IL-12 Stimulates Interferon-Gamma Mediated Inhibition of Tumor-Induced Regulatory T Cell Proliferation and Enhances Tumor Clearance

Abstract

We have recently demonstrated that regulatory T (Treg) cells in the tumor environment (but not in the peripheral lymphoid tissues) utilize granzyme B to suppress anti-tumor immunity (Immunity 2007 Oct; 27(4):635–46), which suggests that Treg cells may be subject to regulation by local factors during anti-tumor immune responses. In order to identify such factors, we have employed an unbiased approach. First, we performed cytokine profiling on the tumor ascites fluid (tumor environment) and plasma samples harvested from naïve and tumor-bearing mice by using rodent Multi-Analyte Profiles (MAPs) provided by Rules-Based Medicine (RBM). Second, we performed gene expression profiling on Treg cells purified from the tumor ascites, spleens of tumor-bearing mice, and spleens of naïve mice by using Affymetrix MOE430v2 arrays. By matching the profiles of local cytokines and their receptor expression on tumor-associated Treg cells, we identified candidate cytokines that may modulate Treg cell function. In addition, we developed an in vitro system to study Treg cell proliferation in the tumor setting. Co-culturing irradiated tumor cells with unfractionated splenocytes (but not purified Treg cells) induces significant expansion of CD4+Foxp3+ Treg cells (2–4 fold in 5 days), suggesting that accessory cells and/or factors produced by them are responsible for Treg expansion. Using this system, we tested candidate cytokines for potential effects on Treg proliferation. Remarkably, IL-12 treatment completely blocked Treg expansion in an IL-12 receptor-dependent fashion. However, absence of IL-12 receptor mRNA in Treg cells suggested an indirect mechanism for this effect. Notably, an Interferon-gamma (IFNg) neutralizing antibody partially abolishes this IL-12 effect, and IFNg treatment exhibits similar (but lesser) inhibition of Treg proliferation. Conversely, Treg cells in IFNg receptor-deficient cultures are partially resistant to IL-12 inhibition, but completely resistant to IFNg inhibition. Mechanistic analyses revealed that IFNg signaling directly causes cell cycle arrest in Treg cells. Furthermore, IL-12 treatment enhances tumor clearance in vivo, and is more efficacious than Treg cell depletion. These findings indicate that IL-12 can stimulate IFNg-mediated inhibition of Treg cell proliferation, which may partially account for the anti-tumor effect of this cytokine. This study provides definitive evidence that an IL-12/IFNg axis can function to inhibit Treg cell proliferation, and that Treg cells, as immune suppressors themselves, are subject to cytokine-mediated suppression during anti-tumor immune responses. Because antigen-based tumor vaccines have been found to activate Treg cells that can considerably dampen antitumor immunity, such Treg-inhibiting cytokines may have the potential to serve as an adjuvant for vaccine-based immunotherapy.