RESUMO
Because of the cytotoxic potential of CD8(+) T cells, maintenance of CD8(+) peripheral tolerance is extremely important. A major peripheral tolerance mechanism is the induction of anergy, a refractory state in which proliferation and IL-2 production are inhibited. We used a TCR transgenic mouse model to investigate the signaling defects in CD8(+) T cells rendered anergic in vivo. In addition to a previously reported alteration in calcium/NFAT signaling, we also found a defect in NF-κB-mediated gene transcription. This was not due to blockade of early NF-κB activation events, including IκB degradation and NF-κB nuclear translocation, as these occurred normally in tolerant T cells. However, we discovered that anergic cells failed to phosphorylate the NF-κB p65 subunit at Ser(311) and also failed to acetylate p65 at Lys(310). Both of these modifications have been implicated as critical for NF-κB transactivation capacity, and thus, our results suggest that defects in key phosphorylation and acetylation events are important for the inhibition of NF-κB activity (and subsequent T cell function) in anergic CD8(+) T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Anergia Clonal , NF-kappa B/metabolismo , Fator de Transcrição RelA/metabolismo , Ativação Transcricional , Acetilação , Animais , Camundongos , Camundongos Transgênicos , NF-kappa B/antagonistas & inibidores , NF-kappa B/genética , Tolerância Periférica/fisiologia , Fosforilação , Transdução de SinaisRESUMO
T lymphocytes play a critical role in cell-mediated immune responses. During activation, extracellular and intracellular signals alter T cell metabolism in order to meet the energetic and biosynthetic needs of a proliferating, active cell, but control of these phenomena is not well defined. Previous studies have demonstrated that signaling from the costimulatory receptor CD28 enhances glucose utilization via the phosphatidylinositol-3-kinase (PI3K) pathway. However, since CD28 ligation alone does not induce glucose metabolism in resting T cells, contributions from T cell receptor-initiated signaling pathways must also be important. We therefore investigated the role of mitogen-activated protein kinase (MAPK) signaling in the regulation of mouse T cell glucose metabolism. T cell stimulation strongly induces glucose uptake and glycolysis, both of which are severely impaired by inhibition of extracellular signal-regulated kinase (ERK), whereas p38 inhibition had a much smaller effect. Activation also induced hexokinase activity and expression in T cells, and both were similarly dependent on ERK signaling. Thus, the ERK signaling pathway cooperates with PI3K to induce glucose utilization in activated T cells, with hexokinase serving as a potential point for coordinated regulation.
Assuntos
Glucose/metabolismo , Sistema de Sinalização das MAP Quinases/imunologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/farmacologia , Butadienos/farmacologia , Antígenos CD28/imunologia , Complexo CD3/imunologia , Linhagem Celular Tumoral , Células Cultivadas , Ensaios Enzimáticos , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glucose/farmacocinética , Glicólise/efeitos dos fármacos , Hexoquinase/genética , Hexoquinase/metabolismo , Imidazóis/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Nitrilas/farmacologia , Piridinas/farmacologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Activation of a naive T cell is a highly energetic event, which requires a substantial increase in nutrient metabolism. Upon stimulation, T cells increase in size, rapidly proliferate, and differentiate, all of which lead to a high demand for energetic and biosynthetic precursors. Although amino acids are the basic building blocks of protein biosynthesis and contribute to many other metabolic processes, the role of amino acid metabolism in T cell activation has not been well characterized. We have found that glutamine in particular is required for T cell function. Depletion of glutamine blocks proliferation and cytokine production, and this cannot be rescued by supplying biosynthetic precursors of glutamine. Correlating with the absolute requirement for glutamine, T cell activation induces a large increase in glutamine import, but not glutamate import, and this increase is CD28-dependent. Activation coordinately enhances expression of glutamine transporters and activities of enzymes required to allow the use of glutamine as a Krebs cycle substrate in T cells. The induction of glutamine uptake and metabolism requires ERK function, providing a link to TCR signaling. Together, these data indicate that regulation of glutamine use is an important component of T cell activation. Thus, a better understanding of glutamine sensing and use in T cells may reveal novel targets for immunomodulation.
