Figure 2

Time course of immunological synapse (IS) formation by ImmTAV-redirected HIV-naïve donor CD8⁺ T cells. SL9-peptide pulsed T2s were (a) stained for surface epitopes using a labelled TCR and imaged by TIRF microscopy for epitope/cell quantification (brightfield and maximum intensity projection (MIP) of labelled TCR shown; one dot represents one cell). These targets were then co-cultured with HIV-naïve donor CD8⁺ T cells (E:T of 1:1) for 5, 15 or 30 min before confocal microscopy imaging and analysis. (b) % of target cells in conjugates with CD8⁺ T cells over time in the presence of HIV-specific ImmTAV (m121, 0.5 nM) or irrelevant TCR-anti-CD3 fusion protein (ImmTAX, m232, 1 nM). C-F all show IS formation in the presence of the HIV ImmTAV, m121. (c) Representative confocal images of conjugates showing CD8⁺ T cell Zap70 (green) localisation in the cytoplasm (left) or in the plasma membrane (right). The percentage of conjugates with Zap70 localised to the cytoplasm (grey) or membrane (black) was calculated at different conjugation times. (d) The percentage Zap70 localised at the IS in CD8⁺ T cells. (e) Confocal images of α-tubulin (green) to define MTOC location (indicated with white arrow) and distance from IS to MTOC (μM). (f) Confocal microscopy images of perforin staining (summed Z stack shown in white) and % conjugates with distal, dispersed or docked perforin in the CD8⁺ T cells. For D & E each dot represents one T2-CD8⁺ T cell conjugate (0.5 nM m121 for c–f); 1 slide (representing 1 donor) was analysed per time-point; magenta = CD8 on differential interference contrast image (DIC). Means shown and data were analysed by one-way ANOVA.