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. 2009 Mar 15;69(6):2497-505.
doi: 10.1158/0008-5472.CAN-08-3031. Epub 2009 Mar 3.

Toll-like receptor 9 activation of signal transducer and activator of transcription 3 constrains its agonist-based immunotherapy

Marcin Kortylewski  1 , Maciej Kujawski, Andreas Herrmann, Chunmei Yang, Lin Wang, Yong Liu, Rosalba Salcedo, Hua Yu
Affiliations

Toll-like receptor 9 activation of signal transducer and activator of transcription 3 constrains its agonist-based immunotherapy

Marcin Kortylewski et al. Cancer Res. .

Abstract

Although toll-like receptor (TLR) agonists, such as CpG, are used as immunotherapeutic agents in clinical trials for cancer and infectious diseases, their effects are limited and the underlying mechanism(s) that restrains CpG efficacy remains obscure. Here, we show that signal transducer and activator of transcription 3 (Stat3) plays a key role in down-modulating immunostimulatory effects of CpG. In the absence of interleukin-6 (IL-6) and IL-10 induction, CpG directly activates Stat3 within minutes through TLR9. Ablating Stat3 in hematopoietic cells results in rapid activation of innate immunity by CpG, with enhanced production of IFN-gamma, tumor necrosis factor-alpha, IL-12, and activation of macrophages, neutrophils, and natural killer cells marked with Stat1 activation. Innate immune responses induced by CpG in mice with a Stat3-ablated hematopoietic system cause potent antitumor effects, leading to eradication of large (>1 cm) B16 melanoma tumors within 72 h. Moreover, ablating Stat3 in myeloid cells increases CpG-induced dendritic cell maturation, T-cell activation, generation of tumor antigen-specific T cells, and long-lasting antitumor immunity. A critical role of Stat3 in mediating immunosuppression by certain cytokines and growth factors in the tumor microenvironment has been recently documented. By demonstrating direct and rapid activation of Stat3 by TLR agonists, we identify a second level of Stat3-mediated immunosuppression. Our results further suggest that targeting Stat3 can drastically improve CpG-based immunotherapeutic approaches.

