Author Affiliations: Peking University People’s Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases, No. 11 Xizhimen South Street, Beijing 100044, China (Kong FY, Feng B, Zhang HH, Rao HY, Wang JH, Cong X and Wei L)

Corresponding Author: Lai Wei, MD, PhD, Peking University People’s Hospital, Peking University Hepatology Institute, No. 11 Xizhimen South Street, Beijing 100044, China (Tel: +86-10-88325727; Email: weilai@pkuph.edu.cn)

 

© 2016, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(16)60054-1

Published online January 7, 2016.

 

 

Acknowledgements: We thank all CHC patients who generously provided blood samples and thank Hong Qin, Qian Jin, Ying Hu, Yan-Hui Chen, Wei Zhang, Kai Deng, Xing-Liang Zhao, Xiang-Yang Li, Hui Hua and He-Chao Li for helping with the specimen handling and experimental process.

Contributors: KFY performed the research, collected and analyzed the data, and wrote the first draft. ZHH, RHY and WJH collected and analyzed the data. CX performed the research. FB and WL contributed to the design and interpretation of the study. WL is the guarantor.

Funding: The work was supported by grants from the National Science and Technology Major Project for Infectious Diseases Control during the 11th Five-Year Plan (2008ZX10002-012 and 2008ZX10002-013) and the 12th Five-Year Plan (2012ZX10002003).

Ethical approval: This study was approved by the ethics committee of Peking University People’s Hospital.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

 

 

BACKGROUND: Chronic hepatitis C virus (HCV) infection causes the skewing and activation of B cell subsets, but the characteristics of IgG⁺ B cells in patients with chronic hepatitis C (CHC) infection have not been thoroughly elucidated. CD4⁺CXCR5⁺ follicular helper T (Tfh) cells, via interleukin (IL)-21 secretion, activate B cells. However, the role of CD4⁺CXCR5⁺ T cells in the activation of IgG⁺ B cells in CHC patients is not clear.

 

METHODS: The frequency of IgG⁺ B cells, including CD27⁻IgG⁺ B and CD27⁺IgG⁺ B cells, the expression of the activation markers (CD86 and CD95) in IgG⁺ B cells, and the percentage of circulating CD4⁺CXCR5⁺ T cells were detected by flow cytometry in CHC patients (n=70) and healthy controls (n=25). The concentrations of serum IL-21 were analyzed using ELISA. The role of CD4⁺CXCR5⁺ T cells in the activation of IgG⁺ B cells was investigated using a co-culture system.

 

RESULTS: A significantly lower proportion of CD27⁺IgG⁺ B cells with increased expression of CD86 and CD95 was observed in CHC patients. The expression of CD95 was negatively correlated with the percentage of CD27⁺IgG⁺ B cells, and it contributed to CD27⁺IgG⁺ B cell apoptosis. Circulating CD4⁺CXCR5⁺ T cells and serum IL-21 were significantly increased in CHC patients. Moreover, circulating CD4⁺CXCR5⁺ T cells from CHC patients induced higher expressions of CD86 and CD95 in CD27⁺IgG⁺ B cells in a co-culture system; the blockade of the IL-21 decreased the expression levels of CD86 and CD95 in CD27⁺IgG⁺ B cells.

 

CONCLUSIONS: HCV infection increased the frequency of CD4⁺CXCR5⁺ T cells and decreased the frequency of CD27⁺IgG⁺ B cells. CD4⁺CXCR5⁺ T cells activated CD27⁺IgG⁺ B cells via the secretion of IL-21.

 

KEY WORDS: chronic hepatitis C; IgG⁺ B cells; CD4⁺CXCR5⁺ T cells; IL-21

Chronic hepatitis C virus (HCV) infection is a major cause of chronic hepatitis C (CHC), cirrhosis, and hepatocellular carcinoma. B cell abnormalities are often noted in CHC patients, and B cell disorders induced by HCV infection include the skewing of B cell subsets with increased numbers of immature B cells and decreased numbers of memory B cells,^[1-4] and an increased activation of naïve and memory B cells with an elevated expression of various activation markers, such as CD86 and CXCR3.^{[5, 6]} IgG⁺ B cells are able to differentiate into IgG antibody-secreting cells (ASCs) and these cells are associated with antigen-specific IgG⁺ memory B cell responses.^[7] However, little is known about the characteristics of IgG⁺ B cells in HCV infection.

