RESUMO
The clonal proliferation of antigen-specific T cells during an immune response critically depends on the differential response to growth factors, such as IL-2. While activated T cells proliferate robustly in response to IL-2 stimulation, naïve (quiescent) T cells are able to ignore the potent effects of growth factors because they possess chromatin that is tightly condensed such that transcription factors, such as STAT5, cannot access DNA. Activation via the T cell receptor (TCR) induces a rapid decondensation of chromatin, permitting STAT5-DNA engagement and ultimately promoting proliferation of only antigen-specific T cells. Previous work demonstrated that the mobilization of intracellular calcium following TCR stimulation is a key event in the decondensation of chromatin. Here we examine PKC-dependent signaling mechanisms to determine their role in activation-induced chromatin decondensation and the subsequent acquisition of competence to respond to IL-2 stimulation. We found that a calcium-dependent PKC contributes to activation-induced chromatin decondensation and that the p38 MAPK and NFκB pathways downstream of PKC each contribute to regulating the proper decondensation of chromatin. Importantly, we found that p44/42 MAPK activity is required for peripheral T cells to gain competence to properly respond to IL-2 stimulation. Our findings shed light on the mechanisms that control the clonal proliferation of antigen-specific peripheral T cells during an immune response.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Interleucina-2/imunologia , Ativação Linfocitária/imunologia , Proteína Quinase C/metabolismo , Linfócitos T/imunologia , Animais , Proliferação de Células/fisiologia , Células Cultivadas , Cromatina/metabolismo , Seleção Clonal Mediada por Antígeno/imunologia , DNA/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Fator de Transcrição STAT5/metabolismo , Transdução de Sinais/imunologia , Fatores de Transcrição/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
During a proper immune response, quiescent T cells become activated upon antigen presentation to their antigen-specific T cell receptor. This leads to clonal proliferation of only those T cells that bear a receptor that recognizes the antigen. Chromatin decondensation is a hallmark of T cell activation and is required for T cells to acquire the ability to proliferate after antigen engagement. This change in chromatin condensation can be detected using antibodies raised against histone proteins. These antibodies cannot bind to their epitopes in naïve T cells as well as they can in activated T cells. We describe how to simultaneously stain T cell-specific surface markers, track viability with a fixable dead cell stain, and measure chromatin status via intracellular staining of Histone H3 proteins. Stained cells are analyzed by flow cytometry and chromatin condensation status is measured as the mean fluorescence intensity (MFI) of the Histone H3 stain. Chromatin decondensation during T cell activation is demonstrated as an increase in the MFI.
Assuntos
Cromatina/química , Citometria de Fluxo/métodos , Linfócitos T/química , Animais , Anticorpos/química , Anticorpos/imunologia , Feminino , Histonas/química , Histonas/imunologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Coloração e Rotulagem/métodos , Linfócitos T/imunologiaRESUMO
Antigen engagement of the T-cell receptor (TCR) induces a rapid and dramatic decondensation of chromatin that is necessary for T-cell activation. This decondensation makes T-cells competent to respond to interleukin-2 providing a mechanism to ensure clonotypic proliferation during an immune response. Using murine T-cells, we investigated the mechanism by which TCR signaling can initiate chromatin decondensation, focusing on the role of calcium mobilization. During T-cell activation, calcium is first released from intracellular stores, followed by influx of extracellular calcium via store operated calcium entry. We show that mobilization of intracellular calcium is required for TCR-induced chromatin decondensation. However, the decondensation is not dependent on the activity of the downstream transcription factor NFAT. Furthermore, we show that the influx of extracellular calcium is dispensable for initiating chromatin decondensation. Finally, we show that mobilization of calcium from intracellular stores is sufficient to induce decondensation, independent of TCR engagement. Collectively, our data suggest that chromatin decondensation in peripheral T-cells is controlled by modulating intracellular calcium levels.