Abstract
Full Text
The Immunological Synapse: A Molecular Machine Controlling T Cell Activation
Arash Grakoui, Shannon K. Bromley, Cenk Sumen, Mark M. Davis, Andrey S. Shaw, Paul M. Allen, and Michael L. Dustin

Supplementary Material


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  • Movie 1
    Motion of single GPI-anchored molecules in glass supported planar bilayers. Free diffusion of GPI anchored molecules in supported planar bilayers is the basis of our approach to imaging the engagement of the fluorescently labeled ligands with cellular receptors. CD58 molecules were tagged with 100 nm fluorescent latex sphere with covalently attached anti-CD58 antibody. Planar bilayers were formed on glass coverslips and were incubated with the anti-CD58 beads. The free beads were washed out and the two-dimensional Brownian motion of the bilayer-attached beads were imaged with a ICCD camera (Solamere Technology Group). Three video frames were integrated for each image frame so that thirty seconds of diffusion are compressed into ten seconds of video. Approximately fifty percent of the particles are mobile. Some particle immobilization may be caused by multivalent binding of the antibody labeled beads. The field is 70 μm across. Similar results were published earlier with colloidal gold labeled lipids [G. M. Lee, A. Ishihara, and K. Jacobson, Proc Natl Acad Sci U S A 88(14): 6274 (1991).]
    [Generated with John Heuser and Hiroshi Hayakawa in the Department of Cell Biology at Washington University School of Medicine.]
  • Movie 2
    Data from Web Movie 1 were processed by running background subtraction and peak detection. This allows the paths and areas covered by the diffusion particles to be more clearly visualized.
    [Generated with John Heuser and Hiroshi Hayakawa in the Department of Cell Biology at Washington University School of Medicine.]
  • Movie 3
    Formation of an immunological Synapse. Animated version of Figure 1 A. IRM images of 2B4 T cells on ICAM-1. The movie runs at one frame per second. Images were taken every thirty seconds for the first five minutes, then every two minutes out to thirty minutes. The last image is at sixty minutes. The initial formation of an outer ring in which the T cell membrane is closely apposed to the bilayer is evident. The close contact zones then move into the center of the contact area over time. This parallels the fluorescence image in Web Movie 4.
  • Movie 4
    Formation of an immunological Synapse. Animated version of Figure 1B. Two color fluorescence image of oregon green Ek(MCC88-103) (green) and Cy5 ICAM-1 in planar bilayer. The density of free Ek(MCC88-103) is 80 molecules/μm2 and the density of free ICAM-1 is 200 molecules/μm2. This is the same contact process as in Web Movie 3. The movie runs at one frame per second. Images were taken every thirty seconds for the first five minutes, then every two minutes out to thirty minutes. The last image is at sixty minutes. Images are corrected for photobleaching by fixing the bilayer fluorescence intensity. Only fluorescence signals above the bilayer intensity are displayed. The initial formation of the central ICAM-1 accumulation and peripheral Ek(MCC88-103) accumulation is evident at the first image acquired at a half minute. The transport of Ek(MCC88-103) to the central position is then followed over time.
  • Movie 5
    Disruption of an Immunological Synapse. Animated sequence of a 2B4 T cell on the same bilayer system as the cell shown in Figure 1. IRM of 2B4 T cell with a poorly organized synapse. After about fifteen minutes the cell crawls away leaving a membrane fragment that is detected by IRM. The fluorescence image of the same cell is in Web Movie 6. We would speculate that the membrane fragment contains shed T cell receptor. TCR shedding during detachment of T cells was predicted in an earlier study [M. L. Dustin et al. (1996). J. Immunol. 157: 2014 (1996)]. This movie provides clues to how T cells resume migration after they have completed the functional interaction with an antigen-presenting cell.
  • Movie 6
    Disruption of an Immunogical Synapse. Animated sequence of a 2B4 T cell on the same bilayer system as the cell shown in Figure 1. Fluorescence images of 2B4 T cell with a poorly organized synapse. After about fifteen minutes the cell crawls away leaving a membrane fragment that is detected by IRM (Web Movie 5). The density of free Ek(MCC88-103) is 80 molecules/?m2 and the density of free ICAM-1 is 200 molecules/μm2. Note that much of the Ek(MCC88-102) cluster is left behind with the membrane fragment. We would speculate that the membrane fragment contains shed T cell receptor. TCR shedding during detachment of T cells was predicted in an earlier study [M. L. Dustin et al. (1996). J. Immunol. 157: 2014 (1996)]. This movie provides clues as to how T cells resume migration after they have completed the functional interaction with an antigen presenting cell.
  • Movie 7
