SlllnmaryInterleukin 12 initiates the differentiation of naive CD4 + T ceils to T helper type 1 (Thl) cells critical for resistance to intracellular pathogens such as Leishmania major. To explore the basis of IL-12 action, we analyzed induction of nuclear factors in Thl cells. IL-12 selectively induced nuclear DNA-binding complexes that contained Star3 and Stat4, recently cloned members of the family of signal transducers and activators of transcription (STATs). While Stat3 participates in signaling for several other cytokines, Stat4 was not previously known to participate in the signaling pathway for any natural ligand. The selective activation of Stat4 provides a basis for unique actions of IL-12 on Thl development. Thus, this study presents the first identification of the early events in IL-12 signaling in T cells and of ligand activation of Stat4.

Recent work has shown that IL-10 induces activation of the JAK-STAT signaling pathway. To define the mechanism underlying signal transducer and activator of transcription (STAT) protein recruitment to the interleukin 10 (IL-10) receptor, the STAT proteins activated by IL-10 in different cell populations were first defined using electrophoretic mobility shift assays. In all cells tested, IL-10 activated Stat1 and Stat3 and induced the formation of three distinct DNA binding complexes that contained different combinations of these two transcription factors. IL-10 also activated Stat5 in Ba/F3 cells that stably expressed the murine IL-10 receptor. Using a structure-function mutagenesis approach, two tyrosine residues (Tyr 427 and Tyr 477 ) in the intracellular domain of the murine IL-10 receptor were found to be redundantly required for receptor function and for activation of Stat3 but not for Stat1 or Stat5. Twelve amino acid peptides encompassing either of these two tyrosine residues in phosphorylated form coprecipitated Stat3 but not Stat1 and blocked IL-10-induced Stat3 phosphorylation in a cell-free system. In contrast, tyrosine-phosphorylated peptides containing Tyr 374 or Tyr 396 did not interact with Stat3 or block Stat3 activation. These data demonstrate that Stat3 but not Stat1 or Stat5 is directly recruited to the ligand-activated IL-10 receptor by binding to specific but redundant receptor intracellular domain sequences containing phosphotyrosine. This study thus supports the concept that utilization of distinct STAT proteins by different cytokine receptors is dependent on the expression of particular ligand-activatable, tyrosine-containing STAT docking sites in receptor intracellular domains. Interleukin-10 (IL-10)1 is a cytokine produced by Th0 and Th2 CD4 ϩ T cells, CD5 ϩ B cells, and macrophages (1-5) that inhibits inflammatory and cell-mediated immune responses while enhancing humoral immunity (6 -9). IL-10 exerts its pleiotropic effects following interaction with a specific IL-10 receptor that is expressed in low numbers on IL-10-responsive cells (1, 10). IL-10 binds to its receptor in a homogeneous manner with high affinity (K a ϭ 9 ϫ 10 9 M Ϫ1 ) (11, 12). The cDNA encoding the ligand-binding polypeptide of the IL-10 receptor was recently cloned, and the receptor polypeptide was characterized as a 110-kDa glycoprotein (1, 10). Based on sequence homology, this protein has been characterized as a member of the type II cytokine receptor family that also contains the IFN␣ and IFN␥ receptor polypeptides (13). This single chain does not reconstitute a functionally active IL-10 receptor when expressed in fibroblasts, indicating that at least one additional polypeptide is required. However, the identity of the accessory molecule(s) has not been established.Receptor proteins belonging to the type I and type II cytokine receptor families are now known to utilize the JAK-STAT family of proteins for signal transduction (14 -22). Currently, this family consists of four Janus family tyrosine kinases (JAK-1, JA...

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Although various molecular mechanisms of STAT protein (signal transducers and activators of transcription) activation have been identified, little is known about the functional role of STAT-dependent transcriptional activation. Herein we report the constitutive nuclear localization, phosphorylation, and DNA-binding activity of STAT proteins in leukemia cells and lymphoma cell lines. With the use of oligonucleotide probes derived from the Fc gamma RI promoter, the beta- casein promoter and a STAT-binding element in the promoter of the Bci-2 gene constitutive activation of STAT proteins was detected in untreated acute T- and C/B-leukemia cells (3 of 5 and 12 of 19 patients, respectively). Supershift analyses using Stats 1–6 specific antisera showed the constitutive DNA binding activity of Stat5 in these cells. Confocal microscopy revealed the nuclear localization of Stat5 and Western blot analyses showed tyrosine phosphorylation of Stat5 in nuclear extracts of acute leukemia cells. In contrast, peripheral blood mononuclear cells did not display constitutive STAT-DNA interaction. Further studies were performed on freshly isolated acute myeloid leukemia cells as well as on cell line derived K562, lymphoblastoid cells (LCL), and Burkitt's lymphoma cells (BL). Fluorescence microscopy, gelshift, and supershift experiments showed the nuclear localization and constitutive DNA-binding activity of Stat5 in K562 cells. Stat1 and Stat3 were constitutively activated in freshly isolated AML cells (10 of 14 patients) and in Epstein Barr virus- positive or interleukin-10 expressing permanent LCL and BL cells. Thus, these data indicate a differential pattern of STAT protein activation in lymphoid or myeloid leukemia and in lymphoma cells.