 

The activation and differentiation of B cells usually depend on T cells, particularly follicular helper T (Tfh) cells, which belong to a subset of CD4⁺ T cells located in germinal centers (GCs).^[8] Tfh cells are characterized by the expression of chemokine (C-X-C motif) receptor 5 (CXCR5), inducible costimulator (ICOS), programmed cell death 1 (PD-1) and the secretion of interleukin (IL)-21.^[9] Circulating CD4⁺CXCR5⁺ T cells share similar phenotypes and functional characteristics with Tfh cells in GCs.^[10] Circulating CD4⁺CXCR5⁺ T cells are also composed of phenotypically and functionally distinct subsets.^[11-15] The investigation of circulating CD4⁺CXCR5⁺ T cells provides some insights into the characteristics of Tfh cells in GCs. The role of CD4⁺CXCR5⁺ T cells in HCV infection has not been well defined. Feng et al^[16] detected a higher frequency of circulating CD4⁺CXCR5⁺ T cells in the peripheral blood of CHC patients; Spaan et al^[17] showed that IL-21-producing CD4⁺CXCR5⁺ T cells are lower in percentage but efficiently support B cell responses in CHC patients. Considering the importance of Tfh cells in the humoral immune response mediated by B cells,^[9] we further highlighted the potential role of CD4⁺CXCR5⁺ T cells in B cells in CHC patients, that may be helpful to better understand the mechanism of B cell abnormalities in patients with HCV infection.

 

This study investigated the frequency and activity of IgG⁺ B cells in CHC patients and healthy controls (HCs). We also analyzed the proportions of CD4⁺CXCR5⁺ T cell subsets and their association with IgG⁺ B cells. Moreover, we tried to elucidate the effect of CD4⁺CXCR5⁺ T cells on the activation of IgG⁺ B cells in HCV infection.

 

 

Seventy HCV-positive patients infected with genotype 1b and 25 gender- and age-matched HCs, who were negative for hepatitis B virus (HBV), HCV, and human immunodeficiency virus (HIV) infections, were recruited in this study (Table 1). All CHC patients were from Guan County in Hebei province and screened for anti-HCV antibodies positive in 1991 or 1998 as described previously.^[18] None of the CHC patients was co-infected with HBV or HIV. Liver fibrosis was evaluated with FibroScan (Echosens, Paris, France) in 65 CHC patients. Significant fibrosis: FibroScan values were between 7.2 kPa and 9.5 kPa; Severe fibrosis: between 9.6 kPa and 12.5 kPa; and cirrhosis: >12.5 kPa.^[18] The FibroScan values of these patients are illustrated in Table 1. None of the patients had cirrhosis. This study was conducted according to the Declaration of Helsinki guidelines and the ethics committee of Peking University People’s Hospital approved the study. All of the subjects provided written informed consent.

 

The tests used for the analyses of HCV antibodies, HCV RNA, and HCV genotypes were performed as described previously.^{[18, 19]} PBMCs were isolated by density gradient centrifugation using Ficoll-Paque Plus (GE Health Bio-science, AB, Sweden) as previously described.^[20] PBMCs were stored in liquid nitrogen until use.

 

B cells (CD19⁺ cells) and memory B cells (CD19⁺CD27⁺ cells) were selected from PBMCs of CHC patients and HCs using B cell isolation kit (Miltenyi Biotec, Bergisch Gladbach, Germany) and memory B cell isolation kit (Miltenyi Biotec) in accordance with the manufacturer’s instructions. Briefly, B cells were isolated from PBMCs by negative selection, then memory B cells were obtained by positive selection from B cells by using antibody-coated magnetic beads. CD4⁺ T cells were isolated using a human CD4⁺ T cell enrichment kit (Stem Cell Technologies, Vancouver, Canada). CD4⁺CXCR5⁺ T cells were isolated using a CXCR5-PE antibody (eBioscience, San Diego, CA, USA) and PE-selection kit (Stem Cell Technologies) with EasySep™ magnet (Stem Cell Technologies) in accordance with the manufacturer’s instructions. The purity of the isolated cell populations was analyzed by flow cytometry and greater than 90%.

 

Phenotypic analyses of B and T cells were performed with the following anti-human monoclonal antibodies (mAbs): CD10-APC, CD19-PerCP, CD3-PerCP, CD21-PE, CD4-FITC, CD8-FITC and IgG-APC. These mAbs were obtained from BD Biosciences (San Jose, CA, USA). PD-1-PE, ICOS-PE, CD38-PE, CD38-APC, CXCR5-APC, CXCR5-PE, CD27-FITC, CD20-PE, IFN-γ-PE, IL-4-PE, IL-17A-PE and IL-21-PE were purchased from eBioscience, and CD86-PE, CXCR3-PE, IL-21R and CD95-PE were obtained from Biolegend (San Diego, CA, USA). Cell staining was performed as described previously.^[20] Fluorescence minus one (FMO) controls were used to set the gates for positive events. Analyses were performed using flow cytometry (BD FACSCalibur, San Jose, CA, USA), and the flow cytometer was properly aligned to control day-to-day variations. Data were analyzed using the FlowJo software (Tree Star, San Carlos, CA, USA).

 

A total of 1×10⁶ PBMCs in 1 mL of RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (FBS) were incubated with 250 ng/mL anti-CD95 (clone EOS9.1; eBioscience) or functional-grade purified mouse IgM at 37 �� for one day. The cells were incubated with CD19-PerCP, CD27-FITC, IgG-APC and Annexin V-PE (eBioscience) antibodies for 15 minutes at room temperature, washed twice with Annexin V in 1×binding buffer (eBioscience), and flow cytometry was performed.

