Journal: The Journal of Clinical Investigation

Article Title: Targeting the innate immunoreceptor RIG-I overcomes melanoma-intrinsic resistance to T cell immunotherapy

doi: 10.1172/JCI131572

Figure Lengend Snippet: (A and B) Human fibrosarcoma cells U3A (STAT1–/–) (A) and U5A (IFNAR2c–/–) (B) were treated with IFNα or IFN-γ for 20 to 24 hours. Controls were left untreated. HLA-I and ICAM-1 surface expression was determined by flow cytometry. Relative MFI given as mean plus SEM of 2 (A) and 3 (B) independent experiments. (C–G) U3A and U5A cells were transfected with 3pRNA or control (ctrl) RNA and subjected to further analyses following an incubation of 20 to 24 hours. (C and D) HLA-I and ICAM-1 surface expression of U3A (C) and U5A (D) cells measured by flow cytometry. Left, representative histogram; right, relative MFI given as mean plus SEM from 3 independent experiments. (E and F) HLA-I APM component expression in U3A (E) and U5A (F) cells analyzed by immunoblot. GAPDH, loading control. Representative data from 3 independent experiments. (G) RIG-I and OAS3 expression in U3A and U5A cells determined by immunoblot. GAPDH, loading control. Representative data from 3 independent experiments. Significantly different experimental groups: *P < 0.05, ***P < 0.005 by 2-tailed paired t test.

Article Snippet: The following anti-human antibodies were used for immunoblot: mouse anti-STAT1 (clone 9H2), rabbit anti-phospho STAT1 (Y701) (clone 58D6), rabbit anti-phospho IRF3 (S396) (clone 4D4G), rabbit anti-GAPDH (clone 14C10), rabbit anti–RIG-I (clone D14G6) were purchased from Cell Signaling; rabbit anti-IRF1 (clone H-205), rabbit anti-IRF3 (clone FL-425), rabbit anti-OAS3 (clone M-190) were obtained from Santa Cruz.

Techniques: Expressing, Flow Cytometry, Transfection, Incubation, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Targeting the innate immunoreceptor RIG-I overcomes melanoma-intrinsic resistance to T cell immunotherapy

doi: 10.1172/JCI131572

Figure Lengend Snippet: (A–D) Melanoma cells Ma-Mel-86c and fibrosarcoma cells U3A (STAT1–/–) and U5A (IFNAR2c–/–) were transfected with 3pRNA (+) or control RNA (–) and subjected to further analysis following an incubation of 20 to 24 hours. (A and B) Representative (p)STAT1 (A), IRF1 (A), (p)IRF3 (B), and NLRC5 (A) immunoblots from 3 independent experiments. GAPDH, loading control. (C and D) Ma-Mel-86c and U3A cells were transfected with siRNA targeting IRF3 (siIRF3) (C), IRF1 (siIRF1) (C), NLRC5 (siNLRC5) (D), or control siRNA (siCtrl) (C and D) 24 hours before 3pRNA (+) or control RNA (–) transfection. Protein expression was analyzed by immunoblot. GAPDH, loading control. Representative data from 3 independent experiments.

Article Snippet: The following anti-human antibodies were used for immunoblot: mouse anti-STAT1 (clone 9H2), rabbit anti-phospho STAT1 (Y701) (clone 58D6), rabbit anti-phospho IRF3 (S396) (clone 4D4G), rabbit anti-GAPDH (clone 14C10), rabbit anti–RIG-I (clone D14G6) were purchased from Cell Signaling; rabbit anti-IRF1 (clone H-205), rabbit anti-IRF3 (clone FL-425), rabbit anti-OAS3 (clone M-190) were obtained from Santa Cruz.

Techniques: Transfection, Incubation, Western Blot, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Targeting the innate immunoreceptor RIG-I overcomes melanoma-intrinsic resistance to T cell immunotherapy

doi: 10.1172/JCI131572

Figure Lengend Snippet: (A) Clinical history of melanoma patient Ma-Mel-61. Horizontal line, time axis; above: diagnosis, therapeutic regimens, death; below: metastases development; arrows indicate cell lines established from metastases Ma-Mel-61b (JAK1-wildtype, JAK1-WT), Ma-Mel-61g (JAK1-mutant, JAK1-G600W) and Ma-Mel-61h (JAK1-mutant, JAK1-G600W). HLA-I surface expression on cell lines established from corresponding lesions was determined by flow cytometry. Representative histograms from 3 independent experiments. (B) Cell lines treated with IFNα-2b or IFN-γ for 48 hours were analyzed for (p)STAT1 and IRF1 expression by immunoblot. GAPDH, loading control. Representative data from 3 independent experiments. (C) Immunohistochemical staining of serial cryostat tissue sections from metastasis Ma-Mel-61g for melanoma marker GP100, HLA-I, B2M, and CD3. Top, tumor margin indicated by the dotted line; bottom, higher magnification of boxed regions; original magnifications: ×2.5 (top), ×10 (bottom). (D–I) Ma-Mel-61h cells were transfected with 3pRNA or control (ctrl) RNA and subjected to further analysis following an incubation of 20 to 24 hours. (D and E) HLA-I and ICAM-1 surface expression measured by flow cytometry. (D) Representative HLA-I dot plot and (E) relative MFI given as mean plus SEM from 3 independent experiments. (F) mRNA expression of APM components analyzed by qPCR. Relative expression given as mean plus SEM from 3 independent experiments. (G) Expression of indicated proteins analyzed by immunoblot. GAPDH, loading control. Representative data from 3 independent experiments. (H and I) Activation of autologous CD8+ T cells by Ma-Mel-61h cells determined by IFN-γ ELISpot assay. (H) Representative ELISpot, (I) mean IFN-γ spots (+ SEM) from 3 independent experiments. without, incubation of T cells without tumor cells. Significantly different experimental groups: *P < 0.05, **P < 0.01, ***P < 0.005 by 2-tailed paired t test.

Article Snippet: The following anti-human antibodies were used for immunoblot: mouse anti-STAT1 (clone 9H2), rabbit anti-phospho STAT1 (Y701) (clone 58D6), rabbit anti-phospho IRF3 (S396) (clone 4D4G), rabbit anti-GAPDH (clone 14C10), rabbit anti–RIG-I (clone D14G6) were purchased from Cell Signaling; rabbit anti-IRF1 (clone H-205), rabbit anti-IRF3 (clone FL-425), rabbit anti-OAS3 (clone M-190) were obtained from Santa Cruz.

Techniques: Mutagenesis, Expressing, Flow Cytometry, Western Blot, Immunohistochemical staining, Staining, Marker, Transfection, Incubation, Activation Assay, Enzyme-linked Immunospot