RESUMO
γδ T cells are a heterogeneous cell population with different subsets playing specialized and often opposing roles during immune responses. A key question is whether γδ thymocytes are determined for their effector function already at an early stage, before their commitment to the γδ T-cell lineage, or are instructed during their later development. Here, we show that the adult Vγ1.1+ and Vγ2+ γδ T-cell subsets both go through a CD73+ CD24+ development stage, and that the gene regulation involved in lineage commitment is shared by both subsets. We demonstrate that the major subset diversification first occurs after the cells have committed to the γδ T-cell lineage, strongly supporting an instructive model for functional programming of γδ T cells. In conclusion, we show that the two major adult γδ T-cell subsets in mice develop through a shared pathway utilizing similar cellular machinery and that they diverge after the CD24+ CD73+ maturity stage.
Assuntos
5'-Nucleotidase/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/fisiologia , Linfócitos T/fisiologia , Timo/imunologia , 5'-Nucleotidase/genética , Animais , Antígeno CD24/metabolismo , Diferenciação Celular , Linhagem da Célula , Células Cultivadas , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
The CD3γ di-leucine-based (diL) receptor-sorting motif plays a central role in TCR down-regulation and in clonal expansion of virus-specific T cells. However, the role of the CD3γ diL motif in T-cell development is not known. In this study, we show that protein kinase C-induced TCR down-regulation is abolished in thymocytes from CD3γLLAA mice with a mutated CD3γ diL motif, and that CD3γLLAA mice have reduced numbers of thymocytes compared with aged-matched wild-type mice. We found that early thymocyte development at the ß-selection checkpoint is impaired resulting in reduced numbers of double negative (DN) 4 cells in CD3γLLAA mice. This was not caused by reduced proliferation but most probably by increased down-regulation of the antiapoptotic molecule Bcl-2 causing enhanced apoptosis during the transition from the DN3 to the DN4 stage. In contrast, proliferation of immature CD8 single positive (ISP) thymocytes was increased resulting in normal numbers of ISP in CD3γLLAA mice. Despite the normal numbers of ISP, CD3γLLAA mice had reduced numbers of double positive and SP thymocytes indicating that the CD3γ diL motif also affected later stages of T-cell development. In accordance, we found that positive and negative selection, differentiation toward CD4 and CD8 SP T cells and the development of nonconventional T cells were affected in CD3γLLAA mice. In conclusion, our study identifies an important role of the CD3γ diL motif in T-cell development most probably mediated by its fine-tuning of pre-TCR and TCR expression, down-regulation and signaling.
Assuntos
Alanina/metabolismo , Complexo CD3/genética , Leucina/metabolismo , Timócitos/imunologia , Alanina/imunologia , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Apoptose , Complexo CD3/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/imunologia , Antígenos CD8/genética , Antígenos CD8/imunologia , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Proliferação de Células , Células Clonais , Regulação da Expressão Gênica , Imunofenotipagem , Leucina/imunologia , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Transdução de Sinais , Timócitos/citologiaRESUMO
BACKGROUND: It has been suggested that interleukin (IL)-17 and IL-22 play important roles in the elicitation of human allergic contact dermatitis; however, the frequencies of T cell subtypes producing IL-17 and IL-22 in human allergic contact dermatitis are unknown. OBJECTIVES: To determine the frequencies of CD4(+) , CD8(+) and γδ T cells producing IL-17, IL-22 and interferon (IFN)-γ in the blood and skin from nickel-allergic patients. PATIENTS/MATERIALS/METHODS: Blood samples were collected from 14 patients and 17 controls, and analysed by flow cytometry. Biopsies were taken from 5 patients and 6 controls, and analysed by immunohistochemistry and flow cytometry of skin lymphocytes. RESULTS: We found an increased frequency of γδ T cells in the blood, but no differences in the distribution of cytokine-producing CLA(+) T cell subtypes in nickel-allergic patients as compared with controls. In nickel-allergic patients, there was massive cellular infiltration dominated by CD4(+) T cells producing IL-17, IL-22 and IFN-γ in nickel-challenged skin but not in vehicle-challenged skin. CONCLUSION: CD4(+) T cells producing IL-17, IL-22 and IFN-γ are important effector cells in the eczematous reactions of nickel-induced allergic contact dermatitis in humans.