Figure 4.

MHCp Antibody Treatment Does Not Disrupt the cSMAC or Microclusters, but Prevents New Microcluster Formation

Images of AND T cell forming an IS on bilayers containing ICAM-1 and 10 agonist MHCp/μm² before (A, D) and after (B, E) treating cells with MHCp antibodies (red; Cy5-Icam-1, green; Oregon Green I-E^K in [A] and [B] and Alexa-488-H57-Fab in [D] and [E]). Scale bar equals 4 μm. TCR-MHCp interactions in the cSMAC are at 80% of initial levels at 10 min after addition of anti-MHCp antibodies based on retention of I-E^k fluorescence (C) and TCR fluorescence (F). A line connects data points for each cell before and after treatment. Some cells have no reduction in TCR-MHCp interaction, yet all cells show reduced Ca²⁺. Microclusters continue to form up to 30-60 min in the mature IS (G, H). Treatment with anti-MHCp blocked formation of new microclusters; however, the existing microclusters were chased into the cSMAC (I, J). Zap-70-GFP was introduced into AND T cells via retroviral transduction. Confocal imaging was performed on these cells interacting with bilayers containing ICAM-1 and 10 agonist MHCp/μm². Microclusters recruit Zap-70 and fade as they approach the cSMAC (K, L). Treatment with anti-MHCp blocked the recruitment of Zap-70 to the microclusters, and the existing ones were chased into the cSMAC by 1-2 min. Tracks shown start in frames (G), (I), (K), and (M) and end in frames (H), (J), (L), and (N). Similar results were obtained from three independent experiments. Scale bar equals 4 μm.