RESUMEN
Although T cell activation can result from signaling via T cell antigen receptor (TCR) alone, physiological T cell responses require costimulation via the coreceptor CD28. Through the use of an N-ethyl-N-nitrosourea-mutagenesis screen, we identified a mutation in Rltpr. We found that Rltpr was a lymphoid cell-specific, actin-uncapping protein essential for costimulation via CD28 and the development of regulatory T cells. Engagement of TCR-CD28 at the immunological synapse resulted in the colocalization of CD28 with both wild-type and mutant Rltpr proteins. However, the connection between CD28 and protein kinase C-θ and Carma1, two key effectors of CD28 costimulation, was abrogated in T cells expressing mutant Rltpr, and CD28 costimulation did not occur in those cells. Our findings provide a more complete model of CD28 costimulation in which Rltpr has a key role.
Asunto(s)
Proteínas Adaptadoras de Señalización CARD/inmunología , Antígenos CD28/inmunología , Proteínas Portadoras/inmunología , Guanilato Ciclasa/inmunología , Proteína Quinasa C/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T Reguladores/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/genética , Citometría de Flujo , Activación de Linfocitos/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos , Microscopía Confocal , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Alineación de Secuencia , Análisis de Secuencia de ADN , Organismos Libres de Patógenos EspecíficosRESUMEN
The RLTPR cytosolic protein, also known as CARMIL2, is essential for CD28 co-stimulation in mice, but its importance in human T cells and mode of action remain elusive. Here, using affinity purification followed by mass spectrometry analysis, we showed that RLTPR acts as a scaffold, bridging CD28 to the CARD11/CARMA1 cytosolic adaptor and to the NF-κB signaling pathway, and identified proteins not found before within the CD28 signaling pathway. We further demonstrated that RLTPR is essential for CD28 co-stimulation in human T cells and that its noncanonical pleckstrin-homology domain, leucine-rich repeat domain, and proline-rich region were mandatory for that task. Although RLTPR is thought to function as an actin-uncapping protein, this property was dispensable for CD28 co-stimulation in both mouse and human. Our findings suggest that the scaffolding role of RLTPR predominates during CD28 co-stimulation and underpins the similar function of RLTPR in human and mouse T cells. Along that line, the lack of functional RLTPR molecules impeded the differentiation toward Th1 and Th17 fates of both human and mouse CD4+ T cells. RLTPR was also expressed in both human and mouse B cells. In the mouse, RLTPR did not play, however, any detectable role in BCR-mediated signaling and T cell-independent B cell responses.