(A) Representative images of maximum intensity projections from Z-stacks (0.19 µm per slice) of WT Jurkat T cells conjugated to SEE pulsed (+ SEE) or non-pulsed (-SEE) antigen presenting cells (APC). Cells were then fixed after incubation at 37 °C for 2, 5, 10, or 15 min, permeabilized and stained for endogenous SNX9 and actin (Phalloidin-488). (B) Mean percentage of SNX9 recruitment towards the synapse at indicated time points of cell shown in A identified in a blinded analysis of 60–100 cells per condition in four independent experiments. (C) Representative live images of WT Jurkat T cells activated on cover glass (anti-CD3ε and anti-CD28), expressing SNX9-mCherry and TCRζ-EGFP. Top panel: maximum intensity projections from Z-stacks (0.19 µm per slice) Bottom panel: shows 3D reconstructions. (D) Representative images of 3D reconstruction and maximum intensity projections of wildtype Jurkat T cells expressing SNX9-EGFP either in resting (PLL-coated cover glass) or under different activating conditions. (E) Mean number of SNX9-positive structures in T cells activated under different conditions in three to four experiments (8–11 cells per experiment). (F) Representative live images of activated Jurkat T cells co-expressing SNX9-EGFP or -mCherry and either TCRζ-mCherry (top), Rab5-mCherry (middle) or CD28-EGFP (bottom). (G) Quantification of the number of SNX9-EGFP/-mCherry, TCRζ-mCherry, Rab5-mCherry and CD28-EGFP positive vesicles in each frame of the z-stack of three to four experiments (5 cells per experiment). (H) Average axial position where the maximum number of CD28, SNX9, TCRζ and Rab5 structures were identified. Dots represent individual experiments. Error bars indicate mean ± SEM. n.s. = not significant, * = p < 0.05, **** = p < 0.0001 from Student’s T-test of means of independent experiments. Scale bar = 5 µm.