Contact Induced Regulatory DC Figure 4. IL-10 induced STAT3 activity is insensitive to JSI-124 inhibition. JSI-124 treated and untreated monocytes (as described in figure 3) were cultured with or without IL-10 (10 ng/ml). After 2 hours, cells were harvested and either lysed and their nuclear extracts were tested for STAT3 activity using Non-Radioactive STAT3 Transcription Factor Assay (left panel) or phosphorylated STAT3 was detected by intracellular immunostaining and flow cytometric analysis (right panel). Dashed line: Isotype control; Filled grey line: monocytes alone; Thick grey line: IL-10 stimulated monocytes; Black line: IL-10 stimulated JSI-124 pretreated monocytes. Comparable results were obtained in three independent experiments. doi:10.1371/journal.pone.0006846.g004 contact-dependent stimulation using co-IP experiments. MCF-7 and monocytes were mixed together and were either lyzed immediately or after 2 hours incubation, followed by JAK2 immuno-precipitation. Phosphorylated-STAT3 was detected in JAK2 immunoprecipitates of MCF-7-monocyte co-cultures but not in control cells (Fig. 6A) or in IL-10 stimulated monocytes (Fig. 6B, left panel) although STAT3 phosphorylation is observed in the latter (Fig. 6B, right panel). Taken together, these data indicate that there are two separate mechanisms for the activation of STAT3 (soluble cytokines versus cell-cell contact) that differ in their usage of JAKs and their susceptibility to inhibition by JSI-124. DC exhibit a different phenotype upon cell:cell interaction as compared to IL-10 treatment Next we examined the effects of tumor secreted cytokines and cell-cell contact on APCs maturation. To test this, DCs were co- cultured with either MCF-7 cells or hMSC or alternatively treated with IL-10 for 24 h followed by examination of maturation markers by flow cytometry. Since MCF-7 cells induce STAT3 activation through both soluble factors and cell-cell contact, hMSC were included in this experiment given that their induction of STAT3 activity is solely dependent on cell-cell contact. As shown in figure 7, hMSC co-culturing had only a slight and statistically insignificant effect on the levels of HLA-DR and CD86 expression, while the levels of CD80 and CCR7 expression were significantly elevated (P = 0.007 and P = 0.003, respectively) compared to DCs single cultures. In contrast to cell-cell interaction, HLA-DR and CD86 levels were significantly reduced (P = 0.007 and P,0.001, respectively) by IL-10 treatment, whereas CD80 and CCR7 expression levels remained low (Fig. 7A). These results were quantified as shown in figure 7B and demonstrate a significant change in the pattern of maturation markers expression between IL-10 treated DCs (soluble) and co-cultures of DCs and hMSC (cell-cell contact), with the relative expression levels of CD86, HLA-DR, CD80 and CCR7 significantly differing between the two treatments. As expected, DC-MCF-7 co-cultures resulted in an intermediate phenotype between that observed in DC- hMSC co-cultures or in DCs treated with IL-10. This is consistent with the findings that MCF-7 cells can induce both soluble- and contact-dependent pathways in parallel. These findings represent a unique developmental stage for DCs upon cell-cell interaction that differs from the immature phenotype induced by IL-10. Dendritic cells and monocytes express the inhibitory receptors ILT3 and ILT4 upon interaction with MCF-7 cells, hMSC or IL-10 Immunoglobulin like transcript (ILT) are receptors structurally and functionally related to killer-cell inhibitory receptors (KIRs), containing long cytoplasmic tails containing immunoreceptor tyrosine based inhibitory motifs (ITIMs) that inhibit cell activation, and they have been shown to be involved in immune regulation. Specifically, ILT3 and ILT4 expression is limited to monocytes, macrophages and DCs. Expression of ILT3 and ILT4 on DC has been associated with a tolerogenic phenotype which was shown to be crucial to the tolerogenic capacity acquired by DCs [16,17]. Interestingly, despite the marked differences in the overall phenotype between the contact- and soluble factor-dependent mechanisms the levels of expression of these two markers for tolerogenic APCs, ILT3 and ILT4 were upregulated in both DCs and monocytes co-cultured with hMSC or MCF-7 as well as when they were treated with IL-10 (Fig. 7C). hMSC:monocytes co-culture actively inhibit T-cell activation Our previous results examining the immunomodulary effect of hMSC suggest that the cell-contact mechanism conferred immunoregulatory properties for APCs. Specifically, hMSC inhibited T-cell activation only upon addition of APCs (DC or monocytes) to the co-culture. The immunoregulatory properties acquired by these APCs upon contact included induction of IL-10 secretion together with reduction in TNF-a and IL-12 secretion by the APCs and inhibition of IFN-c secretion and proliferation of T- cells [12]. In order to determine whether the contact-dependent mechanism converts the APCs into what is effectively a ‘‘deletional or inhibitory APC’’ with active immunoregulatory properties, as opposed to simply being activation incompetent, we examined the ability of these cell-contact conditioned APC to actively suppress T cell activation. In these experiments T cells were activated with anti-CD3 coated beads and soluble anti-CD28 that can stimulate T cells in the absence of any APCs and placed in the upper PLoS ONE | www.plosone.org 5 August 2009 | Volume 4 | Issue 8 | e6846