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A Receptor/Cytoskeletal Movement Triggered by Costimulation During T Cell Activation

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Science  18 Dec 1998:
Vol. 282, Issue 5397, pp. 2266-2269
DOI: 10.1126/science.282.5397.2266

Figures

  • Figure 1

    Bead movement toward the interface. Single frames of a video microscopy experiment (36) of the interaction of 5C.C7 T cells, loaded with 4.5-μm anti–ICAM-1 (YN1) beads, with peptide-loaded CH27 B cell lymphoma cells (35) are shown. The CH27 cell is substantially larger than the T cells. The T cell intracellular calcium concentration is overlaid in a false color scale from blue (low concentration) to red (high concentration) to mark the onset of T cell activation, set to time 0:00 min. Although the beads, one of which is marked with an arrow, are bound to the posterior end of the central T cell before and at the time of its activation, they can be seen to move toward the T cell–B cell interface in subsequent frames. The still frames have been excerpted from movie 1 (37), which can be viewed at Science Online (www.sciencemag.org).

  • Figure 2

    T cell–APC interaction with blocked myosin motor proteins. (A) Single frames of a video microscopy experiment (36) of the interaction of 5C.C7 T cells, loaded with 4.5-μm anti–ICAM-1 (YN1) beads, with peptide-loaded, ICAM-1–GFP–transfected CH27 cells are shown. The bright-field image has been duplicated. In the top panels, the T cell intracellular calcium concentration is overlaid in a false color scale as in Fig. 1. In the bottom panels, the ICAM-1–GFP fluorescence is overlaid in a false color scale from green (low fluorescence) to blue (high fluorescence) to allow the simultaneous investigation of the additional cellular parameter. The accumulation of ICAM-1 at the T cell–APC interface in the absence of all pharmacological agents was described in (9); here, such an interaction is shown in the presence of 2 μM BDM. Although the calcium signal is elevated in a stable manner and ICAM-1–GFP accumulates at the interface, this interface is narrow (as most easily seen by the tightly concentrated ICAM-1–GFP accumulation), and a bead that is bound to an activating T cell, marked with an arrow, does not move. The still frames were excerpted from movie 2 (38), which can be viewed atScience Online (www.sciencemag.org). (B) An even narrower interface at 20 μM BDM, excerpted from a movie of a different experiment.

Tables

  • Table 1

    Occurrence of different types of T cell bead movement (39) during the interaction of a 5C.C7 T cell with APCs loaded with an activating peptide. Unless mentioned, the APCs were CH27 B cell lymphomas. Bead movement was analyzed using three different types of beads (12, 18, 20); only two of these bead types (12, 18) were used for the rest of the experiments. Blocking antibodies and pharmacological agents were used under standard conditions. (40). The number of cells analyzed during wortmannin treatment was small because the majority of the T cells did not establish a polarized phenotype (41). When transfected CHO cells were used as APCs, we found that GFP fusion did not inhibit the function of its fusion partner (9, 22). In the last row, an activating antibody to CD28 was present during the interaction of the T cell and APC; n indicates the number of cells studied (34).

    Type of beads/treatment/APCMovement toward interfaceMovement away from interfaceNo movementn
    Type of beads
    Anti-ICAM74%0%26%81
    Streptavidin (surface biotin)83%7%10%29
    Streptavidin (lipid biotin)64%5%32%22
    Antibody blocking
    anti-ICAM11%5%84%37
    anti-B78%0%92%24
    anti-VCAM62%0%38%21
    anti-CD4875%0%25%20
    Pharmacological agents
    Wortmannin13%0%78%8
    Ni/BAPTA23%0%77%26
    Transfected CHO cells as APCs
    CHO/I-Ek 4%0%96%47
    CHO/I-Ek/ICAM-1–GFP27%7%67%45
    CHO/I-Ek/B7-2–GFP34%2%64%47
    CHO/I-Ek/ICAM-1–GFP (anti-CD28)59%0%41%27
  • Table 2

    Effects of blocking myosin motor proteins. The analysis of T cell activation by peptide loaded CH27 cells in the presence of BDM or noxius toxin (NTX) is shown (42). Narrow interface form denotes activated T cells having a T cell–B cell interface diameter that is smaller than the diameter of the T cell (Fig. 2). Calcium signal back to baseline denotes activated T cells whose intracellular calcium concentration returns to preactivation levels during an observation period of at least 5 min. ICAM cluster formation denotes CH27 cells that show accumulation of ICAM-1–GFP at the T cell–B cell interface after T cell activation (9) (Fig. 2). Bead movement toward the interface (39) refers to activated T cells that show anti–ICAM-1 bead movement. In all cases,n denotes the number of cells analyzed (43).

    Pharmacol. agentInterface formCalcium signalICAM clusteringBead movement
    NarrownBack to baselinenCluster formationnToward interfacen
    BDM (20 mM)93%5568%10836%3319%27
    BDM (10 mM)79%5845%8351%3527%15
    BDM (2 mM)70%4623%7385%3225%28
    Buffer alone14%562%10290%3862%21
    NTX (100 nM)23%479%7685%4063%27

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