased the abundance of a selected number of proteins in CTLs. This included glucose transporters, metabolic enzymes, transcription factors, cytolytic effector molecules, checkpoint receptors and adhesion molecules. While some of these proteins may augment the effector functions of CTLs, others may limit their cytotoxicity. Moreover, we determined that hypoxia could inhibit IL-2-induced proliferation cues and antigen-induced pro-inflammatory cytokine production in CTLs. These data provide a comprehensive resource for understanding the magnitude of the CTL response to hypoxia and emphasise the importance of oxygen-sensing pathways for controlling CD8 + T cells. Additionally, this study provides new understanding about how hypoxia may promote the effector function of CTLs, while contributing to their dysfunction in some contexts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ross, Rollings and Cantrell.)

Phosphoinositide 3-kinase p110 delta (PI3K p110δ) is pivotal for CD8 + T cell immune responses. The current study explores PI3K p110δ induction and repression of antigen receptor and cytokine regulated programs to inform how PI3K p110δ directs CD8 + T cell fate. The studies force a revision of the concept that PI3K p110δ controls metabolic pathways in T cells and reveal major differences in PI3K p110δ regulated transcriptional programs between naïve and effector cytotoxic T cells (CTL). These differences include differential control of the expression of cytolytic effector molecules and costimulatory receptors. Key insights from the work include that PI3K p110δ signalling pathways repress expression of the critical inhibitory receptors CTLA4 and SLAMF6 in CTL. Moreover, in both naïve and effector T cells the dominant role for PI3K p110δ is to restrain the production of the chemokines that orchestrate communication between adaptive and innate immune cells. The study provides a comprehensive resource for understanding how PI3K p110δ uses multiple processes mediated by Protein Kinase B/AKT, FOXO1 dependent and independent mechanisms and mitogen-activated protein kinases (MAPK) to direct CD8 + T cell fate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Spinelli, Marchingo, Nomura, Damasio and Cantrell.)

Background: Acute respiratory distress syndrome (ARDS) is a severe critical condition with a high mortality that is currently in focus given that it is associated with mortality caused by coronavirus disease 2019 (COVID-19). Neutrophils play a key role in the lung injury characteristic of non-COVID-19 ARDS and there is also accumulating evidence of neutrophil mediated lung injury in patients who succumb to infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: We undertook a functional proteomic and metabolomic survey of circulating neutrophil populations, comparing patients with COVID-19 ARDS and non-COVID-19 ARDS to understand the molecular basis of neutrophil dysregulation. Results: Expansion of the circulating neutrophil compartment and the presence of activated low and normal density mature and immature neutrophil populations occurs in ARDS, irrespective of cause. Release of neutrophil granule proteins, neutrophil activation of the clotting cascade and upregulation of the Mac-1 platelet binding complex with formation of neutrophil platelet aggregates is exaggerated in COVID-19 ARDS. Importantly, activation of components of the neutrophil type I interferon responses is seen in ARDS following infection with SARS-CoV-2, with associated rewiring of neutrophil metabolism, and the upregulation of antigen processing and presentation. Whilst dexamethasone treatment constricts the immature low density neutrophil population, it does not impact upon prothrombotic hyperinflammatory neutrophil signatures. Conclusions: Given the crucial role of neutrophils in ARDS and the evidence of a disordered myeloid response observed in COVID-19 patients, this work maps the molecular basis for neutrophil reprogramming in the distinct clinical entities of COVID-19 and non-COVID-19 ARDS., Competing Interests: No competing interests were disclosed., (Copyright: © 2021 Reyes L et al.)

Limiting dysfunctional neutrophilic inflammation while preserving effective immunity requires a better understanding of the processes that dictate neutrophil function in the tissues. Quantitative mass-spectrometry identified how inflammatory murine neutrophils regulated expression of cell surface receptors, signal transduction networks, and metabolic machinery to shape neutrophil phenotypes in response to hypoxia. Through the tracing of labeled amino acids into metabolic enzymes, proinflammatory mediators, and granule proteins, we demonstrated that ongoing protein synthesis shapes the neutrophil proteome. To maintain energy supplies in the tissues, neutrophils consumed extracellular proteins to fuel central carbon metabolism. The physiological stresses of hypoxia and hypoglycemia, characteristic of inflamed tissues, promoted this extracellular protein scavenging with activation of the lysosomal compartment, further driving exploitation of the protein-rich inflammatory milieu. This study provides a comprehensive map of neutrophil proteomes, analysis of which has led to the identification of active catabolic and anabolic pathways that enable neutrophils to sustain synthetic and effector functions in the tissues.

