The Interleukin-1 receptor-associated kinase 3 (IRAK3) is a negative regulator of the pro-inflammatory NF-κB signalling pathway. The expression of IRAK3 is localised to monocytes and macrophages, where its expression is associated with an anti-inflammatory M2-macrophage phenotype. While IRAK3 expression is necessary to attenuate acute inflammatory responses, IRAK3 contributes to cancer development by aiding immune evasion and progression.
The aim of this study was to investigate the effect of IRAK3 in macrophages on their ability to co-stimulate T lymphocytes. CD14+ monocytes and CD3+ T lymphocytes were isolated from fresh buffy coats. CRISPR-Cas9 gene editing technique was employed to knockout IRAK3 in isolated monocytes. Transfected null and IRAK3 knockout macrophages were treated with conditioned media obtained from breast cancer cell lines MCF-7, MDA-MB-231, BT549 and HCC-1937. Conditioned media-treated macrophages were co-cultured with autologous and allogeneic CD3+ T lymphocytes. IFNg and granzyme B concentrations in co-culture supernatants were assessed with ELISA. T lymphocytes were analysed with FACS to immunophenotype and investigate proliferation of CD3+ T lymphocyte sub-populations.
The study concluded that breast cancer conditioned media induces IRAK3 expression in null macrophages, thus confirming that the anti-inflammatory function of IRAK3 can be exploited by cancer cells via secretion of soluble mediators. Most importantly, IRAK3 knockout macrophages treated with triple negative breast cancer cell line conditioned media exhibited M1 macrophage-like activity by promoting proliferation of effector T cells and their secretion of pro-inflammatory cytokines IFNγ and granzyme B. This observed pro-inflammatory function supports the role of IRAK3 as a possible novel cancer immunotherapy target.