Fig. 4
Kv1.3 channel blockade modifies the proteome, targeting the protein synthesis machinery. (a) Mass spectrometry-based quantitative proteomic analysis on CD4+ TEM cells. Cells from three independent donors were stimulated for 24 h with plate-bound OKT3 in the presence or absence of Vm24 (1 nM) and total proteins were analyzed by nano-liquid chromatography coupled to MS/MS. All conditions were normalized to the unstimulated (US) control. Pairwise comparison between the unstimulated and the OKT3-treated group was performed to identify proteins modified upon TCR engagement. Proteins that show at least 1.5-fold change in either direction, and significant difference (p < 0.05) are identified in the upper right and left segments of the volcano plot. Within the OKT3-regulated proteins, a pairwise comparison (p < 0.05) between the OKT3 and the OKT3 + Vm24-treated group was performed to identify proteins (red spots) affected by Kv1.3 channel blockade, in a T cell activation context. Note that these 27 proteins preserved the same trend of change than when cells where stimulated with OKT3 only, yet the amplitude of the change was reduced. (b) Protein–protein interaction network from significantly (p < 0.05) modified proteins affected by Kv1.3 channel blockade in a T cell activation context. The combination of the proteomic data set with the activation markers and the cytokine profile was used to generate a protein physical/functional interaction network and to perform a functional enrichment analysis specific for Biological Process (GO) and KEGG Pathways, using the STRING database. Line thickness on the interaction network indicates the strength of data support. Proteins were clustered and enriched functions are indicated. The expression of (c) IRF4, (d) Hsp90 and (e) CD3e was assessed by flow cytometry to validate proteins identified through the proteomic analysis. Data from three independent individuals are shown as mean ± SEM. Significance of pairwise comparisons between groups is indicated with stars (*p < 0.05, **p < 0.01, ***p < 0.001)