RESUMO
The extraordinary sensitivity of CD8+ T cells to recognize antigen impinges to a large extent on the coreceptor CD8. While several studies have shown that the CD8beta chain endows CD8 with efficient coreceptor function, the molecular basis for this is enigmatic. Here we report that cell-associated CD8alphabeta, but not CD8alphaalpha or soluble CD8alphabeta, substantially increases the avidity of T cell receptor (TCR)-ligand binding. To elucidate how the cytoplasmic and transmembrane portions of CD8beta endow CD8 with efficient coreceptor function, we examined T1.4 T cell hybridomas transfected with various CD8beta constructs. T1.4 hybridomas recognize a photoreactive Plasmodium berghei circumsporozoite (PbCS) peptide derivative (PbCS (4-azidobezoic acid [ABA])) in the context of H-2K(d), and permit assessment of TCR-ligand binding by TCR photoaffinity labeling. We find that the cytoplasmic portion of CD8beta, mainly due to its palmitoylation, mediates partitioning of CD8 in lipid rafts, where it efficiently associates with p56(lck). In addition, the cytoplasmic portion of CD8beta mediates constitutive association of CD8 with TCR/CD3. The resulting TCR-CD8 adducts exhibit high affinity for major histocompatibility complex (MHC)-peptide. Importantly, because CD8alphabeta partitions in rafts, its interaction with TCR/CD3 promotes raft association of TCR/CD3. Engagement of these TCR/CD3-CD8/lck adducts by multimeric MHC-peptide induces activation of p56(lck) in rafts, which in turn phosphorylates CD3 and initiates T cell activation.
Assuntos
Antígenos CD8/metabolismo , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Microdomínios da Membrana/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Sequência de Aminoácidos , Animais , Complexo CD3/metabolismo , Células CHO , Cálcio/metabolismo , Cricetinae , Dados de Sequência Molecular , FosforilaçãoRESUMO
To investigate the role of the coreceptor CD8 and lipid rafts in cytotoxic T lymphocyte (CTL) activation, we used soluble mono-and multimeric H-2Kd-peptide complexes and cloned S14 CTL specific for a photoreactive derivative of the Plasmodium berghei circumsporozoite (PbCS) peptide 252-260 [PbCS(ABA)]. We report that activation of CTL in suspension requires multimeric Kd-PbCS(ABA) complexes co-engaging TCR and CD8. Using TCR ligand photo-cross-linking, we find that monomeric Kd-PbCS(ABA) complexes promote association of TCR/CD3 with CD8/p56lck. Dimerization of these adducts results in activation of p56lck in lipid rafts, where phosphatases are excluded. Additional cross-linking further increases p56lck kinase activity, induces translocation of TCR/CD3 and other signaling molecules to lipid rafts and intracellular calcium mobilization. These events are prevented by blocking Src kinases or CD8 binding to TCR-associated Kd molecules, indicating that CTL activation is initiated by cross-linking of CD8-associated p56lck. They are also inhibited by methyl-beta-cyclodextrin, which disrupts rafts and by dipalmitoyl phosphatidylethanolamine, which interferes with TCR signaling. Because efficient association of CD8 and p56lck takes place in rafts, both reagents, though in different ways, impair coupling of p56lck to TCR, thereby inhibiting the initial and essential activation of p56lck induced by cross-linking of engaged TCR.
Assuntos
Antígenos CD8/metabolismo , Ativação Linfocitária , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Microdomínios da Membrana/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T Citotóxicos/imunologia , beta-Ciclodextrinas , Animais , Antígenos de Protozoários/imunologia , Complexo CD3/metabolismo , Cálcio/metabolismo , Células Cultivadas , Reagentes de Ligações Cruzadas , Ciclodextrinas/farmacologia , Detergentes , Dimerização , Ativação Enzimática , Citometria de Fluxo , Humanos , Ativação Linfocitária/efeitos dos fármacos , Substâncias Macromoleculares , Microdomínios da Membrana/enzimologia , Ácido Palmítico/metabolismo , Plasmodium berghei/imunologia , Transporte Proteico , Transdução de Sinais , Solubilidade , Linfócitos T Citotóxicos/citologia , Linfócitos T Citotóxicos/efeitos dos fármacos , Linfócitos T Citotóxicos/metabolismoRESUMO
Cytotoxic T cell (CTL) activation by antigen requires the specific detection of peptide-major histocompatibility class I (pMHC) molecules on the target-cell surface by the T cell receptor (TCR). We examined the effect of mutations in the antigen-binding site of a Kb-restricted TCR on T cell activation, antigen binding and dissociation from antigen.These parameters were also examined for variants derived from a Kd-restricted peptide that was recognized by a CTL clone. Using these two independent systems, we show that T cell activation can be impaired by mutations that either decrease or increase the binding half-life of the TCR-pMHC interaction. Our data indicate that efficient T cell activation occurs within an optimal dwell-time range of TCR-pMHC interaction. This restricted dwell-time range is consistent with the exclusion of either extremely low or high affinity T cells from the expanded population during immune responses.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Ativação Linfocitária , Proteínas do Nucleocapsídeo , Peptídeos/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/imunologia , Animais , Antígenos/genética , Antígenos/imunologia , Citocinas/biossíntese , Meia-Vida , Antígenos de Histocompatibilidade Classe I/genética , Hibridomas , Cinética , Mutagênese Sítio-Dirigida , Nucleocapsídeo/genética , Nucleocapsídeo/imunologia , Peptídeos/genética , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
To investigate the molecular basis that makes heterodimeric CD8alphabeta a more efficient coreceptor than homodimeric CD8alphaalpha, we used various CD8 transfectants of T1.4 T cell hybridomas, which are specific for H-2Kd, and a photoreactive derivative of the Plasmodium berghei circumsporozoite peptide PbCS 252-260 (SYIPSAEKI). We demonstrate that CD8 is palmitoylated at the cytoplasmic tail of CD8beta and that this allows partitioning of CD8alphabeta, but not of CD8alphaalpha, in lipid rafts. Localization of CD8 in rafts is crucial for its coreceptor function. First, association of CD8 with the src kinase p56lck takes place nearly exclusively in rafts, mainly due to increased concentration of both components in this compartment. Deletion of the cytoplasmic domain of CD8beta abrogated localization of CD8 in rafts and association with p56lck. Second, CD8-mediated cross-linking of p56lck by multimeric Kd-peptide complexes or by anti-CD8 Ab results in p56lck activation in rafts, from which the abundant phosphatase CD45 is excluded. Third, CD8-associated activated p56lck phosphorylates CD3zeta in rafts and hence induces TCR signaling and T cell activation. This study shows that palmitoylation of CD8beta is required for efficient CD8 coreceptor function, mainly because it dramatically increases CD8 association with p56lck and CD8-mediated activation of p56lck in lipid rafts.