Myeloid-derived suppressor cells (MDSCs) have been shown to inhibit T-cell responses in many diseases, but, in hepatitis C virus (HCV) infected patients, MDSCs are still poorly studied. In this assay, we investigated the phenotype and frequency of two new populations of MDSCs denoted as monocytic and granulocytic MDSCs (M-MDSCs and G-MDSCs) in HCV infected patients and analyzed their clinical significance in these patients respectively. We found that the frequency of CD14+
Several studies have shown that persistent HCV infection, which leads to the development of chronic hepatitis C (CHC) or hepatocellular carcinoma (HCC), was associated with impaired T cell responses. It is widely accepted that host immune injury, particularly the impaired T cell responses, plays an important role in HCV persistent infection [
A total of 68 treatment-naive patients with HCV were enrolled from the Third Affiliated Hospital of Sun Yat-Sen University (Guangzhou, China) from April 2012 to July 2010. The population recruited in this study was composed of three groups of subjects, including 56 CHC patients and 12 patients of hepatitis C related liver cirrhosis; 15 healthy controls were randomly selected from the medical center of the Third Affiliated Hospital of Sun Yat-Sen University. All the detailed characteristics of study subjects are presented in Table
Basic characteristics of subjects.
Index | HCV patients |
CHC patients |
Hepatitis C cirrhosis |
Healthy control |
---|---|---|---|---|
Sex |
45/23 | 34/22 | 11/1 | 7/8 |
Age |
42.66 ± 13.17 | 41.20 ± 13.40 | 49.33 ± 9.89 | 30.30 ± 5.40 |
HCV RNA |
5.847 ± 0.122 | 6.017 ± 0.132 | 5.053 ± 0.313 | NA |
HCV genotype | ||||
1b | 20 (29.4%) | NA | ||
6a | 37 (54.4%) | NA | ||
others | 11 (16.2%) | NA | ||
AST |
72.71 ± 6.29 | 65.18 ± 5.57 | 107.83 ± 22.44 | 18.60 ± 6.68 |
ALT |
82.96 ± 6.89 | 84.64 ± 7.29 | 75.08 ± 19.75 | 23.2 ± 5.78 |
NA: not applicable.
The study protocol was approved by the Ethics Review Board of the Third Affiliated Hospital of Sun Yat-Sen University. Written informed consent was obtained from the patients or their families prior to enrollment.
Peripheral blood was drawn (10 mL) into EDTA anticoagulation tubes (Invitrogen, BD) from healthy controls and patients with HCV. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll (Amersham Biosciences) density gradient centrifugation within 4 hours. Cells were washed in RPMI 1640 media (Invitrogen, Grand Island, NY) twice and then resuspended in freeze medium (90% FBS (Life Technologies) and 10% DMSO (Sigma-Aldrich, St. Louis, MO)). Finally, PBMCs were transferred into cryovials (1 mL vial-1), cryopreserved at −80°C, and 72 hours later transferred to the liquid nitrogen.
For analysis, cryovials were removed from the liquid nitrogen, and then were put into the 37°C water bath thawing quickly within 1 min. Then, the cells were resuspended in 10 mL of complete medium (90% RPMI 1640 media, 10% FBS). After being washed twice, the cells were counted by a light microscope after trypan blue dye staining, and then we resuspended the cells and adjusted the concentration to 1 × 106 cells/mL by complete medium.
To determine the frequency and phenotype of CD14+HLA-DR−/low (M-MDSCs) and HLA-DR−/lowCD33+CD11b+CD15+ (G-MDSCs) cells in PBMCs, the following labeled multicolor fluorescence anti-human monoclonal antibodies (mAbs) were used for surface staining: anti-HLA-DR-FITC, anti-CD33-APC, anti-CD11b-PE, anti-CD15-PERCP-Cy5.5, and anti-CD14-PERCP-Cy5.5 (BD Pharmingen, USA). The cells of each sample were incubated with these labeled multicolor fluorescence anti-human monoclonal antibodies at 4°C for 30 minutes. After surface staining, the cells were washed with 2 mL flow staining buffer (PBS plus 1% FBS) at 4°C and centrifuged at 400 g for 5 minutes. Cell pellets were diluted in 300
Plasma HCV RNA was quantified by reverse transcriptase polymerase chain reaction assay (DAAN Gene, Sun Yat-Sen University, China). AST and ALT were tested in the laboratory center of our hospital with Hitachi7170 automatic biochemistry analyzer.
