Figure 4.

Confocal images of TCR, CD8, talin, and PKC-θ distribution of CTL clones. CTL clone CER43 was layered on bilayer containing 200 molecules/μm² Cy3–ICAM-1–GPI with or without unstained agonist MHCp complexes (a–i) or containing 200 molecules/μm² Cy3–ICAM-1–GPI only (j, l–s). In k, the CD8-deficient CTL clone 115ix was layered on a bilayer containing 200 molecules/μm² Cy3-ICAM-1–GPI only. After 30 minutes of incubation, cells were fixed, permeabilized, and stained with the indicated antibodies. Fluorescence was imaged with a laser-scanning confocal microscope. The green signal in j and k is increased to reveal the presence of levels of CD3 staining above the baseline density. (k) CD3 can accumulate in the center of the ring junction without CD8. (b and c) Examples of CD8 accumulations (green) that are filled (b) or have central holes (c). (l and m) Examples of different levels of CD8 in ring junctions, which are not as enriched as in IS. Pairs d/e, n/o, and p/q are the same contacts with talin (green) or ICAM-1 (red) fluorescence; n/o is a ring junction, while p/q is a motile junction, which also shows talin redistribution. Pairs f/g, h/i, and r/s are the same contacts stained for PKC−θ (green) and ICAM-1 (red); f/g shows the classical central localization of PKC-θ compared with h/i, which shows the more frequent localization of PKC-θ to the microaggregates in the pSMAC; r/s shows that PKC-θ was not detected in the ring junctions. The images were processed with a median filter (radius 4 pixels [pixel = .2 μm]). Scale bar: 5 μm.