Department of Pathology; Graduate School of Biomedical Sciences
Amino Acids, Peptides, and Proteins | Cell Biology | Cells | Hemic and Immune Systems | Immunopathology
Circulating naive T cells exist in a quiescent state. After TCR contact with the cognate peptide presented by APCs in secondary lymphoid structures, T cells undergo a period of rapid transcriptional changes that set the stage for fate-determining effector or memory programming. We describe a novel method to analyze TCR signaling pathway activation in nuclei isolated from primary mouse naive T cells after stimulation with natural peptide Ags. We prelabeled cells with cell tracking dye to easily distinguish CD8(+) T cell nuclei from APC nuclei by conventional flow cytometry. Using this approach, we observed clear digital activation of NFAT1 transcription factor in OT-I T cells stimulated with OVA peptide presented by bulk splenocytes. OVA concentration had discrete control over the fraction of the cells that translocated NFAT1, indicating that a distinct threshold amount of TCR signaling is required to switch on NFAT1 in naive T cells. This behavior was cell contact dependent and qualitatively more exact than the NFAT1 response in ionomycin-stimulated naive T cells. These data contribute to our understanding of the digital behavior of TCR signaling components documented in other studies and indicate how T cells might discriminate log-fold changes in Ag availability during an actual infection. Overall, these results highlight the potential of this coculture nuclei isolation protocol to address stimulation-dependent translocation of proteins in primary lymphocytes.
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Copyright © 2018 The Authors. This article is distributed under the terms of the CC BY-NC-ND 4.0 Unported license.
DOI of Published Version
Immunohorizons. 2018 Aug;2(7):208-215. doi: 10.4049/immunohorizons.1800032. Link to article on publisher's site
Gallagher, Michael P.; Conley, James M.; and Berg, Leslie J., "Peptide Antigen Concentration Modulates Digital NFAT1 Activation in Primary Mouse Naive CD8(+) T Cells as Measured by Flow Cytometry of Isolated Cell Nuclei" (2018). Open Access Articles. 3570.
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This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.