 

PBMCs in 200 µL of RPMI 1640 medium were stimulated with 50 ng/mL phorbol-12-myristate-13-acetate (PMA) (Sigma-Aldrich, St. Louis, MO, USA) and 1 µg/mL ionomycin (Sigma-Aldrich) in the presence of 1 µl/mL GolgiStop™ (BD Biosciences) for 4 hours. The cells were harvested, washed twice with PBS containing 0.5% FBS (Gibco), and stained with CD3-PerCP, CD4-FITC and CXCR5-APC for 20 minutes at room temperature in the dark. Cells were fixed and permeabilized with 100 µL of a fixation/permeabilization solution (BD Biosciences) for 20 minutes. The cells were washed twice in 1× BD Perm/Wash™ buffer and incubated at room temperature for 20 minutes with IFN-γ-PE, IL-4-PE, IL-17A-PE and IL-21-PE antibodies or isotype controls.

 

Serum IL-21 and IgG were quantified using commercial human IL-21 and total human IgG Platinum ELISA kits (eBioscience).

 

Isolated CD4⁺CXCR5⁺ T cells or non-CD4⁺CXCR5⁻ T cells were co-cultured with allogeneic B cells (CD19⁺ cells) for one day to test the activation of IgG⁺ B cells, or CD4⁺CXCR5⁺ T cells were co-cultured with allogeneic naive B cells (CD19⁺CD27⁻ cells) or memory B cells (CD19⁺CD27⁺ cells) for one week to detect the differentiation of B cell subsets into plasmablast cells and the expression of IgG in cellular supernatants of a co-culture system. The cells were cultured in RPMI 1640 containing 10% FBS, 20 U/mL penicillin and 250 µg/mL streptomycin. For T cell stimulation, soluble anti-CD3 (1 µg/mL) (clone OKT3; eBioscience) and soluble anti-CD28 (5 µg/mL) (clone CD28.2; eBioscience) were added to the cell culture. In addition, the expression of IgG was described in percentages, where 100% represented the highest amount of IgG produced within each separated individual as described by Havenith et al.^[21] For blocking studies, 10 µg/mL recombinant human IL-21R-Fc chimera and 10 µg/mL mouse IgG1-Fc as control (R&D Systems, Minneapolis, MN, USA) were added to the cell culture.

 

Statistical analyses were performed using the SPSS 16.0 software (Chicago, IL, USA), and the data are presented as the mean±standard deviation (SD). Differences between two groups were analyzed using Student’s t test, two-sample Wilcoxon’s rank-sum (Mann-Whitney) test, or Kruskal-Wallis test, according to the data distribution. The associations between variables were tested using Spearman’s rank-order correlation test in Prism Version 5 software (GraphPad, San Diego, CA, USA). A P<0.05 was considered statistically significant.

 

 

PBMCs from 70 HCV-infected subjects and 25 HCs were investigated to characterize IgG⁺ B cells (CD19⁺IgG⁺ cells) during HCV infection. IgG⁺ B cells were divided into CD27⁻IgG⁺ B and CD27⁺IgG⁺ B cells based on the CD27 expression, which is a convenient marker of memory B cells (Fig. 1A). The frequency of IgG⁺ B cells was significantly lower in the CHC patients than in the HCs, and the percentage of CD27⁺IgG⁺ B cells in the total B cells population was significantly lower in the CHC patients than in the HCs (Fig. 1B). We compared the frequency of memory B cells in both groups because CD27⁺IgG⁺ B cells belong to the memory B cell compartments, and the results were in consistent with previous studies.^{[3, 4]} The percentage of memory B cells in total B cells was reduced in the CHC patients (Fig. 1C). Correlation analysis demonstrated that the percentage of CD27⁺IgG⁺ B cells positively correlated with the frequency of memory B cells in the CHC patients (Fig. 1D). The concentrations of IgG antibody were slightly higher in the CHC patients than in the HCs, but the difference was not significant (Fig. 1E).

 

The differentiation and activation of B cells are often dysregulated in autoimmune diseases and chronic infection.^{[22, 23]} CD27⁺IgG⁺ cells were between the differentiation stage of memory B cells and plasmablasts, and in the process of B cell development from memory B cells to plasmablasts, B cells are associated with a loss of CD20 expression and an increase in CD38 expression.^[24] We determined the frequencies of CD20⁺ B cells and CD38⁺ B cells in the IgG⁺ B subsets of the CHC patients and HCs but did not find the differences in the frequencies of CD20⁺ B cells and CD38⁺ B cells in either CD27⁻IgG⁺ B cells or CD27⁺IgG⁺ B cells between the two groups (Table 2). These results indicated that IgG⁺ B cells from the CHC patients shared a similar differentiation phenotype with those from the HCs.

