Fig 1: Itolizumab increased the tumor cell killing capacity of PBMC by blocking the inhibitory effects associated with CD6-CD318 interaction. (A) PBMC and MDA-MB-231, NCI-H460, SKOV-3 and HCT-116 cell lines (n=4) were pre-incubated with 10 µg/mL of isotype control (IC, green), itolizumab (T1h, blue), or neutralizing antibodies specific for CD318 (brown) or ALCAM (yellow). Effector and target cells were then co-cultured, and tumor cell lysis was measured using 7AAD staining by flow cytometry. (B) CFSE-labeled T-cells (n=6) were activated with antiCD3/CD28 beads and incubated with 10ug/mL of pre-coated human recombinant CD318 (brown), ALCAM (yellow), or PBS (green) as control. (C) Immune cell viability was measured on isotype control treated-PBMC co-cultured with human tumor cell lines MDA-MB-231 (n=10, red) and MCF-7 (n=10, green) using flow cytometry. Representative histograms or dot plots for each condition, and individual viability percentage and CFSE dilution are displayed. Data are depicted as median ± 95% confidence interval. Statistical analysis was performed using the Kruskal-Wallis test and Dunn’s multiple comparisons test with unpaired data. Only statistical significance is shown in the graphs, with *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.