Immune Cell PD-L1 Colocalizes with Macrophages and Is Associated with Outcome in PD-1 Pathway Blockade Therapy

Abstract

Purpose: Programmed death ligand 1 (PD-L1) is expressed in tumor cells and immune cells, and both have been associated with response to anti-PD-1 axis immunotherapy. Here, we examine the expression of PD-L1 to determine which cell type carries the predictive value of the test.

Experimental design: We measured the expression of PD-L1 in multiple immune cells with two platforms and confocal microscopy on three retrospective Yale NSCLC cohorts (425 nonimmunotherapy-treated cases and 62 pembrolizumab/nivolumab/atezolizumab-treated cases). The PD-L1 level was selectively measured in different immune cell subsets using two multiplexed quantitative immunofluorescence panels, including CD56 for natural killer cells, CD68 for macrophages, and CD8 for cytotoxic T cells.

Results: PD-L1 was significantly higher in macrophages in both tumor and stromal compartment compared with other immune cells. Elevated PD-L1 in macrophages was correlated with high PD-L1 level in tumor as well as CD8 and CD68 level (P < 0.0001). High PD-L1 expression in macrophages was correlated with better overall survival (OS; P = 0.036 by cell count/P = 0.019 by molecular colocalization), while high PD-L1 expression in tumor cells was not.

Conclusions: In nearly 500 non-small cell lung cancer (NSCLC) cases, the predominant immune cell type that expresses PD-L1 is CD68⁺ macrophages. The level of PD-L1 in macrophages is significantly associated with the level of PD-L1 in tumor cells and infiltration by CD8⁺ T cells, suggesting a connection between high PD-L1 and "hot" tumors. In anti-PD-1 axis therapy-treated patients, high levels of PD-L1 expression in macrophages are associated with longer OS and may be responsible for the predictive effect of the marker.

Figure 1.

Quantification of PD-L1 localization in tumor and stroma. Four representative cases to demonstrate typical PD-L1 localization patterns with cell counts and confocal images of regions of the counted case (A). Summary pie charts of PD-L1 localization calculated by double positive phenotype cell counts (PD-L1/CK, PD-L1/CD68, PD-L1/CD8, PD-L1/CD56) on the average of 457 NSCLC cases was presented as percentages in both tumor and stromal compartment (B).

Figure 2.

Colocalization of phenotypic markers and PD-L1. Example confocal images of PD-L1 colocalization with tumor cells (A) and with macrophages (B). In Fig. 2A, white arrow is a tumor infiltrating macrophage with CD68 and PD-L1 double staining, but without cytokeratin staining.

Figure 3.

High PD-L1 expression in macrophages was correlated with high CD8 level, high CD68 level and high PD-L1 expression by tumor cells in 457 cases of NSCLC (A). Error bars represent mean with 95% confidence interval. PD-L1 expression in macrophages is not prognostic in this cohort (B).

Figure 4.

PD-L1 level in macrophages predicts patients’ overall survival to anti-PD-1 axis blockade therapy using two different QIF methods. Using InForm to count cells, double positive PD-L1&CK cell (count n=15) was not associated with overall survival (A). Double positive PD-L1/CD68 cells (count n=12) was significantly associated with overall survival of NSCLC patients treated with single drug immunotherapy (B). Using AQUA assessment of PD-L1 in the tumor(C) or stromal (D) compartments was not associated with better outcome on monotherapy, while PD-=L1 in the CD68 compartment was statistically significantly associate with better overall survival(E).