The glutamate/aspartate transporter EAAT1 is crucial for T-cell acute lymphoblastic leukemia proliferation and survival.

T-cell acute lymphoblastic leukemia (T-ALL) is a cancer of the immune system. Approximately 20% of paediatric and 50% of adult T-ALL patients have refractory disease or relapse and die from the disease. To improve patient outcome new therapeutics are needed. With the aim to identify new therapeutic targets, we combined the analysis of T-ALL gene expression and metabolism to identify the metabolic adaptations that T-ALL cells exhibit. We found that glutamine uptake is essential for T-ALL proliferation. Isotope tracing experiments showed that glutamine fuels aspartate synthesis through the TCA cycle and that glutamine and glutamine-derived aspartate together supply three nitrogen atoms in purines and all but one atom in pyrimidine rings. We show that the glutamate-aspartate transporter EAAT1 (SLC1A3), which is normally expressed in the central nervous system, is crucial for glutamine conversion to aspartate and nucleotides and that T-ALL cell proliferation depends on EAAT1 function. Through this work, we identify EAAT1 as a novel therapeutic target for T-ALL treatment.

1,25-Dihydroxyvitamin D3 suppresses CD4+ T-cell effector functionality by inhibition of glycolysis.

In CD4+ T helper cells, the active form of vitamin D3 , 1,25-dihydroxyvitamin D3 (1,25D) suppresses production of inflammatory cytokines, including interferon-gamma (IFN-γ), but the mechanisms for this are not yet fully defined. In innate immune cells, response to 1,25D has been linked to metabolic reprogramming. It is unclear whether 1,25D has similar effects on CD4+ T cells, although it is known that antigen stimulation of these cells promotes an anabolic metabolic phenotype, characterized by high rates of aerobic glycolysis to support clonal expansion and effector cytokine expression. Here, we performed in-depth analysis of metabolic capacity and pathway usage, employing extracellular flux and stable isotope-based tracing approaches, in CD4+ T cells treated with 1,25D. We report that 1,25D significantly decreases rates of aerobic glycolysis in activated CD4+ T cells, whilst exerting a lesser effect on mitochondrial glucose oxidation. This is associated with transcriptional repression of Myc, but not repression of mTOR activity under these conditions. Consistent with the modest effect of 1,25D on mitochondrial activity, it also did not impact CD4+ T-cell mitochondrial mass or membrane potential. Finally, we demonstrate that inhibition of aerobic glycolysis by 1,25D substantially contributes to its immune-regulatory capacity in CD4+ T cells, since the suppression of IFN-γ expression was significantly blunted in the absence of aerobic glycolysis. 1,25-Dihydroxyvitamin D3 (1,25D) suppresses the production of inflammatory cytokines such as interferon-gamma (IFN-γ) by CD4+ T cells, but the underpinning mechanisms are not yet fully defined. Here, we identify that 1,25D inhibits aerobic glycolysis in activated CD4+ T cells, associated with decreased c-Myc expression. This mechanism appears to substantially contribute to the suppression of IFN-γ by 1,25D, since this is significantly blunted in the absence of aerobic glycolysis.

TNF-α signals through ITK-Akt-mTOR to drive CD4+ T cell metabolic reprogramming, which is dysregulated in rheumatoid arthritis.

Upon activation, T cells undergo metabolic reprogramming to meet the bioenergetic demands of clonal expansion and effector function. Because dysregulated T cell cytokine production and metabolic phenotypes coexist in chronic inflammatory disease, including rheumatoid arthritis (RA), we investigated whether inflammatory cytokines released by differentiating T cells amplified their metabolic changes. We found that tumor necrosis factor-α (TNF-α) released by human naïve CD4+ T cells upon activation stimulated the expression of a metabolic transcriptome and increased glycolysis, amino acid uptake, mitochondrial oxidation of glutamine, and mitochondrial biogenesis. The effects of TNF-α were mediated by activation of Akt-mTOR signaling by the kinase ITK and did not require the NF-κB pathway. TNF-α stimulated the differentiation of naïve cells into proinflammatory T helper 1 (TH1) and TH17 cells, but not that of regulatory T cells. CD4+ T cells from patients with RA showed increased TNF-α production and consequent Akt phosphorylation upon activation. These cells also exhibited increased mitochondrial mass, particularly within proinflammatory T cell subsets implicated in disease. Together, these findings suggest that T cell-derived TNF-α drives their metabolic reprogramming by promoting signaling through ITK, Akt, and mTOR, which is dysregulated in autoinflammatory disease.

Uptake of long-chain fatty acids from the bone marrow suppresses CD8+ T-cell metabolism and function in multiple myeloma.

