CONSTITUTIVE ACTIVATION OF CYTOKINE JAK/STAT SIGNALING PATHWAY PROTEINS IN EBV-TRANSFORMED B CELL LINES FROM PATIENTS WITH PTLD

Abstract

127 B cell lymphomas are a major complication of the drug induced immunosuppression required for solid organ transplantation. Epstein-Barr virus(EBV) is the etiologic agent of this post-transplant lymphoproliferative disorder (PTLD). The molecular mechanisms involved in the cellular proliferation of these lymphomas are not yet clearly defined. From PTLD patients, we previously generated EBV infected spontaneous lymphoblastoid cell lines (SLCL) with morphologic and phenotypic similarities to PTLD tumors. SLCL treatment with IL-10 or IL-6 neutralizing antibodies, or with soluble IL-10 receptor inhibits cellular proliferation, indicating that SLCL utilize IL-10 and IL-6 as autocrine growth factors that are required for sustained proliferation. Cytokine receptor binding leads to the autophosphorylation of proteins of the Janus kinase (Jak) family, which then recruit STAT (signal transducers and activators of transcription) cytoplasmic proteins. Jak activated STAT proteins dimerize and translocate to the nucleus to control various cytokine activated genes. To understand the mechanisms by which IL-10 and IL-6 stimulate SLCL we analyzed the expression levels and tyrosine phosphorylation states of the Jak/STAT signal transduction pathway proteins. Three different SLCL, termed JB7, MF4 and VB4 were examined by Western blot and immunoprecipitation. All three SLCL express Jak1, with high levels of this protein particularly evident in the JB7 cell line. Tyk2 was found to be expressed by each of the SLCL at equivalent levels. STAT proteins expressed by these cells include STAT1, STAT2, STAT3, STAT5, and STAT6. STAT4 expression was not detected. To determine which of these proteins may be actively utilized by the SLCL, the tyrosine phosphorylation states of Jak proteins were examined by western blotting with the 4G10 anti-phosphotyrosine antibody. The JB7 line expresses a high level of constitutively tyrosine phosphorylated Jak1. Additionally, all three SLCL, but not the Daudi Burkitt's lymphoma B cell line express a high level of constitutively tyrosine phosphorylated Tyk2. Thus, the cytokine mediated constitutive activation of Jak1 and Tyk2 may be critical for the autonomous growth of SLCL. An understanding of the signal transduction molecules that participate in the cytokine dependent growth of EBV B cell lymphomas may aid in the treatment of PTLD.