NKp46 expression identifies canine CD3 negative lymphocytes as natural killer cells with phenotypic and functional similarity to those in humans.

Abstract

Abstract Canines are a unique model to study adoptive immunotherapy for cancer as they spontaneously develop many cancers with homology to human disease. However canine natural killer (NK) cells are poorly defined, rendering the model less useful for study of innate immune responses. NKp46 (NCR1) is an activating receptor expressed on NK cells and is considered a species-wide marker for NK cells, but its’ expression profile has not been demonstrated in canine. Therefore, we set out to determine whether NKp46 defines canine NK cells phenotypically and functionally. To this end, we generated a monoclonal mouse anti-canine NKp46 antibody by immunizing mice with L-cells expressing a canine NKp46 fusion protein. Canine peripheral blood cells were cultured in vitro with feeder cells expressing membrane bound human IL-21. The resultant cells were assessed for expression of NKp46, T-cell, B-cell, and macrophage markers. Expression of other NK cell genes were analyzed by RT-PCR. Cytotoxicity assays were conducted using a calcein-release assay with cancer cell lines. NKp46 is expressed on CD3− primary and cultured cells. Cultured CD3−/NKp46+ canine lymphocytes displayed spontaneous cytotoxicity against canine tumors and expressed NK cell activating receptors. CD3−/NKp46− (null) cultured cells were evident and had intermediate cytotoxicity between CD3+ and CD3−/NKp46+ cells. NKp46 MFI was inducible in these null cells following activation. We are currently investigating the cytokine, chemokine, and transcription factor profiling of CD3−/NKp46+ cells. We conclude that canine CD3−/NKp46+ cells are NK cells with phenotypic and functional similarity to human NK cells. In addition, canines have peripheral blood NK cell precursors that lack NKp46.