RESUMO
The T cell receptor (TCR) beta chain transmembrane domain contains two evolutionarily conserved tyrosines (Y). In this study, the functional basis for the evolutionary conservation is addressed by mutation of the residues, expression of the mutants in hybridoma and primary T cells, and examination of TCR signaling function. We find that the phenotype of the mutants, both surface expression and ability to signal for IL-2 production, is highly variable in different mouse T hybridoma lines. Although we have not been able to determine the basis for these differences in the hybridomas, expression of the mutants in primary T cells provides a definitive assessment of mutant phenotype. We show that mutation of the N-terminal Y to either leucine (L) or alanine (A) results in low surface expression in primary T cells, while mutation of both N- and C-terminal Y to A or L abrogates surface expression. However, the more conservative mutation of both transmembrane Y to phenylalanine maintained receptor surface expression and assembly while severely disrupting signaling in primary T cells. Our data demonstrate that TCR beta chain transmembrane Y are essential for TCR signal transduction as well as complex assembly. These findings suggest that protein-protein interactions involving membrane-spanning domains are likely relevant for TCR signal transduction mechanisms.
Assuntos
Sequência Conservada/imunologia , Hibridomas/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Subpopulações de Linfócitos T/metabolismo , Tirosina/fisiologia , Animais , Sequência de Bases , Linhagem Celular , Membrana Celular/genética , Membrana Celular/imunologia , Membrana Celular/metabolismo , Células Cultivadas , Regulação da Expressão Gênica/imunologia , Hibridomas/imunologia , Imunofenotipagem , Ativação Linfocitária/genética , Proteínas de Membrana/deficiência , Proteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína/genética , Receptores de Antígenos de Linfócitos T/deficiência , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T alfa-beta/deficiência , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Baço/imunologia , Baço/metabolismo , Subpopulações de Linfócitos T/imunologia , Células Tumorais Cultivadas , Tirosina/genéticaRESUMO
Mutagenic analyses have identified structural motifs important for TCR-mediated signaling in the antigen-binding chains, CD3 and zeta subunits of the TCR complex. In this study, we altered selected residues in the transmembrane and extracellular constant regions of the TCR beta chain and expressed the mutants in a T hybridoma line bearing endogenous receptor. We measured cytokine production and apoptosis in response to antigen or antibody. We found that mutation of one or both of the transmembrane tyrosine residues in the TCR beta chain caused a marked reduction in responsiveness. Mutation of the transmembrane serine to alanine also reduced responses, although less markedly. Immunoprecipitation analyses showed that the TCR beta mutations did not alter association with zeta. These experiments identify a signaling role for the transmembrane domain of the TCR beta chain.
Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Antígenos/farmacologia , Apoptose/fisiologia , Humanos , Interleucina-2/biossíntese , Camundongos , Dados de Sequência Molecular , Mutagênese , Mutação , Estrutura Terciária de Proteína , Receptores de Antígenos de Linfócitos T alfa-beta/biossíntese , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Homologia de Sequência de Aminoácidos , Relação Estrutura-Atividade , Tirosina/metabolismo , Tirosina/fisiologiaRESUMO
The influence of the interaction between CD4 and MHC class II molecules on selection of the T cell repertoire was studied in transgenic mice expressing human or human/mouse hybrid MHC class II beta chains. Either wild-type DR beta chains (DR1 beta) or hybrid beta chains comprising the beta1 domain of DR and the beta2, transmembrane, and intracytoplasmic domains of I-E (DRbeta 1Ebeta2) were introduced into and expressed in transgenic mice as a heterodimer with endogenous I-E alpha. Mice expressing low levels of DR1beta:I-E alpha or those expressing low or higher levels of the hybrid DRbeta 1Ebeta2:I-E alpha were studied. Immunization with a suboptimal dose of influenza nucleoprotein peptide exposed a fivefold lower frequency of DR-restricted, peptide-specific, IL-2-secreting T cells in the mice with low-level expression of DRbeta1 Ebeta2:I-E alpha when compared to mice expressing the same molecule at higher levels. The frequency in DRbeta wild-type mice was only twofold lower than that measured in mice with comparable levels of expression of DRbeta 1Ebeta2. These results suggest that positive selection is sensitive to quantitative variation in MHC class II density, unmasked when antigen is limiting, but is relatively insensitive to qualitative variation in the MHC class II: CD4 interaction.
