RESUMO
Current US Food and Drug Administration-approved chimeric antigen receptor (CAR) T cells harbor the T cell receptor (TCR)-derived ζ chain as an intracellular activation domain in addition to costimulatory domains. The functionality in a CAR format of the other chains of the TCR complex, namely CD3δ, CD3ε and CD3γ, instead of ζ, remains unknown. In the present study, we have systematically engineered new CD3 CARs, each containing only one of the CD3 intracellular domains. We found that CARs containing CD3δ, CD3ε or CD3γ cytoplasmic tails outperformed the conventional ζ CAR T cells in vivo. Transcriptomic and proteomic analysis revealed differences in activation potential, metabolism and stimulation-induced T cell dysfunctionality that mechanistically explain the enhanced anti-tumor performance. Furthermore, dimerization of the CARs improved their overall functionality. Using these CARs as minimalistic and synthetic surrogate TCRs, we have identified the phosphatase SHP-1 as a new interaction partner of CD3δ that binds the CD3δ-ITAM on phosphorylation of its C-terminal tyrosine. SHP-1 attenuates and restrains activation signals and might thus prevent exhaustion and dysfunction. These new insights into T cell activation could promote the rational redesign of synthetic antigen receptors to improve cancer immunotherapy.
Assuntos
Proteômica , Receptores de Antígenos de Linfócitos T , Complexo CD3 , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Membrana Celular/metabolismo , Ativação Linfocitária , Linfócitos TRESUMO
Initiation of T cell antigen receptor (TCR) signaling involves phosphorylation of CD3 cytoplasmic tails by the tyrosine kinase Lck. How Lck is recruited to the TCR to initiate signaling is not well known. We report a previously unknown binding motif in the CD3ε cytoplasmic tail that interacts in a noncanonical mode with the Lck SH3 domain: the receptor kinase (RK) motif. The RK motif is accessible only upon TCR ligation, demonstrating how ligand binding leads to Lck recruitment. Binding of the Lck SH3 domain to the exposed RK motif resulted in local augmentation of Lck activity, CD3 phosphorylation, T cell activation and thymocyte development. Introducing the RK motif into a well-characterized 41BB-based chimeric antigen receptor enhanced its antitumor function in vitro and in vivo. Our findings underscore how a better understanding of the functioning of the TCR might promote rational improvement of chimeric antigen receptor design for the treatment of cancer.
Assuntos
Complexo CD3/metabolismo , Ativação Linfocitária/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Antígenos Quiméricos/imunologia , Motivos de Aminoácidos/imunologia , Animais , Complexo CD3/imunologia , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/imunologia , Camundongos , Receptores de Antígenos de Linfócitos T/imunologiaRESUMO
Most adaptive immune responses require the activation of specific T cells through the T cell antigen receptor (TCR)-CD3 complex. Here we show that cholesterol sulfate (CS), a naturally occurring analog of cholesterol, inhibits CD3 ITAM phosphorylation, a crucial first step in T cell activation. In biochemical studies, CS disrupted TCR multimers, apparently by displacing cholesterol, which is known to bind TCRß. Moreover, CS-deficient mice showed heightened sensitivity to a self-antigen, whereas increasing CS content by intrathymic injection inhibited thymic selection, indicating that this molecule is an intrinsic regulator of thymocyte development. These results reveal a regulatory role for CS in TCR signaling and thymic selection, highlighting the importance of the membrane microenvironment in modulating cell surface receptor activation.
