PD-L1 and PD-1 Are Associated with Clinical Outcomes and Alveolar Immune Cell Activation in Acute Respiratory Distress Syndrome

Abstract

The relationship between the PD-L1 (Programmed Death-Ligand 1)/PD-1 pathway, lung inflammation, and clinical outcomes in acute respiratory distress syndrome (ARDS) is poorly understood. We sought to determine whether PD-L1/PD-1 in the lung or blood is associated with ARDS and associated severity. We measured soluble PD-L1 (sPD-L1) in plasma and lower respiratory tract samples (ARDS1 [n = 59] and ARDS2 [n = 78]) or plasma samples alone (ARDS3 [n = 149]) collected from subjects with ARDS and tested for associations with mortality using multiple regression. We used mass cytometry to measure PD-L1/PD-1 expression and intracellular cytokine staining in cells isolated from BAL fluid (n = 18) and blood (n = 16) from critically ill subjects with or without ARDS enrolled from a fourth cohort. Higher plasma concentrations of sPD-L1 were associated with mortality in ARDS1, ARDS2, and ARDS3. In contrast, higher concentrations of sPD-L1 in the lung were either not associated with mortality (ARDS2) or were associated with survival (ARDS1). Alveolar PD-1^POS T cells had more intracellular cytokine staining than PD-1^NEG T cells. Subjects without ARDS had a higher ratio of PD-L1^POS alveolar macrophages to PD-1^POS T cells than subjects with ARDS. We conclude that sPD-L1 may have divergent cellular sources and/or functions in the alveolar versus blood compartments, given distinct associations with mortality. Alveolar leukocyte subsets defined by PD-L1 or PD-1 cell-surface expression have distinct cytokine secretion profiles, and the relative proportions of these subsets are associated with ARDS.

Keywords: immune checkpoints; macrophage; respiratory distress syndrome.

Figure 1.

Lung and plasma concentrations of sPD-L1 (soluble Programmed Death-Ligand 1), sPD-1 (soluble PD-1), and sPD-L2. Shown are the log₂-transformed levels of ETA, BALF, and plasma sPD-L1, sPD-L2, and sPD-1 from the ARDS1, ARDS2, and ARDS3 cohorts. Displayed are the individual values, mean, and standard deviation for each set.

Figure 2.

Associations between sPD-L1 levels and mortality. We collected lung (endotracheal aspirates in ARDS1 and BALF in ARDS2) and plasma (ARDS1, ARDS2, and ARDS3) samples from subjects with ARDS and measured sPD-L1 levels. Each plot displays the individual values, mean, and SD of log₂-transformed sPD-L1 levels. P values were generated from relative risk regression. The unadjusted and adjusted estimates, as well as the covariates included in each model, are shown in Table E3. Figure E3 displays the results of this analysis for ARDS1 restricted to only subjects with paired lung/blood samples. All samples collected in ARDS2 were paired lung/blood specimens.

Figure 3.

PD-L1/PD-1 cell-surface expression identifies alveolar leukocyte populations that associate with ARDS. We used mass cytometry to analyze alveolar leukocytes isolated from BALF from subjects with (n = 10) or without (n = 8) ARDS. (A) Representative cell population definitions (“gating”) for alveolar leukocytes. Pregating on live, single, CD45^POS cells is shown in Figure E1. We display the individual gating for participants X, Y, and Z, each of whom has different proportions of alveolar macrophage (AM)-PD-L1^POS cells. (B) Uniform manifold approximation and projection (UMAP) plot displaying alveolar leukocyte clustering based on cell-surface protein expression. The cell-surface proteins used for clustering are shown in Figure E4. Color designates assignment of cells to one of the 12 clusters identified by DISCOVE-R. We annotated the clusters based on the normalized expression of each cell-surface marker per cluster (Figure E4). Cells from all patients (n = 18), irrespective of ARDS status, were included in the clustering. (C) UMAPs colored based on marker intensity for each cell-surface protein. (D) The panels show the percentage of PD-L1^POS or PD-1^POS cells as a proportion of their respective parent populations. Shown are individual percentages (dots) and lines connecting individual subjects. Cells from all patients (n = 18), irrespective of ARDS status, were included in these analyses. P values were calculated with paired Wilcoxon tests. (E) The panels show percentages of cell populations as a proportion of their respective parent populations (dots), medians (lines), and interquartile ranges (error bars). P values were calculated with Mann-Whitney tests comparing subjects with or without ARDS. (F) Individual dots show the ratio of the proportion of AM-PD-L1^POS to the proportion of alveolar CD4⁺PD-1^POS or CD8⁺PD-1^POS cells per subject, respectively. Displayed are medians (lines) and interquartile ranges (error bars) for the ratios using either percentage populations or the absolute cell counts for each cell population. The absolute cell numbers for the parent populations are shown in Figure E5. P values were calculated with Mann-Whitney tests comparing subjects with or without ARDS.

