Vamp-7-dependent secretion at the immune synapse regulates antigen extraction and presentation in B-lymphocytes

Abstract

Recognition of surface-tethered antigens (Ags) by B-cells leads to the formation of an immune synapse that promotes Ag uptake for presentation onto MHC-II molecules. Extraction of immobilized Ags at the immune synapse of B-cells relies on the local secretion of lysosomes, which are recruited to the Ag contact site by polarization of their microtubule network. Although conserved polarity proteins have been implicated in coordinating cytoskeleton remodeling with lysosome trafficking, the cellular machinery associated with lysosomal vesicles that regulates their docking and secretion at the synaptic interface has not been defined. Here we show that the v-SNARE protein Vamp-7 is associated with Lamp-1⁺ lysosomal vesicles, which are recruited and docked at the center of the immune synapse of B-cells. A decrease in Vamp-7 expression does not alter lysosome transport to the synaptic interface but impairs their local secretion, a defect that compromises the ability of B-cells to extract, process, and present immobilized Ag. Thus our results reveal that B-cells rely on the SNARE protein Vamp-7 to promote the local exocytosis of lysosomes at the immune synapse, which is required for efficient Ag extraction and presentation.

FIGURE 1:

Vamp-7⁺/Lamp-1⁺ lysosomes are recruited to the IS of B-cells. (A) Immuno­fluorescence staining of lysosomes (Lamp-1) in B-cells expressing Vamp-7–GFP incubated with BCR-ligand⁺ or BCR-ligand^- beads for 2 h. Dashed squares indicate the area where Vamp-7-GFP and Lamp-1 signals were measured. (B) Quantification of Lamp-1 and Vamp-7 mean fluorescence intensity recruited to the antigen-contact site, vvv to the total amount of Lamp-1⁺ lysosomes or Vamp-7⁺ vesicles, respectively, in the entire cell (NFI). n ≥ 36; three independent experiments. (C) Z-planes of confocal images and (E) fluorescence images of B-cells expressing Vamp-7–RFP activated as described and stained for Lamp-1 and Rab6. Scale bar, 3 µm. (D) Pearson’s r for Vamp-7/Rab6 or Vamp-7/Lamp-1 in nonactivated and activated cells (n ≥ 14; two independent experiments). (F) Polarity indexes obtained for Lamp-1⁺ and Vamp-7⁺ vesicles using single-cell analysis. n ≥ 36; three independent experiments. Analysis of variance (ANOVA) followed by Sidak’s multiple comparison test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

FIGURE 2:

Vamp-7⁺ vesicles accumulate at the center of the synapse formed by B-cells. (A) TIRFM images of Vamp-7-RFP⁺ vesicles in B-cells plated for 30 min on antigen-coated slides. Scale bar, 5 µm. Time is shown in minutes:seconds. (B) Quantification of dynamic parameters of Vamp-7⁺ vesicle pools at the central and peripheral regions of the IS. n = 7, total >300 trajectories. Unpaired t test. Speed p = 0.0004, distance p < 0.0001, persistence p = 0.0302. (C) TIRFM images of B-cells expressing Vamp-7–RFP⁺ plated onto Ag-coated slides for 1 h. Vamp-7–RFP or F-actin (phalloidin) and BCR. Scale bar, 5 µm.

FIGURE 3:

Lysosome exocytosis at the IS of B-cells requires Vamp-7. (A) Confocal images of Control and Vamp-7–silenced B-cells incubated with BCR-ligand⁺ beads for 2 h. Cells were stained against γ-tubulin and Lamp-1. Scale bar, 3 μm. Dashed squares show the area around the Ag-coated bead that was magnified. (B, C) MTOC and lysosome polarity indexes and (D) quantification of Lamp-1⁺ rings around Ag-coated beads in control and Vamp-7–silenced cells after 2 h of activation; ≥20 cells/condition; two independent experiments. (B) siCtrl p < 0.0001; siVamp-7 B p < 0.0001. Unpaired t test. (C) siCtrl p = 0.0006; siVamp-7 B p = 0.0029. Unpaired t test. (D) p = 0.0013. Mann–Whitney test. (E) TIRFM images showing the time-projection of cathepsinD-mRFP⁺ vesicles in control and Vamp-7–silenced B-cells. Scale bar, 5 µm. (F, G) Quantification of the percentage and the ratio of lysosomes in the central region (Center) vs. the periphery (Periph) of images shown in E. (F) Twenty cells for siControl and 19 cells for siVamp-7; four independent experiments. ***p < 0.05, ANOVA followed by a Bonferroni multiple comparison test. (G) *p = 0.0295, unpaired t test. (H) Quantification of the percentage of control and Vamp-7–silenced B-cells engaged with CypHer5E Ag-coated beads that were positive for CypHer5E fluorescence (mean fluorescence intensity [MFI] of the bead >10% above background levels) after an incubation of 90 min; >180 cells/condition. p = 6.85 × 10⁻⁷, Kolmogorov–Smirnov test.

FIGURE 4:

Vamp-7 is required for efficient extraction and presentation of immobilized antigens. (A) Representative 3D projections of confocal images of control and Vamp-7–silenced B-cells incubated with BCR-ligand⁺ or BCR-ligand^- beads plus OVA for different time points. Cell–bead conjugates were stained for OVA and Lamp-1. Scale bar, 3 μm. (B) Calculation of the percentage of OVA extracted from beads. Each bar represents the mean ± SEM of data pooled from two independent experiments; >45 cells per condition and per time point. siRNA BCR-ligand⁺ vs. BCR-ligand^- p < 0.0001; siRNA Ctrl BCR-ligand⁺ vs. siRNA Vamp-7 B p < 0.0001. Unpaired t test. (C) Antigen presentation assay and (D) peptide control assay using control and Vamp-7–silenced cells. Mean levels of IL-2 ± SD are shown of representative experiments performed in triplicate. Two independent experiments **p = 0.0015, t test.