Abstract B79: Tumor rejection driven by efficient Treg depletion is associated with a strong change in the tumor microenvironment.
Authors: Salgado Oscar C. , Carretero Rafael , Li Xingrui , Sektioglu Ibrahim M. , Garbi Natalio , Hämmerling Günter J. 
Published: August 2014

Abstract
Efficient regulatory T cell (Treg) depletion leads to tumor rejection in mice. The goal of this study was to understand the mechanisms involved in this rejection process. For the selective and efficient depletion of Tregs, we used the FoxP3.LuciDTR-4 mouse model where Tregs express, among others, the human diphtheria toxin receptor. Our previous studies show that the depletion of more than 90% of Tregs in FoxP3.LuciDTR-4 mice promotes the infiltration of immune cells and subsequent rejection of ovalbumin (OVA)-expressing B16 melanomas.
To unravel the mechanisms involved, we characterized the tumor-infiltrating immune populations and performed a cytokine profile on tumors at the onset of rejection. The analysis revealed a change in the population composition of the tumor-infiltrating leucocytes. There was an increase in T cell numbers, especially CD8+ T cells, and dendritic cells in tumors in rejection. Moreover, the amount of B cells was drastically reduced in tumors being rejected while they were enriched in tumors in progression. Tumor-infiltrating myeloid cells also differed phenotypically between tumors in rejection and tumors in progression.
Inflammatory cytokines related to the type 1 response such as IFN-γ, TNF and IL-12 were significantly more expressed in tumors in rejection as measured at mRNA and protein levels. On the other hand, the expression of some cytokines related to the type 2 response such as IL-5 and IL-10 and of the inhibitory cytokine TGF-β did not change between tumors in rejection and in progression. We also determined that the concentration of molecules related to the M1 macrophage phenotype such as inducible nitric oxide synthase (iNOS), interferon regulatory factor 5 (IRF5), CXCL9 and CXCL10 was significantly higher in tumors in rejection; however, the concentration of most of the molecules related to the M2 macrophage phenotype such as CD163, macrophage mannose receptor 1 (MRC1), Fizz-1 and PPAR-γ was not different between tumors in rejection and tumors in progression. Interestingly, angiogenesis drivers like VEGF and angiotensin 1 (ANG1) were found to be significantly less expressed in tumors in rejection, a fact that correlates with the normalization of the tumor vasculature previously described to be promoted by Treg depletion.
We conclude that Treg depletion provokes a strong change in the tumor microenvironment, not only in terms of the elevated number of immune infiltrating cells but also in terms of the enrichment of certain subpopulations. Furthermore, this change is associated with a striking alteration of the cytokine and chemokine profile that becomes proinflammatory, supports mainly a Type 1-related response and inhibits angiogenesis pathways.
Citation Format: Oscar C. Salgado, Rafael Carretero, Xingrui Li, Ibrahim M. Sektioglu, Natalio Garbi, Günter J. Hämmerling. Tumor rejection driven by efficient Treg depletion is associated with a strong change in the tumor microenvironment. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology: Multidisciplinary Science Driving Basic and Clinical Advances; Dec 2-5, 2012; Miami, FL. Philadelphia (PA): AACR; Cancer Res 2013;73(1 Suppl):Abstract nr B79.