Abstract
Although signals through the T cell receptor (TCR) are essential for the initiation of T helper cell activation, it is unclear what function such signals have during the prolonged T cell–antigen-presenting cell contact. Here we simultaneously tracked TCR-CD3 complex and phosphoinositide 3-kinase activity in single T cells using three-dimensional video microscopy. Despite rapid internalization of most of the TCR-CD3, TCR-dependant signaling was still evident up to 10 h after conjugate formation. Blocking this interaction caused dissolution of the synapse and proportional reductions in interleukin 2 production and cellular proliferation. Thus TCR signaling persists for hours, has a cumulative effect and is necessary for the maintenance of the immunological synapse.
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Acknowledgements
We thank M.F. Kuhns, M.F. Krummel, R. Sciammas and L.C. Wu and for advice and discussions. We thank P.J. Ebert, C. Gerke, M. Krogsgaard, B.F. Lillemeier, Q.-J. Li and M. A. Purbhoo for comments on this manuscript. We thank N. Prado for technical assistance and S.M. Wheaton for final preparation of the manuscript. J.B.H. was a fellow of the Cancer Research Institute and M.G. is a predoctoral fellow of the Howard Hughes Medical Institute. This work was supported by grants from the Howard Hughes Medical Institute and the National Institutes of Health.
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Supplementary Fig. 1.
Antigen specific downregulation of TCR surface expression amounts. (a) Flow cytometry shows downregulation of TCR surface expression amounts. Day 9 5c.c7 T cell blasts were left untreated or pooled with CH27 B cells that had been left untreated or preloaded with MCC peptide (0.4 µM). 30 min after cell pooling, cells were treated with antibody to FcgIII/II, stained with PE-conjugated monoclonal antibody to TCR Vb3 (KJ25) and FITC-conjugated monoclonal antibody to CD4 (RM4-5). TCR expression is shown for CD4 + cells. (b) Kinetics of surface TCR downregulation. Day 9 5c.c7 T cell blasts were pooled as in (a) with CH27 B cells in the absence (negative control) and presence of 0.4µM MCC peptide and incubated for the times indicated. TCR surface expression was analyzed by flow cytometry as in (a). Values refer to mean fluorescence intensities. (PDF 27 kb)
Supplementary Fig. 2.
Selective interference with the 5c.c7 TCR-IEk-MCC interaction as opposed to blocking potential non-antigen mediated interactions involving MHC class II molecules leads to decline in synaptic signaling activity and disruption of the immunological synapse. (a) Sustained PI3K activity depends on the presence of stimulatory pMHC ligands but not irrelevant pMHC complexes. 5c.c7 TCR transgenic T cell blasts expressing PH(AKT)YFP were added to CH27 B cells that had been preloaded with the MCC peptide. About 90% of the T cells were found in conjugates 15 minutes after cell pooling. Three hours after cell pooling monoclonal antibodies (20 µg/ml of the anti-antibodies IAk 10-3.6 and 11-5.2 and 20µg/ml of the antibody to IEk/MCC antibody D4) were added to the conjugates and T cells were inspected for localization of PH(AKT)-YFP 15 minutes later. The ratio of synapse-associated fluorescence intensity to cytoplasmic fluorescence intensity served as a relative measure for PI3K activity. Error bars represent the standard deviation derived from 25 T cells inspected in each group. (b) Sustained calcium signal depends on the presence of stimulatory pMHC ligands but not irrelevant pMHC complexes. 5c.c7 TCR transgenic T cell blasts were added to CH27 B cells that had been preloaded with the MCC peptide (0.4µM). About 90% of the T cells were found in conjugates 15 minutes after cell pooling. Three hours after cell pooling monoclonal antibodies (20 µg/ml of the anti-antibodies IAk 10-3.6 and 11-5.2 and 20µg/ml of the antibody to IEk/MCC antibody D4) and the calcium-sensitive dye fura-2 were added to the conjugates and T cells were inspected for calcium signaling status as described in figure 3 (N/data group=25). (c) The integrity of the immunological synapse depends on the presence of stimulatory pMHC ligands but not irrelevant pMHC complexes. 5c.c7 TCR transgenic T cell blasts were added to CH27 B cells expressing ICAM-1-GFP that had been preloaded with the MCC peptide (0.4µM). About 90% of the T cells were found in conjugates 15 minutes after cell pooling. Three hours after cell pooling monoclonal antibodies (20 µg/ml of the anti-antibodies IAk 10-3.6 and 11-5.2 and 20µg/ml of the antibody to IEk/MCC antibody D4). 15 minutes after antibody addition conjugates were inspected for ICAM-1-GFP surface redistribution as described in figure 3. n=25 (PDF 29 kb)
Supplementary video.
Three-dimensional representations of image stacks (derived from Fig. 2) accentuate gradual internalization of the TCR from the IS. (MOV 559 kb)
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Huppa, J., Gleimer, M., Sumen, C. et al. Continuous T cell receptor signaling required for synapse maintenance and full effector potential. Nat Immunol 4, 749–755 (2003). https://doi.org/10.1038/ni951
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DOI: https://doi.org/10.1038/ni951
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