Deletion of STAT3 in DCs enhances the proliferation of allogeneic T cells.

<p><b>A.</b> An allogeneic mixed lymphocyte reaction (MLR) was used to determine if DCs (stimulator) could induce allogeneic T cell (responder) proliferation. WT and STAT3^−/− DCs were derived using GM-CSF as previously outlined. BMDCs were stimulated with CpG 1668 (500 ng/ml) for 12 hours to increase cell surface expression of MHC-II prior to being γ-irradiated. To induce proliferation, 100,000 DCs were cultured with CFSE-labeled allogeneic T cells at a 1∶1 ratio in 96-well flat bottom wells for 5 days. CFSE intensity of CD8⁺ T cells at day 5 is presented as histograms. The precursor frequency and proliferation index were derived using the proliferation analysis wizard in Modfit LT computer software. <b>B.</b> CpG-matured DCs were irradiated and cultured with 100,000 allogeneic T cells at decreasing ratios of stimulator to responder in 96-well flat bottom wells. Cells were allowed to proliferate for 4 days prior to addition of the nucleotide analogue BrdU (18 hours incubation). Incorporation of BrdU into dividing DNA was determined using a colorimetric ELISA kit. 2-way ANOVA test followed by Tukey-Kramer multiple comparison test were employed to determine statistical significance (*, <i>p</i><0.05 versus wild type).</p

Induction of anti-tumor immunity in response to DC vaccination is independent of STAT3 signaling.

<p><b>A.</b> Diagram illustrating the culture and priming of WT and STAT3 knockout GM-CSF derived BMDCs. Micrograph captured at day 6 of GM-CSF culture demonstrates the formation of loosely adherent cDC clusters. Tumor cell lysate was generated by subjecting GL26 cells to repeated freeze-thaw cycles in liquid nitrogen and a 37°C water bath. DCs were primed with GL26 tumor cell lysate at a 2∶1 ratio of tumor cells to DCs in RPMI-10 for 12 hours at 37°C. After loading, DCs were washed three times with PBS to remove residual tumor lysate. Loaded DCs were mixed with 30 µg of CpG 1668 immediately prior to subcutaneous vaccination. <b>B.</b> DC vaccinations were administered before (prophylactic model) tumor challenge. C57BL/6J mice were vaccinated subcutaneously with 1×10⁶ primed WT DCs (blue line, n = 5), STAT3 KO DCs (green line, n = 5), PBS-control or CpG-control on the indicated days. On day 0, mice were intracranially injected with 20,000 Gl26 glioma cells and followed for survival. <b>C.</b> Animals were monitored daily and euthanized upon signs of morbidity. Survival data is depicted as a Kaplan-Meyer curve and analyzed statistically using the Mantel log-rank test (*, <i>p</i><0.05 versus PBS and CpG control). <b>D.</b> IFNγ ELISPOT assay was used to assess T cell IFNγ secretion from splenocytes of mice at 12 days post tumor implantation. The ELISPOT data showed a significant increase in mice treated WT DCs or STAT3 null DCs treated with CpG compared to CpG treated control mice. <b>E.</b> Quantification of CD3e immunohistochemistry showed significantly more CD3e+ T cells within both DC treatment groups compared to CpG control mice; there was no significant difference between WT DC and STAT3 null DC treated mice. <b>F.</b> Quantification of Iba1⁺ cells within the tumor showed no significant difference Iba1+ microglia between any groups. <b>G.</b> Therapeutic model of tumor bearing mouse vaccination. Mice were vaccinated subcutaneously with 1×10⁶ primed WT DCs (blue line, n = 5), STAT3 KO DCs (green line, n = 5), PBS-control on the indicated days. On day 0, mice were intracranially injected with 20,000 Gl26 glioma cells and followed for survival. <b>H.</b> Mice were monitored for survival and presented as Kaplan-Meyer survival curves. Mantel log-rank test was used to determine statistical significance (*, p<0.05 versus PBS and CpG control).</p

The role of STAT3 signaling in the differentiation and expansion of DCs by Flt3L and GM-CSF.

<p><b>A.</b> WT and STAT3 null bone marrow cells were cultured in the presence of hFlt3L (100 ng/ml) for 8 days then subsequently analyzed by flow cytometry for DC subtypes. Expression of CD45R was used to distinguish pDCs (CD11c⁺/CD45R⁺) from cDCs (CD11c⁺/CD45R⁻). The total number of CD11c⁺ DCs expanded from WT and STAT3 KO bone marrow was quantified from multiple independent bone marrow cultures (*, <i>p</i><0.05; two-tail students t-test). <b>B.</b> Flow cytometry and quantification of GM-CSF-derived (40 ng/ml) BMDCs from WT and STAT3 null bone marrow cells. <b>C.</b> WT and STAT3 deficient mice were injected intracranially with an Ad (1×10⁸pfu) that expresses human soluble Flt3-L. 8 days post injection, animals were euthanized and brains were processed for histology. Flt3 positive and MHC-II positive cells were visualized using immunohistochemistry. Mosaic micrographs of brain sections were captured at 5X and 20X magnifications.</p

Role of STAT3 signaling on phagocytic activity and DCs' maturation.

<p><b>A.</b> Uptake of fluorescently labeled tumor cell remnants were used as a measure of DC phagocytosis. WT and STAT3^−/− bone marrow cells were cultured in the presence of GM-CSF (40 ng/ml) for 6 days to expand the DC pool. BMDCs were then cultured in the presence of PKH-67 labeled GL26 tumor cell lysate for 14 hours then analyzed by flow cytometry. Fluorescence intensity of PKH-67 in CD11c⁺ cells is presented as histograms and indicative of active uptake. Phagocytosis assays were also performed at 4°C to control for uptake by means of passive diffusion. <b>B.</b> WT and STAT3 deficient BMDCs were matured <i>in vitro</i> using CpG. Cell surface expression of MHC-II, CD80, CD86, and CD40 was evaluated in CD11c⁺ GM-CSF derived DCs after an 18 hour stimulation with CpG 1668 (500 ng/ml). <b>C.</b> The median fluorescence intensity of maturation markers was quantified in two independent bone marrow cultures (*, <i>p</i><0.05; two-tail students t-test).</p

Enhanced proliferation of antigen specific T cells in response to STAT3 deficient DCs.

<p><b>A.</b> Antigen specific MLR assay was used to assess antigen processing and presentation by GM-CSF derived BMDCs. WT and STAT deficient BMDCs were cultured with 1 µg/ml ovalbumin for 18 Hrs before being γ-irradiated. DCs were then washed of excess ovalbumin and cultured 1∶1 with 100,000 CFSE labeled OT-1 T cells for 5 days. Peaks of CFSE fluorescence were analyzed by flow cytometry on CD8⁺ OT-1 T cells. The precursor frequency and proliferation index were quantified from three separate MLR assays using non-related mice. Student's t-test was used to determine statistical significance (*, <i>p</i><0.05 versus wild type).</p

Cytokine secretion by WT and STAT3 deficient BMDCs.

<p>Secretion of IL-12p70, IL-10, TNFα, and IL-6 was measured by ELISA from supernatants of 1×10⁶ WT and STAT3^−/− GM-CSF BMDCs stimulated with CpG 1668 (500 ng/ml) for 18 hours in 1 ml of RPMI-10. Cytokine secretion was evaluated from 5 WT and 5 STAT3 KO mice in triplicate wells (*, <i>p</i><0.05; two-tail students t-test).</p