RESUMO
X-linked lymphoproliferative syndrome, characterized by fatal responses to Epstein-Barr virus infection, is caused by mutations affecting the adaptor SAP, which links SLAM family receptors to downstream signaling. Although cytotoxic defects in SAP-deficient T cells are documented, the mechanism remains unclear. We show that SAP-deficient murine CD8(+) T cells exhibited normal cytotoxicity against fibrosarcoma targets, yet had impaired adhesion to and killing of B cell and low-avidity T cell targets. SAP-deficient cytotoxic lymphocytes showed specific defects in immunological synapse organization with these targets, resulting in inefficient actin clearance. In the absence of SAP, signaling through the SLAM family members Ly108 and 2B4 resulted in increased recruitment of the SHP-1 phosphatase, associated with altered SHP-1 localization and decreased activation of Src kinases at the synapse. Hence, SAP and SLAM receptors regulate positive and negative signals required for organizing the T cell:B cell synapse and setting thresholds for cytotoxicity against distinct cellular targets.
Assuntos
Antígenos CD/imunologia , Antígenos Ly/imunologia , Sinapses Imunológicas/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Receptores Imunológicos/imunologia , Linfócitos T Citotóxicos/imunologia , Actinas/análise , Animais , Linfócitos B , Adesão Celular , Linhagem Celular Tumoral , Centrossomo/ultraestrutura , Citoesqueleto/ultraestrutura , Citotoxicidade Imunológica , Fibrossarcoma/patologia , Inositol Polifosfato 5-Fosfatases , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/genética , Linfoma de Células T/patologia , Camundongos , Monoéster Fosfórico Hidrolases/antagonistas & inibidores , Monoéster Fosfórico Hidrolases/fisiologia , Fosforilação , Processamento de Proteína Pós-Traducional , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Família de Moléculas de Sinalização da Ativação Linfocitária , Linfócitos T , Quinases da Família src/metabolismoRESUMO
CD4(+) T cells deficient in signaling lymphocyte activation molecule (SLAM)-associated protein (SAP) exhibit a selective impairment in adhesion to antigen-presenting B cells but not dendritic cells (DCs), resulting in defective germinal center formation. However, the nature of this selective adhesion defect remained unclear. We found that whereas T cell:DC interactions were primarily integrin dependent, T cell:B cell interactions had both an early integrin-dependent phase and a sustained phase that also required SAP. We further found that the SLAM family member CD84 was required for prolonged T cell:B cell contact, optimal T follicular helper function, and germinal center formation in vivo. Moreover, both CD84 and another SLAM member, Ly108, mediated T cell adhesion and participated in stable T cell:B cell interactions in vitro. Our results reveal insight into the dynamic regulation of T cell:B cell interactions and identify SLAM family members as critical components of sustained T cell:B cell adhesion required for productive humoral immunity.
Assuntos
Linfócitos B/metabolismo , Adesão Celular/imunologia , Centro Germinativo/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linfócitos T/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos Ly/imunologia , Antígenos Ly/metabolismo , Linfócitos B/imunologia , Linfócitos B/patologia , Adesão Celular/genética , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células , Células Cultivadas , Centro Germinativo/patologia , Interferon gama/metabolismo , Interleucinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Ativação Linfocitária/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Família de Moléculas de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
The promyelocytic zinc finger transcription factor (PLZF) is required for the development of activated phenotypes in NKT and other innate T lymphocytes. Although strong TCR stimulation has been implicated in the induction of PLZF, the factors regulating PLZF expression are incompletely understood. We show in this study that costimulation of preselection double-positive thymocytes through the signaling lymphocyte activation molecule family receptor Ly108 markedly enhanced PLZF expression compared with that induced by TCR stimulation alone. Costimulation with Ly108 increased expression of early growth response protein (Egr)-2 and binding of Egr-2 to the promoter of Zbtb16, which encodes PLZF, and resulted in PLZF levels similar to those seen in NKT cells. In contrast, costimulation with anti-CD28 failed to enhance Egr-2 binding and Zbtb16 expression. Moreover, mice lacking Ly108 showed decreased numbers of PLZF-expressing CD4(+) T cells. Together, these results support a potential role for Ly108 in the induction of PLZF.
Assuntos
Antígenos Ly/genética , Diferenciação Celular/imunologia , Fatores de Transcrição Kruppel-Like/genética , Timócitos/citologia , Animais , Anticorpos/farmacologia , Antígenos Ly/imunologia , Antígenos CD28/antagonistas & inibidores , Antígenos CD28/imunologia , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Cálcio/metabolismo , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Proteína 2 de Resposta de Crescimento Precoce/genética , Proteína 2 de Resposta de Crescimento Precoce/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/imunologia , Lipopolissacarídeos/farmacologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Transgênicos , Regiões Promotoras Genéticas , Proteína com Dedos de Zinco da Leucemia Promielocítica , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais/efeitos dos fármacos , Timócitos/efeitos dos fármacos , Timócitos/imunologiaRESUMO
Ly108 (CD352) is a member of the signaling lymphocyte activation molecule (SLAM) family of receptors that signals through SLAM-associated protein (SAP), an SH2 domain protein that can function by the recruitment of Src family kinases or by competition with phosphatases. Ly108 is expressed on a variety of hematopoietic cells, with especially high levels on developing thymocytes. We find that Ly108 is constitutively tyrosine phosphorylated in murine thymi in a SAP- and Fyn kinase-dependent manner. Phosphorylation of Ly108 is rapidly lost after thymocyte disaggregation, suggesting dynamic contact-mediated regulation of Ly108. Similar to recent reports, we find at least three isoforms of Ly108 mRNA and protein in the thymus, which are differentially expressed in the thymi of C57BL/6 and 129S6 mice that express the lupus-resistant and lupus-prone haplotypes of Ly108, respectively. Notably, the recently described novel isoform Ly108-H1 is not expressed in mice having the lupus-prone haplotype of Ly108, but is expressed in C57BL/6 mice. We further provide evidence for differential phosphorylation of these isoforms; the novel Ly108-H1does not undergo tyrosine phosphorylation, suggesting that it functions as a decoy isoform that contributes to the reduced overall phosphorylation of Ly108 seen in C57BL/6 mice. Our study suggests that Ly108 is dynamically regulated in the thymus, shedding light on Ly108 isoform expression and phosphorylation.