Figure 3. A homophilic peptide derived from the residual extracellular fragment on CD31^shed inhibits T-cell activation and rescues CD31 signalling.

(A) Proliferative response to TCR engagement of human peripheral blood mononuclear cells in the presence of increasing doses of human CD31 peptide. *p<0.05 vs dose “0”. CD31 peptide inhibits cell proliferation in a dose-dependent manner. (B) Real-time (BIAcore^®) analysis of the human CD31 peptide homophilic interaction at two-fold stepwise dilutions of the peptide (0.63, 1.25, 2.5, 5, 10 and 20 μg/ml). Data are normalized with respect to the control channel (scrambled peptide) and expressed as ΔRU (Resonance Units). (C,D) Flow cytometric assessment of human CD31 pY686 (C) and of SHP2 pY542 (D) on cultured Jurkat cells. Unstim= membrane lysate from untreated Jurkat cells, pervanadate= incubation with sodium pervanadate (Na₃VO₈) at 100μM for 20 minutes, peptide= incubation with the peptide alone (100μg/ml) for 20 minutes, stim= crosslinking of CD3 molecules by a monoclonal mouse anti-human CD3ε + goat-anti-mouse IgG F(ab′)₂ fragments, stim/cross= crosslinking of CD3 with CD31 molecules via their domain 6, stim/peptide= crosslinking of CD3 in the presence of 100μg/ml human CD31 peptide. Quantification of CD31 pY686 was performed on solubilized membrane-bound CD31; SHP2 pY542 was analyzed by intracellular staining. Data are expressed as Median Fluorescent Intensity (MFI). nd= not determined. The crosslinking of the TCR alone and with CD31 molecules induces a rapid and transient phosphorylation of the CD31 inhibitory motifs (ITIM’s) while the peptide treatment is able to sustain phosphorylation for at least 20 minutes after the stimulation (*p<0.001 between “stim peptide” and “stim” at 20 minutes). In parallel, the peptide increases the SHP2 Y542 phosphorylation to an extent comparable to that observed with CD31 antibody-mediated crosslinking. *p<0.001 between “stim peptide” and “stim”.