Assuntos
Membrana Celular/metabolismo , Ésteres do Colesterol/metabolismo , Colesterol/metabolismo , Linfócitos T/fisiologia , Timo/imunologia , Animais , Autoimunidade/genética , Células Cultivadas , Colesterol/análogos & derivados , Seleção Clonal Mediada por Antígeno , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Multimerização Proteica/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais , Sulfotransferases/genéticaRESUMO
The modification of proteins by ubiquitin has a major role in cells of the immune system and is counteracted by various deubiquitinating enzymes (DUBs) with poorly defined functions. Here we identified the ubiquitin-specific protease USP8 as a regulatory component of the T cell antigen receptor (TCR) signalosome that interacted with the adaptor Gads and the regulatory molecule 14-3-3ß. Caspase-dependent processing of USP8 occurred after stimulation of the TCR. T cell-specific deletion of USP8 in mice revealed that USP8 was essential for thymocyte maturation and upregulation of the gene encoding the cytokine receptor IL-7Rα mediated by the transcription factor Foxo1. Mice with T cell-specific USP8 deficiency developed colitis that was promoted by disturbed T cell homeostasis, a predominance of CD8(+) γδ T cells in the intestine and impaired regulatory T cell function. Collectively, our data reveal an unexpected role for USP8 as an immunomodulatory DUB in T cells.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular/imunologia , Endopeptidases/imunologia , Complexos Endossomais de Distribuição Requeridos para Transporte/imunologia , Timócitos/imunologia , Ubiquitina Tiolesterase/imunologia , Proteínas Adaptadoras de Transdução de Sinal/imunologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Linfócitos T CD8-Positivos/metabolismo , Diferenciação Celular/genética , Colite/genética , Colite/imunologia , Endopeptidases/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Proteína Forkhead Box O1 , Fatores de Transcrição Forkhead/imunologia , Fatores de Transcrição Forkhead/metabolismo , Homeostase , Humanos , Células Jurkat , Camundongos , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Receptores de Interleucina-7/imunologia , Receptores de Interleucina-7/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Timócitos/metabolismo , Ubiquitina Tiolesterase/genéticaRESUMO
Vitamin C (ascorbic acid) is a potent antioxidant and a cofactor for various enzymes including histone demethylases and methylcytosine dioxygenases. Vitamin C also exerts direct cytotoxicity toward selected tumor cells including colorectal carcinoma. Moreover, vitamin C has been shown to impact immune cell differentiation at various levels including maturation and/or functionality of T cells and their progenitors, dendritic cells, B cells, and NK cells. γδ T cells have recently attracted great interest as effector cells for cell-based cancer immunotherapy, due to their HLA-independent recognition of a large variety of tumor cells. While γδ T cells can thus be also applied as an allogeneic off-the-shelf product, it is obvious that the effector function of γδ T cells needs to be optimized to ensure the best possible clinical efficacy. Here we review the immunomodulatory mechanisms of vitamin C with a special focus on how vitamin C enhances the effector function of γδ T cells. We also discuss future directions of how vitamin C can be used in the clinical setting to boost the efficacy of adoptive cell therapies.
Assuntos
Ácido Ascórbico , Receptores de Antígenos de Linfócitos T gama-delta , Ácido Ascórbico/farmacologia , Humanos , Receptores de Antígenos de Linfócitos T gama-delta/imunologia , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Animais , Imunoterapia Adotiva/métodos , Linfócitos T/imunologia , Neoplasias/imunologia , Neoplasias/terapia , Neoplasias/tratamento farmacológico , Diferenciação Celular/imunologia , Diferenciação Celular/efeitos dos fármacosRESUMO
Signaling through the T cell receptor (TCR) controls adaptive immune responses. Antigen binding to TCRαß transmits signals through the plasma membrane to induce phosphorylation of the CD3 cytoplasmic tails by incompletely understood mechanisms. Here we show that cholesterol bound to the TCRß transmembrane region keeps the TCR in a resting, inactive conformation that cannot be phosphorylated by active kinases. Only TCRs that spontaneously detached from cholesterol could switch to the active conformation (termed primed TCRs) and then be phosphorylated. Indeed, by modulating cholesterol binding genetically or enzymatically, we could switch the TCR between the resting and primed states. The active conformation was stabilized by binding to peptide-MHC, which thus controlled TCR signaling. These data are explained by a model of reciprocal allosteric regulation of TCR phosphorylation by cholesterol and ligand binding. Our results provide both a molecular mechanism and a conceptual framework for how lipid-receptor interactions regulate signal transduction.