The integration of multiple signalling pathways that co-ordinate T cell metabolism and transcriptional reprogramming is required to drive T cell differentiation and proliferation. One key T cell signalling module is mediated by extracellular signal-regulated kinases (ERKs) which are activated in response to antigen receptor engagement. The activity of ERKs is often used to report antigen receptor occupancy but the full details of how ERKs control T cell activation is not understood. Accordingly, we have used mass spectrometry to explore how ERK signalling pathways control antigen receptor driven proteome restructuring in CD8+ T cells to gain insights about the biological processes controlled by ERKs in primary lymphocytes. Quantitative analysis of >8000 proteins identified 900 ERK regulated proteins in activated CD8+ T cells. The data identify both positive and negative regulatory roles for ERKs during T cell activation and reveal that ERK signalling primarily controls the repertoire of transcription factors, cytokines and cytokine receptors expressed by activated T cells. It was striking that a large proportion of the proteome restructuring that is driven by triggering of the T cell antigen receptor is not dependent on ERK activation. However, the selective targets of the ERK signalling module include the critical effector molecules and the cytokines that allow T cell communication with other immune cells to mediate adaptive immune responses., (© 2021 The Author(s).)

Personalized medicines require understanding the molecular causes of disease. In this issue of Immunity, Gruber et al. reveal that a gain-of-function JAK1 genetic variant results in a mutant protein with mosaic expression that drives multi-organ immune dysregulation via kinase dependent and independent mechanisms. The work highlights how biochemistry can inform therapies to resolve complex immune disorders., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Assays to monitor the metabolic state or nutrient uptake capacity of immune cells at a single cell level are increasingly in demand. One assay, used by many immunologists, employs 2-( N -(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG), a fluorescent analogue of 2-deoxyglucose (2DG), as a substrate for glucose transporters. This molecule has been validated as a substrate for the glucose transporter Glut2 (Slc2a2) in mammalian cells but 2-NDBG selectivity for the glucose transporters expressed by T cells, Glut1 (Slc2a1) and Glut3 (Slc2a3), has never been explored. Nor has the possibility that 2-NBDG might bind to T cells that do not express glucose transporters been assessed. In this technical commentary we interrogate the specificity of 2-NBBG labelling as a readout for glucose transport in T lymphocytes. We compare flow cytometric 2-NBDG staining against well validated radiolabelled glucose transport assays in murine T cells. Our data show there can be a large discordance between glucose transport capacity and 2-NBDG labelling in T cells. We also find that 2-NBDG uptake into murine T cells is not inhibited by competitive substrates or facilitative glucose transporter inhibitors, nor can 2-NBDG competitively block glucose uptake in T cells. Collectively, these data argue that 2-NBDG uptake alone is not a reliable tool for the assessment of cellular glucose transport capacity., Competing Interests: Conflicts of Interest The authors declare that they have no competing interests.

T cell expansion and differentiation are critically dependent on the transcription factor c-Myc (Myc). Herein we use quantitative mass-spectrometry to reveal how Myc controls antigen receptor driven cell growth and proteome restructuring in murine T cells. Analysis of copy numbers per cell of >7000 proteins provides new understanding of the selective role of Myc in controlling the protein machinery that govern T cell fate. The data identify both Myc dependent and independent metabolic processes in immune activated T cells. We uncover that a primary function of Myc is to control expression of multiple amino acid transporters and that loss of a single Myc-controlled amino acid transporter effectively phenocopies the impact of Myc deletion. This study provides a comprehensive map of how Myc selectively shapes T cell phenotypes, revealing that Myc induction of amino acid transport is pivotal for subsequent bioenergetic and biosynthetic programs and licences T cell receptor driven proteome reprogramming., Competing Interests: JM, LS, AH, DC No competing interests declared, (© 2020, Marchingo et al.)

