findings suggest that these are two distinct mechanisms for STAT3 activation that differ in their signaling pathways. Besides cytokines and their receptors, STATs can be activated by other means, for example by growth factor receptors, such as EGF, PDGF, and CSF-1, which all possess an intrinsic tyrosine kinase activity [18], as well as by non-receptor tyrosine kinases such as Src and Abl, and through serpentine receptors such as those for angiotensin II, serotonin, and a-melatonin stimulating hormone [19,4]. In addition, we have previously demonstrated, the induction of STAT3 signaling in APCs upon cell contact with hMSC, suggesting a new pathway for activating STAT3 in APCs. In a different setting, contact-dependent activation of STAT3 has been also demonstrated in normal and cancer breast cells. A study by Vultur et. al. showed a dramatic increase in STAT3 phosphorylation and consequently STAT3 activity with cell density in normal cells. This confluence-dependent activa- tion was eliminated if cell-cell interactions were interrupted through calcium chelation [20]. The contact dependent activation of STAT3 described by Vulture et.al may be due to a mechanism similar to the one we describe here for a heterologous interaction between APCs and their cellular environment, suggesting that a contact dependent activation of STAT3 may be a more general mechanism. Taken together, one can speculate the existence of yet unknown surface molecules participating in intercellular contact that are dependent on JAK2 for their activity. The finding of such surface molecules requires further investigation. DCs play a key role in the initiation and regulation of the immune response, and their ability to initiate immune reaction or induce tolerance is strictly dependent on their maturation state and phenotype. While immature DCs can induce T-cell anergy, mature DCs activate T cells and induce immunity. Nonetheless, it has also been reported that DCs may exist in a semi-mature state in which they are capable of actively inhibiting T cell activity, generating regulatory T-cell and promoting tolerance [2]. In this report, we found that contact- dependent activation of STAT3 induced the generation of functionally regulatory DCs with a seemingly semi-mature or aberrantly-mature phenotype, whereas IL-10 treatment resulted in DCs that express low levels of costimulatory molecules and HLA-DR, consistent with an immature phenotype. This data is in agreement with previous studies that described DCs with regulatory function that varied in their maturation phenotypes [21,22,23]. Among these are two reports describing the generation of regulatory DCs originating from mature cells rather than from immature cells [7,24]. Therefore, it seems that the hallmark of regulatory DCs is their ability to functionally inhibit lymphocyte activation rather than their specific pheno- type and maturation stage. In agreement with this notion, we further demonstrated that despite the different phenotypic outcomes, both IL-10 and cell-cell contact induced the expression of the inhibitory receptors Immunoglobulin like transcript (ILT) 3 and 4, which are characteristic of regulatory DCs, and play a role in their T cell inhibitory activity. Hence, ILT3 and ILT4 expression may account for the inhibitory activity of these cells regardless of their overall phenotype. In response to an inflammatory stimuli, DCs up-regulate the expression of the chemokine receptor, CCR7 and migrate from peripheral tissues to secondary lymphoid organs [25]. While this event is usually linked to the process of DC’s maturation, a previous study has demonstrated that after exposure to apoptotic cells DCs up-regulated CCR7 expression despite the absence of maturation properties [26]. Hence, the authors further speculated that CCR7-expressing immature DCs that ingested apoptotic Contact Induced Regulatory DC material may migrate to peripheral lymph nodes, in order to maintain tolerance [26]. In our study we show that contact- dependent induced regulatory DCs up-regulate the expression of CCR7 thereby enabling these DCs to migrate to lymphoid tissues where they may induce tolerance. Notably, CCR7 is induced only by the contact-dependent mechanism and not by soluble factors, further emphasizing the potential importance of the contact- dependent mechanism in tolerance induction, such as in the case of tumors. In the context of cancer, modulating APCs in a contact- dependent manner may be of special significance in the pre- malignant stage. Physiologically, tumors arise sporadically and between the initiating oncogenic event and tumor progression there might be a long pre-malignant stage. However, data concerning the interaction between tumor cells and the immune system was obtained from models in which the host was exposed to a large number of cells at a single time point. A recent study by Willimsky et.al. [27], has addressed the question of immunoge- nicity of pre-malignant lesions, by using a transgenic mouse model for sporadic cancer. The authors of this study concluded that tolerance occurs already at the pre-malignant stage, before tumor progression. Specifically, they showed the accumulation of immature myeloid cells at the pre-malignant stage, and suggest they play a role in creating T-cell unresponsiveness. Given our results, one may speculate that at the pre-malignant stage physical interactions between resident APCs and tumor cells that activate STAT3 play a significant role in tumor escape, long before tumors are visible and large enough to secrete significant amounts of APCs-modulating factors. Materials and Methods Cells Cells were purified from the venous blood of healthy donors. Either, CD4+ T cells or CD14+ cells were isolated by negative selection using the RosetteSepTM enrichment cocktail (StemCell Technologies, Vancouver, Canada). hMSC were obtained from discarded bone tissues from patients undergoing total hip replacement surgeries, under approval of Hadassah Medical Center Helsinki Ethics Committee following an informed consent. The hMSC were separated from other bone-marrow residing cells by plastic adherence, and were then grown under tissue culture conditions, as previously described [13]. The cells were main- tained in a low-glucose DMEM medium supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, and penicillin/ streptomycin (Biological Industries, Beit-Haemek, Israel). For DC generation, CD14+ cells were plated in RPMI 1640 (Biological Industries) containing 1% autologous plasma, 0.1 mg/ml IL-4 and 0.1 mg/ml GM-CSF (PeproTech, Rocky Hill, NJ). Every 2 days 0.3 ml were removed and 0.5 ml media containing plasma and cytokines were added. By day 7, .90% of the cells were CD14- and CD11c+ immature DCs. For JAK blockade freshly isolated CD14+ cells or DC were pre-incubated with JAK2 inhibitors, JSI- 124 (cucurbitacin I; Calbiochem,). Human breast adenocarcinoma (MCF7) and human prostate adenocarcinoma (PC3), cell lines were obtained from the American Type Culture Collection (ATCC). MCF7 cells were cultured in DMEM with 10% heat- inactivated fetal calf serum, 2 mM glutamine, and penicillin/ streptomycin (Biological Industries, Beit-Haemek, Israel) at 37uC and 5% CO2. PC3 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Biological Industries, Beit- Haemek, Israel) supplemented with 10% heat-inactivated fetal calf serum (Biological Industries), 2 mM glutamine, and penicillin/ streptomycin, at 37uC and 5% CO2. PLoS ONE | www.plosone.org 8 August 2009 | Volume 4 | Issue 8 | e6846