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. 2021 Apr 29;22(9):4710.
doi: 10.3390/ijms22094710.

Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma

Dong-Jun Park  1 , Pil-Soo Sung  1   2 , Gil-Won Lee  1 , Sungwoo Cho  1 , Sung-Min Kim  1 , Byung-Yoon Kang  1 , Wonhee Hur  1 , Hyun Yang  1   3 , Soon-Kyu Lee  1   2 , Sung-Hak Lee  4 , Eun-Sun Jung  5 , Chang-Ho Seo  6 , Joseph Ahn  6 , Ho-Joong Choi  6 , Young-Kyoung You  6 , Jeong-Won Jang  1   2 , Si-Hyun Bae  1   3 , Jong-Young Choi  1   2 , Seung-Kew Yoon  1   2
Affiliations

Preferential Expression of Programmed Death Ligand 1 Protein in Tumor-Associated Macrophages and Its Potential Role in Immunotherapy for Hepatocellular Carcinoma

Dong-Jun Park et al. Int J Mol Sci. .

Abstract

A predictive biomarker of immune checkpoint inhibitor (ICI)-based treatments in hepatocellular carcinoma (HCC) has not been clearly demonstrated. In this study, we focused on the infiltration and programmed death ligand 1 (PD-L1) expression of tumor-associated macrophages (TAMs) in the tumor microenvironment of HCC. Immunohistochemistry demonstrated that PD-L1 was preferentially expressed on CD68+ macrophages in the tumor microenvironment of HCC, suggestive of its expression in TAMs rather than in T cells or tumor cells (P < 0.05). A co-culture experiment using activated T cells and M2 macrophages confirmed a significant increase in T cell functionality after the pretreatment of M2 macrophages with anti-PD-L1. Syngeneic mouse model experiments demonstrated that TAMs expressed PD-L1 and tumors treated with anti-PD-L1 showed smaller diameters than those treated with IgG. In these mice, anti-PD-L1 treatment increased activation markers in intratumoral CD8+ T cells and reduced the size of the TAM population. Regarding nivolumab-treated patients, three of eight patients responded to the anti-PD-1 treatment. The percentage of Ki-67-positive CD4+ and CD8+ T cells was higher in responders than non-responders after nivolumab. Overall, PD-L1 expression on TAMs may be targeted by immune-based HCC treatment, and ICI treatment results in the reinvigoration of exhausted CD8+ T cells in HCC.

Keywords: PD-L1; anti-PD-L1; hepatocellular carcinoma; tumor-associated macrophages.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Patterns and correlations of CD3, CD68, and PD-L1-expressing cells in human HCC tissues: (A) a representative pattern of CD3, CD68, and PD-L1 expression in human tissues acquired through liver resection; (B,C) the number of CD3+ T cells, CD68+ macrophages, and PD-L1+ cells located in intratumoral and peritumoral region. *** P < 0.001; (D–E) correlation of CD3+ T cells, CD68+ macrophages, serum AFP, and PD-L1+ cells located in peritumoral and intratumoral region (n = 33). Abbreviations: AFP, alpha fetoprotein; HCC, hepatocellular carcinoma; PD-L1, programmed death ligand 1.
Figure 2
Figure 2
Functional enhancement of CD8+ and CD4+ T cells after co-culture with anti-PD-L1-treated macrophages: (A,B) expression and MFI of (A) IFN-γ, and (B) TNF-α, in CD8+ T cells when CD3+ T cells were co-cultured with PD-L1-blocked macrophages (n = 3) * P < 0.05, ** P < 0.01; (C,D) expression and MFI of (C) IFN-γ, and (D) TNF-α in CD4+ T cells when CD3+ T cells were co-cultured with PD-L1-blocked macrophages (n = 3) * P < 0.05, ** P < 0.01; (E) experiment schedule for separation of T cells and macrophages from the tissues acquired by hepatic resection; (F,G) differential expression of IFN-γ and TNF-α in CD8+ and CD4+ T cells when CD3+ T cells were co-cultured with PD-L1-blocked CD206+ macrophages and control cells from human tissues acquired through liver resection. Abbreviations: IFN-γ, interferon-γ; MFI, mean fluorescence intensity; PD-L1, programmed death ligand 1; TNF-α, tumor necrosis factor-α.
Figure 3
Figure 3
In vivo effect on intratumoral T cells and macrophages following anti-PD-L1 treatment in HCC syngeneic model; (A) expression of PD-L1 on non-treated Hepa1-6 cells, IFN-γ treated Hepa1-6, spleen macrophages, and CD11b+ F4/80+ macrophages; (B) PD-1 expression on CD8+ T cells in the spleen and tumor (left). PD-L1 expression on CD11b+ F4/80+ macrophages in the spleen and the tumor (right). (n = 5) ***P < 0.001; (C) experimental schedule in the syngeneic HCC mouse model. In total, 100 µg of anti-PD-L1 antibody was intraperitoneally injected. (D0; Day0, D3; Day3, D8; Day8, D14; Day14, D15; Day15); (D) serial alteration in the size of the tumors generated from Hepa1-6 cells (1 × 107 cells) after treatment with anti-PD-L1 antibody (absolute tumor volume; left, relative tumor volume; right). * P < 0.05; (E) increased activation of T cells when anti-PD-L1 on the CD11b+ F4/80+ macrophage surface was blocked. (n = 5). *** P < 0.001; (F) decreased the number of CD11b+ F4/80+ macrophages per 1g tumor weight when anti-PD-L1 on the CD11b+ F4/80+ macrophage surface was blocked. (n = 5). * P < 0.05. Abbreviations: HCC, hepatocellular carcinoma; IFN-γ, interferon-gamma; PD-1, programmed death-1; PD-L1, programmed death ligand 1.
Figure 4
Figure 4
Ki-67 expression in peripheral CD8+ and CD4+ T cells before and after nivolumab treatment in patients with unresectable HCC: (A,B) differential expression of Ki-67 in CD8+ and CD4+ T cells from CR + PR (n = 3) and SD + PD (n = 5) groups. * P < 0.05, ** P < 0.01; (C,D) CD3, CD68, and PD-L1 expression patterns in patient tissues with CR (C) or PD (D) after nivolumab administration. Abbreviations: CR, complete response; HCC, hepatocellular carcinoma; PD, progressive disease; PD-L1, programmed death ligand 1; PR, partial response; SD, stable disease.
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