The ability of dendritic cells (DCs) to stimulate and regulate T cells is critical to effective anti-tumor immunity. Therefore, it is important to fully recognize any inherent factors which may influence DC function under experimental conditions, especially in laboratory mice since they are used so heavily to model immune responses. The goals of this report are to 1) briefly summarize previous work revealing how DCs respond to various forms of physiological stress and 2) to present new data highlighting the potential for chronic mild cold stress inherent to mice housed at the required standard ambient temperatures to influence baseline DCs properties in naïve and tumor-bearing mice. As recent data from our group shows that CD8+ T cell function is significantly altered by chronic mild cold stress and since DC function is crucial for CD8+ T cell activation, we wondered whether housing temperature may also be influencing DC function. Here we report that there are several significant phenotypical and functional differences among DC subsets in naïve and tumor-bearing mice housed at either standard housing temperature or at a thermoneutral ambient temperature, which significantly reduces the extent of cold stress. The new data presented here strongly suggests that, by itself, the housing temperature of mice can affect fundamental properties and functions of DCs. Therefore differences in basal levels of stress due to housing should be taken into consideration when interpreting experiments designed to evaluate the impact of additional variables, including other stressors on DC function.

185 The immune system plays a key role in preventing and controlling tumor growth and metastasis. In overcoming endogenous anti-tumor immunity, cancers elicit a number of immunosuppressive networks.One key network is the blockade of myeloid progenitor - to - dendritic cell (DC) differentiation, resulting in loss of mature DC (critical in the induction of T cell mediated immune responses) and accumulation of immature, actively immunosuppressive myeloid derived suppressor cells (MDSC) in cancer patients, thus compromising the ability to maintain existing and initiate novel anti-tumor immune responses. This is mediated by tumor derived factors (e.g. VEGF, IL-6) that inhibit DC differentiation by driving STAT3 hyperactivation. The downstream target(s) of this STAT3 signaling, however, have not been defined. Previous work in our lab has identified protein kinase C βII (PKCβII) as being essential in myeloid progenitor to DC differentiation and demonstrated that PKCβII inhibition prevents DC differentiation. These observations and others lead us to hypothesize that tumor driven activation of STAT3 inhibits DC differentiation by down regulating PKCβII expression. To determine if PKCβII expression in DC-progenitors is decreased by cancer, myeloid cells were enriched peripheral blood mononuclear cells (by lymphoid cell depletion) from stage III/IV breast cancer patients (known to have reduced numbers of DCs) and “normal” age-matched donors and gene expression analyzed by qPCR. PKCβII levels in myeloid cells from cancer patients were significantly reduced versus “normal” donors (on average reduced 62%, p < 0.05). This comports with previous in vitro work demonstrating that culture in tumor conditioned media (TCM) decreases PKCβII protein levels and significantly decreases PKCβII mRNA levels and promoter activity, and that this decrease in PKCβII expression was accompanied by significant impairments in DC differentiation in myeloid progenitor-like cell lines long used to model DC differentiation. We've previously shown that TCM drives STAT3 activation and that genetically activated STAT3 (courtesy of a constitutively active STAT3 mutant, CA-STAT3) significantly decreases PKCβII expression. To determine if STAT3 was acting directly on the PKCβ promoter to impair its activity, the promoter was analyzed in silico for STAT3 binding sites. This analysis uncovered 4 putative STAT3 binding sites in close proximity to one another and the start site of PKCβ transcription. To determine if STAT3 binds to any of these sites in response to TCM, quantitative chromatin immunoprecipitation was conducted. TCM rapidly (within 15 minutes) drove >4-fold greater STAT3 binding to the PKCβ promoter (p < 0.05). To determine if this STAT3 binding was responsible for decreased PKCβ promoter activity, we generated reporter constructs containing site-specific mutations designed to ablate the putative STAT3 binding