Identification of PD-1 ligands: PD-L1 and PD-L2 on macrophages in lung cancer milieu by flow cytometry

Background: The efficacy of immune checkpoint inhibitors (ICIs) remains unexpected and in some patients the resistance to anti-programmed death-1 (anti-PD-1) and anti-programmed death ligand 1 (anti-PD-L1) agents is observed. One of possible explanation may be PD-L2 activity. PD-1 ligands: PD-L1 and PD-L2 are present on cancer cells but also, not without significance, on alveolar macrophages (AMs) contributing to immune-suppression in the tumor microenvironment. The aim of this study was to analyse PD-L2, PD-L1 expression on AMs in bronchoalveolar lavage fluid (BALF) in relation to PD-1 positive T lymphocytes.

Methods: Seventeen patients with lung cancer were investigated. BALF cells from the lung with cancer (clBALF) and from the opposite "healthy" lung (hlBALF) and peripheral blood (PB) lymphocytes were investigated. Flow cytometry method was used.

Results: We found that 100% of CD68+ AMs from the clBALF were PD-L1 and PD-L2-positive. Unexpectedly, fluorescence minus one (FMO) PD-L1 and PD-L2 stained controls and isotype controls also showed strong autofluorescence. The hlBALF AMs exhibited a similar PD-L1 and PD-L2 autofluorescence. The median proportion of PD-1+ T lymphocytes was higher in the clBALF than the hlBALF and PB (28.9 vs. 23.4% vs. 15.6%, P=0.0281).

Conclusions: We discussed the opportunities of exploring the PD-1-PD-L1/PD-L2 pathway in the lung cancer environment, which may help to find new potential biomarkers for immunotherapy. We concluded that precise identification by flow cytometry of macrophages in the BALF is possible, but our study showed that the autofluorescence of macrophages did not allow to assess a real expression of PD-L2 as well as PD-L1 on AMs.