Background: Successful immunotherapy will require alteration of the tumour microenvironment and/or decreased immune suppression. Tumour-associated macrophages (TAMs) are one major factor affecting tumour microenvironment. We hypothesised that altering TAM phenotype would augment the efficacy of immunotherapy.Methods:We and others have reported that 5,6-Dimethylxanthenone-4- acetic-acid (DMXAA, Vadimezan) has the ability to change TAM phenotypes, inducing a tumour microenvironment conducive to antitumour immune responses. We therefore combined DMXAA with active immunotherapies, and evaluated anti-tumour efficacy, immune cell phenotypes (flow cytometry), and tumour microenvironment (RT-PCR).Results:In several different murine models of immunotherapy for lung cancer, DMXAA-induced macrophage activation significantly augmented the therapeutic effects of immunotherapy. By increasing influx of neutrophils and anti-tumour (M1) macrophages to the tumour, DMXAA altered myeloid cell phenotypes, thus changing the intratumoural M2/non-M2 TAM immunoinhibitory ratio. It also altered the tumour microenvironment to be more pro-inflammatory. Modulating macrophages during immunotherapy resulted in increased numbers, activity, and antigen-specificity of intratumoural CD8 + T cells. Macrophage depletion reduced the effect of combining immunotherapy with macrophage activation, supporting the importance of TAMs in the combined effect.Conclusion:Modulating intratumoural macrophages dramatically augmented the effect of immunotherapy. Our observations suggest that addition of agents that activate TAMs to immunotherapy should be considered in future trials.