Figure 5. Cell:cell contact but not conditioned media or IL-10 induce JAK2 phosphorylation. Monocytes were either left untreat- ed or treated with JSI-124 (as indicated above), and then were treated with either IL-10 (10 ng/ml), MCF-7’s conditioned media or were co- cultured with MCF-7, for 2 hours. Untreated cells in the case of co- cultures were obtained by mixing MCF-7 and monocytes without incubation. Cell extracts were subjected to SDS-PAGE and anti- phosphorylated JAK2 immunoblotting (upper panels). Immunoblotting of anti-phosphorylated STAT3 demonstrates induction of STAT3 activation by all treatments regardless of JAK2 phosphorylation (middle panels) and that JSI-124 treatment selectively blocks contact- and JAK2- dependent STAT3 phosphorylation. Anti-STAT3 immunoblotting reveals relative amounts of protein in each lane (lower panels). Similar results were obtained in three independent experiments. doi:10.1371/journal.pone.0006846.g005 chamber of a transwell. Monocytes alone or in contact with hMSC were placed in the bottom chamber (to avoid contact between the monocytes and T cells that can contribute to T cell activation) and their effect on bead-stimulated T cell activation was determined by IFN-c secretion. Here again hMSC were used and not cancer cells in-order to determine the exclusive contribution of the contact depended effect and to avoid the additional effect of soluble factor secreted by the cancer cells over the 72 h period. Inhibition of IFN-c secretion was observed only when hMSC and monocyte were co-cultured in the lower chamber but not when each cell type was cultured alone (Supporting data Fig. S2). This data support our previous reports that upon cell-cell contact the APCs are converted into functionally regulatory cells via STAT3 activation and that this T cell inhibitory activity is partially mediated by a soluble factor secreted only upon cell-cell interaction [12,13]. Discussion Cancer cells utilize different mechanisms to evade immune surveillance. One way is by influencing APCs within the tumor Contact Induced Regulatory DC Figure 6. phosphorylated-STAT3 co-immunoprecipitates with JAK2 upon MCF7:monocyte cell contact but not IL-10 treat- ment. A: Monocytes were either co-cultured with MCF-7, for 2 hours or mixed with MCF-7 without incubation as a control, and then lysed. B: monocytes were either left untreated or were treated with IL-10 (10 ng/ ml) for 2 hours, and then lysed. Cell extracts were subjected to immunoprecipitation by anti-JAK2 antibodies and the precipitates were separated on SDS-PAGE. Anti-phosphorylated STAT3 immunoblotting demonstrates that phosphorylated-STAT3 associates with JAK2 upon contact-dependent activation but not IL-10 treatment (upper panels). Anti-JAK2 immunoblotting reveals relative amounts of protein in each lane (lower panels). In B (right panels), total lysates are shown to demonstrate STAT3 is phosphorylated upon IL-10 treatment although did not co-precipitate with JAK2, and phosphorylation of the latter is not induced by IL-10. Similar results were obtained in three experiments. doi:10.1371/journal.pone.0006846.g006 bed rendering them tolerogenic. Recent studies have shown that tumors secrete factors that inhibit DCs maturation through activation of STAT3 in DCs [7]. Here we show that in addition to releasing soluble factors, tumor cells, physically interact with APCs, consequently inducing the activation of STAT3. This induction results in APCs with a distinct phenotype, which are capable of inhibiting T-cell activation. Thus, tumor cells have at least two pathways by which they can alter APCs maturation and function. The first, described previously, is mediated by tumor- derived factors such as VEGF and IL-10, that promote the generation of immature DCs that lack the T cell stimulatory activity characteristic of mature DCs [7]. The second pathway, demonstrated in this study, is a novel cell contact induced and JAK2-dependent pathway that generates tolerogenic DCs with a distinct altered-mature phenotype that actively suppress T-cell activation. Hence, although the two pathways converge on activation of STAT3 they subsequently diverge in their down- stream effect on APC phenotype. In the current study, by simply washing the cells just prior to adding the APC we eliminated the contribution of tumor-secreted factors, thereby revealing the contact-dependent activation of STAT3. Second, we found that the cell contact-dependent pathway is sensitive to treatment with the JAK2 inhibitor JSI- 124, distinguishing it from the soluble factor pathway. Third, phosphorylation of JAK2 and physical interactions between JAK2 and phosphorylated-STAT3 are contact dependent. These PLoS ONE | www.plosone.org 6 August 2009 | Volume 4 | Issue 8 | e6846