title: CD4+CD25+CD127(low) Regulatory T Cells Play Predominant Anti-Tumor Suppressive Role in Hepatitis B Virus-Associated Hepatocellular Carcinoma.
abstract: BACKGROUND
  Hepatocellular carcinoma (HCC) is the second leading cause of cancer death worldwide and hepatitis B is one of the commonest causes. T regulatory cells (Tregs) are strong immunomodulators and are likely to play a major role in HCC development. HBV infection is reported to induce expansion of Tregs. We investigated the CD4+CD25+CD127(-ve)FoxP3+ Tregs in HBV-related HCC as compared to non-HBV-HCC.
  
  
  METHODS
  Whole blood immunophenotyping was analyzed by multicolor flow cytometry in patients with HBV-related HCC (HBV-HCC, n = 17), non-HBV-HCC (n = 22; NASH = 16, alcohol-related = 6), and chronic hepatitis B infection (CHBV; n = 10). Tregs functionality was checked by in vitro suppression assays using CD4+ CD25+ CD127(low) Tregs. Levels of serum alpha-fetoprotein (AFP), expression of FoxP3, IL-10, PD1, TGF-β, and Notch in Tregs, and liver explants were analyzed by flow cytometry, immunohistochemistry, and quantitative RT-PCR.
  
  
  RESULTS
  CD4+CD25+(hi) and Foxp3 expression in CD4+CD25+(hi)CD127(low) was significantly increased (P = 0.04, P = 0.007) in HBVHCC compared to non-HBVHCC and CHBV patients. HBVHCC also showed high IL-10 and TGF-β secreting CD4 + CD25 + (hi)Tregs. The PD1 expression in CD4 + CD25+(hi) was significantly decreased in the HBVHCC than non-HBVHCC. In HBVHCC, AFP levels were significantly high (median 941, range 2-727940) than non-HBVHCC (median 13.5, range 2-18,900). In HBVHCC, patients with high AFP (range; 3982-727940 ng/ml) showed positive correlation with Foxp3 expression in CD4+CD25+(hi) CD127(low) (r = 0.857, P = 0.014). Reduced PD1 expression in HBVHCC also had negative correlation with FOXP3 in CD4+CD25+(hi) CD127(low) (r = -0.78, P = 0.04). However, AFP levels in non-HBVHCC showed negative correlation with (R = -0.67, P = 0.005) with CD4+CD25+(hi) Tregs.
  
  
  CONCLUSIONS
  Our results demonstrate that CD4+ CD25+(hi) Tregs from HBVHCC patients have decreased expression of PD1, resulting in higher IL-10 and TGF-β secretion. Increased suppressive ability of Tregs in HBV-related HCC confers increased anti-tumor suppressive response than in non-HBV-HCC. Modulation of Tregs and PD1 may serve as useful therapeutic targets.
authors.affiliation: Department of Research, Institute of Liver and Biliary Sciences , New Delhi , India.
authors.forename: Shreya
authors.initials: S
authors.lastname: Sharma
authors.affiliation: Department of Research, Institute of Liver and Biliary Sciences , New Delhi , India.
authors.forename: Ritu
authors.initials: R
authors.lastname: Khosla
authors.affiliation: Department of Research, Institute of Liver and Biliary Sciences , New Delhi , India.
authors.forename: Paul
authors.initials: P
authors.lastname: David
authors.affiliation: Department of Pathology, Institute of Liver and Biliary Sciences , New Delhi , India.
authors.forename: Archana
authors.initials: A
authors.lastname: Rastogi
authors.affiliation: Department of Research, Institute of Liver and Biliary Sciences , New Delhi , India.
authors.forename: Ashish
authors.initials: A
authors.lastname: Vyas
authors.affiliation: Department of Research, Institute of Liver and Biliary Sciences , New Delhi , India.
authors.forename: Dileep
authors.initials: D
authors.lastname: Singh
authors.affiliation: Department of Hepatology, Institute of Liver and Biliary Sciences , New Delhi , India.
authors.forename: Ankit
authors.initials: A
authors.lastname: Bhardwaj
authors.affiliation: Department of Hepatology, Institute of Liver and Biliary Sciences , New Delhi , India.
authors.forename: Amrish
authors.initials: A
authors.lastname: Sahney
authors.affiliation: Department of Hepatology, Institute of Liver and Biliary Sciences , New Delhi , India.
authors.forename: Rakhi
authors.initials: R
authors.lastname: Maiwall
authors.affiliation: Department of Hepatology, Institute of Liver and Biliary Sciences , New Delhi , India.
authors.forename: Shiv Kumar
authors.initials: SK
authors.lastname: Sarin
authors.affiliation: Department of Research, Institute of Liver and Biliary Sciences , New Delhi , India.
authors.forename: Nirupma
authors.initials: N
authors.lastname: Trehanpati
keywords: T regulatory cells; alpha-fetoprotein; hepatitis B virus; hepatocellular carcinoma; transforming growth factor-β