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

Hikmat Assi,Daniel Zamler,Maria G Castro,Sarah Suprise,Jaclyn Espinosa,Pedro R Lowenstein,Michael Sofroniew,Robert Doherty,Christopher Wheeler

doi:10.1371/journal.pone.0096318

Abstract

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.

Highlights

Constitutive activation of signal transducer and activator of transcription-3 (STAT3) has been implicated as a central mechanism of tumor-induced immunosuppression
Our results demonstrate a function of STAT3 in modulating dendritic cells (DCs) activity in vitro, we found no differences in the induction of anti-tumor immune responses elicited by WT or STAT3 null DCs
Inducible hematopoietic STAT3 knockout mouse model To circumvent issues of embryonic lethality associated with total ablation of the STAT3 gene, an inducible conditional knockout was generated in mice using the Mx1-Cre system as illustrated in figure 1A and previously described [13,21]

Summary

Introduction

Constitutive activation of signal transducer and activator of transcription-3 (STAT3) has been implicated as a central mechanism of tumor-induced immunosuppression. STAT3 mediates the expression of genes such as Cyclin-D, Bcl-xl, and survivin, which promote the growth and survival of individual tumor cells [9,10]. Wang et al demonstrated the increased secretion of pro-inflammatory cytokines and chemokines such as TNF-a, IL-6, RANTES, and IFN-b in B16 melanoma cells after transfecting a dominant negative mutant of STAT3 [11]. These factors have pleiotropic immune stimulatory activity and are critical for inducing the activation and migration of dendritic cells (DCs). Hyperactivation of STAT3 in CT26 or C6 tumor cells was implicated for the abnormal differentiation of DCs in cultures containing conditioned media [12]

Methods

Results

Conclusion