sites identified above. Elimination of site #4 (widely conserved among mammalian species) almost completely abrogated the ability of TCM or IL-6 or CA-STAT3 (see below) to inhibit PKCβ promoter activity, demonstrating that STAT3 binding to the PKCβ promoter negatively regulates PKCβ promoter activity and PKCβII expression. TCM contains many factors known to drive STAT3 activation, including high amounts of IL-6 (commonly greater than 100 ng/ml in our model). To determine if these effects are dependent on these high IL-6 levels, a myeloid progenitor cell line was cultured in TCM that had been pre-incubated with an IL-6 depleting antibody (TCM-αIL-6) or isotype control (TCM-iso). Cells grown in TCM-iso had decreased (approximately 50%) PKCβII levels compared both to cells grown in normal media and to cells grown in TCM-αIL-6, demonstrating that TCM driven PKCβII down regulation is IL-6 dependent (in this model). Furthermore, high levels of IL-6 significantly reduced PKCβ promoter activity (comparable to the affects seen with TCM;) in the dual luciferase reporter assay system (p < 0.05). These results demonstrate that tumors down regulate myeloid PKCβII expression by driving hyperactive STAT3 signaling, resulting in STAT3 binding to and negatively regulating the PKCβ promoter, resulting in impaired DC differentiation. These effects are mediated, at least in part, by IL-6. This work identifies several potential avenues to block or reverse tumor mediated suppression of DC differentiation in cancer. Disclosures: No relevant conflicts of interest to declare.

To better understand how Dendritic Cell (DC) subsets are differentiating, we investigated two essential arms of DC differentiation and how they interact to mediate this process. The transcription factors Interferon Regulatory Factor 4 and 8 (IRF4 and IRF8) are critical for DC differentiation, but have varying roles depending on the stimuli. While IRF8 is required for FLT3-L induced plasmacytoid DC differentiation, and IRF4 is required for GM-CSF induced conventional DC differentiation, it is not well characterized how these two molecules are differentially regulated. We found that Protein Kinase C βII (PKCβII) is activated by both FLT3-L and GM-CSF, and that it is required for DC differentiation when either of these stimuli is used to generate DCs from progenitor cells. Since PKCβII and these IRFs are contributing to DC differentiation we hypothesized they were interacting. We discovered that PKCβII upregulates IRF8 when activated by FLT3-L or phorbol ester (PMA, a known activator of PKCβII), and IRF8 upregulation is lost with the addition of a PKCβII inhibitor. Likewise, IRF4 upregulation is induced by PKCβII in cells treated with GM-CSF or PMA, and lost in the presence of a PKCβII inhibitor. We have also found that in IRF8 knockout DCs there are higer levels of PKCβII, suggesting a feedback mechanism by which these IRFs can be autoregulated. Thus, PKCβII regulating these IRFs may be the key to understanding and manipulating DC differentiation.

Tumor mediated blockade of dendritic cell (DC) differentiation is a large component of cancer induced immunosuppression, contributing to tumor outgrowth. Tumors impair DC differentiation via factors that hyperactivate STAT3 in DC progenitors, though the mechanisms by which this occurs are largely unknown. PKCβII is essential in DC differentiation, and we test here if tumor driven STAT3 hyperactivation downregulates PKCβII expression, and if this is the mechanism underlying impaired DC differentiation. Myeloid cells from tumor bearing mice have significantly decreased PKCβII expression. Culture in tumor conditioned media (TCM) significantly decreased PKCβII expression and significantly impaired phorbol ester driven DC differentiation in human monocytes and in a human cell line model (KG1). In KG1, this was dependent on PKCβII downregulation: enforced PKCβII expression preserved phorbol ester driven DC differentiation in the face of TCM. Interestingly, PKCβII overexpression and/or activation also antagonized TCM-driven STAT3 activation. STAT3 drives this decreased PKCβII expression: TCM induced STAT3 activation and drove 4.5 fold higher STAT3 binding to the PKCβ promoter (compared to media control, p&lt;0.001). Mutation of a STAT3 consensus binding site in the promoter eliminates the ability of STAT3 to impair PKCβ promoter activity. Together, these observations argue that tumor driven STAT3 hyperactivation inhibits DC differentiation by downregulating PKCβII.