 

To investigate the activation of IgG⁺ B subsets, we first determined whether peripheral B cell disorders are characterized by increased numbers of activated B cells in HCV infection. We divided B cells into mature and immature subsets based on the CD10 expression. Mature B cells (CD10⁻ B cells) were further classified into naïve B cells (CD27⁻ B cells) and memory B cells (CD27⁺ B cells). CD21 was used to identify the activated subsets (CD21⁻cells) in B cells.^[2] The frequency of immature B cells in peripheral blood was higher in the CHC patients than in the HCs. The tissue-like memory B cells (CD27⁻CD21⁻ B cells) and activated memory B cells (CD27⁺CD21⁻ B cells) were increased in the CHC patients (Table 3). We also found that the frequencies of activated CD27⁻IgG⁺ B cells (CD27⁻IgG⁺CD21⁻ B cells) and activated CD27⁺IgG⁺ B cells (CD27⁺IgG⁺CD21⁻ B cells) were increased in the CHC patients (Table 3). Taken together, these increased percentages of activated B cell subsets likely reflected the systemic activating effects of HCV infection on B cells.

 

Activated B cells express distinct activation markers. We first detected whether CXCR3 expression on IgG⁺ B subsets contributed to the migration of B cells from peripheral to inflammation sites on IgG⁺ B subsets.^[4] Our data showed that CXCR3 expression on IgG⁺ B subsets was not different between the CHC patients and HCs (Fig. 2A and B). Activated B cells also expressed high levels of the co-stimulatory molecules CD86 and CD95.^{[6, 25]} We found that the expressions of CD86 and CD95 were increased on CD27⁺IgG⁺ B cells from the CHC patients (Fig. 2C and D).

 

Correlation analysis revealed that CD95 expression on CD27⁺IgG⁺ B cells was negatively correlated with the frequency of CD27⁺IgG⁺ B cells in the CHC patients (Fig. 2E). CD95, which is also known as Fas, interacts with its ligand FasL and induces apoptosis through the extrinsic apoptosis signaling pathway.^[26] The apoptosis frequencies of CD27⁺IgG⁺ B cells from the CHC patients were slightly higher than those from the HCs when the cells were incubated with medium and mouse IgM, but no significant differences were found. However, CD27⁺IgG⁺ B cells from the CHC patients incubated with CD95 antibody significantly increased apoptosis frequency compared with those incubated with medium or mouse IgM. Furthermore, anti-CD95 induced apoptosis frequency in CD27⁺IgG⁺ B cells from the CHC patients compared with those from the HCs (Fig. 2F). Taken together, these results indicated that spontaneous apoptosis of CD27⁺IgG⁺ B cells is comparable in the CHC patients and HCs, but CD27⁺IgG⁺ B cells from the CHC patients are prone to apoptosis at the presence of anti-CD95.

 

CD4⁺CXCR5⁺ T cells are crucial for B cell activation and differentiation.^[9] Our flow cytometry analysis showed that the percentage of circulating CD4⁺CXCR5⁺ T cells was significantly higher in the CHC patients than in the HCs (Fig. 3A and B). Recent studies demonstrated that the coexpression of CXCR5 with ICOS and/or PD-1 is a useful phenotypic profile to distinguish this T cell subset.^{[16, 17]} Our results revealed that the frequencies of PD-1⁺CD4⁺CXCR5⁺ T cells and ICOS⁺CD4⁺CXCR5⁺ T cells were also increased in the CHC patients (Fig. 3C and D). Circulating CD4⁺CXCR5⁺ T cells comprised three subsets, Tfh1, Tfh2 and Tfh17 cells, according to the expression of IFN-γ, IL-4 and IL-17.^[10] The frequencies of Tfh1 and Tfh2 cells were increased in CHC patients, but the proportion of Tfh17 cells was not significantly increased in the CHC patients compared with that in the HCs (Fig. 3E). IL-21 secreted by CD4⁺CXCR5⁺ T cells promotes the activation and differentiation of B cells. Our data showed that IL-21⁺CD4⁺CXCR5⁺ T cells and serum IL-21 were significantly higher in the CHC patients than in the HCs (Fig. 4A and B). The frequency of circulating IL-21⁺CD4⁺CXCR5⁺ T cells was also positively correlated with serum IL-21 concentration in the CHC patients (Fig. 4C), but there was no significant difference in the expression of IL-21R on IgG⁺ B subsets between the two groups (Fig. 4D-F).

 

The frequency of circulating CD4⁺CXCR5⁺ T cells was positively correlated with CD95 expression on CD27⁺IgG⁺ B cells in the CHC patients (Fig. 5A). A similarly positive correlation was observed between the proportion of ICOS⁺CD4⁺CXCR5⁺ T cells and the expression of CD86 in CD27⁺IgG⁺ B cells from the CHC patients (Fig. 5B). The frequency of PD-1⁺CD4⁺CXCR5⁺ T cells was positively correlated with the frequency of CD27⁺CD21⁻IgG⁺ B cells and the expression of CD86 on CD27⁺IgG⁺ B cells in the CHC patients (Fig. 5C and D). The frequency of IL-21⁺CD4⁺CXCR5⁺ T cells was also positively correlated with CD86 and CD95 expression on CD27⁺IgG⁺ B cells in the CHC patients (Fig. 5E and F).