    Relationship of Ca2+ mobilization to Immunological Synapse formation. 2B4 T cells were loaded with Fura-2 and visualized by interference reflection microscopy (IRM) to identify junctions and by fluorescence ratio imaging to determine the Ca2+ concentration in the cytoplasm. The substrate was 8 molecules/μm2 Ek(MCC88-102) and 150 molecules/μm2 ICAM-1. The images were acquired every 30 seconds and T cells are settling on the surface and starting to form synapses during the first 5 frames. It is apparent that T cells display a spike in intracellular Ca2+ concentration within 30 seconds of initial contact. Thus, MHC-peptide engagement in the outer ring of close contact does result in rapid signaling in this system. Based on other studies examining kinetics of the Ca2+ signal this was the expected result. However, it was important to confirm that this holds true under conditions of our imaging experiments. Sustained Ca2+ increases were observed down to 0.2 molecules/μm2 of Ek(MCC88-102). Thus, formation of the immunological synapse was correlated with sustained increases in cytoplasmic Ca2+ concentration.
  • Movie 8
    Formation of an immunological Synapse using wt Hb64-76. Animated version of Figure 3A. 3.L2 T cells were incubated with planar bilayer containing Ek and ICAM-1 at 200 molec./μm2 each. The Ek was loaded with Hb64-76 peptide at 80 molec./μm2. Fluorescence images of MHC-peptide (green) and IRM (dark) are shown. Hb64-76 peptide formed immunological synapses and stopped T cell migration. The movie runs at one frame per second. Images were taken every minute after two minute incubation with the bilayer for thirty minutes total.
  • Movie 9
    Formation of an immunological Synapse using N72T (weak agonist). Animated version of Figure 3A. 3.L2 T cells were incubated with planar bilayer containing Ek and ICAM-1 at 200 molec./μm2 each. The Ek was loaded with N72T peptide at 80 molec./μm2. Fluorescence images of MHC-peptide (green) and IRM (dark) are shown. N72T stopped T cell migration and accumulated MHC-peptide complexes. The movie runs at one frame per second. Images were taken every minute after two minute incubation with the bilayer for thirty minutes total.
  • Movie 10
    Formation of an immunological Synapse using N72I (strong antagonist). Animated version of Figure 3A. 3.L2 T cells were incubated with planar bilayer containing Ek and ICAM-1 at 200 molec./μm2 each. The Ek was loaded with N72I peptide at 80 molec./μm2. Fluorescence images of MHC-peptide (green) and IRM (dark) are shown. N72I peptide did not stop T cell migration and formed immunological synapses transiently. Formation of the MHC-peptide clusters were kinetically much delayed compared to strong agonist ligands. The movie runs at one frame per second. Images were taken every minute after two minute incubation with the bilayer for thirty minutes total.
  • Movie 11
    Formation of an immunological Synapse using N72A (weak antagonist). Animated version of Figure 3A. 3.L2 T cells were incubated with planar bilayer containing Ek and ICAM-1 at 200 molec./μm2 each. The Ek was loaded with N72A peptide at 80 molec./?m2. Fluorescence images of MHC-peptide (green) and IRM (dark) are shown. N72A peptide did not stop T cell migration and failed to form immunological synapses. The movie runs at one frame per second. Images were taken every minute after two minute incubation with the bilayer for thirty minutes total.
  • Movie 12
    Formation of an immunological Synapse using N72E (null ligand). Animated version of Figure 3A. 3.L2 T cells were incubated with planar bilayer containing Ek and ICAM-1 at 200 molec./μm2 each. The Ek was loaded with N72E peptide at 80 molec./μm2. Fluorescence images of MHC-peptide (green) and IRM (dark) are shown. However, no clustering of the MHC-peptide complex is detected and the T cells move rapidly across the bilayer. Therefore, a single T cell is not in the view of the bilayer in a one minute time interval. The movie runs at one frame per second. Images were taken every minute after two minute incubation with the bilayer for thirty minutes total.
  • Movie 13
    Formation of an immunological Synapse using wt 3.L2 T cells. Animated version of Figure 4A. 3.L2 T cells were incubated with planar bilayer containing Ek and ICAM-1 at 200 molec./μm2 each. The Ek was loaded with Hb64-76 peptide at 80 molec./μm2. Fluorescence images of MHC-peptide (green), ICAM-1 (red) and IRM (dark) are shown. The movie runs at one frame per second. Images were taken every minute after two minute incubation with the bilayer for thirty minutes total.
  • Movie 14
    Formation of an immunological Synapse using 3.L2 CD4-/- T cells. Animated version of Figure 4B. 3.L2 CD4-/- T cells were incubated with planar bilayer containing Ek and ICAM-1 at 200 molec./μm2 each. The Ek was loaded with Hb64-76 peptide at 80 molec./μm2. Fluorescence images of MHC-peptide (green), ICAM-1 (red) and IRM (dark) are shown. 3.L2 CD4-/- T cells move rapidly on the bilayer and fail to induce formation of a bona fide immunological synapse. The movie runs at one frame per second. Images were taken every minute after two minute incubation with the bilayer for thirty minutes total.
  • Movie 15
    Formation of an immunological Synapse using wt 3.L2 T cells in the presence of anti-CD4 mAb (GK1.5). 3.L2 T cells were incubated with 10 μg/ml of GK1.5 mAb for 10 minutes prior to interaction with the bilayer. Afterwards, 3.L2 T cells were incubated with planar bilayer containing Ek and ICAM-1 at 200 molec./μm2 each. The Ek was loaded with Hb64-76 peptide at 80 molec./μm2. Fluorescence images of MHC-peptide (green), ICAM-1 (red) and IRM (dark) are shown. However, no clustering of the MHC-peptide complex is detected. The movie runs at one frame per second. Images were taken every minute after two minute incubation with the bilayer for thirty minutes total.