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Figures

Figure 1
Figure 1
TLR9 receptor activation induces immediate and direct activation of Stat3 in dendritic cells. A, TLR ligand-specific Stat3 activation in primary splenic DCs. CD11c+ DCs freshly isolated from pooled splenocytes (n=3) were treated (3h) with TLR ligands (1 μg/ml) as indicated. Stat3 activation was assessed by Western blotting to detect phospho-Stat3 or total Stat3 and β-actin for loading control. B, Stimulation with CpG induced rapid and prolonged activation of Stat3 but not Stat1 or Stat5 in splenic DCs (enriched from pooled splenocytes; n=3). Kinetics of Stat phosphorylation was assessed by Western blotting as in Fig. 1A. C, Delayed kinetics of CpG-induced IL-6 expression in splenic DCs. Intracellular cytokine staining and FACS analysis of IL-6 and IL-10 production in splenic CD11c+ DCs after 4 or 24h of in vitro incubation in the presence or absence of CpG (1 μg/ml). D, Stat3 activation is mediated by TLR9 independent of IL-10 expression. Western blots detecting tyrosine-phosphorylated Stat3 in DCs enriched from pooled TLR9−/− and IL-10−/− splenocytes or from the matching WT controls (WT1, C57BL/6 strain; WT2, Balb/C) (n=4 mice/group). All results were confirmed by at least two independent experiments.
Figure 2
Figure 2
Stat3 ablation sensitizes DCs to CpG stimulation resulting in increased production of Th1 proinflammatory mediators. A, Splenic DCs were incubated (48h) with or without CpG. Conditioned culture media were analyzed using antibody arrays to detect cytokine and chemokine secretion. B, Quantification of antibody array analyses showing increased CpG-induced cytokine and chemokine secretion in Stat3−/− mice compared to Stat3+/+ counterparts. Dot intensities were measured by densitometry and normalized to positive controls (shown in the box), mean±SEM, (3 independent experiments, n=3–4 mice/group).
Figure 3
Figure 3
CpG ODN triggers rapid eradication of large B16 tumors in mice with Stat3 ablation. Mice with Stat3+/+ or Stat3−/− hematopoietic cells were challenged with B16 melanoma cells (1×105, s.c.). Mice with established tumors were treated with a single peritumoral injection of CpG ODN (5 μg). A, Tumor size before and after CpG treatment. B-C, Changes in tumor volume within 3d post-CpG treatment (2 independent experiments, n=4 mice/group, with either larger, ~10 mm (B) or smaller, ~5 mm (C), average tumor diameter at the time of CpG ODN injection, mean±SEM. D, Two-photon intravital imaging of vasculature stained using dextran-rhodamine within tumor or tumor-draining lymph node in Stat3+/+ or Stat3−/− mice 18h post CpG ODN treatment. Images composed of multiple scans from 52 μm z-dimensional stack (2 independent experiments; bar=100 μm).
Figure 4
Figure 4
Local CpG treatment and Stat3 ablation induce potent antitumor innate immunity. A–B, B16 tumors show strongly increased infiltration by neutrophils (A) and iNOS-expressing macrophages (B) in Stat3−/− relative to Stat3+/+ mice. Fluorescence microscopy of cryosections of tumor specimens detected with antibodies specific for neutrophils (7/4, green) or macrophages (Mac3, red) together with iNOS (green), 2 independent experiments, n=3–4 mice/group. C, CpG induces high levels of NO secretion from Stat3-negative myeloid cells. Splenic Stat3+/+ or Stat3−/− CD11b+ myeloid cells isolated from tumor-bearing mice were treated with CpG in vitro. Nitrate concentrations in culture media were assessed after 48h; mean±SEM, combined from 2 independent experiments performed in triplicate. D, Stat3 deletion promotes CpG-induced tumor infiltration by activated NK lymphocytes. Left two panels - FACS analysis of CD49b+ NK cell numbers among tumor-infiltrating lymphocytes after excluding CD4+ and CD8+ T cell populations (2 independent experiments, n=3–4 mice/group). Right two panels, Stat1 activation in NK cells isolated from tumor-draining lymph nodes collected from 3–4 mice, 24h after peritumoral ODN injection, as assessed by FACS, using intracellular staining with antibodies specific for tyrosine-phosphorylated Stat1 (2 independent experiments).
Figure 5
Figure 5
Ablating Stat3 promotes TLR9-induced dendritic cells maturation and antigen-specific T cell responses. Stat3 deletion enhances CpG-induced tumor DC maturation in vivo. A, The phenotypic analysis of CD11c+ DCs accumulated in tumor-draining lymph nodes of Stat3+/+ and Stat3−/− mice 48h post-CpG injection. The maturation of CD11c+ DCs was increased by Stat3 ablation as shown by a greater percentage of double-positive MHC class IIhi and CD86hi DCs (top two rows), as well as higher expression of CD80 and CD40 on DCs (bottom two rows) as seen by FACS analyses (3 independent experiments, n=3–4 mice/group). B, Stat1 is activated in Stat3−/− DCs from tumor-draining lymph nodes following CpG but not control GpC ODN treatment. Intracellular flow cytometric analysis of DCs isolated from tumor-draining lymph nodes using an antibody to detect tyrosine-phosphorylated Stat1 (phospho-Stat1) (3 mice/group). C, Stat3 deletion promotes tumor antigen-specific T cell responses.Expression of the early lymphocyte activation marker CD69 was analyzed by flow cytometry on CD8+ T cells 24 h after injecting CpG or control GpC ODNs; 3 independent experiments using lymph node cell suspensions, n=3–4 mice/group. D, Stat3−/− mice mount a stronger T-cell response against an endogenous B16 tumor-antigen than their Stat3+/+ counterparts, following treatment with CpG ODN. IFNγ production in T cells derived from tumor-draining lymph node was assessed by ELISPOT assay; mean±SEM of p15E-specific IFNγ-producing cells (2 independent experiments, cells pooled from n=4 mice/group).
Figure 6
Figure 6
Stat3 targeting in vivo improves TLR9-mediated antitumor immunity. A, Single peritumoral injection of CpG results in long-term complete rejection of tumors in Stat3−/− but not in Stat3+/+ mice through CD4- and CD8-mediated immunity. Mice with established B16 tumors were treated with CpG ODNs. Depleting antibodies against CD4+ and CD8+ T cells or control rat IgG were given to the indicated group of mice (3 independent experiments, n=6 mice/group). Statistically significant differences between the CpG-treated Stat3−/− group of mice with or without CD4/CD8 T cell depletion are indicated with asterisks; **, p<0.01 and *, p<0.05. B–D, Growth of B16F10 (B) and C4 (C) tumors is significantly inhibited when peritumoral CpG ODN treatment is combined with systemic inhibition of Stat3 activity by a Stat3 inhibitor, CPA7. Mice with established tumors (average diameter 4–8 mm) were treated with CPA7, followed by peritumoral CpG injection a day later. The treatment was repeated twice weekly. Shown are the results of three (B) and two (C) independent experiments. D, Combination of Stat3 targeting with CpG-treatment generates concomitant antitumor immunity. Surviving mice treated as in Fig. 6B, were re-challenged with the same number of B16F10 cells injected in the opposite flank two weeks after the first tumor challenge. Statistically significant differences between the group treated with combination of CPA7 with CpG comparing to controls treated only with CpG only are indicated with asterisks; **, p<0.01 and *, p<0.05.
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