Assuntos
MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Glutamina/metabolismo , Ativação Linfocitária/imunologia , Linfócitos T/imunologia , Alanina Transaminase/metabolismo , Sistema A de Transporte de Aminoácidos/genética , Sistema A de Transporte de Aminoácidos/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Transporte Biológico/efeitos dos fármacos , Linhagem Celular Tumoral , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/antagonistas & inibidores , MAP Quinases Reguladas por Sinal Extracelular/genética , Flavonoides/farmacologia , Citometria de Fluxo , Glutamato Desidrogenase/metabolismo , Glutaminase/genética , Glutaminase/metabolismo , Glutamina/farmacocinética , Glutamina/farmacologia , Ácidos Cetoglutáricos/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/citologia , Linfócitos T/metabolismoRESUMO
The Bcl-2-associated X protein (Bax) and Bcl-2-antagonist/killer (Bak) are essential regulators of lymphocyte apoptosis, but whether they play a role in viable T cell function remains unclear. Here, we report that T cells lacking both Bax and Bak display defects in antigen-specific proliferation because of Ca(2+)-signaling defects. Bax(-/-), Bak(-/-) T cells displayed defective T cell receptor (TCR)- and inositol-1,4,5-trisphosphate (IP(3))-dependent Ca(2+) mobilization because of altered endoplasmic reticulum (ER) Ca(2+) regulation that was reversed by Bax's reintroduction. The ability of TCR-dependent Ca(2+) signals to stimulate mitochondrial NADH production in excess of that utilized for ATP synthesis was dependent on Bax and Bak. Blunting of Ca(2+)-induced mitochondrial NADH elevation in the absence of Bax and Bak resulted in decreased reactive-oxygen-species production, which was required for T cell proliferation. Together, the data establish that Bax and Bak play an essential role in the control of T cell proliferation by modulating ER Ca(2+) release.
Assuntos
Apoptose/imunologia , Sinalização do Cálcio , Retículo Endoplasmático/imunologia , Linfócitos T/imunologia , Proteína Killer-Antagonista Homóloga a bcl-2/fisiologia , Proteína X Associada a bcl-2/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Cálcio/metabolismo , Proliferação de Células , Metabolismo Energético , Homeostase , Inositol 1,4,5-Trifosfato/metabolismo , Camundongos , Camundongos Mutantes , Mitocôndrias/metabolismo , NAD/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Proteína Killer-Antagonista Homóloga a bcl-2/genética , Proteína X Associada a bcl-2/genéticaRESUMO
We generated three populations of macrophages (Mphi) in vitro and characterized each. Classically activated Mphi (Ca-Mphi) were primed with IFN-gamma and stimulated with LPS. Type II-activated Mphi (Mphi-II) were similarly primed but stimulated with LPS plus immune complexes. Alternatively activated Mphi (AA-Mphi) were primed overnight with IL-4. Here, we present a side-by-side comparison of the three cell types. We focus primarily on differences between Mphi-II and AA-Mphi, as both have been classified as M2 Mphi, distinct from Ca-Mphi. We show that Mphi-II more closely resemble Ca-Mphi than they are to AA-Mphi. Mphi-II and Ca-Mphi, but not AA-Mphi, produce high levels of NO and have low arginase activity. AA-Mphi express FIZZ1, whereas neither Mphi-II nor Ca-Mphi do. Mphi-II and Ca-Mphi express relatively high levels of CD86, whereas AA-Mphi are virtually devoid of this costimulatory molecule. Ca-Mphi and Mphi-II are efficient APC, whereas AA-Mphi fail to stimulate efficient T cell proliferation. The differences between Ca-Mphi and Mphi-II are more subtle. Ca-Mphi produce IL-12 and give rise to Th1 cells, whereas Mphi-II produce high levels of IL-10 and thus, give rise to Th2 cells secreting IL-4 and IL-10. Mphi-II express two markers that may be used to identify them in tissue. These are sphingosine kinase-1 and LIGHT (TNF superfamily 14). Thus, Ca-Mphi, Mphi-II, and AA-Mphi represent three populations of cells with different biological functions.