T cells demonstrate impaired function in multiple myeloma (MM) but suppressive mechanisms in the bone marrow microenvironment remain poorly defined. We observe that bone marrow CD8+ T-cell function is decreased in MM compared with controls, and is also consistently lower within bone marrow samples than in matched peripheral blood samples. These changes are accompanied by decreased mitochondrial mass and markedly elevated long-chain fatty acid uptake. In vitro modeling confirmed that uptake of bone marrow lipids suppresses CD8+ T function, which is impaired in autologous bone marrow plasma but rescued by lipid removal. Analysis of single-cell RNA-sequencing data identified expression of fatty acid transport protein 1 (FATP1) in bone marrow CD8+ T cells in MM, and FATP1 blockade also rescued CD8+ T-cell function, thereby identifying this as a novel target to augment T-cell activity in MM. Finally, analysis of samples from cohorts of patients who had received treatment identified that CD8+ T-cell metabolic dysfunction resolves in patients with MM who are responsive to treatment but not in patients with relapsed MM, and is associated with substantial T-cell functional restoration.

Canagliflozin impairs T cell effector function via metabolic suppression in autoimmunity.

Augmented T cell function leading to host damage in autoimmunity is supported by metabolic dysregulation, making targeting immunometabolism an attractive therapeutic avenue. Canagliflozin, a type 2 diabetes drug, is a sodium glucose co-transporter 2 (SGLT2) inhibitor with known off-target effects on glutamate dehydrogenase and complex I. However, the effects of SGLT2 inhibitors on human T cell function have not been extensively explored. Here, we show that canagliflozin-treated T cells are compromised in their ability to activate, proliferate, and initiate effector functions. Canagliflozin inhibits T cell receptor signaling, impacting on ERK and mTORC1 activity, concomitantly associated with reduced c-Myc. Compromised c-Myc levels were encapsulated by a failure to engage translational machinery resulting in impaired metabolic protein and solute carrier production among others. Importantly, canagliflozin-treated T cells derived from patients with autoimmune disorders impaired their effector function. Taken together, our work highlights a potential therapeutic avenue for repurposing canagliflozin as an intervention for T cell-mediated autoimmunity.

Succinate uptake by T cells suppresses their effector function via inhibition of mitochondrial glucose oxidation.

Succinate dehydrogenase (SDH) loss-of-function mutations drive succinate accumulation in tumor microenvironments, for example in the neuroendocrine tumors pheochromocytoma (PC) and paraganglioma (PG). Control of innate immune cell activity by succinate is described, but effects on T cells have not been interrogated. Here we report that exposure of human CD4+ and CD8+ T cells to tumor-associated succinate concentrations suppresses degranulation and cytokine secretion, including of the key anti-tumor cytokine interferon-γ (IFN-γ). Mechanistically, this is associated with succinate uptake-partly via the monocarboxylate transporter 1 (MCT1)-inhibition of succinyl coenzyme A synthetase activity and impaired glucose flux through the tricarboxylic acid cycle. Consistently, pharmacological and genetic interventions restoring glucose oxidation rescue T cell function. Tumor RNA-sequencing data from patients with PC and PG reveal profound suppression of IFN-γ-induced genes in SDH-deficient tumors compared with those with other mutations, supporting a role for succinate in modulating the anti-tumor immune response in vivo.

CD4+ T-cell clones recognizing human lymphoma-associated antigens: generation by in vitro stimulation with autologous Epstein-Barr virus-transformed B cells.

Epstein-Barr virus (EBV)-specific T-cell preparations, generated by stimulating immune donor lymphocytes with the autologous virus-transformed B-lymphoblastoid cell line (LCL) in vitro, can be used to target EBV-positive malignancies. Although these preparations are enriched for EBV antigen-specific CD8(+) T cells, most also contain a CD4(+) T-cell population whose specificity is unknown. Here, we show that, although CD4(+) T-cell clones derived from such cultures recognize HLA class II-matched LCLs but not mitogen-activated B lymphoblasts, many (1) do not map to any known EBV antigen, (2) can be raised from EBV-naive as well as EBV-immune persons, and (3) can recognize a broad range of human B lymphoma-derived cell lines irrespective of EBV genome status, providing those lines to express the relevant HLA class II-restricting allele. Importantly, such CD4(+) clones not only produce IFNgamma but are also cytotoxic and can control the outgrowth of HLA-matched lymphoma cells in cocultivation assays. We infer that such CD4(+) T cells recognize cellular antigens that are preferentially up-regulated in EBV-transformed but not mitogen-activated B lymphoblasts and that are also expressed in a range of B-cell malignancies. Such antigens are therefore of potential value as targets for CD4(+) T cell-based immunotherapy.

Control of T Cell Metabolism by Cytokines and Hormones.

