Programmed death ligand 1 expression in human intrahepatic cholangiocarcinoma and its association with prognosis and CD8+ T-cell immune responses

Abstract

Background: Agents targeting the programmed death ligand 1 (PD-L1)/programmed death receptor 1 immune checkpoint exhibited promising clinical outcomes in a variety of malignant tumors, including intrahepatic cholangiocarcinoma (ICC). However, the relationship between PD-L1 expression and CD8⁺ T-cell immune responses is not well defined in ICC.

Patients and methods: We investigated PD-L1 expression immunohistochemistry in formalin-fixed, paraffin-embedded tissues from 192 ICC patients undergoing curative resection and correlated our results with the clinicopathologic features and prognosis. We also quantified CD8⁺ T-cell infiltration in ICC specimens and evaluated the relationship between PD-L1 expression and CD8⁺ T-cell infiltration. After incubating human ICC cell lines (HCCC9810 and RBE) with interferon (IFN)-γ, we measured the PD-L1 expression of these ICC cells by Western blot and flow cytometry.

Results: Only 34 patients (17.7%) showed ≥5% membranous PD-L1 expression on tumor cells, and tumoral PD-L1 overexpression (≥5%) was significantly associated with superior overall survival (P=0.012) and disease-free survival (P=0.018). A significant positive association was found between PD-L1 expression and the presence of CD8⁺ T-cells. In fresh frozen ICC specimens, IFN-γ was found to be significantly correlated with PD-L1 and CD8A gene expression, as evaluated by reverse transcription-polymerase chain reaction. Moreover, stimulation of the HCCC9810 and RBE cells with recombinant IFN-γ, secreted by CD8⁺ T-cells rapidly induced PD-L1 upregulation in these cell lines in vitro.

Conclusion: Tumor PD-L1 overexpression is mainly stimulated by activated CD8⁺ T-cells pre-existing in the ICC microenvironment, and PD-L1 is a favorable prognostic factor for the patients. These observations suggest that anti-PD-L1/programmed death receptor 1 therapy may benefit ICC patients with tumor cell PD-L1 expression and the presence of CD8⁺ T-cells.

Keywords: CD8+ T-cell; IFN-γ; PD-L1; adaptive immune resistance; tumor microenvironment.

Figure 1

Patterns of PD-L1 expression in primary ICC surgical specimens stained with anti-PD-L1 monoclonal antibody. Notes: PD-L1 staining was detected in a membranous pattern in ICC tissues. (A and B) Samples displaying pattern 1 exhibited negative expression in ICC cells. (A) Original magnification, ×100. (B) Original magnification of the boxed area shown in (A), ×200. (C and D) Samples exhibiting pattern 2 displayed diffuse staining. (C) Original magnification, ×200. (D) Original magnification of the boxed area shown in (C), ×400. (E and F) Samples displaying pattern 3 exhibited regional PD-L1 expression. (E) Original magnification, ×100. (F) Original magnification of the boxed area shown in (E), ×200. Abbreviations: ICC, intrahepatic cholangiocarcinoma; PD-L1, programmed death ligand 1.

Figure 2

PD-L1 overexpression is correlated with superior OS and DFS of ICC patients. Notes: Kaplan–Meier curves for the analysis of ICC patients. (A) OS and (B) DFS according to PD-L1 protein levels. P-values were calculated by log-rank test. Abbreviations: DFS, disease-free survival; ICC, intrahepatic cholangiocarcinoma; OS, overall survival; PD-L1, programmed death ligand 1.

Figure 3

Immunohistochemical staining of human ICC tissues using anti-PD-L1 and CD8 monoclonal antibody. Notes: (A) ICC tissue sections were analyzed by IHC for PD-L1 expression on tumor cells and CD8⁺ T-cell infiltration. PD-L1 positivity was defined as ≥5%, and the number of CD8⁺ T-cells was assessed in five distinct microscopic fields (×200). (B) Tumors were classified as PD-L1+ (≥5%) and PD-L1− (<5%) and analyzed for the amount of CD8 (P-value<0.001). (C) Correlation studies were performed for PD-L1 and CD8A in 54 ICC tissues by linear regression. β-Actin was used as an internal control. r: Spearman’s correlation coefficient. (D) Correlation studies were performed for PD-L1 and CD8A in the 149 ICC patients from National Center for Biotechnology Information Gene Expression Omnibus database (GSE33327) by linear regression. r: Spearman’s correlation coefficient. Abbreviations: ICC, intrahepatic cholangiocarcinoma; IHC, immunohistochemistry; PD-L1, programmed death ligand 1.

Figure 4

PD-L1 expression was upregulated in ICC by IFN-γ. Notes: (A) Correlation studies were performed for (left) PD-L1 and (right) IFN-γ CD8A and INF-γ in 54 ICC tissues. β-Actin was used as an internal control. r: Spearman’s correlation coefficient. (B) Correlation studies were performed for (left) PD-L1 and (right) IFN-γ CD8A and INF-γ in the 149 ICC patients from GSE33327. r: Spearman’s correlation coefficient. (C and D) After HCCC9810 and RBE cells were treated with recombinant IFN-γ (0, 10 and 20 ng/mL) for 24 hours, PD-L1 was examined by (C) Western blot and (D) flow cytometry. Abbreviations: ICC, intrahepatic cholangiocarcinoma; IFN, interferon; PD-L1, programmed death ligand 1; RBE, human ICC cell line.