Assuntos
Imunidade Adaptativa , Colesterol/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Linfócitos T/imunologia , Regulação Alostérica , Antígenos/imunologia , Antígenos/metabolismo , Antígenos de Histocompatibilidade/metabolismo , Humanos , Células Jurkat , Ativação Linfocitária , Modelos Imunológicos , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Conformação Proteica , Estabilidade Proteica , Transdução de SinaisRESUMO
Signaling via the T cell receptor (TCR) is critical during the development, maintenance, and activation of T cells. Quantitative aspects of TCR signaling have an important role during positive and negative selection, lineage choice, and ability to respond to small amounts of antigen. By using a mutant mouse line expressing a hypomorphic allele of the CD3ζ chain, we show here that the strength of pre-TCRmediated signaling during T cell development determines the diversity of the TCRß repertoire available for positive and negative selection, and hence of the final αßTCR repertoire. This finding uncovers an unexpected, pre-TCR signalingdependent and repertoireshaping role for ß-selection beyond selection of in-frame rearranged TCRß chains. Our data furthermore support a model of pre-TCR signaling in which the arrangement of this receptor in stable nanoclusters determines its quantitative signaling capacity.
Assuntos
Receptores de Antígenos de Linfócitos T alfa-beta , Linfócitos T , Animais , Complexo CD3/genética , Diferenciação Celular , Camundongos , Camundongos Mutantes , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Transdução de Sinais , Linfócitos T/imunologiaRESUMO
CCR5 is not only a coreceptor for HIV-1 infection in CD4+ T cells, but also contributes to their functional fitness. Here, we show that by limiting transcription of specific ceramide synthases, CCR5 signaling reduces ceramide levels and thereby increases T-cell antigen receptor (TCR) nanoclustering in antigen-experienced mouse and human CD4+ T cells. This activity is CCR5-specific and independent of CCR5 co-stimulatory activity. CCR5-deficient mice showed reduced production of high-affinity class-switched antibodies, but only after antigen rechallenge, which implies an impaired memory CD4+ T-cell response. This study identifies a CCR5 function in the generation of CD4+ T-cell memory responses and establishes an antigen-independent mechanism that regulates TCR nanoclustering by altering specific lipid species.
Assuntos
Antígenos/imunologia , Linfócitos T CD4-Positivos/imunologia , Ceramidas/imunologia , Memória Imunológica , Receptores CCR5/deficiência , Animais , Antígenos/genética , Linfócitos T CD4-Positivos/citologia , Ceramidas/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , Receptores CCR5/imunologiaRESUMO
The αß T-cell receptor (TCR) is a multiprotein complex controlling the activation of T cells. Although the structure of the complete TCR is not known, cumulative evidence supports that the TCR cycles between different conformational states that are promoted either by thermal motion or by force. These structural transitions determine whether the TCR engages intracellular effectors or not, regulating TCR phosphorylation and signaling. As for other membrane receptors, ligand binding selects and stabilizes the TCR in active conformations, and/or switches the TCR to activating states that were not visited before ligand engagement. Here we review the main models of TCR allostery, that is, ligand binding at TCRαß changes the structure at CD3 and ζ. (a) The ITAM and proline-rich sequence exposure model, in which the TCR's cytoplasmic tails shield each other and ligand binding exposes them for phosphorylation. (b) The membrane-ITAM model, in which the CD3ε and ζ tails are sequestered inside the membrane and again ligand binding exposes them. (c) The mechanosensor model in which ligand binding exerts force on the TCR, inducing structural changes that allow signaling. Since these models are complementary rather than competing, we propose a unified model that aims to incorporate all existing data.
Assuntos
Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Animais , Humanos , Ligantes , Ativação Linfocitária , Modelos Biológicos , Ligação Proteica , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
During T cell development, Lck gene expression is temporally controlled by its proximal and distal promoters. The pLckCre transgenic mouse available from The Jackson Laboratory, in which the proximal promoter of Lck drives Cre expression, is a commonly used Cre driver line to recombine genes flanked by loxP sites in T cells. pLckCre drives recombination early in thymocyte development and is frequently used to delete genes in αß and γδ T cells. We found that pLckCre failed to efficiently delete floxed genes in γδ T cells in contrast to a complete deletion in conventional as well as unconventional αß T cells. Mechanistically, γδ T cells inefficiently transcribed the endogenous proximal Lck promoter compared with αß T cells during adult thymic development. A small population of γδ T cells that had activated pLckCre was detected, many of which were located in nonlymphoid organs as well as precommitted IL-17- or IFN-γ-producing γδ T effector cells. In newborn thymi, both pLckCre and endogenous Lck proximal promoter expression were substantially enhanced, giving rise to an elevated fraction of γδ T cells with recombined floxed genes that were increased in unique γδ T subsets, such as the IL-17-producing γδ T cells. Our data point out striking differences in Lck transcription between perinatal and adult γδ T cell development. Taken together, the data presented in this study shed new light on γδ T cell development and stimulate a reanalysis of data generated using the pLckCre transgenic mice.