Dynamic, coordinated changes in metabolic pathway activity underpin the protective and inflammatory activity of T cells, through provision of energy and biosynthetic precursors for effector functions, as well as direct effects of metabolic enzymes, intermediates and end-products on signaling pathways and transcriptional mechanisms. Consequently, it has become increasingly clear that the metabolic status of the tissue microenvironment directly influences T cell activity, with changes in nutrient and/or metabolite abundance leading to dysfunctional T cell metabolism and interlinked immune function. Emerging evidence now indicates that additional signals are integrated by T cells to determine their overall metabolic phenotype, including those arising from interaction with cytokines and hormones in their environment. The impact of these on T cell metabolism, the mechanisms involved and the pathological implications are discussed in this review article.

Epitope-specific evolution of human CD8(+) T cell responses from primary to persistent phases of Epstein-Barr virus infection.

Primary virus infection often elicits a large CD8(+) T cell response which subsequently contracts to a smaller memory T cell pool; the relationship between these two virus-specific populations is not well understood. Here we follow the human CD8(+) T cell response to Epstein-Barr virus (EBV) from its primary phase in infectious mononucleosis (IM) through to the persistent carrier state. Using HLA-A2.1 or B8 tetramers specific for four lytic cycle and three latent cycle epitopes, we find marked differences in the epitope-specific composition of the T cell populations between the two phases of infection. The primary response is dominated by lytic epitope specificities which are severely culled (and in one case extinguished) with resolution of the acute infection; in contrast latent epitope specificities are less abundant, if present at all, in acute IM but often then increase their percentage representation in the CD8 pool. Even comparing epitopes of the same type, the relative size of responses seen in primary infection does not necessarily correlate with that seen in the longer term. We also follow the evolution of phenotypic change in these populations and show that, from a uniform CD45RA(-)RO(+)CCR7(-) phenotype in IM, lytic epitope responses show greater reversion to a CD45RA(+)RO(-) phenotype whereas latent epitope responses remain CD45RA(-)RO(+) with a greater tendency to acquire CCR7. Interestingly these phenotypic distinctions reflect the source of the epitope as lytic or latent, and not the extent to which the response has been amplified in vivo.

Characterization of the CD4+ T cell response to Epstein-Barr virus during primary and persistent infection.

The CD8+ T cell response to Epstein-Barr virus (EBV) is well characterized. Much less is known about the evolution of the CD4+ T cell response. Here we show that EBV stimulates a primary burst of effector CD4+ T cells and this is followed by a period of down-regulation. A small population of EBV-specific effector CD4+ T cells survives during the lifelong persistent phase of infection. The EBV-specific effector CD4+ T cells accumulate within a CD27+ CD28+ differentiation compartment during primary infection and remain enriched within this compartment throughout the persistent phase of infection. Analysis of CD4+ T cell responses to individual epitopes from EBV latent and lytic cycle proteins confirms the observation that the majority of the effector cells express both CD27 and CD28, although CD4+ T cells specific for lytic cycle antigens have a greater tendency to express CD45RA than those specific for the latent antigens. In clear contrast, effector CD4+ T cells specific for cytomegalovirus (CMV) accumulate within the CD27- CD28+ and CD27- CD28- compartments. There are striking parallels in terms of the differentiation of CD8+ T cells specific for EBV and CMV. The results challenge current ideas on the definition of memory subsets.

CD4 and CD8 T cell responses to tumour-associated Epstein-Barr virus antigens in nasopharyngeal carcinoma patients.

Nasopharyngeal carcinoma (NPC), an Epstein-Barr virus (EBV)-associated tumour common in Southern Chinese populations, is a potentially important target for T cell-based immunotherapy. The tumour cells are HLA class I- and II-positive and express a limited subset of EBV latent proteins, namely the nuclear antigen EBNA1 and the latent membrane proteins LMP2 and (in some cases) LMP1. To ask whether the tumour develops in the presence of a potentially protective host response or in its absence, we set out to determine the prevailing levels of CD4+ and CD8+ T cell memory to these proteins in NPC patients at tumour diagnosis. We first screened healthy Chinese donors against Chinese strain EBNA1, LMP1 and LMP2 sequences in Elispot assays of interferon-gamma release and identified the immunodominant CD4+ and CD8+ epitope peptides presented by common Chinese HLA alleles. Then, comparing 60 patients with >70 healthy controls on peptide epitope mini-panels, we found that T cell memory to CD4 epitopes in all three proteins was unimpaired in the blood of patients at diagnosis. In most cases NPC patients also showed detectable responses to CD8 epitopes relevant to their HLA type, the one consistent exception being the absence in patients of a B*4001-restricted response to LMP2. We infer that NPC arises in patients whose prevailing levels of T cell memory to tumour-associated EBV proteins is largely intact; the therapeutic goal must therefore be to re-direct the existing memory repertoire more effectively against antigen-expressing tumour cells.