Quantitative mass spectrometry reveals how CD4 + and CD8 + T cells restructure proteomes in response to antigen and mammalian target of rapamycin complex 1 (mTORC1). Analysis of copy numbers per cell of >9,000 proteins provides new understanding of T cell phenotypes, exposing the metabolic and protein synthesis machinery and environmental sensors that shape T cell fate. We reveal that lymphocyte environment sensing is controlled by immune activation, and that CD4 + and CD8 + T cells differ in their intrinsic nutrient transport and biosynthetic capacity. Our data also reveal shared and divergent outcomes of mTORC1 inhibition in naïve versus effector T cells: mTORC1 inhibition impaired cell cycle progression in activated naïve cells, but not effector cells, whereas metabolism was consistently impacted in both populations. This study provides a comprehensive map of naïve and effector T cell proteomes, and a resource for exploring and understanding T cell phenotypes and cell context effects of mTORC1.

AMPK acts downstream of the tumor suppressor LKB1, yet its role in cancer has been controversial. AMPK is activated by biguanides, such as metformin and phenformin, and metformin use in diabetics has been associated with reduced cancer risk. However, whether this is mediated by cell-autonomous AMPK activation within tumor progenitor cells has been unclear. We report that T-cell-specific loss of AMPK-α1 caused accelerated growth of T cell acute lymphoblastic leukemia/lymphoma (T-ALL) induced by PTEN loss in thymic T cell progenitors. Oral administration of phenformin, but not metformin, delayed onset and growth of lymphomas, but only when T cells expressed AMPK-α1. This differential effect of biguanides correlated with detection of phenformin, but not metformin, in thymus. Phenformin also enhanced apoptosis in T-ALL cells both in vivo and in vitro. Thus, AMPK-α1 can be a cell-autonomous tumor suppressor in the context of T-ALL, and phenformin may have potential for the prevention of some cancers., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

Immune activated T lymphocytes modulate the activity of key metabolic pathways to support the transcriptional reprograming and reshaping of cell proteomes that permits effector T cell differentiation. The present study uses high resolution mass spectrometry and metabolic labelling to explore how murine T cells control the methionine cycle to produce methyl donors for protein and nucleotide methylations. We show that antigen receptor engagement controls flux through the methionine cycle and RNA and histone methylations. We establish that the main rate limiting step for protein synthesis and the methionine cycle is control of methionine transporter expression. Only T cells that respond to antigen to upregulate and sustain methionine transport are supplied with methyl donors that permit the dynamic nucleotide methylations and epigenetic reprogramming that drives T cell differentiation. These data highlight how the regulation of methionine transport licenses use of methionine for multiple fundamental processes that drive T lymphocyte proliferation and differentiation., Competing Interests: LS, AH, AB, LS, JH, AM, XL, ST, PT, JR, JL, AL, DC No competing interests declared, (© 2019, Sinclair et al.)

Natural killer (NK) cells are lymphocytes with important anti-tumour functions. Cytokine activation of NK cell glycolysis and oxidative phosphorylation (OXPHOS) are essential for robust NK cell responses. However, the mechanisms leading to this metabolic phenotype are unclear. Here we show that the transcription factor cMyc is essential for IL-2/IL-12-induced metabolic and functional responses in mice. cMyc protein levels are acutely regulated by amino acids; cMyc protein is lost rapidly when glutamine is withdrawn or when system L-amino acid transport is blocked. We identify SLC7A5 as the predominant system L-amino acid transporter in activated NK cells. Unlike other lymphocyte subsets, glutaminolysis and the tricarboxylic acid cycle do not sustain OXPHOS in activated NK cells. Glutamine withdrawal, but not the inhibition of glutaminolysis, results in the loss of cMyc protein, reduced cell growth and impaired NK cell responses. These data identify an essential role for amino acid-controlled cMyc for NK cell metabolism and function.

The tryptophan metabolite kynurenine has critical immunomodulatory properties and can function as an aryl hydrocarbon receptor (AHR) ligand. Here we show that the ability of T cells to transport kynurenine is restricted to cells activated by the T-cell antigen receptor or proinflammatory cytokines. Kynurenine is transported across the T-cell membrane by the System L transporter SLC7A5. Accordingly, the ability of kynurenine to activate the AHR is restricted to T cells that express SLC7A5. We use the fluorescence spectral properties of kynurenine to develop a flow cytometry-based assay for rapid, sensitive and quantitative measurement of the kynurenine transport capacity in a single cell. Our findings provide a method to assess the susceptibility of T cells to kynurenine, and a sensitive single cell assay to monitor System L amino acid transport.