Figure 4.

PD-L1/PD-1 is associated with alveolar immune cell cytokine secretion. We stimulated alveolar immune cells with either LPS or PMA-ionomycin and compared intracellular cytokine staining between AMs, CD4⁺, and CD8⁺ T-cell populations based on PD-L1/PD-1 expression. Representative gating is shown in Figure E1. (A) x-axis displays CD4⁺, CD8⁺, or AMs classified as PD-1^POS/NEG or PD-L1^POS/NEG, respectively. y-axis displays the percentage CD4⁺, CD8⁺, or AMs positively staining for each respective cytokine (percentages are reported as the difference between stimulated and unstimulated [baseline] samples per individual). The lines connect populations from the same individual. Cells from all patients (n = 18), irrespective of ARDS status, were included in these analyses. P values were calculated with paired Wilcoxon tests comparing PD-1^POS versus PD-1^NEG cells or PD-L1^POS versus PD-L1^NEG, respectively, per subject. (B) The panels show the relationship between the proportion of CD4⁺PD-1^POS, CD8⁺PD-1^POS, or AM-PD-L1^POS cells and sPD-L1 levels in the BALF in subjects with ARDS (n = 10). Shown are individual values (dots) linear regression best-fit line (straight lines). r = Pearson’s correlation coefficient. (C) In separate experiments, we cocultured AMs from patients with hypoxemic respiratory failure (n = 3) with peripheral blood T cells from a single healthy donor for 48 hours under three experimental conditions: 1) anti-CD3 antibody (stimulant); 2) anti-CD3 antibody + anti–PD-L1 antibody (treatment); and 3) anti-CD3 antibody + isotype control. We measured IFN-γ and TNF-α levels in the cell culture supernatants at the end of the experiment. Lines connect measurements from an individual subject. AMs from the individual with the highest IFN-γ and TNF-α levels were cultured directly after sample collection. The AMs from the other two individuals were analyzed after cryopreservation. IFN-γ levels were numerically higher in treatment versus control in two of three experiments. TNF-α levels were numerically higher in treatment versus control in three of three experiments.

Figure 5.

PD-1 expression is higher on alveolar compared with peripheral blood leukocytes. Paired alveolar cells from BALF were simultaneously collected from five subjects who had peripheral blood mononuclear cells analyzed. We stimulated cells with either LPS (1 μg/ml) or PMA-ionomycin (50/500 ng/ml) for 4 hours and then assessed intracellular cytokine staining. (A) Individual dots represent the percentage of a cell population as a proportion of their respective parent population in paired samples. Lines connect samples collected from the same subject. P values were calculated with paired t tests comparing peripheral blood versus alveolar samples for each subject. (B) We compared the percentage of CD4⁺PD-1^POS, CD8⁺PD-1^POS, and AM-PD-L1^POS cells as a proportion of each of their respective parent populations in peripheral blood compared with alveolar samples. Lines connect samples collected from the same subject. P values were calculated with paired t tests comparing peripheral blood versus alveolar samples for each subject. PBM = peripheral blood monocytes.

Figure 6.

A proposed schematic for cell-surface and soluble PD-L1 in ARDS. (A) Observations from association studies of alveolar macrophages and T cells during steady state. (B) Observations from association studies of alveolar macrophages and T cells during ARDS. (C) Potential functional role of PD-L1/PD-1 in the alveolar space. This figure was generated using BioRender.