Tumoral B7x promotes pulmonary metastases independent of direct Neuropilin-1 interaction

Abstract

The B7 ligand and CD28 receptor family of proteins are critical in the maintenance of normal immune homeostasis, specifically with regard to T cells. The ligation between B7 ligands and its corresponding CD28 receptor(s) typically expressed on T cells lead to either co-stimulation or co-inhibition during both the activation and effector phase. B7x (B7-H4/B7S1/VTCNI) is a co-inhibitory B7 ligand that functions to inhibit T cell activation, cellular proliferation, and pro-inflammatory cytokine secretion in activated CD4+ and CD8+ T cells; in addition to modulating the innate arm of the immune system through the ligation of an unidentified receptor(s). In sharp contrast to other B7 ligands that are commonly expressed on lymphoid tissues, B7x mRNA is more widely present in non-lymphoid peripheral tissues, though protein expression is limited. However, several studies have demonstrated that B7x is highly overexpressed on the majority of solid tumors and when compared to B7x negative cancer cells correlates to more distant metastases and poor clinical outcome. Therefore, we sought to elucidate the immunological mechanism of how tumoral B7x promotes metastatic development and if Neuropilin-1, a reported binding partner of B7x, is involved this pathway.;To investigate the immunological effects of tumor-expressed B7x on metastatic development and disease severity we first engineered the colorectal carcinoma cell line, CT26, with a murine stem cell virus (MSCV) retrovirus to stably express murine B7x. After generating this cell line, we intravenously (i.v.) injected control CT26 [MSCV] or CT26 [B7x] cells into wild-type mice to assess disease development and the immunological consequences in a pulmonary metastasis model. Our results demonstrated that mice injected with CT26 [B7x] developed more tumor nodules and had a higher overall mortality compared to CT26 [MSCV] control mice. Furthermore, after analyzing the tumor infiltrating leukocytes in the lungs of both groups of mice, we observed a significant decrease in the CD45+ population, but a sharp increase in the CD4 +Foxp3+ T cells (Tregs) in CT26 [B7x] mice compared to control mice. Similarly, there was a significant increase in the immunosuppressive myeloid derived suppressor cells (MDSCs) and M2 tumor associated macrophages (TAMs) populations in CT26 [B7x] mice. This collective accumulation of Tregs, MDSCs, and TAMs due to tumoral B7x expression promoted an increase in antigen-specific exhausted T cells and a decrease in cytolytic function and pro-inflammatory cytokine expression in CD4+ and CD8+ T cells. This suggested that tumor-expressed B7x generated a highly immunosuppressive tumor microenvironment that suppressed antigen-specific anti-tumor immunity.;To better understand the functional consequences of the B7x pathway we evaluated the ability of B7x to interact with a potential binding partner, Neuroplin-1. Our data revealed that Neuropilin-1 was highly expressed on Tregs and to a higher degree on M2 macrophages when compared to MI macrophages. However, when we assessed the ability of B7x to bind to Neuropilin-1 through surface plasmon resonance we were unable to detect any signs of an interaction, though Neuropilin-1 was able to bind to its confirmed binding partners vascular endothelial growth factor (VEGF) and semaphorin 3a (SEMA3A) in a dose dependent fashion. These results were also confirmed through cellular binding assays utilizing flow cytometric analyses. These results highlighted the fact that B7x did not interact with Neuropilin-1.;Overall, our studies signified that B7x is an essential co-inhibitory B7 ligand that functions to enhance pulmonary metastases and limit effective anti-tumor immunity, resulting in reduced overall survival. We implicated tumoral B7x responsible for the significant increase in Tregs, MDSCs, and TAMs observed in the tumor microenvironment as contributors to this acceleration in disease. Though specific mechanistic details of the B7x pathway in cancer immunology were not precisely elucidated, due to the current in identification of its receptor following confirmation that B7x did not bind to Neuropilin-1. This study supported the importance of the B7x pathway in tumor immunology and features it and its unknown cognate receptor(s) as promising targets in the field.