Altered TCR Signaling from Geometrically Repatterned Immunological Synapses
Kaspar D. Mossman1,2,3,
Jay T. Groves1,2,3*, and
Michael L. Dustin4*
1 Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA.
2 Department of Chemistry, University of California, Berkeley, CA 94720, USA.
3 Physical Biosciences and Materials Sciences Divisions, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA.
4 Department of Pathology, New York University School of Medicine, and the Program in Molecular Pathogenesis, Skirball Institute of Biomolecular Medicine, 540 First Avenue, New York, NY 10016, USA.
Fig. 1.. Diagram of a hybrid live T cellsupported membrane junction. Receptors on the cell surface engage cognate ligands in the supported membrane and become subject to constraints on mobility imposed by physical barriers. The cytoskeleton is represented schematically to reflect the active source of central organization observed in our experiments.
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Fig. 2.. Synapse formation is altered by geometrical constraints of the substrate. T cells were incubated with fluorescently labeled anti-TCR H57 Fab (green) before being introduced to supported bilayers containing GPI-linked pMHC (unlabeled) and ICAM-1 (red). Chromium lines are visible in brightfield, although they are only 100 nm across, verified by electron microscopy. Images are at 10 min after cells were introduced. IS on unpatterned substrate (A), 2-µm parallel lines (B), 5-µm square grid (C), and concentric hexagonal barriers (spacing 1 µm) (D). TCR distribution (grayscale) on 1-µm square grid (E). Transport map of (E) formed by drawing arrows toward the TCR cluster within the enhanced grid (F).
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Fig. 3.. TCR-specific phosphotyrosine (pY) signaling in native and repatterned synapses. T cells, which had been incubated with fluorescently labeled anti-TCR H57 Fab, were allowed to interact with pMHC-ICAM membranes for either 2 or 5 min before being fixed and stained for pY. (A) Synapse on unpatterned membrane at 2 min. TCR clusters are distributed, and relatively enhanced pY staining colocalizes with each cluster. The diffuse ring of pY staining in the periphery is likely associated with cortical actin. (B) Synapse on a 2-µm chromium grid at 2 min. (C) Synapse on unpatterned membrane at 5 min. (D) Synapse on a 2-µm chromium grid at 5 min. (E) Statistical results for % TCR colocalization with pY. Black, cells off pattern; gray, cells on 2-µm grids. Results are from three independent experiments at 2 min (a minimum of 9 cells per experiment both on and off patterns; total 31 on, 51 off) and four independent experiments at 5 minutes (a minimum of 7 cells per experiment on and off patterns; total 39 on, 53 off). Data from the 1-min time point (not shown) had extremely high standard deviation because cell population was not well synchronized. (F) Intracellular calcium is elevated in cells on grids. T cells were loaded with the ratiometric calcium-sensitive dye fura-2 and allowed to interact with pMHC-ICAM membranes. Fura-2 fluorescence emission ratio was integrated from 5 min to 20 min in cells on and off 2-µm grids (five independent experiments; total 49 on, 57 off).
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