Figure 4: IFNγ-resistant melanoma evolves into a T-cell-resistant lesion.

(a) Mutation defined by targeted sequencing on DNA from Ma-Mel-61h cells and autologous blood cells as wild-type (WT) control (ctrl). Plots of aligned sequencing reads in the location where the JAK1 c.1798G>T, p.G600W mutation was identified. WT sequence shown on the bottom, arrow highlights mutation or corresponding wild-type (WT) site. Number of sequencing reads notated on the left; %, frequency of mutations in reads. (b) Lysates from IFNγ-treated (48 h) Ma-Mel-61b, Ma-Mel-61g and Ma-Mel-61h cells analysed by western blot for expression of IRF1 and HLA class I heavy chains; GAPDH, loading control. Representative data from n=3 independent experiments. (c) HLA class I and CD54 surface expression on IFNγ-treated (48 h) Ma-Mel-61g and Ma-Mel-61h cells, measured by flow cytometry. Representative data from n=3 independent experiments. (d) Immunohistochemical staining of serial cryostat tissue sections from metastasis Ma-Mel-61g for melanoma marker HMB45 and HLA class I. (e) Ma-Mel-61h and Ma-Mel-61g cells, transfected with expression plasmids encoding wild-type JAK1 or mutant JAK1-G600W, analysed for specific mRNA expression by quantitative reverse transcription–PCR in the presence of absence or IFNγ (48 h). Ma-Mel-61b cells served as a control (ctrl). Relative expression levels given as means (+s.e.m.) from n=2 independent experiments. (f) HLA class I surface expression on IFNγ-treated (48 h) Ma-Mel-61h-JAK1 and Ma-Mel-61h-JAK1-G600W transfectants, measured by flow cytometry. Representative data from n=2 independent experiments. (g) Ma-Mel-61h and Ma-Mel-61g cells, transfected with expression plasmids encoding wild-type JAK1 (WT) or mutant JAK1-G600W (M), analysed for recognition by autologous CD8⁺ T cells in the presence or absence of IFNγ (48 h). T-cell activation measured as IFNγ release by ELISpot assay. Representative data from n=2 independent experiments.