T-cell hybridoma activation does not lead to significant cell shape and CD43 polarization or lipid raft coalescence.

Hybridoma- or column-purified splenic T cells were conjugated with beads coated with anti-CD3 and anti-CD28, fixed, and stained for CD43. (A,D) Examples of 3D9 hybridoma-bead conjugation where CD43 does not modulate away from the interaction site. (B,E) Examples of D0.11.10 hybridoma-bead complexes. The red CD43 staining is clearly visible at the cell-bead contact area. (C,F) Unprimed T cells activated with the same beads. During the activation, the cells change shape and CD43 accumulates away from the bead contact site, toward the uropodlike structure. At the right image the cell not conjugated with the bead demonstrates the typical CD43 distribution on unstimulated cells. The arrows denote location of the beads. (G-I) T-cell hybridomas or column-purified spleen T cells were conjugated with beads coated with anti-CD3 and anti-CD28, fixed, and permeabilized as described in “Materials and methods.” Rafts were visualized with FITC-labeled cholera toxin B subunit. (G) An example of 3D9 hybridoma cell-bead conjugate. (H) An example of a D0.11.10 hybridoma cell-bead complex. Both photographs show dispersed noncoalescent lipid rafts after activation of the T-cell hybridomas. (I) Unprimed T cells activated at the same time show redistribution of lipid rafts and their coalescence at the cell-bead interface. The results are representative of at least 3 experiments. The arrows indicate location of the beads. Magnification × 1000.