Identification Of Pd-L2 On Macrophages In Lung Cancer Milieu-Pilot Study

The efficacy of immune checkpoint inhibitors remains unexpected and in some patients the resistance to anti- PD-1, PD-L1 agents is observed. Possible explanation is may be PD-L2 activity. The ligands PD-L1 and PD-L2 are present on cancer cells but not-without significance- on macrophages (AMs) contributing to the immune-suppressive tumor microenvironment. The aim of this study was to evaluate PD-L2 expression on AMs in the bronchoalveolar lavage fluid (BALF) from lung affected by cancer (cBALF) and healthy lung (hBALF) in relation to PD-L1 AMs and PD-1 T lymphocytes. Patients with confirmed lung cancer were investigated. Flow cytometry method with a panel of monoclonal antibodies was used. We found that 100% of CD68+ macrophages from cBALF were PD-L1 and PD-L2 -positive. Unexpectedly, fluorescence minus one (FMO) PD-L1 and PD-L2 stained controls also showed strong autofluorescence and similar percentages of these cells fell within the same FLs channel that counted the anti-PD-L1 and PD-L2 stained population. The hBALF AMs exhibited similar PD-L1 and PD-L2 autofluorescence. The evaluation of expression of PD-1 on lymphocytes was much eastier: the median proportion of PD-1+ T cells was higher in cBALF then hlBALF and PB (33.3 vs. 17.9 vs 19.5%, p< 0.05). The geometric mean was higher in BALF then PB (1024 vs. 1196 vs. 408). Our study shows that macrophages autofluorescence did not allow to assess real expression of PD-L2 as well as PD-L1 on AMs. We conclude that the reported expression of PD-L1 and PD-L2 macrophages by flow cytometry should be combined with other qualitative methods to avoid false-positive results.