 

Fig. 6A and B showed that anti-CD3 plus anti-CD28-stimulated non-CD4⁺CXCR5⁻ T cells did not have significant effect on CD86 and CD95 expressions on CD27⁻IgG⁺ B cells and CD27⁺IgG⁺ B cells in a co-culture system between the CHC patients and HCs. However, the expressions of CD86 and CD95 on CD27⁺IgG⁺ B cells were significantly higher in the CHC patients than in the HCs, when we substituted CD4⁺CXCR5⁻ T for non-CD4⁺CXCR5⁺ T cells in the co-culture system (Fig. 6C and D). When we added IL-21R-Fc chimera to the co-culture system, the expressions of CD86 and CD95 on CD27⁺IgG⁺ B cells were decreased significantly under the stimulation of anti-CD3 plus anti-CD28 in the CHC patients (Fig. 6E and F).

 

We also examined the role of CD4⁺CXCR5⁺ T cells on plasma cell differentiation and IgG production from B cells. The frequency of plasmablast cells (CD19⁺CD20-CD38⁺ cells) and the production of IgG were detected after the co-culture of CD4⁺CXCR5⁺ T cells with isolated allogeneic naïve B cells or memory B cells for one week, but we did not find significant difference in the frequency of plasmablast cells or the production of IgG in the co-culture system of CD4⁺CXCR5⁺ T cells with naïve B cells or memory B cells between the CHC patients and HCs (Fig. 7A-E). Taken together, these results indicated that the influence of CD4⁺CXCR5⁺ T cells on the function of B cells is primarily reflected in CD27⁺IgG⁺ B cell activation with an increase in distinct activation markers, and IL-21 is responsible for the activation of CD27⁺IgG⁺ B cells that are induced by CD4⁺CXCR5⁺ T cells in CHC patients.

 

 

The present study identified the reduced frequency, but increased activation, of CD27⁺IgG⁺ B cells with an increase of distinct markers in the CHC patients. CD4⁺CXCR5⁺ T cells also activated CD27⁺IgG⁺ B cells through IL-21, and the blockade of IL-21 decreased the activity of CD27⁺IgG⁺ B cells.

 

IgG⁺ B cells are responsible for IgG production, and these cells differentiate into IgG-ASC after stimulation. IgG⁺ B cell abnormalities are associated with autoimmunity diseases and chronic infection.^{[7, 27, 28]} We found a significantly lower frequency of CD27⁺IgG⁺ B cells in total B cells in the CHC patients, suggesting that the reduction of CD27⁺IgG⁺ B cells was associated with HCV infection. Furthermore, we detected a strong correlation between CD27⁺IgG⁺ B cells and memory B cells in the CHC patients, which implied that the factors responsible for the decline of CD27⁺IgG⁺ B cells may be associated with decreased memory B cells in HCV infection.

 

Possible reasons for the decreased memory B cells include increased plasmablast differentiation,^[3] increased migration from the peripheral blood to the inflamed liver,^[4] and increased activation-induced B cell apoptosis.^[3] We did not find that the rate of differentiation of IgG⁺ B subsets into plasmablasts was increased, but the activation of CD27⁻IgG⁺ B cells and CD27⁺IgG⁺ B cells was increased in the CHC patients. Tissue-like memory B cells and activated memory B cells were also elevated in the CHC patients, which was consistent with the result previously reported.^{[1, 29]} The result suggested that several B subsets were associated with HCV-induced immune activation, and abnormal immune activation was related to reductions in CD27⁺IgG⁺ B cells. Our results demonstrated that the expression of CXCR3 on subsets of IgG⁺ B cells was similar in the CHC patients and HCs. Mizuochi et al^[4] suggested that a higher expression of CXCR3 contributes to the migration of memory B cells from the peripheral blood into the liver. Our data indicated that the reduction of CD27⁺IgG⁺ B cells was not associated with CXCR3-mediated migration. We further identified the increases in CD86 and CD95 expression on CD27⁺IgG⁺ B cells, and CD95 was associated with a reduction in CD27⁺IgG⁺ B cells. CD86 was a critical co-stimulatory molecule for the interaction of T cells with B cells,^[30] and the increased expression of CD86 implied that the activation of CD27⁺IgG⁺ B cells may be dependent on T cells. CD95 is a death receptor that initiates the extrinsic pathways of apoptosis for the removal of activated immune cells after these cells performed their function or as a result of incomplete or inappropriate activation.^[26] Our results indicated that the increased expression of CD95 activated CD27⁺IgG⁺ B cells highly susceptible to extrinsic apoptosis through interaction with CD95 ligand. Taken together, the reduction of CD27⁺IgG⁺ B cells was primarily associated with the apoptosis that was mediated by the interaction of CD95 with its ligand in the CHC patients.