Assuntos
Antígenos CD4/fisiologia , Antígenos HLA-DR/fisiologia , Linfócitos T/fisiologia , Animais , Células Cultivadas , Antígenos HLA-DR/análise , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Camundongos TransgênicosRESUMO
Conflicting results of the effects of Ag presentation by MHC class II-expressing T cells have been described. In some studies class II-expressing T cells have been shown to act as effective APCs, while others have reported that the recognition of Ag on the surface of another T cell inactivates IL-2 production. In this study we have investigated the mechanisms involved in Ag presentation by T cells. The results obtained suggest that 1) lack of costimulation is not responsible for the inhibitory effects of T cell Ag presentation on IL-2 production; the provision of costimulation by immobilized anti-CD28 Ab or by the addition of accessory cells failed to reverse the effects of T cell Ag presentation, but restored the response to immobilized anti-CD3; 2) T cell Ag presentation induced a minimal increase in intracellular Ca2+ compared with that induced by antigen-pulsed B cells; this difference in the calcium response is not explained by quantitative differences in ligand density between B cells and T cells; and 3) despite the weak calcium signal, T cell presentation supported IL-4 release in the absence of IL-2 production. Taken together these data suggest that T cell Ag presentation leads to altered TCR/CD3-transduced signals, which biases the T cell towards a Th2 phenotype.
Assuntos
Apresentação de Antígeno , Células Apresentadoras de Antígenos/imunologia , Interleucina-2/antagonistas & inibidores , Interleucina-2/biossíntese , Interleucina-4/metabolismo , Transdução de Sinais/imunologia , Linfócitos T/imunologia , Animais , Apresentação de Antígeno/efeitos dos fármacos , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/metabolismo , Linfócitos B/imunologia , Linfócitos B/metabolismo , Cálcio/metabolismo , Linhagem Celular , Espaço Extracelular/metabolismo , Humanos , Interleucina-12/farmacologia , Interleucina-2/metabolismo , Ativação Linfocitária , Camundongos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismoRESUMO
DAP.3 transfectants expressing native H-2E molecules with or without human LFA-3 and ICAM-1 (intercellular adhesion molecule-1) failed to induce proliferation by human peripheral blood T cells. Introduction of sequence from the DR beta 2 domain into the H-2E molecule led to the induction of detectable proliferation, which was substantially augmented by co-expression of human LFA-3 and ICAM-1 to levels comparable to those induced by DAP.3 cells co-expressing wild-type DR alloantigens with human LFA-3/ICAM-1. In marked contrast, cells expressing native H-2A molecules together with human accessory molecules provoked strong primary proliferative responses. The results of Ab inhibition experiments confirmed that this was caused by direct xenorecognition. In limiting dilution assays the frequency of anti-H-2A, IL-2-secreting, CD4+ human T cells was only fivefold lower than that measured against a DR alloantigen expressed on the same background. No measurable frequency was recorded against H-2E-expressing cells. Evidence to suggest that this difference was a result of isotype-specific differences in the interaction with CD4 was provided using transfectants expressing DR alloantigens with either the H-2E or H-2A beta 2 domain. DR molecules with the H-2A beta 2 domain stimulated a substantially stronger response than those with the H-2E beta 2 domain. These results challenge the view that xenogeneic T cell responses between evolutionarily distant species are weak; further emphasize the influence of the interaction between the T cell co-receptor molecule CD4, with its MHC class II molecular ligand on the strength of primary xenoresponses; and suggest that MHC class II isotypes may differ substantially in their interaction with CD4.