Fumarates suppress Th1 responses by blocking IL-12 and IL-23 production by dendritic cells via distinct pathways.

Purpose Abnormalities in the constitutive and IFN-γ–inducible HLA class I surface antigen expression of tumor cells is often associated with an impaired expression of components of the antigen processing machinery (APM). Hence, we analyzed whether there exists a link between the IFN-γ signaling pathway, constitutive HLA class I APM component expression, and IFN-γ resistance. Experimental Design The basal and IFN-γ–inducible expression profiles of HLA class I APM and IFN-γ signal transduction cascade components were assessed in melanoma cells by real-time PCR (RT-PCR), Western blot analysis and/or flow cytometry, the integrity of the Janus activated kinase (JAK) 2 locus by comparative genomic hybridization. JAK2 was transiently overexpressed in JAK2− cells. The effect of IFN-γ on the cell growth was assessed by XTT [2,3-bis(2-methoxy-4-nitro-S-sulfophenynl)-H-tetrazolium-5-carboxanilide inner salt] assay. Results The analysis of 8 melanoma cell lines linked the IFN-γ unresponsiveness of Colo 857 cells determined by lack of inducibility of HLA class I surface expression on IFN-γ treatment to a deletion of JAK2 on chromosome 9, whereas other IFN-γ signaling pathway components were not affected. In addition, the constitutive HLA class I APM component expression levels were significantly reduced in JAK2− cells. Furthermore, JAK2-deficient cells were also resistant to the antiproliferative effect of IFN-γ. Transfection of wild-type JAK2 into JAK2− Colo 857 not only increased the basal APM expression but also restored their IFN-γ sensitivity. Conclusions Impaired JAK2 expression in melanoma cells leads to reduced basal expression of MHC class I APM components and impairs their IFN-γ inducibility, suggesting that malfunctional IFN-γ signaling might cause HLA class I abnormalities.

Supplementary Figures S1-S2 from Association of IFN-γ Signal Transduction Defects with Impaired HLA Class I Antigen Processing in Melanoma Cell Lines

Supplementary Figures S1-S2 from Association of IFN-γ Signal Transduction Defects with Impaired HLA Class I Antigen Processing in Melanoma Cell Lines

<div>Abstract<p><b>Purpose:</b> Abnormalities in the constitutive and IFN-γ–inducible HLA class I surface antigen expression of tumor cells is often associated with an impaired expression of components of the antigen processing machinery (APM). Hence, we analyzed whether there exists a link between the IFN-γ signaling pathway, constitutive HLA class I APM component expression, and IFN-γ resistance.</p><p><b>Experimental Design:</b> The basal and IFN-γ–inducible expression profiles of HLA class I APM and IFN-γ signal transduction cascade components were assessed in melanoma cells by real-time PCR (RT-PCR), Western blot analysis and/or flow cytometry, the integrity of the Janus activated kinase (JAK) 2 locus by comparative genomic hybridization. JAK2 was transiently overexpressed in JAK2⁻ cells. The effect of IFN-γ on the cell growth was assessed by XTT [2,3-bis(2-methoxy-4-nitro-<i>S</i>-sulfophenynl)-<i>H</i>-tetrazolium-5-carboxanilide inner salt] assay.</p><p><b>Results:</b> The analysis of 8 melanoma cell lines linked the IFN-γ unresponsiveness of Colo 857 cells determined by lack of inducibility of HLA class I surface expression on IFN-γ treatment to a deletion of JAK2 on chromosome 9, whereas other IFN-γ signaling pathway components were not affected. In addition, the constitutive HLA class I APM component expression levels were significantly reduced in JAK2⁻ cells. Furthermore, JAK2-deficient cells were also resistant to the antiproliferative effect of IFN-γ. Transfection of wild-type JAK2 into JAK2⁻ Colo 857 not only increased the basal APM expression but also restored their IFN-γ sensitivity.</p><p><b>Conclusions:</b> Impaired JAK2 expression in melanoma cells leads to reduced basal expression of MHC class I APM components and impairs their IFN-γ inducibility, suggesting that malfunctional IFN-γ signaling might cause HLA class I abnormalities. <i>Clin Cancer Res; 17(9); 2668–78. ©2011 AACR</i>.</p></div>

<div>Abstract<p><b>Purpose:</b> Abnormalities in the constitutive and IFN-γ–inducible HLA class I surface antigen expression of tumor cells is often associated with an impaired expression of components of the antigen processing machinery (APM). Hence, we analyzed whether there exists a link between the IFN-γ signaling pathway, constitutive HLA class I APM component expression, and IFN-γ resistance.</p><p><b>Experimental Design:</b> The basal and IFN-γ–inducible expression profiles of HLA class I APM and IFN-γ signal transduction cascade components were assessed in melanoma cells by real-time PCR (RT-PCR), Western blot analysis and/or flow cytometry, the integrity of the Janus activated kinase (JAK) 2 locus by comparative genomic hybridization. JAK2 was transiently overexpressed in JAK2⁻ cells. The effect of IFN-γ on the cell growth was assessed by XTT [2,3-bis(2-methoxy-4-nitro-<i>S</i>-sulfophenynl)-<i>H</i>-tetrazolium-5-carboxanilide inner salt] assay.</p><p><b>Results:</b> The analysis of 8 melanoma cell lines linked the IFN-γ unresponsiveness of Colo 857 cells determined by lack of inducibility of HLA class I surface expression on IFN-γ treatment to a deletion of JAK2 on chromosome 9, whereas other IFN-γ signaling pathway components were not affected. In addition, the constitutive HLA class I APM component expression levels were significantly reduced in JAK2⁻ cells. Furthermore, JAK2-deficient cells were also resistant to the antiproliferative effect of IFN-γ. Transfection of wild-type JAK2 into JAK2⁻ Colo 857 not only increased the basal APM expression but also restored their IFN-γ sensitivity.</p><p><b>Conclusions:</b> Impaired JAK2 expression in melanoma cells leads to reduced basal expression of MHC class I APM components and impairs their IFN-γ inducibility, suggesting that malfunctional IFN-γ signaling might cause HLA class I abnormalities. <i>Clin Cancer Res; 17(9); 2668–78. ©2011 AACR</i>.</p></div>