 

The mechanisms involved in B cell activation in HCV infection are not well elucidated. Rosa et al^[5] suggested that the engagement of CD81 by HCV E2 protein is responsible for the activation of naïve B cells. Moorman et al^[31] showed that abnormal B cell activation is associated with core-mediated TALL-1 over-expression and SOCS-1 suppression. However, CD27⁺IgG⁺ B cells are switched to memory B cells, which are required for T cell co-stimulation in vivo.^[32] Moreover, our results also showed that the activation of CD27⁺IgG⁺ B cells relies on T cells. Although CD4⁺CXCR5⁺ T cells are associated with HCV infection,^{[16, 17]} the relationship of CD4⁺CXCR5⁺ T cells with CD27⁺IgG⁺ B cell activation was not investigated in the CHC patients in our study. The expressions of ICOS and PD-1 in circulating CD4⁺CXCR5⁺ T cells were lower than Tfh cells in the GCs of tonsils,^[33] but circulating CD4⁺CXCR5⁺ T cells exhibited similar features of Tfh cells in GCs, and these cells were associated with B cell responses. Analysis of circulating CD4⁺CXCR5⁺ T cells is a favorable surrogate strategy for the investigation of Tfh cells in GCs. We found that circulating CD4⁺CXCR5⁺ T cells, PD-1⁺CD4⁺CXCR5⁺ T cells and ICOS⁺CD4⁺CXCR5⁺ T cells were significantly higher in the CHC patients, and these results are consistent with Feng et al.^[16] We also demonstrated that increased percentages of CD4⁺CXCR5⁺ T subsets, specifically the IL21⁺CD4⁺CXCR5⁺ T subsets, were significantly related to the expressions of CD86 and CD95 on CD27⁺IgG⁺ B cells. Based on these results, we proposed that the upregulation of CD4⁺CXCR5⁺ T cells contributes to CD27⁺IgG⁺ B cell activation in CHC patients.

 

Spaan et al^[17] demonstrated that the frequency of CD4⁺CXCR5⁺ T cells in blood is not different between HCs and CHC patients, and the frequency of IL-21⁺CD4⁺CXCR5⁺ T cells or serum IL-21 was lower in CHC patients, but these observations were not replicated in our study. Our finding was consistent with Feng et al^[16] who observed the increase of circulating CD4⁺CXCR5⁺ T cells and IL-21 in CHC patients. Many reasons may be involved in these differences. First, the differences between the study of Spaan et al and our study may be due to the divergent genetic background of the study population because previous research indicated that IL-21 gene polymorphisms affect serum IL-21 levels in viral infection and autoimmune disease.^[34,35] Second, the age of the cohort was different. The subjects in the Spaan et al’s study were younger than ours, and the report from Zhou et al^[36] showed that the percentage of blood CXCR5⁺CD4⁺ cells is increased in the elderly population. A smaller number of CHC patients was also included in Spaan et al’s study to analyze the frequency of CD4⁺CXCR5⁺ T cell subsets. Furthermore, half of the patients in Spaan et al’s study had advanced fibrosis, but most patients in our study did not have fibrosis or only had mild liver fibrosis. The variable levels of fibrosis might have differential effect on the frequency of CD4⁺CXCR5⁺ T cells.

 

The effect of CD4⁺CXCR5⁺ T cells on B cell activation and differentiation is primarily dependent on IL-21.^[37] IL-21 may play a critical role in CD27⁺IgG⁺ B cell activation induced by CD4⁺CXCR5⁺ T cells. As expected, our results indicated that CD4⁺CXCR5⁺ T cells participated in CD27⁺IgG⁺ B cell activation with increased CD86 and CD95 expressions, through the production of IL-21 in an in vitro co-culture system. This study also explored the role of CD4⁺CXCR5⁺ T cells on plasmablast cell differentiation and IgG production from naïve B cells and memory B cells, but no evidence was observed for the dysfunction of CD4⁺CXCR5⁺ T cells on plasmablast cells differentiation and IgG production from B cells. These results suggested that the abnormality of CD4⁺CXCR5⁺ T cells primarily influenced the activation of CD27⁺IgG⁺ B cells with an increased expression of CD86 and CD95 in CHC patients. The identification of CD4⁺CXCR5⁺ T cells and IL-21 as important factors for CD27⁺IgG⁺ B cell activation may aid the design of more selective strategies to tackle CD27⁺IgG⁺ B cell abnormalities.

 

In conclusion, in our study the percentages of activated CD27⁺IgG⁺ B cells significantly increased in CHC patients, and CD4⁺CXCR5⁺ T cells mediated the activation of CD27⁺IgG⁺ B cells through IL-21. Moreover, Tfh1 and Tfh 2 cells significantly increased in CHC patients. Among the CD4⁺CXCR5⁺ T subclasses, Tfh1 cells lacked the capacity to help B cells, but Tfh2 and Tfh17 cells were associated with B cell immune responses.^[10] Further studies are warranted to clarify the role of different CD4⁺CXCR5⁺ T subclasses in CD27⁺IgG⁺ B cell activation in CHC patients.