The discovery of interleukin-2 (IL-2) changed the molecular understanding of how the immune system is controlled. IL-2 is a pleiotropic cytokine, and dissecting the signaling pathways that allow IL-2 to control the differentiation and homeostasis of both pro- and anti-inflammatory T cells is fundamental to determining the molecular details of immune regulation. The IL-2 receptor couples to JAK tyrosine kinases and activates the STAT5 transcription factors. However, IL-2 does much more than control transcriptional programs; it is a key regulator of T cell metabolic programs. The development of global phosphoproteomic approaches has expanded the understanding of IL-2 signaling further, revealing the diversity of phosphoproteins that may be influenced by IL-2 in T cells. However, it is increasingly clear that within each T cell subset, IL-2 will signal within a framework of other signal transduction networks that together will shape the transcriptional and metabolic programs that determine T cell fate.

Interleukin-2 (IL-2) and Janus kinases (JAKs) regulate transcriptional programs and protein synthesis to promote the differentiation of effector CD8 + cytotoxic T lymphocytes (CTLs). Using high-resolution mass spectrometry, we generated an in-depth characterization of how IL-2 and JAKs configure the CTL proteome to control CTL function. We found that IL-2 signaling through JAK1 and JAK3 (JAK1/3) increased the abundance of a key subset of proteins to induce the accumulation of critical cytokines and effector molecules in T cells. Moreover, IL-2 maintained the concentration of proteins that support core metabolic processes essential for cellular fitness. One fundamental insight was the dominant role for IL-2 in stimulating effector T cells to detect microenvironmental cues. IL-2-JAK1/3 signaling pathways thus increased the abundance of nutrient transporters, nutrient sensors, and critical oxygen-sensing molecules. These data provide key insights into how IL-2 promotes T cell function and highlight signaling mechanisms and transcription factors that integrate oxygen sensing to transcriptional control of CD8 + T cell differentiation., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

This study explores the regulation and importance of System L amino acid transport in a murine model of T-cell acute lymphoblastic leukemia (T-ALL) caused by deletion of phosphatase and tensin homolog deleted on chromosome 10 (PTEN). There has been a strong focus on glucose transport in leukemias but the present data show that primary T-ALL cells have increased transport of multiple nutrients. Specifically, increased leucine transport in T-ALL fuels mammalian target of rapamycin complex 1 (mTORC1) activity which then sustains expression of hypoxia inducible factor-1α (HIF1α) and c-Myc; drivers of glucose metabolism in T cells. A key finding is that PTEN deletion and phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P 3 ) accumulation is insufficient to initiate leucine uptake, mTORC1 activity, HIF1α or c-Myc expression in T cells and hence cannot drive T-ALL metabolic reprogramming. Instead, a key regulator for leucine transport in T-ALL is identified as NOTCH. Mass spectrometry based proteomics identifies SLC7A5 as the predominant amino acid transporter in primary PTEN -/- T-ALL cells. Importantly, expression of SLC7A5 is critical for the malignant transformation induced by PTEN deletion. These data reveal the importance of regulated amino acid transport for T-cell malignancies, highlighting how a single amino acid transporter can have a key role.

Chemical strategies to using small molecules to stimulate hypoxia inducible factors (HIFs) activity and trigger a hypoxic response under normoxic conditions, such as iron chelators and inhibitors of prolyl hydroxylase domain (PHD) enzymes, have broad-spectrum activities and off-target effects. Here we disclose VH298, a potent VHL inhibitor that stabilizes HIF-α and elicits a hypoxic response via a different mechanism, that is the blockade of the VHL:HIF-α protein-protein interaction downstream of HIF-α hydroxylation by PHD enzymes. We show that VH298 engages with high affinity and specificity with VHL as its only major cellular target, leading to selective on-target accumulation of hydroxylated HIF-α in a concentration- and time-dependent fashion in different cell lines, with subsequent upregulation of HIF-target genes at both mRNA and protein levels. VH298 represents a high-quality chemical probe of the HIF signalling cascade and an attractive starting point to the development of potential new therapeutics targeting hypoxia signalling.