Assuntos
Citocinas/imunologia , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Animais , Arginase/biossíntese , Arginase/imunologia , Antígeno B7-2/imunologia , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Citocinas/biossíntese , Citocinas/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/classificação , Macrófagos/enzimologia , Camundongos , Camundongos Endogâmicos BALB C , Fator de Crescimento Neural/biossíntese , Fator de Crescimento Neural/imunologia , Óxido Nítrico/biossíntese , Óxido Nítrico/imunologia , Fosfotransferases (Aceptor do Grupo Álcool)/biossíntese , Fosfotransferases (Aceptor do Grupo Álcool)/imunologia , Proteínas/imunologia , Células Th1/imunologia , Células Th2/imunologia , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/biossíntese , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/imunologiaRESUMO
CTLA-4 and PD-1 are receptors that negatively regulate T-cell activation. Ligation of both CTLA-4 and PD-1 blocked CD3/CD28-mediated upregulation of glucose metabolism and Akt activity, but each accomplished this regulation using separate mechanisms. CTLA-4-mediated inhibition of Akt phosphorylation is sensitive to okadaic acid, providing direct evidence that PP2A plays a prominent role in mediating CTLA-4 suppression of T-cell activation. In contrast, PD-1 signaling inhibits Akt phosphorylation by preventing CD28-mediated activation of phosphatidylinositol 3-kinase (PI3K). The ability of PD-1 to suppress PI3K/AKT activation was dependent upon the immunoreceptor tyrosine-based switch motif located in its cytoplasmic tail, adding further importance to this domain in mediating PD-1 signal transduction. Lastly, PD-1 ligation is more effective in suppressing CD3/CD28-induced changes in the T-cell transcriptional profile, suggesting that differential regulation of PI3K activation by PD-1 and CTLA-4 ligation results in distinct cellular phenotypes. Together, these data suggest that CTLA-4 and PD-1 inhibit T-cell activation through distinct and potentially synergistic mechanisms.
Assuntos
Antígenos de Diferenciação/fisiologia , Antígenos de Superfície/fisiologia , Proteínas Reguladoras de Apoptose/fisiologia , Ativação Linfocitária/fisiologia , Linfócitos T/fisiologia , Antígenos CD , Antígenos CD28/metabolismo , Complexo CD3/metabolismo , Antígeno CTLA-4 , Ativação Enzimática , Regulação da Expressão Gênica , Humanos , Técnicas In Vitro , Ácido Okadáico/farmacologia , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Fosfatidilinositóis/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Receptor de Morte Celular Programada 1 , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
The role of the NKG2D immunoreceptor and its ligands in antitumor immune response is incompletely understood. Here, we report that effector immune cells infiltrating ovarian carcinoma are mostly CD8+ lymphocytes lacking CD28 but expressing the NKG2D costimulatory receptor. Human ovarian carcinoma expresses the novel NKG2D ligand lymphocyte effector cell toxicity-activating ligand (Letal). Letal was found to be an independent prognosticator of improved survival in advanced ovarian cancer. Higher levels of tumor-derived Letal were associated with stronger lymphocyte infiltration. Letal exerted marked costimulatory effects and induced type-1 polarization in CD8+CD28- tumor-infiltrating lymphocytes ex vivo. Letal engagement increased the expression of the glucose transporter Glut-1, enhanced glucose up-take, and protected CD8+ lymphocytes from cisplatin-induced killing. Letal also down-regulated the expression of Fas in CD8+ cells and rendered them resistant to Fas ligand-induced apoptosis. Our results indicate that Letal promotes tumor immune surveillance by promoting the survival and intratumoral expansion of antitumor cytotoxic lymphocytes. We propose that Letal could be used for the ex vivo expansion of apoptosis-resistant tumor-reactive cytotoxic lymphocytes for adoptive transfer.
Assuntos
Antígenos CD28/metabolismo , Linfócitos T CD8-Positivos/imunologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Ovarianas/imunologia , Receptores Imunológicos/metabolismo , Adenocarcinoma Mucinoso/imunologia , Adenocarcinoma Mucinoso/patologia , Animais , Apoptose , Carcinoma Endometrioide/imunologia , Carcinoma Endometrioide/patologia , Sobrevivência Celular , Cistadenocarcinoma Seroso/imunologia , Cistadenocarcinoma Seroso/patologia , Citotoxicidade Imunológica , Feminino , Humanos , Ligantes , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Subfamília K de Receptores Semelhantes a Lectina de Células NK , Neoplasias Ovarianas/patologia , Receptores de Células Matadoras NaturaisRESUMO
Lymphocyte activation initiates a program of cell growth, proliferation, and differentiation that increases metabolic demand. Although T cells increase glucose uptake and glycolysis during an immune response, the signaling pathways that regulate these increases remain largely unknown. Here we show that CD28 costimulation, acting through phosphatidylinositol 3'-kinase (PI3K) and Akt, is required for T cells to increase their glycolytic rate in response to activation. Furthermore, CD28 controls a primary response pathway, inducing a level of glucose uptake and glycolysis in excess of that needed to maintain cellular ATP/ADP levels or macromolecular synthesis. These data suggest that CD28 costimulation functions to increase glycolytic flux, allowing T cells to anticipate energetic and biosynthetic needs associated with a sustained response.