Immune synapse components ALCAM and ICAM1 are retrograde transport cargoes that rely on EndoA3 and retromer

A Illustration of the SNAP-tag-based BG-labelled antibody uptake assay to study membrane protein endocytosis and retrograde transport.

B Confocal images of GalT-GFP-SNAP (green) and TMR-Star (red) in HeLa cells stably expressing the Golgi-resident GFP-fused SNAP-tag construct (HeLa GalT-GFP-SNAP). Actin (phalloidin, white) and nuclei (DAPI, blue) were also stained. Fluorescence intensity profile was made along the dashed line region in enlarged cropped area, and shows the colocalization of both signals. Scale bar: 20 μm.

C-F Retrograde transport of ALCAM and ICAM-1. Continuous BG-labelled anti-ALCAM (C and E) and anti-ICAM1 (D and F) antibody uptake for 4h at 37°C in HeLa GalT-GFP-SNAP cells. (C-D) Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72h with siRNAs: negative control (siCtrl) or against retromer subunits (siVPS35 and siVPS26A). Immunodetection made with anti-SNAP, anti-VPS35, anti-VPS26A and anti-α-Tubulin (loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex is shown as fractions of siCtrl condition (histogram). Quantification of VPS35 and VPS26A depletion is shown in Fig. S1C. (E-F) Western blot analysis of HeLa GalT-GFP-SNAP cells transfected for 72h with siRNAs: negative control (siCtrl) or against EndoA3 (siEndoA3). Immunodetection made with anti-SNAP, anti-EndoA3 and anti-clathrin heavy chain (CHC, loading control) antibodies. Quantification of the covalent IgG-SNAP-GFP-GalT complex (IB:anti-SNAP) is shown as fractions of siCtrl condition (histogram). Quantification of EndoA3 depletion is shown in Fig. S1H.

Data information: In (B), images are from a single experiment. Quantification data (C-F) are pooled from three independent experiments. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 (C-D, RM one-way ANOVA with Dunnett’s multiple comparison test; E-F, one sample t test). Source data are available online for this figure.

EndoA3-dependent CIE mediates the uptake of immune synapse components ALCAM and ICAM1 in cancer cells

A-B Live-cell TIRF images of EndoA3-GFP (stable) and ICAM1-mScarlet (transient) in HeLa (A) and LB33-MEL (B) cells. Time series show enlarged cropped areas extracted from Movie S1 (A) and Movie S2 (B). White arrows indicate dynamic co-distribution of both signals. Kymograph in (B) was made along dashed line region in the 2nd cropped area (B; Movie S3). Scale bars: 10 μm (full size image), 2 μm (enlarged cropped areas).

C-E Continuous uptake of anti-ALCAM antibody for 15 min at 37°C in the following LB33-MEL cell lines: wild-type (WT, D), stably transfected with empty plasmid (Φ, D), or stably expressing EndoA3-GFP (LB33-MEL EndoA3+, C-E). In (E), cells were transfected with siRNAs: negative control (siCtrl) or against EndoA3 (siEndoA3). Quantification of EndoA3 depletion by western blots in Fig. S3A. (C) Airyscan images of Anti-ALCAM (red) and EndoA3-GFP (green). White arrowheads show colocalization between ALCAM and EndoA3. Scale bars: 10 μm (full size image), 1 μm (enlarged cropped areas). (D-E) Quantifications of anti-ALCAM internalization, expressed as fractions of WT condition (D) or siCtrl condition (E). (D) n cells: WT, n = 270; Φ, n = 279; EndoA3+, n = 274. (E) n cells: siCtrl, n = 350; siEndoA3, n = 234. Representative image examples in Fig. S3B-C.

Data information: All images (A-C) are representative of two independent experiments. In (D-E), data are pooled from three independent experiments. Data are presented as median and quartiles. ns, not significant. ****P < 0.0001 (D, Kruskal-Wallis test with Dunn’s multiple comparison test; E, Mann-Whitney test). Source data are available online for this figure.

Inhibition of EndoA3-dependent endocytosis and retrograde transport impairs T cell activation

A Scheme of flow cytometry analysis of cytokine production inside CD8 T cells stimulated by siRNA transfected EndoA3+ LB33-MEL cells.

B-C Flow cytometry analysis of CD8 T cell intracellular cytokine production after co-culture for 4h at 37°C with: no LB33-MEL (T only), EndoA3+ LB33-MEL transfected with negative control siRNA (siCtrl) or with siRNA against EndoA3 (siEndoA3). Quantification of EndoA3 depletion is shown in Fig. S4A. (B) Representative examples and quantifications (scatter plots) of the percentages of cytokine producing CD8 T cells (up, IL-2; middle, TNFα; bottom, IFNγ) after being stimulated by siCtrl or siEndoA3 treated LB33-MEL EndoA3+ cells. (C) Representative examples and quantifications (scatter plots) of the absolute amount of cytokines produced by CD8 T cells (up, IL-2; middle, TNFα; bottom, IFNγ; presented as median fluorescence intensity, MFI) after stimulation by siCtrl or siEndoA3 treated LB33-MEL EndoA3+ cells.

D Scheme of ELISA analysis of cytokine secretion from CD8 T cells stimulated with siRNA transfected EndoA3+ LB33-MEL cells.