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , Dermatite Alérgica de Contato/imunologia , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucinas/metabolismo , Níquel/imunologia , Biomarcadores/metabolismo , Biópsia , Linfócitos T CD8-Positivos/metabolismo , Estudos de Casos e Controles , Dermatite Alérgica de Contato/etiologia , Dermatite Alérgica de Contato/patologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Contagem de Linfócitos , Níquel/efeitos adversos , Pele/imunologia , Pele/metabolismo , Pele/patologia , Subpopulações de Linfócitos T/metabolismo , Interleucina 22RESUMO
TCR and cytokine receptor signaling play key roles in the complex homeostatic mechanisms that maintain a relative stable number of T cells throughout life. Despite the homeostatic mechanisms, a slow decline in naive T cells is typically observed with age. The CD3gamma di-leucine-based motif controls TCR down-regulation and plays a central role in fine-tuning TCR expression and signaling in T cells. In this study, we show that the age-associated decline of naive T cells is strongly accelerated in CD3gammaLLAA knock-in mice homozygous for a double leucine to alanine mutation in the CD3gamma di-leucine-based motif, whereas the number of memory T cells is unaffected by the mutation. This results in premature T cell population senescence with a severe dominance of memory T cells and very few naive T cells in middle-aged to old CD3gamma mutant mice. The reduced number of naive T cells in CD3gamma mutant mice was caused by the combination of reduced thymic output, decreased T cell apoptosis, and increased transition of naive T cells to memory T cells. Experiments with bone marrow chimeric mice confirmed that the CD3gammaLLAA mutation exerted a T cell intrinsic effect on T cell homeostasis that resulted in an increased transition of CD3gammaLLAA naive T cells to memory T cells and a survival advantage of CD3gammaLLAA T cells compared with wild-type T cells. The experimental observations were further supported by mathematical modeling of T cell homeostasis. Our study thus identifies an important role of CD3gamma-mediated TCR down-regulation in T cell homeostasis.
Assuntos
Complexo CD3/imunologia , Homeostase/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T/imunologia , Timo/imunologia , Animais , Apoptose/imunologia , Complexo CD3/genética , Complexo CD3/metabolismo , Regulação para Baixo/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/metabolismo , Timo/metabolismoRESUMO
T cell receptor (TCR) structure and function have been thoroughly studied for decades. Production and analyses of knock-out and knock-in mice with mutations in the CD3 chains have contributed significantly to these studies. The generation of such gene-modified mice relies on the availability of suitable embryonic stem (ES) cell lines. Traditionally, ES cell lines from the 129 mouse strains have been used followed by backcrossing to the C57BL/6 strain. In the present study, we demonstrate the existence of polymorphisms in the CD3 genes from mice of the 129 and C57BL/6 strains. These polymorphisms result in amino acid substitutions in the ectodomains of both the CD3delta and CD3epsilon chains in 129 mice compared to C57BL/6 mice. The amino acid substitutions do not change the stoichiometry or surface expression level of the TCR complex in 129 T cells but cause reduced anti-CD3 antibody binding to 129 T cells. Further, when stimulated with mitogenic anti-CD3 antibodies, T cells from the 129 strains show reduced expression of the activation marker CD69, Ca(2+) flux, IL-2 production and proliferative responses compared to C57BL/6 T cells. These findings demonstrate that polymorphisms of the CD3delta and epsilon ectodomains exist in mice, and that some of these polymorphisms lead to amino acid substitutions which cause structural changes and affect anti-CD3 antibody binding. Thus, functional T cell studies should be interpreted with caution when anti-CD3 antibodies are used for stimulation of T cells derived from gene-modified mice originating from 129 ES cell lines.
