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

Abstract

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.

Keywords: Macrophages; PD-L1; PD-L2; bronchoalveolar lavage fluid (BALF); lung cancer microenvironment.

Figure 1

FACS analysis of BALF cells with specific antibodies for AMs and PD-L1 or PD-L2 expression: (A) CD45+ bright/CD68+ bright/HLA-DR+ bright on SSC/FSC macrophage population was gated (green population) and “PD-L1 positive population AMs” (red population: approximately 100%) and “PD-L2 positive population AMs” (blue population: approximately 100%) were determined. (B) FMO controls for PD-L1-PE minus and PD-L2-PerCp-Cy5.5 minus were analyzed. The percentages of autofluorescent population were shown in the SSC/FL1 (PE) (red population 100%) and in the SSC/FL1 (PerCp-Cy5.5) (green population 100%) plots and histograms. (C) Isotypes controls for PD-L1-PE and PD-L2-PerCp-Cy5.5 were evaluated. The percentage of autofluorescent population were shown in the SSC/FL1 (PE isotypes controls) (red population 100%) and in the SSC/FL1 (PerCp-Cy5.5 isotypes control) (green population 100%) plots and histograms. BALF, bronchoalveolar lavage fluid; AM, alveolar macrophage; FMO, fluorescence minus one.

Figure 2

FACS analysis of PD-1 expression on T lymphocytes in clBALF from example patient with lung cancer. (A) Gating strategy of T cells. (B) PD-1 expression on CD3+ T cells read as % and GMF intensity. (C) PD-1 expression on CD8+ T cells read as % and GMF intensity. (D) PD-1 expression on CD4+ T cells read as % and GMF intensity. BALF, bronchoalveolar lavage fluid; clBALF, BALF from the lung with cancer; hlBALF, BALF from the opposite “healthy” lung; GMF, geometric mean fluorescence.

Figure 3

The differences between PD-1 expression on T lymphocytes (CD4+ and CD8+) in clBALF, hlBALF and PB patients with lung cancer. (A) PD-1 expression on T cells read as % and GMF intensity. (B) PD-1 expression on CD8+ T cells read as % and GMF. (C) PD-1 expression on CD4+ T cells read as % and GMF. The median values and quartile q25–q75 were shown on graphs (*, P<0.05). BALF, bronchoalveolar lavage fluid; clBALF, BALF from the lung with cancer; hlBALF, BALF from the opposite “healthy” lung; PB, peripheral blood; GMF, geometric mean fluorescence.