 

 

1 Sugalski JM, Rodriguez B, Moir S, Anthony DD. Peripheral blood B cell subset skewing is associated with altered cell cycling and intrinsic resistance to apoptosis and reflects a state of immune activation in chronic hepatitis C virus infection. J Immunol 2010;185:3019-3027. PMID: 20656924

2 Holz LE, Yoon JC, Raghuraman S, Moir S, Sneller MC, Rehermann B. B cell homeostasis in chronic hepatitis C virus-related mixed cryoglobulinemia is maintained through naïve B cell apoptosis. Hepatology 2012;56:1602-1610. PMID: 22556016

3 Racanelli V, Frassanito MA, Leone P, Galiano M, De Re V, Silvestris F, et al. Antibody production and in vitro behavior of CD27-defined B-cell subsets: persistent hepatitis C virus infection changes the rules. J Virol 2006;80:3923-3934. PMID: 16571809

4 Mizuochi T, Ito M, Saito K, Kasai M, Kunimura T, Morohoshi T, et al. Possible recruitment of peripheral blood CXCR3+ CD27+ CD19+ B cells to the liver of chronic hepatitis C patients. J Interferon Cytokine Res 2010;30:243-252. PMID: 20377416

5 Rosa D, Saletti G, De Gregorio E, Zorat F, Comar C, D’Oro U, et al. Activation of naïve B lymphocytes via CD81, a pathogenetic mechanism for hepatitis C virus-associated B lymphocyte disorders. Proc Natl Acad Sci U S A 2005;102:18544-18549. PMID: 16339892

6 Oliviero B, Cerino A, Varchetta S, Paudice E, Pai S, Ludovisi S, et al. Enhanced B-cell differentiation and reduced proliferative capacity in chronic hepatitis C and chronic hepatitis B virus infections. J Hepatol 2011;55:53-60. PMID: 21145853

7 Moir S, De Ravin SS, Santich BH, Kim JY, Posada JG, Ho J, et al. Humans with chronic granulomatous disease maintain humoral immunologic memory despite low frequencies of circulating memory B cells. Blood 2012;120:4850-4858. PMID: 23074274

8 Craft JE. Follicular helper T cells in immunity and systemic autoimmunity. Nat Rev Rheumatol 2012;8:337-347. PMID: 22549246

9 Tangye SG, Ma CS, Brink R, Deenick EK. The good, the bad and the ugly - TFH cells in human health and disease. Nat Rev Immunol 2013;13:412-426. PMID: 23681096

10 Morita R, Schmitt N, Bentebibel SE, Ranganathan R, Bourdery L, Zurawski G, et al. Human blood CXCR5(+)CD4(+) T cells are counterparts of T follicular cells and contain specific subsets that differentially support antibody secretion. Immunity 2011;34:108-121. PMID: 21215658

11 He J, Tsai LM, Leong YA, Hu X, Ma CS, Chevalier N, et al. Circulating precursor CCR7(lo)PD-1(hi) CXCR5(+)CD4(+) T cells indicate Tfh cell activity and promote antibody responses upon antigen reexposure. Immunity 2013;39:770-781. PMID: 24138884

12 Locci M, Havenar-Daughton C, Landais E, Wu J, Kroenke MA, Arlehamn CL, et al. Human circulating PD-1+CXCR3-CXCR5+ memory Tfh cells are highly functional and correlate with broadly neutralizing HIV antibody responses. Immunity 2013;39:758-769. PMID: 24035365

13 Schmitt N, Ueno H. Blood Tfh cells come with colors. Immunity 2013;39:629-630. PMID: 24138878

14 Pissani F, Streeck H. Emerging concepts on T follicular helper cell dynamics in HIV infection. Trends Immunol 2014;35:278- 286. PMID: 24703588

15 Schmitt N, Bentebibel SE, Ueno H. Phenotype and functions of memory Tfh cells in human blood. Trends Immunol 2014;35:436-442. PMID: 24998903

16 Feng J, Hu X, Guo H, Sun X, Wang J, Xu L, et al. Patients with chronic hepatitis C express a high percentage of CD4(+)CXCR5(+) T follicular helper cells. J Gastroenterol 2012;47:1048-1056. PMID: 22426636

17 Spaan M, Kreefft K, de Graav GN, Brouwer WP, de Knegt RJ, ten Kate FJ, et al. CD4+ CXCR5+ T cells in chronic HCV infection produce less IL-21, yet are efficient at supporting B cell responses. J Hepatol 2015;62:303-310. PMID: 25281860

18 Rao HY, Sun DG, Yang RF, Liu F, Wang J, Feng B, et al. Outcome of hepatitis C virus infection in Chinese paid plasma donors: a 12-19-year cohort study. J Gastroenterol Hepatol 2012;27:526-532. PMID: 21871021

