Increased infiltration of regulatory T cells in hepatocellular carcinoma of patients with hepatitis B virus pre-S2 mutant

Chiao-Fang Teng,Ting Wang,John Wang,Tsai-Chung Li,Tzu-Hua Wu,Yi-Hsuan Wen,Han-Chieh Wu,Woei-Cherng Shyu,Ih-Jen Su,Da-Ching Liao,Long-Bin Jeng

doi:10.1038/s41598-020-80935-5

Abstract

Hepatocellular carcinoma (HCC) is a frequent and deadly human cancer worldwide that is intimately associated with chronic hepatitis B virus (HBV) infection. Pre-S2 mutant is a HBV oncoprotein that plays important roles in HCC development and is linked to poor prognosis in HCC patients. However, the profiles of tumor-infiltrating lymphocytes in HCC tissues of pre-S2 mutant-positive patients remain unknown. In this study, we performed fluorescent immunohistochemistry staining to detect the infiltration of ‘anti-tumor’ cytotoxic T lymphocytes (CTLs) and ‘pro-tumor’ regulatory T cells (Tregs) in pre-S2 mutant-positive and -negative HCC patients. We showed that pre-S2 mutant-positive patients had a significantly higher infiltration of CD4+CD25+ cells and forkhead box P3 (Foxp3)-expressing cells but similar CTLs and lower granzyme B-expressing cells in HCC tissues compared with pre-S2 mutant-negative patients. Moreover, the percentage of pre-S2 plus pre-S1 + pre-S2 deletion (pre-S2 mutant) was positively correlated with the density of CD4+CD25+ cells and Foxp3-expressing cells but negatively with granzyme B-expressing cells in HCC tissues. Considering that increased intratumoral Tregs have been shown to promote tumor immune evasion, our data may provide new insights into the pathogenesis of HBV pre-S2 mutant-induced HCC and suggest that therapeutics targeting Tregs may be a promising strategy for treating pre-S2 mutant-positive high-risk patient population.

Highlights

As one of the most common and lethal human cancers, hepatocellular carcinoma (HCC) kills approximately 700,000 people each year worldwide[1,2]
We have identified that the hepatitis B virus (HBV)-related Hepatocellular carcinoma (HCC) patients with either deletion spanning pre-S2 gene segment or high percentage of pre-S2 plus pre-S1 + pre-S2 deletion have a worse prognosis than those without[21]
Transforming growth factor-β1 (TGF-β1) is important to suppress the activities of cytotoxic T lymphocytes (CTLs) through inhibiting the expression of cytotoxic genes, such as granzyme B41

Summary

Introduction

As one of the most common and lethal human cancers, hepatocellular carcinoma (HCC) kills approximately 700,000 people each year worldwide[1,2]. Chronic HBV carriers and HBV-related HCC patients, who carry pre-S2 mutant in liver tissues or blood, have been significantly associated with a higher risk of HCC development and recurrence after surgical resection, r espectively[13,14,15,16,17,18]. We have recently developed a next-generation sequencing (NGS)-based platform for quantitative detection of pre-S deletions in plasma and identified that HCC patients with either deletion spanning preS2 gene segment or high percentage of pre-S2 plus pre-S1 + pre-S2 deletion, who were defined as the pre-S2 mutant-positive HCC patients, have a poorer recurrence-free survival after surgical resection[19,20,21]. Increased levels of Tregs are observed in patients with chronic HBV infection and are linked to impaired immune functions of CTLs, promoting the progression of HCC34. The infiltration of CTLs and Tregs in tumor tissues of these two groups of patients was detected by fluorescent immunohistochemistry (IHC) staining and was further comparatively analyzed

Methods

Results

Conclusion