Summary Transcriptional regulatory networks direct the development of specialized cell types. The transcription factors Stat4 and T-bet are required for the development of T helper 1 cells, although the hierarchy of activity by these factors has not been clearly defined. In this report we show that these factors are not in a linear pathway and that each factor plays a unique role in programming chromatin architecture for Th1 gene expression, with subsets of genes depending on Stat4, T-bet, or both for expression in Th1 cells. T-bet is not able to transactivate expression of Stat4-dependent genes in the absence of endogenous Stat4 expression. Thus, T-bet requires Stat4 to achieve complete Th1 fate determination.

Blocking the function of Stat (signal transducer and activator of transcription) proteins, which are critical for antiviral responses, has evolved as a common mechanism for pathogen immune evasion. The poxvirus-encoded phosphatase H1 is critical for viral replication, and may play an additional role in the evasion of host defense by dephosphorylating Stat1 and blocking interferon (IFN)-stimulated innate immune responses. Vaccinia virus (VACV) H1 can inhibit the phosphorylation of the transcription factor Stat1 after IFN-γ stimulation of epithelial cells, greatly attenuating IFN-induced biological functions. In this study, we demonstrate that VACV infection is capable of inhibiting the phosphorylation of Stat1 and Stat2 after stimulation of fibroblasts or bone marrowderived macrophages with either type I or type II IFNs, but did not inhibit the activation of Stat3 or Stat5 in either cell type. By using recombinant proteins for in vitro assays, we observe that variola virus H1 is more active than VACV H1, although it has similar selectivity for Stat targets. Differential effects of VACV infection were observed on the induction of IFN-stimulated genes, with complete inhibition of some genes by VACV infection, while others were less affected. Despite the IFN-γ-induced expression of some genes in VACV-infected cells, IFN-γ was unable to rescue the VACV-mediated inhibition of MHC class II antigen presentation. Moreover, VACV infection can affect the IFN-induced expression of Stat1-dependent and Stat1-independent genes, suggesting that the virus may target additional IFN-activated pathways. Thus, VACV targets multiple signaling pathways in the evasion of antiviral immune responses.

Production of interferon ␥ (IFN-␥ IntroductionNon-Hodgkin lymphoma is the most common of the hematologic malignancies. 1 The incidence of non-Hodgkin lymphoma has been rising steadily for the past 50 years, and approximately 60 000 new cases are currently diagnosed annually in the United States. Because of this increased incidence, non-Hodgkin lymphoma is one of the few cancers for which mortality has increased in the past 20 years. 2 High-dose therapy and autologous hematopoietic stem cell transplantation is the treatment of choice for eligible patients with non-Hodgkin or Hodgkin lymphoma that is refractory to or has relapsed after conventional chemotherapy. [3][4][5][6] Nevertheless, greater than 50% to 60% of patients with relapsed or refractory lymphoma will ultimately develop progressive disease after autologous stem cell transplantation. Novel strategies are needed to reduce the risk of recurrent lymphoma after high-dose therapy. One approach is posttransplantation immunotherapy to eliminate chemotherapy-resistant tumor cells. 7 Immunotherapeutic approaches under investigation include posttransplantation administration of monoclonal antibodies, immunostimulatory cytokines, and cancer vaccines. [8][9][10] In attempts to stimulate effective antitumor immune responses, we have investigated the use of interleukin 12 (IL-12) after autologous stem cell transplantation. IL-12 plays an important role in the regulation of innate and adaptive immune responses. 11 In preclinical tumor models, IL-12 therapy induces regression of established primary tumors, inhibits the formation of distant metastases, and prolongs the survival of tumor-bearing mice. [12][13][14][15] In several animal models production of interferon ␥ (IFN-␥) in vivo has been shown to be necessary, but not sufficient, for the antitumor effects of 14,16,17 IL-12 mediates its immunologic effects by binding to a specific dimeric cell surface receptor complex, composed of ␤1 and ␤2 chains, which results in activation of the Janus kinases Jak2 and Tyk2. 18,19 The subsequent activation of signal transducer and activator of transcription 4 (STAT4) is required for normal responses to 20,21 Our previous studies have shown that IL-12 can be safely given in biologically active doses after autologous stem cell transplantation. 22 However, serum IFN-␥ levels during IL-12 therapy after transplantation were found to be an order of magnitude lower than those seen in patients with solid tumors not receiving a transplant who received the same doses of 23 Moreover, posttransplantation patient peripheral blood mononuclear cells (PBMCs) were shown to be intrinsically defective in their ability to produce IFN-␥ Patients, materials, and methods Patient and control subject samplesThe study was approved by the Institutional Review Board at Indiana University Medical Center, and written informed consent was obtained from each subject prior to collection of blood samples. Blood samples were obtained from patients with non-Hodgkin and Hodgkin lymphoma who were scheduled to und...

Immunostimulatory cytokines can enhance anti-tumor immunity and are part of the therapeutic armamentarium for cancer treatment. We have previously reported that post-transplant lymphoma patients have an acquired deficiency of signal transducer and activator of transcription 4, which results in defective IFNγ production during clinical immunotherapy. With the goal of further improving cytokine-based immunotherapy, we examined the effects of a soybean peptide called lunasin that synergistically works with cytokines on natural killer (NK) cells. Peripheral blood mononuclear cells of healthy donors and post-transplant lymphoma patients were stimulated with or without lunasin in the presence of IL-12 or IL-2. NK activation was evaluated, and its tumoricidal activity was assessed using in vitro and in vivo tumor models. Chromatin immunoprecipitation assay was performed to evaluate the histone modification of gene loci that are regulated by lunasin and cytokine. Adding lunasin to IL-12- or IL-2-stimulated NK cells demonstrated synergistic effects in the induction of IFNG and GZMB involved in cytotoxicity. The combination of lunasin and cytokines (IL-12 plus IL-2) was capable of restoring IFNγ production by NK cells from post-transplant lymphoma patients. In addition, NK cells stimulated with lunasin plus cytokines displayed higher tumoricidal activity than those stimulated with cytokines alone using in vitro and in vivo tumor models. The underlying mechanism responsible for the effects of lunasin on NK cells is likely due to epigenetic modulation on target gene loci. Lunasin represents a different class of immune modulating agent that may augment the therapeutic responses mediated by cytokine-based immunotherapy.