Supplemental Figure 1. Immunological Synapse formation in the presence of CD2 and CD28. (A to D) Immunological synapse formation by antigen primed 3A9 T cells in the presence of CD80; (A) Oregon green Ak (HEL48-61) accumulation (green); (B) Cy5 ICAM-1 accumulation (blue); (C) Cy3-CD80 accumulation (red); (D) Overlay. Accumulated CD80 co-clusters with the MHC-peptide complex. (E-H) Immunological synapse formation by antigen primed 2B4 T cell in the presence of CD48. (E) Oregon green Ek(MCC88-103) accumulation (green); (B) Cy3 ICAM-1 accumulation (blue); (G) Cy5 labeled CD48 (red); (H) Overlay. Accumulated CD48 defines a unique domain within the immunological synapse. However, neither CD80 nor CD48 perturb the fundamental pattern of MHC-peptide and ICAM-1 accumulation in the immunological synapse.


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Supplemental Figure 2. Proliferation of 5CC7 T cells in response to altered peptide ligands presented by glass suppported planar bilayers. 5CC7 is a distinct T cell receptor with equivalent specificity to 2B4 [P. A. Patten et al. J. Immunol 150(6): 2281 (1993)]. T cells from 5CC7 T cell receptor transgenic mice were prepared identically to the T cells from 2B4 mice. Bilayers were formed on 5 mm round coverlips in 96 well plates. Bilayers contained 200 molecules/?m2 of Ek-GPI that was loaded with the indicated ligands at 40% efficiency. Antigen primed T cells were cultured without exogenous IL-2 for 6 days prior to initiation of the cultures on bilayers. After 48 hours of interaction, proliferation was measured by pulsing with 3H thymidine for 10 hours. Data are mean ± S.D. of quadruplicate determinations and are representative of two experiments. Web Figure 2 is a companion to Figure 3. Proliferation correlates with the density of MHC-peptide clusters.


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