E Quantification of IFNγ secretion (detected by ELISA) from CD8 T cells, cultured alone (T only) or co-cultured for 20h with the following LB33-MEL cell lines: wild-type LB33-MEL cells (WT), LB33-MEL cells stably transfected with empty (Φ) or EndoA3-GFP encoding plasmid (EndoA3+), treated with negative control siRNA (siCtrl) or with EndoA3-targeting siRNA (siEndoA3). Data are presented as fractions of WT siCtrl condition. The absolute concentration of secreted IFNγ in the supernatant of WT siCtrl condition is 2203 ± 201 (mean ± SEM) pg/mL.

F Quantification of IFNγ secretion (detected by ELISA) from CD8 T cells, cultured alone (T only) or co-cultured for 20h at 37°C with EndoA3+ LB33-MEL cells transfected with siRNAs: negative control (siCtrl), or against retromer subunits (siVPS35 or siVPS26A). Data are presented as fractions of siCtrl condition. The absolute concentration of secreted IFNγ in the supernatant of siCtrl condition is 4077 ± 99.62 (mean ± SEM) pg/mL. Quantification of VPS35 and VPS26A depletion is shown in Fig. S4B.

Data information: In (B-C), data are pooled from five independent experiments. In (E), data are pooled from six independent experiments. In (F), data are pooled from three independent experiments. In (E-F), data are presented as mean ± SEM. ns, not significant. *P < 0.05, **P < 0.01, ***P < 0.001 (B-C, paired t test; E, RM one-way ANOVA with Tukey’s multiple comparison test; F, RM one-way ANOVA with Dunnett’s multiple comparison test). Source data are available online for this figure.

Inhibition of EndoA3-mediated endocytosis and retrograde transport affects ICAM1 recruitment to and structure of immune synapses

A Live-cell Airyscan images of an immune synapse-like conjugate formed between a CD8 T cell (red) and an adherent stable HLA-A*68012-expressing HeLa cell line transiently expressing ICAM1-EGFP (green). Time series show enlarged cropped areas extracted from Movie S6. The dashed line contours delineate the contact area between the HeLa cell and the CD8 T cell. White arrowheads show two ICAM1-positive vesicular structures moving towards the contact area. Scale bars: 10 μm (full size image), 2 μm (enlarged cropped areas).

B Airyscan images of an immune synapse-like conjugate formed between a CD8 T cell (stained for CD45, yellow) and a stable HLA-A*68012-expressing HeLa cell (stained for actin, green) transiently expressing ICAM1-mScarlet (red) in suspension. Cell segmentation (white contour) was based on the actin staining for further quantifications (C). Scale bar: 5 μm.

C Quantification of relative ICAM1 recruitment to the vicinity of CD8 T cell when an immune synapse-like conjugate is formed between a CD8 T cell and a stable HLA-A*68012-expressing HeLa cell transiently expressing ICAM1-mScarlet in suspension. HLA-A*68012-expressing HeLa cells were transfected for 72h with siRNAs: negative control (siCtrl) or against EndoA3 (siEndoA3). Western blot analysis of EndoA3 depletion is shown in Fig. S5E. n conjuagates: siCtrl, n = 85; siEndoA3, n = 75.

D,F Airyscan images of immune synapse-like conjugates formed between CD8 T cells (stained for CD45, yellow) and stable EndoA3-GFP-expressing LB33-MEL cells (EndoA3-GFP, green) transfected for 72h with different siRNAs: negative control (siCtrl), siRNAs against EndoA3 (siEndoA3, D) or against VPS26A (siVPS26A, F). Actin (phalloidin, red) and nuclei (DAPI, blue) were also stained. Two examples are displayed per condition. Scale bar: 5 μm.

E,G Quantifications of the sizes of immune synapse-like conjugates formed between CD8 T cells and stable EndoA3-GFP-expressing LB33-MEL cells transfected with different siRNAs from images in panels D and F. Western blot analyses of EndoA3 and VPS26A depletion are shown in Fig. S6C and S6D. (E) n conjugates: siCtrl, n = 48; siEndoA3, n = 58. (G) n conjugates: siCtrl, n = 74; siVPS26A, n = 78.

H Working model. Immune synapse components ALCAM and ICAM1 are endocytosed into cancer cells through EndoA3-mediated CIE and are subsequently transported to the TGN in a retromer-dependent retrograde manner. Upon immune synapse formation, immune synapse components, such as ALCAM and ICAM1, are efficiently recruited to the contact area with CD8 T cells, likely from the TGN by polarized re-distribution, which can stabilize the immune synapse and promote CD8 T cell activation (left). Consequently, disruption of EndoA3-mediated CIE or retromer-dependent retrograde transport in cancer cells impair the efficient recruitment of immune synapse components to the contact area with CD8 T cells. In this situation, CD8 T cells expand their contact areas with cancer cells to try to compensate the loss of immune synapse components. The compensation is insufficient, which ultimately results in impaired T cell activation (right).

Data information: In (A), (B), (D) and (F), images are representative of three independent experiments. In (C), (E) and (G), data are pooled from three independent experiments. Data are presented as median and quartiles. *P < 0.05, **P < 0.01, ***P < 0.001 (C, Kolmogorov-Smirnov test; E and G, Mann-Whitney test). Source data are available online for this figure.