Impact of metabolic resources and infiltrating immune cells on protective responses against solid tumors

The tumor microenvironment is composed of different cell types, such as cancer cells, endothelial, fibroblast, stromal cells, and also leukocytes, including γδ T lymphocytes, which participate in anti-tumor immunity. Currently the main strategy for immunotherapy using gamma delta (γδ) T cells is based on the unique reactivity of its dominant subtype in the peripheral blood, Vgamma9Vdelta2 T cells (Vγ9Vδ2 T cells), to antigens of non-peptidic origin (phosphoantigens). Here we investigated the role of systemic cholesterol, namely LDL-cholesterol, in the selective activation and effector functions of T Vγ9Vδ2 cells, particularly in breast cancer. Our results clearly show that LDL-cholesterol was internalized by Vγ9Vδ2 T cells, which was followed by the down-regulation of the LDL receptor (LDL-R), the major cholesterol-carrying lipoprotein receptor. Thereafter, we used human plasma to confirm the effect of the LDL-cholesterol on Vγ9Vδ2 T cell functions. The role of metabolic changes within the immune microenvironment is a timely topic now that there are a variety of cellular therapies utilized to treat cancer patients. In order to understand the role of LDL-cholesterol in the metabolic viability of Vγ9Vδ2 T cells, several parameters were evaluated. In the presence of LDL-cholesterol, the metabolic functions of γδ T cells were significantly inhibited as shown by decreases in mitochondrial mass, mitochondrial DNA, Adenosine triphosphate (ATP) and reactive oxygen species (ROS). Upon elucidation of the decreased metabolic viability involved in the negative regulation of Vγ9Vδ2 T cell activation by LDL-cholesterol, we next dissected the mechanisms involved in the recognition and interaction with tumor cells. Among the potential cytotoxic receptors, we demonstrated that the expression of Natural Killer group 2D (NKG2D) and DNAX Accessory Molecule-1 (DNAM-1), involved in the recognition of tumor cell ligands, and the potent anti-tumor cytokine interferon gamma (IFN-g), were all impaired in the presence of LDL-cholesterol, thereby affecting the cytotoxic capacity of the Vγ9Vδ2 T cells against breast cancer cells in vitro. Consequently, we conducted in vivo studies to support these findings in a tumor context. As a first approach, we evaluated the anti-tumor capacity of Vγ9Vδ2 T cells previously incubated in the presence or absence of LDL-cholesterol in immunodeficient mice bearing human breast tumors. On the other hand, since obesity is characterized by overproduction of inflammatory cytokines, such as TNF alpha and interleukin-1, which may lead to macrophage activation, we used a second approach to evaluate the impact of a cholesterol-rich microenvironment on the antitumor functions of murine γδ lymphocytes in a syngeneic breast cancer model. In both experiments, the negative impact of LDL-cholesterol on the antitumor functions of γδ T lymphocytes was confirmed, leading to increased tumor growth. The influence of cholesterol on tumor growth had been demonstrated in previous studies, where mice on a fat diet developed larger tumors compared to mice exposed to "normal" diets. One of the possible explanations offered by our study is that "bad" cholesterol, when at high levels, has an inhibitory effect on γδ T cells that are normally mobilized to destroy tumor cells. Moreover, our work is the first to describe how human Vγ9Vδ2 T cells may be suppressed by high LDL-cholesterol. This new finding can help in the development of immunotherapy against breast cancer, namely towards preserving the lymphocyte activation and functions required to eliminate tumor cell