The advances in genetics and biochemistry that have taken place over the last 10 years led to significant advances in experimental and clinical immunology. In turn, this has led to the development of new mathematical models to investigate qualitatively and quantitatively various open questions in immunology. In this study we present a review of some research areas in mathematical immunology that evolved over the last 10 years. To this end, we take a step-by-step approach in discussing a range of models derived to study the dynamics of both the innate and immune responses at the molecular, cellular and tissue scales. To emphasise the use of mathematics in modelling in this area, we also review some of the mathematical tools used to investigate these models. Finally, we discuss some future trends in both experimental immunology and mathematical immunology for the upcoming years.

Interleukin-2 (IL-2) is a fundamental cytokine that controls proliferation and differentiation of T cells. Here, we used high-resolution mass spectrometry to generate a comprehensive and detailed map of IL-2 protein phosphorylations in cytotoxic T cells (CTL). The data revealed that Janus kinases (JAKs) couple IL-2 receptors to the coordinated phosphorylation of transcription factors, regulators of chromatin, mRNA translation, GTPases, vesicle trafficking, and the actin and microtubule cytoskeleton. We identified an IL-2-JAK-independent SRC family Tyr-kinase-controlled signaling network that regulates ∼10% of the CTL phosphoproteome, the production of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), and the activity of the serine/threonine kinase AKT. These data reveal a signaling framework wherein IL-2-JAK-controlled pathways coordinate with IL-2-independent networks of kinase activity and provide a resource toward the further understanding of the networks of protein phosphorylation that program CTL fate., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Sustained glucose and glutamine transport are essential for activated T lymphocytes to support ATP and macromolecule biosynthesis. We found that glutamine and glucose also fuel an indispensable dynamic regulation of intracellular protein O-GlcNAcylation at key stages of T cell development, transformation and differentiation. Glucose and glutamine are precursors of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a substrate for cellular glycosyltransferases. Immune-activated T cells contained higher concentrations of UDP-GlcNAc and increased intracellular protein O-GlcNAcylation controlled by the enzyme O-linked-β-N-acetylglucosamine (O-GlcNAc) glycosyltransferase as compared with naive cells. We identified Notch, the T cell antigen receptor and c-Myc as key controllers of T cell protein O-GlcNAcylation via regulation of glucose and glutamine transport. Loss of O-GlcNAc transferase blocked T cell progenitor renewal, malignant transformation and peripheral T cell clonal expansion. Nutrient-dependent signaling pathways regulated by O-GlcNAc glycosyltransferase are thus fundamental for T cell biology., Competing Interests: statement The authors have no competing interests, or other interests that might be perceived to influence the results and discussion reported in this paper.

We used high-resolution mass spectrometry to map the cytotoxic T lymphocyte (CTL) proteome and the effect of the metabolic checkpoint kinase mTORC1 on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes, and mTORC1 selectively repressed and promoted expression of a subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for negative control of the production of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) by mTORC1 in CTLs. mTORC1 repressed PtdIns(3,4,5)P3 production and determined the requirement for mTORC2 in activation of the kinase Akt. Our unbiased proteomic analysis thus provides comprehensive understanding of CTL identity and the control of CTL function by mTORC1.

Myc controls the metabolic reprogramming that supports effector T cell differentiation. The expression of Myc is regulated by the T cell antigen receptor (TCR) and pro-inflammatory cytokines such as interleukin-2 (IL-2). We now show that the TCR is a digital switch for Myc mRNA and protein expression that allows the strength of the antigen stimulus to determine the frequency of T cells that express Myc. IL-2 signalling strength also directs Myc expression but in an analogue process that fine-tunes Myc quantity in individual cells via post-transcriptional control of Myc protein. Fine-tuning Myc matters and is possible as Myc protein has a very short half-life in T cells due to its constant phosphorylation by glycogen synthase kinase 3 (GSK3) and subsequent proteasomal degradation. We show that Myc only accumulates in T cells exhibiting high levels of amino acid uptake allowing T cells to match Myc expression to biosynthetic demands. The combination of digital and analogue processes allows tight control of Myc expression at the population and single cell level during immune responses., (© 2015 The Authors. Published under the terms of the CC BY 4.0 license.)