Assuntos
Integrases/genética , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/genética , Regiões Promotoras Genéticas/genética , Receptores de Antígenos de Linfócitos T gama-delta/genética , Animais , Diferenciação Celular/genética , Interleucina-17/genética , Camundongos , Camundongos Transgênicos , Subpopulações de Linfócitos T/imunologiaRESUMO
Activation of T cells by agonistic peptide-MHC can be inhibited by antagonistic ones. However, the exact mechanism remains elusive. We used Jurkat cells expressing two different TCRs and tested whether stimulation of the endogenous TCR by agonistic anti-Vß8 antibodies can be modulated by ligand-binding to the second, optogenetic TCR. The latter TCR uses phytochrome B tetramers (PhyBt) as ligand, the binding half-life of which can be altered by light. We show that this half-life determined whether the PhyBt acted as a second agonist (long half-life), an antagonist (short half-life) or did not have any influence (very short half-life) on calcium influx. A mathematical model of this cross-antagonism shows that a mechanism based on an inhibitory signal generated by early recruitment of a phosphatase and an activating signal by later recruitment of a kinase explains the data.
Assuntos
Optogenética , Receptores de Antígenos de Linfócitos T/antagonistas & inibidores , Anticorpos/metabolismo , Membrana Celular/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Meia-Vida , Humanos , Células Jurkat , Ligantes , Modelos Biológicos , Receptores de Antígenos de Linfócitos T/metabolismoRESUMO
Following T-cell antigen receptor (TCR) engagement, rearrangement of the actin cytoskeleton supports intracellular signal transduction and T-cell activation. The non-catalytic region of the tyrosine kinase (Nck) molecule is an adapter protein implicated in TCR-induced actin polymerization. Further, Nck is recruited to the CD3ε subunit of the TCR upon TCR triggering. Here we examine the role of actin polymerization in the recruitment of Nck to the TCR. To this end, Nck binding to CD3ε was quantified in Jurkat cells using the proximity ligation assay. We show that inhibition of actin polymerization using cytochalasin D delayed the recruitment of Nck1 to the TCR upon TCR triggering. Interestingly, CD3ε phosphorylation was also delayed. These findings suggest that actin polymerization promotes the recruitment of Nck to the TCR, enhancing downstream signaling, such as phosphorylation of CD3ε.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Complexo CD3/metabolismo , Ativação Linfocitária , Proteínas Oncogênicas/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Linfócitos T/enzimologia , Citoesqueleto de Actina/imunologia , Actinas/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Complexo CD3/imunologia , Citocalasina D/farmacologia , Humanos , Células Jurkat , Ativação Linfocitária/efeitos dos fármacos , Proteínas Oncogênicas/genética , Fosforilação , Polimerização , Ligação Proteica , Complexo Receptor-CD3 de Antígeno de Linfócitos T/genética , Complexo Receptor-CD3 de Antígeno de Linfócitos T/imunologia , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Fatores de Tempo , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Despite being mutated in cancer and RASopathies, the role of the activation segment (AS) has not been addressed for B-Raf signaling in vivo. Here, we generated a conditional knock-in mouse allowing the expression of the B-Raf(AVKA) mutant in which the AS phosphoacceptor sites T599 and S602 are replaced by alanine residues. Surprisingly, despite producing a kinase-impaired protein, the Braf(AVKA) allele does not phenocopy the lethality of Braf-knockout or paradoxically acting knock-in alleles. However, Braf(AVKA) mice display abnormalities in the hematopoietic system, a distinct facial morphology, reduced ERK pathway activity in the brain, and an abnormal gait. This phenotype suggests that maximum B-Raf activity is required for the proper development, function, and maintenance of certain cell populations. By establishing conditional murine embryonic fibroblast cultures, we further show that MEK/ERK phosphorylation and the immediate early gene response toward growth factors are impaired in the presence of B-Raf(AVKA). Importantly, alanine substitution of T599/S602 impairs the transformation potential of oncogenic non-V600E B-Raf mutants and a fusion protein, suggesting that blocking their phosphorylation could represent an alternative strategy to ATP-competitive inhibitors.
