Antibiotics aerosolization improves anti PD-1 immunotherapy efficacy via lung microbiota modulation

Several studies demonstrated that lungs harbor a resident microbiota, which is modified by the presence of tumor. Lung microbiota plays a role in regulating pulmonary immune tolerance, influencing APCs and Tregs recruitment and activation. We reported that lung microbiota manipulation by local delivery of antibiotics/probiotics can revert the lung immunosuppressive status and prevent B16 melanoma metastases implantation. Here, we assessed whether perturbing the tumor-associated microbiota by aerosolized antibiotics, by reducing the immunosuppression, may improve immune checkpoint inhibitors effectiveness in preclinical models of lung tumor or metastases. C57BL/6 mice were intravenously (i.v.) injected with LLC1 lung cancer cells, or B16 melanoma or MC38 colon carcinoma cells, histotypes that can metastasize to lungs in human. Mice were aerosolized with vancomycin (50 mg)/neomycin (100 mg) (5 days/week) and/or intraperitoneally administered with anti PD-1 antibody (200 µg/mouse twice a week), starting 7 days after tumor injection. The antitumor immune response was evaluated by measuring cytotoxic activity of lung immune infiltrate and analyzing the expansion/activation of specific subsets in the spleen and lungs by flow cytometry and single cell RNA (sc-RNA) sequencing. Antibiotics aerosolization significantly reduced the number of lung tumor foci in all the three cancer models. This reduction was associated with an increased cytotoxicity of lung infiltrating immune effector cells and a reduction of Tregs. Myeloid-derived immune subsets were variably modulated in each different model. Combination of antibiotics aerosol with anti-PD-1 treatment improved the anti-tumor efficacy of the checkpoint inhibitor in mice i.v. injected with MC38 colon (p= 0,0002) or LLC1 Lewis lung carcinomas (p= 0,0255), compared to antibody alone. FACS analysis highlighted that combining antibiotics aerosol to anti PD-1 therapy enhanced T cells activation and promoted the expansion of stem cells memory and terminally differentiated effector memory T cells in the spleen of both LLC1 and MC38 models. Moreover, in LLC1 tumor injected mice, the combinatorial treatment expanded NKT-like cells. Sc-RNA sequencing of lung-derived CD3+ T cells revealed the expansion of memory T cells subsets and NKT at the tumor site. These results reveal that the modulation of lung microbiota may represent a strategy to reduce immunosuppression in the lung and improve the efficacy of immune-based therapies.