Infection with hepatitis B virus (HBV) results in disparate degrees of tissue injury: the virus can either replicate without pathological consequences or trigger immune-mediated necroinflammatory liver damage. We investigated the potential for myeloid-derived suppressor cells (MDSCs) to suppress T cell-mediated immunopathology in this setting. Granulocytic MDSCs (gMDSCs) expanded transiently in acute resolving HBV, decreasing in frequency prior to peak hepatic injury. In persistent infection, arginase-expressing gMDSCs (and circulating arginase) increased most in disease phases characterized by HBV replication without immunopathology, whilst L-arginine decreased. gMDSCs expressed liver-homing chemokine receptors and accumulated in the liver, their expansion supported by hepatic stellate cells. We provide in vitro and ex vivo evidence that gMDSCs potently inhibited T cells in a partially arginase-dependent manner. L-arginine-deprived T cells upregulated system L amino acid transporters to increase uptake of essential nutrients and attempt metabolic reprogramming. These data demonstrate the capacity of expanded arginase-expressing gMDSCs to regulate liver immunopathology in HBV infection.

T follicular helper (Tfh) cells are essential in the induction of high-affinity, class-switched antibodies. The differentiation of Tfh cells is a multi-step process that depends upon the co-receptor ICOS and the activation of phosphoinositide-3 kinase leading to the expression of key Tfh cell genes. We report that ICOS signaling inactivates the transcription factor FOXO1, and a Foxo1 genetic deletion allowed for generation of Tfh cells with reduced dependence on ICOS ligand. Conversely, enforced nuclear localization of FOXO1 inhibited Tfh cell development even though ICOS was overexpressed. FOXO1 regulated Tfh cell differentiation through a broad program of gene expression exemplified by its negative regulation of Bcl6. Final differentiation to germinal center Tfh cells (GC-Tfh) was instead FOXO1 dependent as the Foxo1(-/-) GC-Tfh cell population was substantially reduced. We propose that ICOS signaling transiently inactivates FOXO1 to initiate a Tfh cell contingency that is completed in a FOXO1-dependent manner., (Copyright © 2015 Elsevier Inc. All rights reserved.)

The focus of the present study was to characterize the phosphoproteome of cytotoxic T cells and to explore the role of the serine threonine kinase PKD2 (Protein Kinase D2) in the phosphorylation networks of this key lymphocyte population. We used Stable Isotope Labeling of Amino acids in Culture (SILAC) combined with phosphopeptide enrichment and quantitative mass-spectrometry to determine the impact of PKD2 loss on the cytotoxic T cells phosphoproteome. We identified 15,871 phosphorylations on 3505 proteins in cytotoxic T cells. 450 phosphosites on 281 proteins were down-regulated and 300 phosphosites on 196 proteins were up-regulated in PKD2 null cytotoxic T cells. These data give valuable new insights about the protein phosphorylation networks operational in effector T cells and reveal that PKD2 regulates directly and indirectly about 5% of the cytotoxic T-cell phosphoproteome. PKD2 candidate substrates identified in this study include proteins involved in two distinct biological functions: regulation of protein sorting and intracellular vesicle trafficking, and control of chromatin structure, transcription, and translation. In other cell types, PKD substrates include class II histone deacetylases such as HDAC7 and actin regulatory proteins such as Slingshot. The current data show these are not PKD substrates in primary T cells revealing that the functional role of PKD isoforms is different in different cell lineages., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Protein kinase D2 (PKD2) is a serine and threonine kinase that is activated in T cells by diacylglycerol and protein kinase C in response to stimulation of the T cell receptor (TCR) by antigen. We quantified the activation of PKD2 at the single-cell level and found that this kinase acts as a sensitive digital amplifier of TCR engagement, enabling CD8(+) T cells to match the production of inflammatory cytokines to the quality and quantity of TCR ligands. There was a digital response pattern of PKD2 activation in response to TCR engagement, such that increasing the concentration and potency of TCR ligands increased the number of cells that exhibited activated PKD2. However, for each cell that responded to TCR stimulation, the entire cellular pool of PKD2 (~400,000 molecules) was activated. Moreover, PKD2 acted as an amplification checkpoint for antigen-stimulated digital cytokine responses and translated the differential strength of TCR signaling to determine the number of naïve CD8(+) T cells that became effector cells. Together, these results provide insights into PKD family kinases and how they act digitally to amplify signaling networks controlled by the TCR.