Assuntos
Complexo CD3/genética , Ativação Linfocitária , Polimorfismo de Nucleotídeo Único , Linfócitos T/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Complexo CD3/metabolismo , Linhagem Celular , Células-Tronco Embrionárias , Técnicas de Introdução de Genes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Camundongos Knockout , Dados de Sequência Molecular , Muromonab-CD3/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
CD3zeta is a subunit of the T-cell antigen receptor (TCR) complex required for its assembly and surface expression that also plays an important role in TCR-mediated signal transduction. We report here a patient with T(-)B(+)NK(+) severe combined immunodeficiency (SCID) who was homozygous for a single C insertion following nucleotide 411 in exon 7 of the CD3zeta gene. The few T cells present contained no detectable CD3zeta protein, expressed low levels of cell surface CD3epsilon, and were nonfunctional. CD4(+)CD8(-)CD3epsilon(low), CD4(-)CD8(+)CD3epsilon(low), and CD4(-)CD8(-)CD3epsilon(low) cells were detected in the periphery, and the patient also exhibited an unusual population of CD56(-)CD16(+) NK cells with diminished cytolytic activity. Additional studies demonstrated that retrovirally transduced patient mutant CD3zeta cDNA failed to rescue assembly of nascent complete TCR complexes or surface TCR expression in CD3zeta-deficient MA5.8 murine T-cell hybridoma cells. Nascent transduced mutant CD3zeta protein was also not detected in metabolically labeled MA5.8 cells, suggesting that it was unstable and rapidly degraded. Taken together, these findings provide the first demonstration that complete CD3zeta deficiency in humans can cause SCID by preventing normal TCR assembly and surface expression.
Assuntos
Linfócitos B/imunologia , Complexo CD3/genética , Células Matadoras Naturais/imunologia , Mutagênese Insercional , Receptores de Antígenos de Linfócitos T/genética , Imunodeficiência Combinada Severa/genética , Complexo CD3/imunologia , Antígenos CD4/imunologia , Antígenos CD8/imunologia , Linhagem Celular , Éxons/genética , Éxons/imunologia , Feminino , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/imunologia , Humanos , Lactente , Complexos Multiproteicos/genética , Complexos Multiproteicos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Retroviridae , Imunodeficiência Combinada Severa/imunologia , Linfócitos T/imunologia , Transdução GenéticaRESUMO
During thymopoiesis, two major types of mature T cells are generated that can be distinguished by the clonotypic subunits contained within their T-cell receptor (TCR) complexes: alphabeta T cells and gammadelta T cells. Although there is no consensus as to the exact developmental stage where alphabeta and gammadelta T-cell lineages diverge, gammadelta T cells and precursors to the alphabeta T-cell lineage (bearing the pre-TCR) are thought to be derived from a common CD4- CD8- double-negative precursor. The role of the TCR in alphabeta/gammadelta lineage commitment has been controversial, in particular whether different TCR isotypes intrinsically favor adoption of the corresponding lineage. Recent evidence supports a signal strength model of lineage commitment, whereby stronger signals promote gammadelta development and weaker signals promote adoption of the alphabeta fate, irrespective of the TCR isotype from which the signals originate. Moreover, differences in the amplitude of activation of the extracellular signal-regulated kinase- mitogen-activated protein kinase-early growth response pathway appear to play a critical role. These findings will be placed in context of previous analyses in an effort to more precisely define the signals that control T-lineage fate during thymocyte development.
Assuntos
Linhagem da Célula , Células-Tronco Hematopoéticas/citologia , Linfopoese , Transdução de Sinais , Subpopulações de Linfócitos T/citologia , Animais , Fatores de Transcrição de Resposta de Crescimento Precoce/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células-Tronco Hematopoéticas/enzimologia , Humanos , Modelos Imunológicos , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/enzimologia , Timo/citologia , Timo/enzimologia , Fatores de TempoRESUMO
The role of the T cell antigen receptor complex (TCR) in alphabeta/gammadelta lineage commitment remains controversial, in particular whether different TCR isoforms intrinsically favor adoption of a certain lineage. Here, we demonstrate that impairing the signaling capacity of a gammadeltaTCR complex enables it to efficiently direct thymocytes to the alphabeta lineage. In the presence of a ligand, a transgenic gammadeltaTCR mediates almost exclusive adoption of the gammadelta lineage, while in the absence of ligand, the same gammadeltaTCR promotes alphabeta lineage development with efficiency comparable to the pre-TCR. Importantly, attenuating gammadeltaTCR signaling through Lck deficiency causes reduced ERK1/2 activation and Egr expression and diverts thymocytes to the alphabeta lineage even in the presence of ligand. Conversely, ectopic Egr overexpression favors gammadelta T cell development. Our data support a model whereby gammadelta versus alphabeta lineage commitment is controlled by TCR signal strength, which depends critically on the ERK MAPK-Egr pathway.
Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Subpopulações de Linfócitos T/citologia , Subpopulações de Linfócitos T/imunologia , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Proteínas Imediatamente Precoces/biossíntese , Proteínas Imediatamente Precoces/genética , Proteínas Inibidoras de Diferenciação , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Fosforilação , Proteínas/genética , Proteínas/metabolismo , Receptores de Antígenos de Linfócitos T gama-delta/genética , Transdução de Sinais , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
TCR internalization takes place both in resting T cells as part of constitutive TCR cycling, after PKC activation, and during TCR triggering. It is still a matter of debate whether these pathways represent distinct pathways. Thus, some studies have indicated that ligand-induced TCR internalization is regulated by mechanisms distinct from those involved in constitutive internalization, whereas other studies have suggested that the ligand-induced TCR internalization pathway is identical with the constitutive pathway. To resolve this question, we first identified requirements for constitutive TCR cycling. We found that in contrast to PKC-induced TCR internalization where both CD3gamma-S(126) and the CD3gamma leucine-based internalization motif are required, constitutive TCR cycling required neither PKC nor CD3gamma-S(126) but only the CD3gamma leucine-based motif. Having identified these requirements, we next studied ligand-induced internalization in cells with abolished constitutive TCR cycling. We found that ligand-induced TCR internalization was not dependent on constitutive TCR internalization. Likewise, constitutive internalization and recycling of the TCR were independent of an intact ligand-induced internalization of the TCR. In conclusion, ligand-induced TCR internalization and constitutive cycling of the TCR represents two independent pathways regulated by different mechanisms.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Motivos de Aminoácidos , Complexo CD3/fisiologia , Regulação para Baixo , Humanos , Células Jurkat , Ligantes , Proteína Quinase C/fisiologia , Receptores de Antígenos de Linfócitos T/químicaRESUMO
One of the earliest events following TCR triggering is TCR down-regulation. However, the mechanisms behind TCR down-regulation are still not fully known. Some studies have suggested that only directly triggered TCR are internalized, whereas others studies have indicated that, in addition to triggered receptors, nonengaged TCR are also internalized (comodulated). In this study, we used transfected T cells expressing two different TCR to analyze whether comodulation took place. We show that TCR triggering by anti-TCR mAb and peptide-MHC complexes clearly induced internalization of nonengaged TCR. By using a panel of mAb against the Ti beta chain, we demonstrate that the comodulation kinetics depended on the affinity of the ligand. Thus, high-affinity mAb (K(D) = 2.3 nM) induced a rapid but reversible comodulation, whereas low-affinity mAb (K(D) = 6200 nM) induced a slower but more permanent type of comodulation. Like internalization of engaged TCR, comodulation was dependent on protein tyrosine kinase activity. Finally, we found that in contrast to internalization of engaged TCR, comodulation was highly dependent on protein kinase C activity and the CD3 gamma di-leucine-based motif. Based on these observations, a physiological role of comodulation is proposed and the plausibility of the TCR serial triggering model is discussed.
