RESUMO
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.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/imunologia , Antígenos CD28/imunologia , Proteínas de Transporte/imunologia , Guanilato Ciclase/imunologia , Proteína Quinase C/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte/genética , Citometria de Fluxo , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas dos Microfilamentos , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Alinhamento de Sequência , Análise de Sequência de DNA , Organismos Livres de Patógenos EspecíficosRESUMO
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.