Regression of Epstein-Barr virus-induced B-cell transformation in vitro involves virus-specific CD8+ T cells as the principal effectors and a novel CD4+ T-cell reactivity.

T-cell memory to Epstein-Barr virus (EBV) was first demonstrated through regression of EBV-induced B-cell transformation to lymphoblastoid cell lines (LCLs) in virus-infected peripheral blood mononuclear cell (PBMC) cultures. Here, using donors with virus-specific T-cell memory to well-defined CD4 and CD8 epitopes, we reexamine recent reports that the effector cells mediating regression are EBV latent antigen-specific CD4+ and not, as previously assumed, CD8+ T cells. In regressing cultures, we find that the reversal of CD23+ B-cell proliferation was always coincident with an expansion of latent epitope-specific CD8+, but not CD4+, T cells; furthermore CD8+ T-cell clones derived from regressing cultures were epitope specific and reproduced regression when cocultivated with EBV-infected autologous B cells. In cultures of CD4-depleted PBMCs, there was less efficient expansion of these epitope-specific CD8+ T cells and correspondingly weaker regression. The data are consistent with an effector role for epitope-specific CD8+ T cells in regression and an auxiliary role for CD4+ T cells in expanding the CD8 response. However, we also occasionally observed late regression in CD8-depleted PBMC cultures, though again without any detectable expansion of preexisting epitope-specific CD4+ T-cell memory. CD4+ T-cell clones derived from such cultures were LCL specific in gamma interferon release assays but did not recognize any known EBV latent cycle protein or derived peptide. A subset of these clones was also cytolytic and could block LCL outgrowth. These novel effectors, whose antigen specificity remains to be determined, may also play a role in limiting virus-induced B-cell proliferation in vitro and in vivo.

Herpesvirus-specific CD8 T cell immunity in old age: cytomegalovirus impairs the response to a coresident EBV infection.

Aging in humans is associated with increased infections and the reduced proliferative capacity of T cells, part of the more global phenomenon termed immune senescence. The etiology of immune senescence is unknown but the accumulation of virus-specific memory T cells may be a contributory factor. We have examined CD8 T cell responses to two persistent herpesvirus infections, CMV and EBV, and to a recurrent virus infection, influenza, in different age cohorts of healthy donors using HLA-peptide tetramers and intracellular cytokine detection. Of these, CMV appears to be the most immunogenic, with the CD8 T cell response representing over 10% of the CD8 pool in many elderly donors. Interestingly, the effect of age upon EBV-specific responses depends upon donor CMV sero-status. In CMV seropositive donors, the magnitude of the EBV-specific immune response is stable with age, but in CMV seronegative donors, the response to EBV increases significantly with age. By contrast, the influenza-specific CD8 T cell immune response decreases with age, independent of CMV status. The functional activity of the herpesvirus-specific immune response decreases in elderly donors, although the characteristic phenotypes of CMV- and EBV-specific memory populations are retained. This demonstrates that aging is associated with a marked accumulation of CMV-specific CD8 T cells together with a decrease in immediate effector function. Moreover, infection with CMV can reduce prevailing levels of immunity to EBV, another persistent virus. These results suggest that carriage of CMV may be detrimental to the immunocompetent host by suppressing heterologous virus-specific immunity during aging.

Epstein-Barr virus-specific CD8(+) T cells that re-express CD45RA are apoptosis-resistant memory cells that retain replicative potential.

During acute infection, latent and lytic Epstein-Barr virus (EBV) epitope-specific CD8(+) T cells have a CD45RO(+) CD45RA(-) phenotype. However, after resolution of the infection, a large proportion of these cells, particularly those specific for lytic viral epitopes, re-express the CD45RA molecule. The role of CD8(+) CD45RA(+) T cells in ongoing immunity to EBV and other viruses is unknown. We now demonstrate that, relative to their CD45RO(+) counterparts, the EBV-specific CD8(+) T cells that revert to CD45RA expression after acute infectious mononucleosis are not in cell cycle, have longer telomeres, and are more resistant to apoptosis partly because of increased Bcl-2 expression. However, the EBV-specific CD8(+) CD45RA(+) T cells have shorter telomeres than the total CD8(+) CD45RA(+) T-cell pool and predominantly express low levels of the CCR7 chemokine receptor, indicating that they are not naive cells. In addition, EBV-specific CD8(+) CD45RA(+) T cells can be induced to proliferate and exhibit potent cytotoxic activity against target cells loaded with specific peptide. Our results strongly suggest, therefore, that EBV-specific CD8(+) CD45RA(+) T cells represent a stabilized virus-specific memory pool and not terminally differentiated effector cells. The identification of mechanisms that enable stable virus-specific CD8(+) T cells to persist after acute infection may lead to the enhancement of antiviral immunity in immunocompromised and elderly persons.