Basal levels of TCR and cytokine signaling pathways in samples taken from patients in the NRA study of tremelimumab administered together with MART-1_26–35 peptide pulsed dendritic cells (DC).

The MFI of phosphoproteins was measured in CD3+CD4+ (T helper), CD3+CD4− (mainly representing CD8+ CTLs) and CD3−CD14+ (monocyte) cell subsets. PBMC were obtained from patients before (Pre) and after (Post) receiving the combined therapy. a) pLck (pY505), b) pZAP70 (pY292) and c) pLAT (pY171) representative of proximal T cell receptor (TCR) activation, which was only analyzed in CD3 gates including CD4 and CD8 cells. d) pAKT (pT308), e) p38 (pT180/pY182), f) pERK1/2 (T202/204), correspond to the main intracellular signaling pathways downstream of surface receptors, including the TCR. g) Cyclin D1 and h) bcl2 were measured as whole protein content for surrogate evidence for modulation of the cell cycle and apoptosis, respectively. i) Phospho-STAT1 (pY701), j) pSTAT3 (pY705), k) pSTAT5 (Y694) and l) pSTAT6 (Y641) were studied as a measure of STAT signaling downstream of cytokine receptors. In all cases, p values were calculated using a two-sided paired t-test, and significant results are denoted with a line comparing pre- and post-dosing samples with an asterisk to represent the significance level as follows: *p < 0.05; **p < 0.01; ***p < 0.001. Y-axis = MFI - mean fluorescent intensity. Each dot represents the average of six replicates in most instances, and twenty-four replicates for pSTAT 1 and pSTAT5. The horizontal bar in each scatter column indicates medians. The vertical bar in each scatter column indicates standard error.