Cancer Res Treat.  2021 Jan;53(1):162-171. 10.4143/crt.2020.704.

Spatial Distribution and Prognostic Implications of Tumor-Infiltrating FoxP3^- CD4⁺ T Cells in Biliary Tract Cancer

• Kim HD
• Kim JH
• Ryu YM
• Kim D
• Lee S
• Shin J
• Hong SM
• Kim KH
• Jung D
• Song G
• Hwang DW
• Lee JH
• Song KB
• Ryoo BY
• Jeong JH
• Kim Kp
• Kim SY
• Yoo C

• KMID: 2510658
• DOI: http://doi.org/10.4143/crt.2020.704

Abstract

Purpose
The clinical implications of tumor-infiltrating T cell subsets and their spatial distribution in biliary tract cancer (BTC) patients treated with gemcitabine plus cisplatin were investigated.
Materials and Methods
A total of 52 BTC patients treated with palliative gemcitabine plus cisplatin were included. Multiplexed immunohistochemistry was performed on tumor tissues, and immune infiltrates were separately analyzed for the stroma, tumor margin, and tumor core.
Results
The density of CD8⁺ T cells, FoxP3^- CD4⁺ helper T cells, and FoxP3⁺ CD4⁺ regulatory T cells was significantly higher in the tumor margin than in the stroma and tumor core. The density of LAG3- or TIM3-expressing CD8⁺ T cell and FoxP3^- CD4⁺ helper T cell infiltrates was also higher in the tumor margin. In extrahepatic cholangiocarcinoma, there was a higher density of T cell subsets in the tumor core and regulatory T cells in all regions. A high density of FoxP3^- CD4⁺ helper T cells in the tumor margin showed a trend toward better progression-free survival (PFS) (p=0.092) and significantly better overall survival (OS) (p=0.012). In multivariate analyses, a high density of FoxP3^- CD4⁺ helper T cells in the tumor margin was independently associated with favorable PFS and OS.
Conclusion
The tumor margin is the major site for the active infiltration of T cell subsets with higher levels of LAG3 and TIM3 expression in BTC. The density of tumor margin-infiltrating FoxP3^- CD4⁺ helper T cells may be associated with clinical outcomes in BTC patients treated with gemcitabine plus cisplatin.

Keyword

Biliary tract neoplasms; Multiplexed immunohistochemistry; Tumor margin; CD4 helper T cells

Figure

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  Fig. 1. Quantification of the infiltration of T cell subsets according to the spatial distribution. (A) Representative image of multiplexed immunohistochemistry (IHC) demonstrating the selection of regions of interest and fluorescence imaging. (B) Spatial distribution patterns of CD8+ T cells, FoxP3− CD4+ helper T cells, and FoxP3+ CD4+ regulatory T cells (Treg) in the tumor core, tumor margin, and stroma. ***p < 0.001; ns, not significant.

• 

  Fig. 2. Spatial distribution of LAG3- and TIM3-expressing T cell subsets. (A, B) The density of LAG3- and TIM3-expressing CD8+ T cells, FoxP3− CD4+ helper T cells, and FoxP3+ CD4+ regulatory T cells (Treg) in the tumor core, tumor margin, and stroma. *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant.

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  Fig. 3. Comparison of the density of T cell subset infiltrates according to the primary tumor site. (A-C) The density of CD8+ T cells (A), FoxP3− CD4+ helper T cells (B), and FoxP3+ CD4+ regulatory T cells (Treg) (C) according to the primary tumor site. EHCCA, extrahepatic cholangiocarcinoma; GB, gall bladder; IHCCA, intrahepatic cholangiocarcinoma. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 4. Survival outcomes of biliary tract cancer patients according to the density of FoxP3− CD4+ helper T cells. (A, B) Progression-free survival (A) and overall survival (B) of biliary tract cancer patients treated with gemcitabine plus cisplatin according to the density of FoxP3− CD4+ helper T cells in the tumor core, tumor margin, and stroma.

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