Assuntos
Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Animais , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Ativação Enzimática/genética , Ativação Enzimática/fisiologia , Feminino , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/efeitos da radiação , Masculino , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Mutação , Fosforilação , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
BACKGROUND: Upon engagement of the T-cell receptor (TCR), the Src-family protein tyrosine kinase p56Lck phosphorylates components of the TCR (e.g. the TCRζ chains), thereby initiating T-cell activation. The enzymatic activity of Lck is primarily regulated via reversible and dynamic phosphorylation of two tyrosine residues, Y394 and Y505. Lck possesses an additional highly conserved tyrosine Y192, located within the SH2 domain, whose role in T-cell activation is not fully understood. METHODS: Knock-in mice expressing a phospho-mimetic (Y192E) form of Lck were generated. Cellular and biochemical characterization was performed to elucidate the function of Y192 in primary T cells. HEK 293T and Jurkat T cells were used for in vitro studies. RESULTS: Co-immunoprecipitation studies and biochemical analyses using T cells from LckY192E knock-in mice revealed a diminished binding of LckY192E to CD45 and a concomitant hyperphosphorylation of Y505, thus corroborating previous data obtained in Jurkat T cells. Surprisingly however, in vitro kinase assays showed that LckY192E possesses a normal enzymatic activity in human and murine T cells. FLIM/FRET measurements employing an LckY192E biosensor further indicated that the steady state conformation of the LckY192E mutant is similar to Lckwt. These data suggest that Y192 might regulate Lck functions also independently from the Lck/CD45-association. Indeed, when LckY192E was expressed in CD45-/-/Csk-/- non-T cells (HEK 293T cells), phosphorylation of Y505 was similar to Lckwt, but LckY192E still failed to optimally phosphorylate and activate the Lck downstream substrate ZAP70. Furthermore, LckY19E was recruited less to CD3 after TCR stimulation. CONCLUSIONS: Taken together, phosphorylation of Y192 regulates Lck functions in T cells at least twofold, by preventing Lck association to CD45 and by modulating ligand-induced recruitment of Lck to the TCR. MAJOR FINDINGS: Our data change the current view on the function of Y192 and suggest that Y192 also regulates Lck activity in a manner independent of Y505 phosphorylation. Video Abstract.
Assuntos
Antígenos Comuns de Leucócito/metabolismo , Ativação Linfocitária/imunologia , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/química , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Linfócitos T/imunologia , Tirosina/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Células HEK293 , Humanos , Cinética , Camundongos Endogâmicos C57BL , Fosforilação , Conformação Proteica , Receptores de Antígenos de Linfócitos T/metabolismo , Baço/imunologia , Relação Estrutura-Atividade , Especificidade por Substrato , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
In the current issue of Immunity, Martínez-Martín et al. (2011) describe the central supramolecular activation cluster (cSMAC) as a site of clathrin-independent T cell receptor (TCR) internalization and trogocytosis. Further, they identify small Rho GTPases TC21 and RhoG as key mediators of these processes.
