The Effect of Arginase on Canine T-Lymphocyte Functions and its Modulation by All-Trans Retinoid Acid (ATRA) in Canine Monocyte-Derived Macrophages

SIMPLE SUMMARY: Vast improvements in our understanding of the host’s immune response to tumor cells has dramatically improved cancer treatment in the last 20 years. Naturally occurring cancers in domesticated dogs closely model the immune response to cancers in humans. Therefore, understanding similarities and differences between the immune systems of domesticated dogs and people is critical to translating results relating to immune-targeting cancer therapies between species. To this end, this study evaluated the ability of all-trans retinoic acid, a vitamin A derivative, to decrease the production of pro-cancer substances from a particular immune cell (macrophages). These macrophages were developed in vitro from the blood of dogs with naturally occurring cancers. Our data revealed the ability of all-trans retinoic acid (ATRA) to decrease macrophage production of substances that have been previously reported to increase tumor cell invasion and spread as well as inhibit an effective anti-tumor immune response. We also confirmed the inhibitory activity of one of these substances on critical anti-cancer immune cells, T-lymphocytes. While additional studies are needed to show a direct link between ATRA treatment, macrophage inhibition, and subsequent T-lymphocyte stimulation, these preliminary findings suggest a potential role for ATRA in modulating cancer immunity in dogs. ABSTRACT: Immunosuppressive myeloid cells in the tumor microenvironment play a major role in suppressing tumor immunity via the production of arginase, IL-10, and others. The objectives of this study were to determine the ability of all-trans retinoic acid (ATRA) to decrease the expression of arginase and other soluble mediators by canine monocyte-derived macrophages (MDMs) and to determine the inhibitory activity of arginase on canine T-lymphocytes. The immunomodulatory ability of ATRA (2 µM) on canine MDMs was evaluated via reverse transcription quantitative PCR (RT-qPCR), flow cytometry, arginase activity assay, and enzyme-linked immunoassay (ELISA). Arginase effects on T-lymphocyte phenotype and proliferation were then evaluated by flow cytometry. ATRA consistently decreased MDM expression of IL6, TGFB1, NOS2, ARG1, and CIITA transcripts, by approximately 2–4-fold, although this did not reach statistical significance for ARG1 or CIITA. Furthermore, arginase activity was decreased in ATRA-treated MDMs while the MDM phenotype remained unchanged. Arginase decreased the expression of granzyme B on CD8+ T-lymphocytes and inhibited CD4+ and CD8+ T-lymphocyte proliferation. These findings suggested that ATRA could inhibit canine MDM production of soluble inflammatory/immunosuppressive mediators. These data also revealed that arginase decreased canine T-lymphocyte proliferation and granzyme B expression. Further studies are needed to determine whether ATRA could reverse the immunosuppressive effects of myeloid cells on canine T-lymphocytes in vivo.