Figure 4.
Figure 4. VAMP8-carrying recycling endosomes accumulate and fuse at immune synapses. (A–E) Bead-stimulated human CD8+ T cells were transfected with VAMP8-TFP and mCherry-Rab11a encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and mCherry-Rab11a in a transfected CTL in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15, paired t test, P > 0.05). (C) Mean VAMP8-TFP and mCherry-Rab11a vesicle accumulation over time in the TIRF plane per cell (n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 16, paired t test, P > 0.05). (E) Mean cumulative fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15). (F and G) Bead-stimulated human CD8+ T cells were transfected with constructs encoding VAMP8-TFP or VAMP8-3×FLAG-TFP and analyzed by flow cytometry 24 h after transfection. CTLs were stimulated with anti-CD3 and anti-CD28 antibodies and then surface stained with the anti-FLAG antibody. (F) Schematic representations of the constructs indicating the SNARE domain (SD) and transmembrane (TM) domains are depicted. Histograms from one representative donor show FLAG staining on TFP+ CTL, as indicated. Data are representative of three independent experiments performed on multiple donors. SBD, syntaxin binding domain. (G) Graphs depict FLAG mean fluorescent intensity (MFI) relative to unstimulated, VAMP8-3×FLAG-TFP–transfected CTLs in five individual donors. Mean and SDs are indicated (paired t test, **, P < 0.01). (H) Bead-stimulated human CD8+ T cells were transfected with VAMP8-pHluorin-mCherry (mCh) encoding constructs and imaged 24 h after transfection. Selected live-cell TIRF microscopy frames of two individual CTL vesicles are shown, as indicated. Graphs below depict respective pHluorin and mCherry vesicle MFI over time (experiments were repeated with three individual donors and n = 10 cells). Bars, 0.15 µm. Error bars show means and SDs.

VAMP8-carrying recycling endosomes accumulate and fuse at immune synapses. (A–E) Bead-stimulated human CD8+ T cells were transfected with VAMP8-TFP and mCherry-Rab11a encoding constructs and imaged 24 h after transfection. (A) Selected live-cell TIRF microscopy images of VAMP8-TFP and mCherry-Rab11a in a transfected CTL in contact with an anti-CD3– and anti-CD28–coated coverslip. Bar, 2.5 µm. (B) Mean dwell time of VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15, paired t test, P > 0.05). (C) Mean VAMP8-TFP and mCherry-Rab11a vesicle accumulation over time in the TIRF plane per cell (n = 15). (D) Mean fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 16, paired t test, P > 0.05). (E) Mean cumulative fluorescence dispersion events for VAMP8-TFP and mCherry-Rab11a vesicles in the TIRF plane per cell (n = 15). (F and G) Bead-stimulated human CD8+ T cells were transfected with constructs encoding VAMP8-TFP or VAMP8-3×FLAG-TFP and analyzed by flow cytometry 24 h after transfection. CTLs were stimulated with anti-CD3 and anti-CD28 antibodies and then surface stained with the anti-FLAG antibody. (F) Schematic representations of the constructs indicating the SNARE domain (SD) and transmembrane (TM) domains are depicted. Histograms from one representative donor show FLAG staining on TFP+ CTL, as indicated. Data are representative of three independent experiments performed on multiple donors. SBD, syntaxin binding domain. (G) Graphs depict FLAG mean fluorescent intensity (MFI) relative to unstimulated, VAMP8-3×FLAG-TFP–transfected CTLs in five individual donors. Mean and SDs are indicated (paired t test, **, P < 0.01). (H) Bead-stimulated human CD8+ T cells were transfected with VAMP8-pHluorin-mCherry (mCh) encoding constructs and imaged 24 h after transfection. Selected live-cell TIRF microscopy frames of two individual CTL vesicles are shown, as indicated. Graphs below depict respective pHluorin and mCherry vesicle MFI over time (experiments were repeated with three individual donors and n = 10 cells). Bars, 0.15 µm. Error bars show means and SDs.

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