The anti-diabetic drug metformin regulates T-cell responses to immune activation and is proposed to function by regulating the energy-stress-sensing adenosine-monophosphate-activated protein kinase (AMPK). However, the molecular details of how metformin controls T cell immune responses have not been studied nor is there any direct evidence that metformin acts on T cells via AMPK. Here, we report that metformin regulates cell growth and proliferation of antigen-activated T cells by modulating the metabolic reprogramming that is required for effector T cell differentiation. Metformin thus inhibits the mammalian target of rapamycin complex I signalling pathway and prevents the expression of the transcription factors c-Myc and hypoxia-inducible factor 1 alpha. However, the inhibitory effects of metformin on T cells did not depend on the expression of AMPK in T cells. Accordingly, experiments with metformin inform about the importance of metabolic reprogramming for T cell immune responses but do not inform about the importance of AMPK.

T lymphocyte proliferation and differentiation are controlled by signaling pathways initiated by the T cell antigen receptor. Here we explore how key serine-threonine kinases and their substrates mediate T cell signaling and coordinate T cell metabolism to meet the metabolic demands of participating in an immune response.

Krüppel-like factor 2 (KLF2) is a transcription factor that is highly expressed in quiescent T lymphocytes and downregulated in effector T cells. We now show that antigen receptor engagement downregulates KLF2 expression in a graded response determined by the affinity of T cell antigen receptor (TCR) ligand and the integrated activation of protein kinase B and the MAP kinases ERK1/2. The present study explores the importance of KLF2 downregulation and reveals that the loss of KLF2 controls a select portion of the CD8 effector T cell transcriptional program. In particular, KLF2 loss is required for CD8 T cells to express the inflammatory chemokine receptor CXCR3 and for maximum clonal expansion of T cells. KLF2 thus negatively controls the ability of CD8 T cells to respond to the CXCR3 ligand CXCL10. Strikingly, the KLF2 threshold for restraining expression of CXCR3 is very low and quite distinct to the KLF2 threshold for restraining T cell proliferation. KLF2 is thus an analogue (tunable) not a digital (on/off) cellular switch where the magnitude of KLF2 expression differentially modifies the T cell responses.

T lymphocytes must regulate nutrient uptake to meet the metabolic demands of an immune response. Here we show that the intracellular supply of large neutral amino acids (LNAAs) in T cells was regulated by pathogens and the T cell antigen receptor (TCR). T cells responded to antigen by upregulating expression of many amino-acid transporters, but a single System L ('leucine-preferring system') transporter, Slc7a5, mediated uptake of LNAAs in activated T cells. Slc7a5-null T cells were unable to metabolically reprogram in response to antigen and did not undergo clonal expansion or effector differentiation. The metabolic catastrophe caused by loss of Slc7a5 reflected the requirement for sustained uptake of the LNAA leucine for activation of the serine-threonine kinase complex mTORC1 and for expression of the transcription factor c-Myc. Control of expression of the System L transporter by pathogens is thus a critical metabolic checkpoint for T cells.

The adenosine monophosphate-activated protein kinase (AMPK) is activated by antigen receptor signals and energy stress in T cells. In many cell types, AMPK can maintain energy homeostasis and can enforce quiescence to limit energy demands. We consequently evaluated the importance of AMPK for controlling the transition of metabolically active effector CD8 T lymphocytes to the metabolically quiescent catabolic memory T cells during the contraction phase of the immune response. We show that AMPKα1 activates rapidly in response to the metabolic stress caused by glucose deprivation of CD8 cytotoxic T lymphocytes (CTLs). Moreover, AMPKα1 restrains mammalian target of rapamycin complex 1 activity under conditions of glucose stress. AMPKα1 activity is dispensable for proliferation and differentiation of CTLs. However, AMPKα1 is required for in vivo survival of CTLs following withdrawal of immune stimulation. AMPKα1(null) T cells also show a striking defect in their ability to generate memory CD8 T-cell responses during Listeria monocytogenes infection. These results show that AMPKα1 monitors energy stress in CTLs and controls CD8 T-cell memory., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)