CD4+CD25+CD127low regulatory T cells play predominant anti-tumor suppressive role in hepatitis B virus-associated hepatocellular carcinoma

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. Patients and 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; NAS H = 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-beta, 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-beta 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. Conclusion: Our results demonstrate that CD4+ CD25+(hi) Tregs from HBVHCC patients have decreased expression of PD1, resulting in higher 11,10 and TGF-beta 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.