All data were analyzed by SPSS Statistics 17.0 and all figures were made by Prizm 5.0 statistical analysis software (GraphPad Software). Quantitative data normally distributed are expressed as mean ± standard, followed by the
To analyze circulating MDSCs subsets in patients of HCV infection, we first analyzed the expression differences of M-MDSCs and G-MDSCs between HCV infected patients and healthy volunteers. Representative flow cytometry plots of these two distinct cell populations were presented in Figures
Phenotypic analysis of MDSCs subsets in patients with HCV infection. PBMCs were obtained from patients (
M-MDSCs
G-MDSCs
Frequency of two MDSCs subpopulation in HCV patients and healthy control. (a) M-MDSCs from 68 HCV patients and 15 healthy donors. (b) G-MDSCs from 68 HCV patients and 15 healthy donors. (c) Frequency of M-MDSCs and G-MDSCs population in PMBC of HCV patients.
HCV patients were divided into CHC groups and hepatitis C related liver cirrhosis groups based on their different disease progression. The frequency of M-MDSCs in liver cirrhosis patients was higher than CHC patients, but there was no statistical significance (2.995% versus 2.120%,
Frequency of two MDSCs subpopulation in CHC patients compared to hepatitis C cirrhosis patients. (a) M-MDSCs from 56 CHC patients and 12 hepatitis C cirrhosis patients. (b) G-MDSCs of 56 CHC patients and 12 hepatitis C cirrhosis patients.
We also determined whether the increased frequency of MDSCs in HCV infected patients was associated with their clinical parameters (Table
MDSCs were involved in the immune tolerance of various diseases, such as cancers, autoimmune diseases, and acute and chronic infection diseases [
In tumors, two main subsets of MDSCs were identified. One was referred to as M-MDSCs with the expression of CD14, which have a common phenotype and morphology but distinct function compared to monocytes. The other was G-MDSCs with the expression of CD15, which have common phenotype and significant suppressive activity compared to granulocytes [
In this study, we observed a significant elevation of M-MDSCs in peripheral blood of HCV infected patients compared to healthy controls. What is more is that the frequency of M-MDSCs was also higher than G-MDSCs in HCV infected patients, while the expression of G-MDSCs in HCV patients was as low as healthy controls. These results suggested that HCV infection could cause the proliferation of M-MDSCs. As we all know the density gradient centrifugation has been a traditional method to separate PBMCs, such as lymphocytes and monocytes, from polymorphonuclear cells. Most granulocytes, mature CD15 positive cells, deposit to the cell pellet after Ficoll centrifugation because of their high density. Nevertheless, it is possible that some granulocytes with slightly lower density persisted and accounted for the HLA-
HCV is prone to cause persistent infection and result in HCV related cirrhosis. In addition to the virus mutation, the impaired immune system plays a main role in liver damage. The frequency of M-MDSCs or G-MDSCs in CHC patients was nearly equivalent to CHC patients with liver cirrhosis in this study, which might indicate that these two MDSCs subsets make little sense to the disease progression from chronic CHC to cirrhosis. What is more is that we found that the frequency of M-MDSCs was associated with the age of patients compared to healthy volunteers. Then we divided the HCV patients into age > 40 yr group and age ≤ 40 yr group and found that M-MDSCs in the former group was significantly higher than that of the latter group. It may be related to the duration of HCV infection. As it has found that the younger age of the patients, the shorter duration they may be infected. In other studies, It has been reported that the frequency of M-MDSCs is correlated with the clinical biochemical parameters of HCV patients, including HCV viral load and the level of ALT and AST which reflect liver injury [
With the complexity and heterogeneity of MDSCs, it is difficult to accurately analyze the phenotype of MDSCs subpopulations in human and their clinical significance. Here we have demonstrated a significant accumulation of M-MDSCs rather than G-MDSCs in peripheral blood of HCV infected patients and we also explored the clinical significance of M-MDSCs and G-MDSCs in HCV infected patients. This observation may help to shed light on the possible role of M-MDSCs in HCV infected patients and lay a foundation for the immune targeted therapy to hepatitis C.
Myeloid-derived suppressor cells
Hepatitis C virus
Granulocytic myeloid-derived suppressor cells
Monocytic myeloid-derived suppressor cells
Chronic hepatitis C
Hepatocellular carcinoma
Dendritic cells
Regulatory T cells
Programmed death 1
T cell immunoglobulin and mucin domain 3
Reactive oxygen species
Peripheral blood mononuclear cells
Human immunodeficiency virus
Renal cell carcinoma.
The authors declare no conflict of interests.
Gang Ning and Lanhui She are equal contributors.
This research is supported by Grants of National Natural Science Foundation of China (Grant no. 31370907) and Natural Science Fund from Government Agency, Guangdong province, China (Grant no. S2012010009155), and Medical Study Fund from Government Agency, Guangdong province (Grant number: B2012195). We are grateful to the patients for their participation in this study.