Fig. 3

In vitro antimelanoma activity by CSPG4-CAR.CIK against melanoma cells, independently from HLA class I expression. Patient-derived Mel (n = 10) were characterized by flow cytometry for CSPG4 expression and for the presence of MICA/B and ULBP2-5–6 ligands. The same Mel were then utilized as targets to test in vitro the antitumor activity of CSPG4-CAR.CIK. A Flow cytometry histograms showing CSPG4 expression on Mel. B Mean expression level of CSPG4 (82 ± 17%), MIC A/B (11 ± 16%), and ULBP2-5–6 (56 ± 18%) on melanoma cells, with each symbol representing a single Mel. C Specific tumor lysis of Mel with high (n = 7) or low/absent (n = 3) HLA-I expression, co-cultured with CSPG4-CAR.CIK, CD19-CAR.CIK or unmodified CIK at different E/T ratios. Percentage of tumor cell killing was determined by flow cytometry following a 48-h incubation period. The extent of CSPG4-CAR.CIK-mediated killing was significantly higher compared to unmodified (p < 0.001) and CD19-CAR CIK (p < 0.0001), utilized as controls. No significant difference was observed when comparing unmodified CIK with CD19-CAR.CIK (p = 0.123), or the activity of CSPG4-CAR.CIK vs either high- or low-HLA melanoma (p = 0.995) at all E:T ratio. Statistical significance showed for each E:T ratio refers to the comparison between the killing mediated by CSPG4-CAR.CIK and the killing mediated by unmodified CIK. Comparisons between other groups are indicated in the figure legend. Statistical analysis was performed by two-way ANOVA multiple comparison test. Results are shown as mean ± SD