Assessing the role of STAT3 in DC differentiation and autologous DC immunotherapy in mouse models of GBM.

Cellular microenvironments, particularly those found in tumors, elicit a tolerogenic DC phenotype which can attenuate immune responses. Central to this process is the STAT3-mediated signaling cascade. As a transcription factor and oncogene, STAT3 promotes the expression of genes which allow tumor cells to proliferate, migrate and evade apoptosis. More importantly, activation of STAT3 in tumor infiltrating immune cells has been shown to be responsible, in part, for their immune-suppressed phenotype. The ability of STAT3 to orchestrate a diverse set of immunosuppressive instructions has made it an attractive target for cancer vaccines. Using a conditional hematopoietic knockout mouse model of STAT3, we evaluated the impact of STAT3 gene ablation on the differentiation of dendritic cells from bone marrow precursors. We also assessed the impact of STAT3 deletion on phagocytosis, maturation, cytokine secretion and antigen presentation by GM-CSF derived DCs in vitro. In addition to in vitro studies, we compared the therapeutic efficacy of DC vaccination using STAT3 deficient DCs to wild type counterparts in an intracranial mouse model of GBM. Our results indicated the following pleiotropic functions of STAT3: hematopoietic cells which lacked STAT3 were unresponsive to Flt3L and failed to differentiate as DCs. In contrast, STAT3 was not required for GM-CSF induced DC differentiation as both wild type and STAT3 null bone marrow cells gave rise to similar number of DCs. STAT3 also appeared to regulate the response of GM-CSF derived DCs to CpG. STAT3 null DCs expressed high levels of MHC-II, secreted more IL-12p70, IL-10, and TNFα were better antigen presenters in vitro. Although STAT3 deficient DCs displayed an enhanced activated phenotype in culture, they elicited comparable therapeutic efficacy in vivo compared to their wild type counterparts when utilized in vaccination paradigms in mice bearing intracranial glioma tumors

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