Fig 2: PODXL is expressed in human monocyte-derived immature DCs. (A) Determination of PODXL expression in total lysates from human monocytes and monocyte-derived immature DCs from two donors and CD4+ T cells from one donor by Western blot analysis using a specific mouse anti-human PODXL monoclonal antibody. GAPDH was used as loading control. The blot shown is representative of two independent experiments. (B) Flow cytometry gating and overlay histograms depicting the surface expression of PODXL on monocytes (CD14+ CD209-) and immature DCs (CD14- CD209+) detected by flow cytometry using a biotinylated goat anti-human PODXL antibody (red line) or an isotype control (grey line) followed by PE-conjugated streptavidin from one representative donor. The graph shows PODXL surface expression (?MFI= PODXL MFI - isotype control MFI) on monocytes and immature DCs from three donors. *p < 0.05, paired t test. (C) Expression of PODXL in monocyte-derived immature DCs by fluorescence microscopy. Monocyte-derived immature DCs were deposited onto glass slides using a cytospin and incubated in complete culture medium for 30 min. Cell were stained with a goat anti-PODXL polyclonal antibody followed by an anti-goat Cy2-labelled secondary antibody (green). Images were acquired with a 63x objective using a confocal fluorescence microscope and are representative of ten rounded and twelve elongated immature DCs photographed from three independent experiments. Example shows maximal intensity z-projections of confocal fluorescence sections. DIC, differential interference contrast. Scale bar corresponds to 5µm. (D) Determination of PODXL expression in cell lysates from different myelomonocytic cell lines by Western blot analysis using a specific anti-PODXL monoclonal antibody. GAPDH was used as loading control. The blot shown is representative of two independent experiments. (E) Overlay histograms showing the surface expression of PODXL on four different myeloid cell lines determined by flow cytometry using a biotinylated goat anti-human PODXL antibody (red line) or an isotype control (grey line) followed by PE-conjugated streptavidin from one representative experiment. Graph shows mean ± SD of PODXL surface expression (?MFI= PODXL MFI - isotype control MFI) from four independent experiments. ***p < 0.001, ****p < 0.0001, one-way ANOVA. (F) Analysis of PODXL mRNA level by RT-PCR in four myelomonocytic cell lines and in T cells, NK cells, monocytes, monocyte-derived immature and mature DCs. Results are depicted relative to THP-1 cells. *p < 0.05, Krustal-Wallis test.

Fig 3: PODXL expressed in APCs perturbs CD4+ T cell-centrosome polarization to the contact site. (A) Raji PODXL or Raji control cells pulsed with SEA were incubated with CD4+ T cells at a ratio of 1:1 for 30 min or 60 min. Then, cells were stained with a monoclonal antibody against ?-tubulin to visualize the centrosome (red) and Hoechst 33342 for nuclei staining (blue). Confocal fluorescence microscopy images show separate and merged channels of maximal intensity z-projections of z-sections from a representative experiment performed at 60 min of incubation. Arrows point to T-cell centrosome in each T-cell. DIC: differential interference contrast. Scale bars correspond to 5 µm. (B) Quantification of T-cell centrosome polarization in Raji-CD4+ T cells conjugates. Scatter graph depicts the distance of CD4+ T cell centrosome to Raji-cell contact site in each conjugate for a total of 41 (Raji Ctrl, 30 min), 37 (Raji PODXL, 30 min), 51 (Raji Ctrl, 60 min), and 45 (Raji PODXL, 60 min) conjugates from three independent experiments. The results are shown as mean ± SD. *p < 0.05, two-way ANOVA followed by Holm-Šídák multiple comparisons test. Bar graph represents the percentage of conjugates in which T-cell centrosome localizes at a distal (=3 µm), intermediate (1-3 µm) or proximal (=1 µm) distance from Raji-cell contact site for the indicated conditions.

Fig 4: PODXL expression is positively regulated by IL-4 through MEK/ERK and JAK3/STAT6 signaling pathways in myeloid cells. THP-1 cells were stimulated with IL-4 (400 U/ml) and GM-CSF (800 U/ml) cytokines (A) for 96 h or with PMA (100 ng/ml) (B) for 48 h followed by a resting period of 48 (h) Afterwards, total cell lysates were subjected to Western blotting using a specific monoclonal antibody against PODXL. Actin was used as loading control. Bar graphs represent the mean ± SD of PODXL levels relative to those of unstimulated cells of four independent experiments. *p < 0.05, **p < 0.01, one-way ANOVA. (C) PODXL surface expression was analyzed by flow cytometry using a biotinylated goat anti-human PODXL antibody followed by PE-conjugated streptavidin. Overlay histograms show surface expression of PODXL (red line) and isotype control (grey line) from one representative donor. Graph shows PODXL surface expression (?MFI= PODXL MFI - isotype control MFI) of four independent experiments. **p < 0.01, ***p < 0.001, one-way ANOVA. (D, E) Effect of signaling pathway inhibitors on IL-4-induced PODXL expression in THP-1 cells. Cells were incubated with 400 U/ml IL-4 in the presence of increasing concentration of MEK/ERK signaling pathway inhibitor (PD98059) (D) or JAK3 signaling pathway inhibitor (PF956980) (E) for 96 (h) Then, cell lysates were subjected to Western blot analysis with a monoclonal anti-PODXL antibody. Actin was used as loading control. Bar graphs represent the mean ± SD of PODXL levels relative to those of unstimulated and untreated cells of three (D) or four (E) independent experiments. A representative blot is shown below each graph. *p < 0.05, **p < 0.01, ***p < 0.001, one-way ANOVA.

Fig 5: PODXL expressed in APCs enhances both APC-CD4+ T cell and APC-CD8+ T cell interactions. (A) Raji cells overexpressing PODXL (red) or Raji-Ctrl cells (blue) were pulsed or not with SEA. Afterwards, CD4+ T cells isolated from PBMCs and pre-labelled with CMTMR were mixed with Raji-PODXL or Raji-Ctrl at a ratio of 1:1 and incubated for different time points at 37°C. The formation of conjugates between Raji cells (GFP+) and CD4+ T cells (CMTMR+) was detected as CMTMR+ and GFP+ events by flow cytometry. A representative density plot out of seven independent experiments is shown. Graph shows mean ± SD of the percentage of conjugates (CMTMR+GFP+) from the total Raji cells (GFP+) of four independent experiments. *p < 0.05, **p < 0.01, ***p <0.001, ****p < 0.0001, two-way ANOVA. (B) Raji-PODXL or Raji-Ctrl cells were mixed with isolated CD4+ T and CD8+ T at a ratio of 1:1 and incubated for 30 min. CD4+ and CD8+ T cells were detected using anti-CD4-APCH7 and anti-CD8-APCH7 monoclonal antibodies by flow cytometry. The percentage of Raji-CD4+ T cell (GFP+APCH7+) or Raji-CD8+ T cell (GFP+APCH7+) out of the total Raji cells was determined. Bar graph represents the mean ± SD of five independent experiments. *p < 0.05, RM one-way ANOVA. (C) Raji-PODXL or Raji-Ctrl cells were mixed with CMTMR-labeled Jurkat cells at a ratio of 1:1 and incubated at different time points. The percentage of Raji-Jurkat cell conjugates was determined by flow cytometry as in (A). Bar graph represents the mean ± SD of five independent experiments. **p < 0.01, two-way ANOVA.