RESUMO
Although memory T cells respond more vigorously to stimulation and they are more sensitive to low doses of antigen than naive T cells, the molecular basis of this increased sensitivity remains unclear. We have previously shown that the T cell receptor (TCR) exists as different-sized oligomers on the surface of resting T cells and that large oligomers are preferentially activated in response to low antigen doses. Through biochemistry and electron microscopy, we now showed that previously stimulated and memory T cells have more and larger TCR oligomers at the cell surface than their naive counterparts. Reconstitution of cells and mice with a point mutant of the CD3ζ subunit, which impairs TCR oligomer formation, demonstrated that the increased size of TCR oligomers was directly responsible for the increased sensitivity of antigen-experienced T cells. Thus, we propose that an "avidity maturation" mechanism underlies T cell antigenic memory.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Memória Imunológica , Oligodesoxirribonucleotídeos , Receptores de Antígenos de Linfócitos T/imunologia , Animais , Complexo CD3/genética , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Citometria de Fluxo , Humanos , Immunoblotting , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/imunologia , Receptores de Antígenos de Linfócitos T/genéticaRESUMO
The activity of the αß TCR is controlled by conformational switches. In the resting conformation, the TCR is not phosphorylated and is inactive. Binding of multivalent peptide-MHC to the TCR stabilizes the active conformation, leading to TCR signaling. These two conformations allow the TCRs to be allosterically regulated. We review recent data on heterotropic allostery where peptide-MHC and membrane cholesterol serve opposing functions as positive and negative allosteric regulators, respectively. In resting T cells cholesterol keeps TCRs in the resting conformation that otherwise would become spontaneously active. This regulation is well described by the classical Monod-Wyman-Changeux model of allostery. Moreover, the observation that TCRs assemble into nanoclusters might allow for homotropic allostery, in which individual TCRs could positively cooperate and thus enhance the sensitivity of T cell activation. This new view of TCR regulation will contribute to a better understanding of TCR functioning.
Assuntos
Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/imunologia , Regulação Alostérica , Animais , Humanos , Modelos Moleculares , Conformação Proteica , Transdução de Sinais/imunologiaRESUMO
The T-cell receptor (TCR)-CD3 complex, expressed on T cells, determines the outcome of a T-cell response. It consists of the TCR-αß heterodimer and the non-covalently associated signalling dimers of CD3εγ, CD3εδ and CD3ζζ. TCR-αß binds specifically to a cognate peptide antigen bound to an MHC molecule, whereas the CD3 subunits transmit the signal into the cytosol to activate signalling events. Recruitment of proteins to specialized localizations is one mechanism to regulate activation and termination of signalling. In the last 25 years a large number of signalling molecules recruited to the TCR-CD3 complex upon antigen binding to TCR-αß have been described. Here, we review knowledge about five of those interaction partners: Lck, ZAP-70, Nck, WASP and Numb. Some of these proteins have been targeted in the development of immunomodulatory drugs aiming to treat patients with autoimmune diseases and organ transplants.
Assuntos
Proteínas de Transporte/metabolismo , Complexo Receptor-CD3 de Antígeno de Linfócitos T/metabolismo , Transdução de Sinais , Subpopulações de Linfócitos T/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Complexo CD3/química , Complexo CD3/genética , Complexo CD3/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/genética , Humanos , Proteína Tirosina Quinase p56(lck) Linfócito-Específica/metabolismo , Proteínas de Membrana/metabolismo , Mutação , Proteínas do Tecido Nervoso/metabolismo , Proteínas Oncogênicas/metabolismo , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Complexo Receptor-CD3 de Antígeno de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/química , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/metabolismo , Subpopulações de Linfócitos T/imunologia , Proteína da Síndrome de Wiskott-Aldrich/metabolismo , Proteína-Tirosina Quinase ZAP-70/metabolismoRESUMO
Ligand binding to the TCR causes a conformational change at the CD3 subunits to expose the CD3ε cytoplasmic proline-rich sequence (PRS). It was suggested that the PRS is important for TCR signaling and T cell activation. It has been shown that the purified, recombinant SH3.1 domain of the adaptor molecule noncatalytic region of tyrosine kinase (Nck) can bind to the exposed PRS of CD3ε, but the molecular mechanism of how full-length Nck binds to the TCR in cells has not been investigated so far. Using the in situ proximity ligation assay and copurifications, we show that the binding of Nck to the TCR requires partial phosphorylation of CD3ε, as it is based on two cooperating interactions. First, the SH3.1(Nck) domain has to bind to the nonphosphorylated and exposed PRS, that is, the first ITAM tyrosine has to be in the unphosphorylated state. Second, the SH2(Nck) domain has to bind to the second ITAM tyrosine in the phosphorylated state. Likewise, mutations of the SH3.1 and SH2 domains in Nck1 resulted in the loss of Nck1 binding to the TCR. Furthermore, expression of an SH3.1-mutated Nck impaired TCR signaling and T cell activation. Our data suggest that the exact pattern of CD3ε phosphorylation is critical for TCR functioning.