RESUMEN
The adapter protein Src homology 2 (SH2) domain-containing leukocyte protein of 76 kDa (SLP-76) is critical for multiple aspects of T cell development and function. Through its protein-binding domains, SLP-76 serves as a platform for the assembly of multiple enzymes and adapter proteins that function together to activate second messengers required for TCR signal propagation. The N terminus of SLP-76, which contains three tyrosines that serve as docking sites for SH2 domain-containing proteins, and the central proline-rich region of SLP-76 have been well studied and are known to be important for both thymocyte selection and activation of peripheral T cells. Less is known about the function of the C-terminal SH2 domain of SLP-76. This region inducibly associates with ADAP and HPK1. Combining regulated deletion of endogenous SLP-76 with transgenic expression of a SLP-76 SH2 domain mutant, we demonstrate that the SLP-76 SH2 domain is required for peripheral T cell activation and positive selection of thymocytes, a function not previously attributed to this region. This domain is also important for T cell proliferation, IL-2 production, and phosphorylation of protein kinase D and IκB. ADAP-deficient T cells display similar, but in some cases less severe, defects despite phosphorylation of a negative regulatory site on SLP-76 by HPK1, a function that is lost in SLP-76 SH2 domain mutant T cells.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Diferenciación Celular/inmunología , Activación de Linfocitos/inmunología , Fosfoproteínas/fisiología , Linfocitos T/citología , Linfocitos T/inmunología , Dominios Homologos src/inmunología , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Diferenciación Celular/genética , Humanos , Células Jurkat , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Dominios Homologos src/genéticaRESUMEN
The adaptor protein Src homology 2 domain-containing leukocyte-specific protein of 76 kDa (SLP-76) is central to the organization of intracellular signaling downstream of the T-cell receptor (TCR). Evaluation of its role in mature, primary T cells has been hampered by developmental defects that occur in the absence of WT SLP-76 protein in thymocytes. Here, we show that following tamoxifen-regulated conditional deletion of SLP-76, mature, antigen-inexperienced T cells maintain normal TCR surface expression but fail to transduce TCR-generated signals. Conditionally deficient T cells fail to proliferate in response to antigenic stimulation or a lymphopenic environment. Mice with induced deletion of SLP-76 are resistant to induction of the CD4+ T-cell-mediated autoimmune disease experimental autoimmune encephalomyelitis. Altogether, our findings demonstrate the critical role of SLP-76-mediated signaling in initiating T-cell-directed immune responses both in vitro and in vivo and highlight the ability to analyze signaling processes in mature T cells in the absence of developmental defects.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Linfocitos T CD4-Positivos/inmunología , Fosfoproteínas/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Linfocitos T CD4-Positivos/metabolismo , Proliferación Celular , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosfoproteínas/genética , Reacción en Cadena de la Polimerasa , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/metabolismo , Tamoxifeno/farmacologíaRESUMEN
The requirements for tonic T-cell receptor (TCR) signaling in CD8(+) memory T-cell generation and homeostasis are poorly defined. The SRC homology 2 (SH2)-domain-containing leukocyte protein of 76 kDa (SLP-76) is critical for proximal TCR-generated signaling. We used temporally mediated deletion of SLP-76 to interrupt tonic and activating TCR signals after clearance of the lymphocytic choriomeningitis virus (LCMV). SLP-76-dependent signals are required during the contraction phase of the immune response for the normal generation of CD8 memory precursor cells. Conversely, LCMV-specific memory CD8 T cells generated in the presence of SLP-76 and then acutely deprived of TCR-mediated signals persist in vivo in normal numbers for more than 40 weeks. Tonic TCR signals are not required for the transition of the memory pool toward a central memory phenotype, but the absence of SLP-76 during memory homeostasis substantially alters the kinetics. Our data are consistent with a model in which tonic TCR signals are required at multiple stages of differentiation, but are dispensable for memory CD8 T-cell persistence.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Linfocitos T CD8-positivos/inmunología , Diferenciación Celular , Memoria Inmunológica , Fosfoproteínas/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal , Animales , Western Blotting , Linfocitos T CD8-positivos/virología , Proliferación Celular , Femenino , Citometría de Flujo , Activación de Linfocitos , Coriomeningitis Linfocítica/inmunología , Coriomeningitis Linfocítica/patología , Coriomeningitis Linfocítica/virología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones TransgénicosRESUMEN
BACKGROUND: Ezrin/radixin/moesin (ERM) proteins are highly homologous proteins that function to link cargo molecules to the actin cytoskeleton. Ezrin and moesin are both expressed in mature lymphocytes, where they play overlapping roles in cell signaling and polarity, but their role in lymphoid development has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: We characterized ERM protein expression in lymphoid tissues and analyzed the requirement for ezrin expression in lymphoid development. In wildtype mice, we found that most cells in the spleen and thymus express both ezrin and moesin, but little radixin. ERM protein expression in the thymus was differentially regulated, such that ezrin expression was highest in immature thymocytes and diminished during T cell development. In contrast, moesin expression was low in early thymocytes and upregulated during T cell development. Mice bearing a germline deletion of ezrin exhibited profound defects in the size and cellularity of the spleen and thymus, abnormal thymic architecture, diminished hematopoiesis, and increased proportions of granulocytic precursors. Further analysis using fetal liver chimeras and thymic transplants showed that ezrin expression is dispensable in hematopoietic and stromal lineages, and that most of the defects in lymphoid development in ezrin(-/-) mice likely arise as a consequence of nutritional stress. CONCLUSIONS/SIGNIFICANCE: We conclude that despite high expression in lymphoid precursor cells, ezrin is dispensable for lymphoid development, most likely due to redundancy with moesin.
Asunto(s)
Proteínas del Citoesqueleto/genética , Regulación del Desarrollo de la Expresión Génica , Linfocitos T/citología , Timo/crecimiento & desarrollo , Timo/metabolismo , Animales , Recuento de Células , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/metabolismo , Hematopoyesis , Masculino , Proteínas de la Membrana/genética , Ratones , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Bazo/citología , Bazo/metabolismo , Células del Estroma/metabolismo , Linfocitos T/metabolismo , Timo/citología , Factores de Tiempo , Regulación hacia ArribaRESUMEN
The intracellular signaling mechanisms regulating the generation and long-term persistence of memory T cells in vivo remain unclear. In this study, we used mouse models with conditional deletion of the key T cell receptor (TCR)-coupled adaptor molecule SH2-domain-containing phosphoprotein of 76 kDa (SLP-76), to analyze signaling mechanisms for memory CD4 T cell generation, maintenance, and homeostasis. We found that ablation of SLP-76 expression after T cell priming did not inhibit generation of phenotypic effector or memory CD4 T cells; however, the resultant SLP-76-deficient memory CD4 T cells could not produce recall cytokines in response to TCR-mediated stimulation and showed decreased persistence in vivo. In addition, SLP-76-deficient memory CD4 T cells exhibited reduced steady-state homeostasis and were impaired in their ability to homeostatically expand in vivo in response to the gamma(c) cytokine IL-7, despite intact proximal signaling through the IL-7R-coupled JAK3/STAT5 pathway. Direct in vivo deletion of SLP-76 in polyclonal memory CD4 T cells likewise led to impaired steady-state homeostasis as well as impaired homeostatic responses to IL-7. Our findings demonstrate a dominant role for SLP-76-dependent TCR signals in regulating turnover and perpetuation of memory CD4 T cells and their responses to homeostatic cytokines, with implications for the selective survival of memory CD4 T cells following pathogen exposure, vaccination, and aging.