Assuntos
Complexo CD3/fisiologia , Dipeptídeos/fisiologia , Leucina/fisiologia , Proteína Quinase C/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Motivos de Aminoácidos/fisiologia , Animais , Anticorpos Monoclonais/farmacologia , Especificidade de Anticorpos , Linhagem Celular Tumoral , Regulação para Baixo/genética , Regulação para Baixo/imunologia , Antígenos de Histocompatibilidade Classe II/fisiologia , Humanos , Células Jurkat , Ligantes , Camundongos , Peptídeos/farmacologia , Ligação Proteica/imunologia , Proteínas Tirosina Quinases/fisiologia , Receptores de Antígenos de Linfócitos T alfa-beta/antagonistas & inibidores , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Subpopulações de Linfócitos T/enzimologia , Subpopulações de Linfócitos T/imunologia , Subpopulações de Linfócitos T/metabolismo , TransfecçãoRESUMO
Modulation of TCR expression levels is a central event during T cell development and activation, and it probably plays an important role in adjusting T cell responsiveness. Conflicting data have been published on down-regulation and degradation rates of the individual TCR subunits, and several divergent models for TCR down-regulation and degradation have been suggested. The aims of this study were to determine the rate constants for constitutive and ligand-induced TCR degradation and to determine whether the TCR subunits segregate or are processed as an intact unit during TCR down-regulation and degradation. We found that the TCR subunits in nonstimulated Jurkat cells were degraded with rate constants of approximately 0.0011 min(-1), resulting in a half-life of approximately 10.5 h. Triggering of the TCR by anti-TCR Abs resulted in a 3-fold increase in the degradation rate constants to approximately 0.0033 min(-1), resulting in a half-life of approximately 3.5 h. The subunits of the TCR complex were down-regulated from the cell surface and degraded with identical kinetics, and most likely remained associated during the passage throughout the endocytic pathway from the cell surface to the lysosomes. Similar results were obtained in studies of primary human Vbeta8+ T cells stimulated with superantigen. Based on these results, the simplest model for TCR internalization, sorting, and degradation is proposed.
Assuntos
Receptores de Antígenos de Linfócitos T/metabolismo , Biotinilação , Complexo CD3/metabolismo , Dimerização , Endocitose , Humanos , Células Jurkat , Ligantes , Subunidades Proteicas , Receptores de Antígenos de Linfócitos T/química , Linfócitos T/metabolismoRESUMO
TCR down-regulation plays an important role in modulating T cell responses both during T cell development and in mature T cells. At least two distinct pathways exist for down-regulation of the TCR. One pathway is activated following TCR ligation and is dependent on tyrosine phosphorylation. The other pathway is dependent on protein kinase C (PKC)-mediated activation of the CD3 gamma di-leucine-based receptor-sorting motif. Previous studies have failed to demonstrate a connection between ligand- and PKC-induced TCR down-regulation. Thus, although an apparent paradox, the dogma has been that ligand- and PKC-induced TCR down-regulations are not interrelated. By analyses of a newly developed CD3 gamma-negative T cell variant, freshly isolated and PHA-activated PBMC, and a mouse T cell line, we challenged this dogma and demonstrate in this work that PKC activation and the CD3 gamma di-leucine-based motif are indeed required for efficient ligand-induced TCR down-regulation.
Assuntos
Complexo CD3/química , Regulação para Baixo , Leucina/química , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Linfócitos T/imunologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Complexo CD3/genética , Linhagem Celular , Células Cultivadas , Deleção de Genes , Humanos , Células Jurkat , Cinética , Ligantes , Camundongos , Dados de Sequência Molecular , Proteína Quinase C/fisiologia , Homologia de SequênciaRESUMO
The T-cell receptor (TCR) is a multimeric receptor composed of the Ti alpha beta heterodimer and the noncovalently associated CD3 gamma delta epsilon and zeta(2) chains. All of the TCR chains are required for efficient cell surface expression of the TCR. Previous studies on chimeric molecules containing the di-leucine-based endocytosis motif of the TCR subunit CD3 gamma have indicated that the zeta chain can mask this motif. In this study, we show that successive truncations of the cytoplasmic tail of zeta led to reduced surface expression levels of completely assembled TCR complexes. The reduced TCR expression levels were caused by an increase in the TCR endocytic rate constant in combination with an unaffected exocytic rate constant. Furthermore, the TCR degradation rate constant was increased in cells with truncated zeta. Introduction of a CD3 gamma chain with a disrupted di-leucine-based endocytosis motif partially restored TCR expression in cells with truncated zeta chains, indicating that the zeta chain masks the endocytosis motif in CD3 gamma and thereby stabilizes TCR cell surface expression.