Fig. 3

Phenotypic characterization of murine 2D2 CD4⁺Vα3.2⁺c-Met⁺ polarized Th1 T cells in vitro. A Relative expression of c‐Met mRNA by qRT-PCR in undifferentiated 2D2 CD4⁺Vα3.2⁺ T cells (d0), and FACS‐sorted CD4⁺Vα3.2⁺c‐Met⁻ and c‐Met⁺ T cells after in vitro polarization (d6) were assessed. B Representative flow cytometric plots that showed live 2D2 CD4⁺Vα3.2⁺ T cells (7AAD⁻CD45⁺CD4⁺, not shown) were analyzed during 6 days of in vitro Th1 differentiation (left panels), and mean percentages of c‐Met⁺ cells are shown (right panel). c-Met isotype control antibody (top panels) was used to define c-Met expression on 2D2 CD4⁺Vα3.2⁺ T lymphocytes. C Protein expression levels of c‐Met by Western blot in bulk 2D2 CD4⁺Vα3.2⁺ T cells undifferentiated (d0), versus Th1 differentiated (d6), and FACS‐sorted 2D2 CD4⁺Vα3.2⁺c‐Met⁺ and c‐Met⁻ T cells (d6). The expression of β-actin protein was assessed in all samples as control. Data are representative of three independent experiments. D Representative flow cytometric plots for the sorting of 2D2 CD4⁺Vα3.2⁺c-Met⁻ and CD4⁺Vα3.2⁺c-Met⁺ T cells before differentiation (d0, left panels) and after differentiation (d6, middle panels) of sorted CD4⁺Vα3.2⁺c-Met⁻ (blue) or CD4⁺Vα3.2⁺c-Met⁺ (red) T cells (middle panels). Quantification of CD4⁺Vα3.2⁺c-Met⁺ T cells proportion was shown on the right panel. E Representative flow cytometric histograms (top panels) and Gmean/isotype quantification (lower panel) of CD44, CD69, IFNγ and TNFα expression, gated on 2D2 CD4⁺Vα3.2⁺c‐Met⁺ and c‐Met⁻ T cells at day 6 post-Th1 differentiation. F Quantifications IFNγ and TNFα production by ELISA in supernatant from 2D2 CD4⁺ Vα3.2⁺c‐Met⁻ and c‐Met⁺ T cells, co-cultured for 48 h with MOG_35–55-loaded DCs 6 day post-Th1 differentiation (triplicate). G Representative flow cytometric histograms (top panels) and Gmean/Isotype quantification (bottom panels) at day 6 post-Th1 differentiation of the expression of Itgα4 and ItgαL (VLA4 and LFA-1 integrins subunits, respectively) on 2D2 CD4⁺Vα3.2⁺c‐Met⁻ and c‐Met⁺ T cells. H Representative flow cytometric histograms (top panels) and Gmean/Isotype quantification (lower panel) at day 6 post-Th1 polarization of CXCR3, CCR2, CCR5, CCR6, and CCR8 chemokine receptors gated on 2D2 CD4⁺Vα3.2⁺c‐Met⁻ or c‐Met⁺ T cells. I Quantifications of CCL3, CXCL2 and CXCL10 production by multiplex assay in supernatant from 2D2 CD4⁺Vα3.2⁺c‐Met⁻ and c‐Met⁺ T cells, co-cultured for 48 h with MOG_35–55-loaded DCs at day 6 post-Th1 differentiation (triplicate). Data are representative of three independent experiments and mean values ± SEM are shown; *p ≤ 0.05, **p ≤ 0.01 ***p ≤ 0.001, ****p ≤ 0.0001, unpaired two-tailed Student’s t test for two groups (A, D, E, G, H) or two-way ANOVA followed by Tukey’s post hoc test for multiple groups (B, F, I)