Changes in immune profile affect disease progression in hepatocellular carcinoma

Abstract

Objective: Hepatocellular carcinoma (HCC) as a chronic liver condition is largely associated with immune responses. Previous studies have revealed that different subsets of lymphocytes play fundamental roles in controlling or improving the development and outcome of solid tumors like HCC. Hence, this study aimed to investigate whether immune system changes were related to disease development in HCC patients. Methods: Peripheral blood mononuclear cells were isolated from 30 HCC patients and 30 healthy volunteers using Ficoll density centrifugation. The isolated cells were stained with different primary antibodies and percentages of different immune cells were determined by flow cytometry. Results: HCC patients indicated significant reductions in the numbers of CD4⁺ cells, Tbet+IFNγ+cells, and GATA+IL-4+cells in peripheral blood in comparison with healthy individuals (p < 0.05). There was no significant change in IL-17+RORγt+cells between patient and healthy groups. In contrast, Foxp3+CD127^lowcell frequency was significantly higher in patients than healthy subjects (p < 0.0001). The numbers of Th1, Th2, and Th17 cells were significantly lower in HCC patients than healthy control (p < 0.0001), although the reduction in Th2 cell numbers was not statistically significant. On the contrary, Treg percentage showed a significant increase in patients compared to healthy subjects (p < 0.0001). Other data revealed that Th1, Th2, and Th17 cell frequencies were significantly higher in healthy individuals than patients with different TNM stages of HCC, with the exception of Th2 in patients with stage II HCC (p < 0.01-0.05). Treg percentage was significantly increased in patients with different TNM stages (p < 0.0001). Among all CD4⁺ T cells, the frequency of Th2 cell was significantly associated with TNM stages of HCC (p < 0.05). Conclusion: Our data provide further evidence to show that immune changes may participate in determining HCC progression and disease outcome. However, it should be mentioned that more investigations are needed to clarify our results and explain possible impacts of other immune cells on the pathogenesis of HCC.

Keywords: T helper cells; cellular immunity; hepatocellular carcinoma; immune system.

Figure 1.

The frequencies of CD4⁺, Tbet+IFNγ+, GATA3+IL-4+, RORγt+IL-17+, Foxp3+CD127low cells in peripheral blood of patient and healthy subjects. PBMCs of Hepatocellular carcinoma patients (n = 30) and healthy controls (n = 30) were stained with monoclonal antibodies. The frequencies of the stained cells were measured using flow cytometry (A, B, C, D, and E) and then analyzed (F, G, H, I, and J). Data are shown as mean ± SD. Two groups with non-normal distributions were compared by Mann–Whitney test, while unpaired t-test was used in case of normal distributions. ^****p < 0.0001, ^*p < 0.05.

Figure 2.

The frequencies of circulating different CD4⁺ T subsets of patient and healthy subjects. PBMCs of Hepatocellular carcinoma patients (n = 30) and healthy controls (n = 30) were stained with monoclonal antibodies. The numbers of Th1, Th2, Th17 cells, and Tregs were measured using flow cytometry (A, B, C, and D) and then analyzed (E, F, G, and H). Values are shown as mean ± SD. Unpaired t-test and Mann–Whitney test were used to compare two groups with normal and non-normal distributions, respectively. ^****p < 0.0001.

Figure 3.

The frequencies of immune cells in different TNM stages of Hepatocellular carcinoma (HCC). The percentages of Th1, Th2, Th17 cells, and Tregs in PBMCs of HCC patients with stage II (n = 9), stage IIIA (n = 15), and stage IIIB (n = 6) and healthy controls (n = 30) were investigated by using flow cytometry (A, B, C, and D) and then analyzed (E, F, G, and H). Data are shown as mean ± SEM. Unpaired t-test and Mann–Whitney test were used to compare two groups with normal and non-normal distributions, respectively. ^****p < 0.0001, ^**p < 0.01, ^*p < 0.05.