Abstract Rationale Lung cancer is the leading cause of cancer deaths worldwide, and, though treatments have advanced, the prognosis remains poor when a surgical cure cannot be obtained. Immunotherapies have demonstrated increasing efficacy, but lung cancer immunology remains poorly understood. Here we developed two lung cancer cell lines of varying antigenicity and employed them in a murine model, with the immune response assayed by high-dimensional flow cytometry. Methods Mouse lung cancer cells were harvested from an autochthonic model bearing a Kras mutation and p53 knockout. One cell line was maintained in its original identity and another was generated by introducing a GFP marker and OVA peptide expression to augment immunogenicity. These respective cell lines were inoculated to WT mice intratracheally to induce orthotopic lung cancer. BAL and lung parenchymal digests were collected at 2- and 4-weeks post-inoculation (4 animals per group/time point). After staining with a broad panel of markers designed to elicit viability, cell identity, functional receptors, and secreted cytokines, data was collected on an Aurora flow cytometer (Cytek) and analyzed in Flowjo (Becton Dickinson). Immune cells and their subsets were identified after clustering, and functional markers were evaluated either by MFI or staining above FMO. Results Both cancer lines provoked marked changes in the immune landscape of the lung, but changes were more pronounced with the unaltered version, apparently due to more severe disease as judged by animal weights (p 0.05). Immunologic changes were also more prominent at 4-weeks than at 2-weeks, given progression of disease. The modified line demonstrated uptake by alveolar macrophages at 2 weeks, but at 4 weeks also by cDC2s, neutrophils, monocytes, and B cells. Cell counts revealed expansion of cDC2, neutrophils, interstitial macrophages, CD4+ T cells, T-regs, and double-negative T cells (p 0.05). Myeloid and B cells demonstrated an activated phenotype by MHCII and costimulatory molecule expression (CD14, CD83, CD86, CD200; p 0.05). PD-L1 expression was increased generally, but was notably decreased on cDC1s (p 0.05). T cells, including CD4+, CD8+, T-reg, NKT, gamma/delta, and double-negative, expressed elevated activation markers (CD69, CTLA-4, PD-1; p 0.05). CD8+, gamma/delta, and NKT cells produced increased IFNg (p 0.05). Conclusions The reporter-modified cancer cell line informed uptake by antigen-presenting cells, but also produced less severe disease and inflammation, likely due to more efficient clearance, given augmented immunogenicity. Immune responses also gave evidence of likely pathogenic tolerance induction via cDC2 and Tregs, confirming a potential role for immunotherapy in our model. This abstract is funded by: NIH
Eccles et al. (Fri,) studied this question.