19 Rao H, Wei L, Lopez-Talavera JC, Shang J, Chen H, Li J, et al. Distribution and clinical correlates of viral and host genotypes in Chinese patients with chronic hepatitis C virus infection. J Gastroenterol Hepatol 2014;29:545-553. PMID: 24090188

20 Guo Z, Zhang H, Rao H, Jiang D, Cong X, Feng B, et al. DCs pulsed with novel HLA-A2-restricted CTL epitopes against hepatitis C virus induced a broadly reactive anti-HCV-specific T lymphocyte response. PLoS One 2012;7:e38390. PMID: 22701633

21 Havenith SH, Remmerswaal EB, Idu MM, van Donselaar-van der Pant KA, van der Bom N, Bemelman FJ, et al. CXCR5+CD4+ follicular helper T cells accumulate in resting human lymph nodes and have superior B cell helper activity. Int Immunol 2014;26:183-192. PMID: 24291746

22 Moir S, Fauci AS. B cells in HIV infection and disease. Nat Rev Immunol 2009;9:235-245. PMID: 19319142

23 Sang A, Zheng YY, Morel L. Contributions of B cells to lupus pathogenesis. Mol Immunol 2014;62:329-338. PMID: 24332482

24 Moir S, Fauci AS. Pathogenic mechanisms of B-lymphocyte dysfunction in HIV disease. J Allergy Clin Immunol 2008;122:12-21. PMID: 18547629

25 Mizuno T, Zhong X, Rothstein TL. Fas-induced apoptosis in B cells. Apoptosis 2003;8:451-460. PMID: 12975576

26 Strasser A, Jost PJ, Nagata S. The many roles of FAS receptor signaling in the immune system. Immunity 2009;30:180-192. PMID: 19239902

27 Joo H, Coquery C, Xue Y, Gayet I, Dillon SR, Punaro M, et al. Serum from patients with SLE instructs monocytes to promote IgG and IgA plasmablast differentiation. J Exp Med 2012;209:1335-1348. PMID: 22689824

28 Shirota Y, Yarboro C, Fischer R, Pham TH, Lipsky P, Illei GG. Impact of anti-interleukin-6 receptor blockade on circulating T and B cell subsets in patients with systemic lupus erythematosus. Ann Rheum Dis 2013;72:118-128. PMID: 22858586

29 Doi H, Tanoue S, Kaplan DE. Peripheral CD27-CD21- B-cells represent an exhausted lymphocyte population in hepatitis C cirrhosis. Clin Immunol 2014;150:184-191 PMID: 24434272

30 Lim TS, Goh JK, Mortellaro A, Lim CT, Hämmerling GJ, Ricciardi-Castagnoli P. CD80 and CD86 differentially regulate mechanical interactions of T-cells with antigen-presenting dendritic cells and B-cells. PLoS One 2012;7:e45185. PMID: 23024807

31 Moorman J, Dong ZP, Ni L, Zhang C, Borthwick T, Yao ZQ. Abnormal B-cell activation associated with TALL-1 over-expression and SOCS-1 suppression during chronic hepatitis C virus infection. Immunology 2009;128:227-235. PMID: 19740379

32 Perez-Andres M, Paiva B, Nieto WG, Caraux A, Schmitz A, Almeida J, et al. Human peripheral blood B-cell compartments: a crossroad in B-cell traffic. Cytometry B Clin Cytom 2010;78:S47-60. PMID: 20839338

33 Amé-Thomas P, Maby-El Hajjami H, Monvoisin C, Jean R, Monnier D, Caulet-Maugendre S, et al. Human mesenchymal stem cells isolated from bone marrow and lymphoid organs support tumor B-cell growth: role of stromal cells in follicular lymphoma pathogenesis. Blood 2007;109:693-702. PMID: 16985173

34 Li N, Zhu Q, Li Z, Han Q, Chen J, Lv Y, et al. IL21 and IL21R polymorphisms and their interactive effects on serum IL-21 and IgE levels in patients with chronic hepatitis B virus infection. Hum Immunol 2013;74:567-573. PMID: 23354321

35 Lan Y, Luo B, Wang JL, Jiang YW, Wei YS. The association of interleukin-21 polymorphisms with interleukin-21 serum levels and risk of systemic lupus erythematosus. Gene 2014;538:94-98. PMID: 24434811

36 Zhou M, Zou R, Gan H, Liang Z, Li F, Lin T, et al. The effect of aging on the frequency, phenotype and cytokine production of human blood CD4+CXCR5+T follicular helper cells: comparison of aged and young subjects. Immun Ageing 2014;11:12. PMID: 25177353

37 Vogelzang A, McGuire HM, Yu D, Sprent J, Mackay CR, King C. A fundamental role for interleukin-21 in the generation of T follicular helper cells. Immunity 2008;29:127-137. PMID: 18602282