(a) Motility speeds of naïve CD4 and CD8 T-cells and acDCs in collagen gels with different chemokines. Note the faster motility of T-cells compared to acDCs and similar motility using homeostatic chemokines (CCL19, CCL21 and CXCL12). Representative of 2 independent repeats (Video 2, Table 3). (b) Snapshots from a time-lapse movie (Video 3) following the interactions of 1G4-expressing naïve CD8 T-cells (magenta) with antigen-loaded acDCs (green) in a collagen gel containing CXCL12, and the upregulation of CD69 is visible at T = 140 min following the accumulation of an anti-CD69 antibody on the surface of the cells (cyan, also in cyan are irrelevant CD4 T-cells). Representative of 3 independent repeats. (c) Snapshots from a time-lapse movie (Video 4) showing three different 1G4-expressing CD8 T-cells (magenta), loaded with calcium dye Fluo4-AM (green), interacting with an antigen loaded acDC (100 nM NY-ESO-9V, cyan) in 3D collagen and fluxing calcium (green) upon binding. Note the lower flux in the second and third contacts suggesting lower antigen availability, being sequestered by the primary synapse. (d) Speed of 1G4-expressing naïve CD8 T-cells upon culture in collagen gels containing CXCL12, with acDC either loaded with high affinity (100 nM NY-ESO-9V) or low affinity (100 nM NY-ESO-9L) peptide or unloaded (null). Note the deceleration of the cells upon engaging with high affinity peptide (3 μm/min compared to 12 μm/min for both null and 9L). Representative of 2 independent repeats. (Videos 7–9). (e) The cells from (c) extracted from the collagen gel and run on a flow cytometr to look at CD69 and CD25 as activation markers. Note the good activation despite the absence of T-cell arrest with low affinity peptide (NY-ESO-9L). Representative of >3 independent repeats. Scale bars = 10 μm. (****, p<0.0001, ANOVA).