1735 The immune system plays a key role in preventing and controlling tumor growth. Cancer frequently induces a state of immune suppression in patients mediated, in part, through inhibition of dendritic cell (DC) differentiation. This results in the accumulation of actively immunosuppressive myeloid derived suppressor cells (MDSCs) and a loss of DCs (critical in the induction of T cell mediated immune responses), thus compromising the ability to initiate anti-tumor immune responses. This is mediated by tumor derived factors (TDFs)(e.g. VEGF) that inhibit DC differentiation by driving STAT3 hyperactivation. The downstream target(s) of this STAT3 signaling that inhibits DC differentiation, however, has not been defined. Previous work in our lab has identified protein kinase C βII (PKC βII) as being essential in myeloid progenitor to DC differentiation and demonstrated that PKC βII inhibition (signaling or expression) prevents DC differentiation. We've also found that PKC βII positively regulates its own expression and that, under certain circumstances, the PKCβ promoter is negatively regulated. These observations lead us to hypothesize that TDF activation of Stat3 inhibits DC differentiation by down regulating PKC βII expression. We've previously shown that culture in tumor conditioned media (TCM) decreased PKC βII protein levels and significantly reduced PKC βII mRNA transcript levels in KG1, a myeloid progenitor-like cell line long used to model DC differentiation. We've also previously seen that decreased PKC βII expression following culture in TCM significantly impaired DC differentiation, compared to cells grown in the absence of tumor conditions; however, the mechanism by which Stat3 signaling down regulated PKC βII expression remained unclear. We now show that culture in TCM reduced PKCβ promoter driven transcription 7-fold, compared to cells grown in normal media (p<0.01). Given the previously described importance of Stat3 hyperactivation in tumor-mediated suppression of DC differentiation, and since PKC βII down regulation appears to occur at the promoter level, we examined the role of Stat3 in regulating PKC βII expression. Culture in TCM rapidly (<5 min.) induced Stat3 phosphorylation, an indication of activation. By chromatin immunoprecipitation, we found that TCM treatment induces direct interaction between Stat3 and the PKCβ promoter, suggesting that Stat3 signaling may act to decrease PKC βII expression. To directly test the role of Stat3 signaling in regulation of PKC βII expression, we generated a series of clones stably expressing wild type (WT) or constitutive active (CA) Stat3 constructs in K562, a second DC progenitor-like cell line. We've previously seen that clones stably expressing the CA-Stat3 construct have decreased PKC βII protein levels and significantly decreased PKC βII mRNA levels, compared to the parental cell line and WT-Stat3 clones. We now show that this decrease in PKC βII expression was dependent on constitutive Stat3 signaling, as pharmacologic Stat3 inhibition restored PKC βII expression to levels seen in the parental cell line. Consistent with the proposed model and our previous work, decreased PKC βII expression in clones expressing CA-Stat3 resulted in significantly inhibited phorbol ester driven DC differentiation (p<0.05)(as measured by allogenic T cell proliferation, a key measure of DC differentiation). Interestingly, we've also found that PKC βII antagonizes Stat3 signaling: myeloid progenitor-like cells (KG1a) overexpressing PKC βII do not exhibit Stat3 activation in response to TCM: PKC βII overexpression or activation led to down regulation of the receptors for G-CSF, IL-6, and VEGF, TDFs demonstrated to inhibit myeloid progenitor to DC differentiation. These findings suggest a novel mechanism by which PKC βII negatively regulates the potential of a cell to respond to (tumor derived) inflammatory cytokines. This work demonstrates that tumor driven Stat3 hyperactivation down regulates PKCβ promoter activity, resulting in decreased PKC βII protein levels. In agreement with our previous work, this decrease in PKC βII expression impairs a cell's potential to undergo DC differentiation. Additionally, this work suggests that PKC βII signaling impairs a cell's potential to signal via Stat3 in response to TDFs, perhaps providing an avenue by which to block or reverse tumor mediated suppression of DC differentiation in cancer. Disclosures: No relevant conflicts of interest to declare.