The Induction of APC with a Distinct Tolerogenic Phenotype via Contact-Dependent STAT3 Activation Devorah Gur-Wahnon 1 , Zipora Borovsky1, Meir Liebergall2, Jacob Rachmilewitz1* 1 Goldyne Savad Institute of Gene Therapy, Hadassah-Hebrew University Medical Center, Jerusalem, Israel, 2 Department of Orthopedic Surgery, Hadassah-Hebrew University Medical Center, Jerusalem, Israel Abstract Background: Activation of the signal transducer and activator of transcription 3 (STAT3) within antigen presenting cells (APCs) is linked to abnormal APCs differentiation and function. We have previously shown that STAT3 is activated within APC by a novel contact-dependent mechanism, which plays a key role in mediating the immunomodulatory effects of hMSC. In order to better understand the underlying mechanisms that control APC maturation in a contact dependent manner, we extended our observation to tumor cells. Tumors were shown to secrete a variety of tumor-derived factors that activate STAT3 within infiltrating APCs. We now tested whether tumor cells can activate STAT3 within APC using the contact-dependent mechanism, in addition to soluble factors, and compared these two STAT3 activating pathways. Principal Findings: We demonstrate that in addition to tumor-derived secreted factors tumor cells activate STAT3 by a mechanism that is based on cell-cell interaction. We further demonstrate that these two STAT3 activating mechanisms differ in their JAK usage and their susceptibility to JSI-124 inhibition thereby representing two distinct pathways. Significantly, although both pathways activate STAT3, they modulate DCs maturation in a different manner that results in disparate phenotypic outcomes. Whereas the soluble-dependent pathway results in an immature phenotype, the contact-dependent pathway results in an apparently mature phenotype. Albeit their mature-like phenotype these latter cells express the tolerogenic markers ILT3 and ILT4 and possess T cell inhibitory activity. Significance: This data suggests that, in at least certain cellular microenvironments, cell:cell interactions represent a novel way to activate STAT3 signaling, uncouple APC activation events and consequently regulate immunity and tolerance. Significantly, we have now demonstrated that this contact-dependent signaling pathway differs from that mediated by soluble factors and cytokines, inducing disparate phenotypic outcome, suggesting these two mechanisms have different and possibly complementary biological functions. Citation: Gur-Wahnon D, Borovsky Z, Liebergall M, Rachmilewitz J (2009) The Induction of APC with a Distinct Tolerogenic Phenotype via Contact-Dependent STAT3 Activation. PLoS ONE 4(8): e6846. doi:10.1371/journal.pone.0006846 Editor: Derya Unutmaz, New York University School of Medicine, United States of America Received June 10, 2009; Accepted August 3, 2009; Published August 31, 2009 Copyright: 2009 Gur-Wahnon et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by The Israel Science Foundation (grant No. 337/08). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: rjacob@cc.huji.ac.il Introduction Antigen-presenting cells (APCs), and specifically dendritic cells (DCs), are the most potent inducers of the immune response. DCs in the periphery capture and process antigens in their immature state followed by a maturation process in response to a spectrum of stimuli, allowing them to induce both innate and adaptive responses [1]. Only upon receiving maturation signals, DCs migrate to lymphoid organs, secrete cytokines and express co-stimulatory molecules that are required for lymphocyte activation [1]. In recent years, however, there is growing evidence suggesting that DCs not only initiate T cell responses but are also involved in silencing T cell immune responses. These functions of DCs are thought to be mainly dependent on their activation and differentiation state. For example, terminally differen- tiated mature DCs can efficiently induce the development of effector T cells, whereas immature DCs or partially matured DCs are involved in maintenance of peripheral tolerance. Hence, APCs and specifically DCs orchestrate a range of immune responses including induction and suppression of T cell activation [1,2]. Regulation of DCs maturation occurs through the function of Janus activated kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway [3]. The JAK family of tyrosine kinases and STAT are important components of diverse signal transduction pathways that are actively involved in cellular survival, proliferation, differentiation and apoptosis. Four members of the Jak family have been identified in mammalian cells, Jak1, Jak2, Jak3 and Tyk2 [4]. Cytokine receptor-ligand binding induces receptor oligomerization and phophorylation, followed by Jak activation. Activated Jaks phosphorylate receptors on target tyrosine residues, generating docking sites for STATs, which are subsequently recruited and phosphorylated by activated Jaks. Dimerized STATs then translocate to the nucleus, where they modulate expression of target genes [5]. One of these proteins STAT3, has been implicated as a negative regulator of the immune response [6]. Mice devoid of the STAT3 gene in macrophages and neutrophils have enhanced inflammatory activity, leading to the development of chronic colitis [6]. STAT3 has been recently proposed as an important molecule that mediates tumor induced immunosupression. STAT3 is PLoS ONE | www.plosone.org 1 August 2009 | Volume 4 | Issue 8 | e6846