RESUMEN
The immunoreceptor tyrosine-based activation motifs (ITAMs) in the CD3 chains associated with the T cell receptor (TCR) are crucial for TCR signaling. To probe the role of the CD3gamma-ITAM in T cell development, we created knock-in mice in which the CD3gamma chain of the TCR complex is replaced by a mutant signaling-deficient CD3gamma chain, lacking the CD3gamma-ITAM. This mutation results in considerable impairment in positive selection in the polyclonal TCR repertoire. When CD3gamma-deltaITAM mice are crossed to mice expressing transgenic F5 TCRs, their thymocytes are completely unable to perform positive selection in vivo in response to intrathymic ligands. Also, the in vitro positive selection response of double-positive (DP) thymocytes with F5-CD3gamma-deltaITAM mutant receptors to their agonist ligand and many of its variants is severely impaired or abrogated. Yet, the binding and dissociation constants of agonist ligands for the F5 receptor are not affected by the CD3gamma-deltaITAM mutation. Furthermore, DP thymocytes with mutant receptors can respond to agonist ligand with normal antigen sensitivity and to normal levels, as shown by their ability to induce CD69 up-regulation, TCR down-regulation, negative selection, and ZAP70 and c-Jun NH2-terminal kinase activation. In sharp contrast, induction of extracellular signal-regulated kinase (ERK) activation and linker for activation of T cells (LAT) phosphorylation are severely impaired in these cells. Together, these findings underscore that intrinsic properties of the TCR-CD3 complex regulate selection at the DP checkpoint. More importantly, this analysis provides the first direct genetic evidence for a role of the CD3gamma-ITAM in TCR-driven thymocyte selection.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Complejo CD3/metabolismo , Diferenciación Celular/inmunología , Proteínas de la Membrana , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/inmunología , Timo/metabolismo , Secuencias de Aminoácidos/fisiología , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Complejo CD3/genética , Complejo CD3/inmunología , Antígenos CD4/biosíntesis , Antígenos CD8/biosíntesis , Proteínas Portadoras/metabolismo , Células Cultivadas , Cruzamientos Genéticos , Activación Enzimática/efectos de los fármacos , Citometría de Flujo , Técnicas In Vitro , Proteínas Quinasas JNK Activadas por Mitógenos , Lectinas Tipo C , Ligandos , Ratones , Ratones Mutantes , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Mutación , Fosfoproteínas/metabolismo , Fosforilación , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/citología , Timo/inmunología , Proteína Tirosina Quinasa ZAP-70RESUMEN
Progression of immature CD4(-)CD8(-) thymocytes beyond the beta-selection checkpoint to the CD4(+)CD8(+) stage requires activation of the pre-TCR complex; however, few of the DNA-binding proteins that serve as molecular effectors of those pre-TCR signals have been identified. We demonstrate in this study that members of the early growth response (Egr) family of transcription factors are critical effectors of the signals that promote this developmental transition. Specifically, the induction of three Egr family members (Egr1, 2, and 3) correlates with pre-TCR activation and development of CD4(-)CD8(-) thymocytes beyond the beta-selection checkpoint. Enforced expression of each of these Egr factors is able to bypass the block in thymocyte development associated with defective pre-TCR function. However, Egr family members may play somewhat distinct roles in promoting thymocyte development, because there are differences in the genes modulated by enforced expression of particular Egr factors. Finally, interfering with Egr function using dominant-negative proteins disrupts thymocyte development from the CD4(-)CD8(-) to the CD4(+)CD8(+) stage. Taken together, these data demonstrate that the Egr proteins play an essential role in executing the differentiation program initiated by pre-TCR signaling.