Interaction Between TFH Cells And B Cells In Chronic Hepatitis B

CHAPTER ONE THE STUDY FOR THE PHENOTYPE AND PROPORTION OF CIRCULATING TFH CELLS AND CIRCULATING B CELLS IN CHRONIC HEPATISIS B PATIENTSObjectiveTo understand the change of Circulating Tfh cells and B cells in proportion and phenotype, before or after the hepatitis B e antigen serum conversion, in hepatitis B patients undering anti-virus therapy.Methods and materialsthe subjects were consist of the CHB patients treated with anti-virus drug and obtained hepatitis B e antigen serum conversion(HBEAG-),the CHB patients without therapy (HBEAG+), the healthy donors were enrolled as control(Healthy), and the CHB patients with liver cirrhosis and spleen function hyperfunction who were treated with Splenectomy surgery(CHB-sf). Heparinized peripheral venous blood were taken from all subjects and the spleen were taken from the CHB-sf patients after informed consent. Flow cytometry was used to detect the proportions and expression of Tfh cells and B cells in the peripheral blood and spleen cell. ELISA was used to the level of serum IL-21 in all subjects. Clinical parameters of all patients was collectedand sorted.The All data were analyzed using SPSS forWindows software (version 17.0; SPSS). The Kolmogorov-Smirnov test was used for the normality test of each variable. All data are provided as means and SDs for normal distributions. Levene's test was used to test for the homogeneity of the variable. One-way anal-ysis of variance (ANOVA) was used to test the differences in means between various individuals. Nonparametric statistical analysis was performed using the Kruskal-Wallis H test, and Correlation analysis was evaluated by the Spearman rank correlation test. For all tests, two-sided P< 0.05 was considered statistically significant. Results1 Detection the expression of spleen cell and PBMC by FCM Gated on CD4+CXCR5+Tfh cells and CD19+B cells,detecting the expression of CCR7, ICOS,IL-21R and PD-1 molecules on them, the results showed that circular Tfh express higher CCR7 molecules than spleen-Tfh, On the contrary, the levels of PD-1,ICOS molecules expressed on circular Tfh were lower than spleen-Tfh, both the two kinds of Tfh cells expressed the similar IL-21R on themselves; In addition, the levels of CD38 andIL-21R molecules expressed on circular B cells were higher than spleen B cells, whereas the PD-1 molecules was converse, and the CD27 molecules was similar in both B cells.2 Detection the cytokine levels in Tfh cells were detected Peripheral blood mononuclear cells were isolated and cultured with antibody stained by fluorescence, gated on CD4+CXCR5+Tfhcells, by Flow cytometric analysis. The results showed that Circular Tfh cells could secrete higher levels of IFN– gamma(2.23±0.82), in addition, the cytokine of IL - 21 (0.95 + 0.31), IL - 4 (0.8 + 0.35) and IL - 17 (0.93 + 0.48) could be detected too.3 Detection the expression of cytokine in Circulating Tfh cells and B cells by FCM The fresh peripheral blood from volunteers and CHB patients were cultured with antibody stained by fluorescence, the percent of circular Tfh cells, circular B cells and the associated molecules expressed on them were detected by FCM, the results showed, Compared with healthy volunteers, CHB patients express higher circular CCR7+Tfh cells and lower plasma cell (P < 0.05); In both CHB patients groups,the patients with antiviral treatment expressed higher circular plasma cells than without treatment ones, and the levels of circular Tfh cells and circular CCR7+ Tfh cells were similar;Three groups of people expressing the same levels of total B lymphocytes frequency and IL-21R positive plasma cells;Further analysis on the associated molecules expressed on the B cells, the results showed that the level of PD-1 the expressed on plasma cells from CHB patients higher significantly than the healthy volunteers, whereas the difference was not significant in both CHB patients groups; In addition, the level of IL-21R expressed on plasma cells in three groups were also similar.4 Detection the level of serum IL-21 by ELISA all the serum samples were collected from healthy volunteers and patients, and detected the levels of IL-21,the results showed that, compared with healthy volunteers(407.29±65.75), the levels of serum IL-21 in the CHB patients were higher, whereas whereas the difference was not significant in both CHB patients groups.5 Correlation analysis between the Clinical and laboratory parameters To understand the relationship between the laboratory parameters and the degree of hepatisis, we analized the Correlation and the result showed that the IL-21R and PD-1 molecules expressed on plasma cells was negatively associated with HBV-DNA and ALT, the IL-21R expressed on CCR7+Tfh cells was positively associated with GGT,whereas the percent of circular Tfh cells and CCR7+Tfh cells showed no correlation with the liver function parameters.ConclusionThe molecules expressed the circular Tfh(B)cells and the spleen-Tfh(B)cells were different significantly, the CCR7+ molecules was higher but the PD-1, ICOS was lower in the circular Tfh, in addition, the circular Tfh cells could secreted amount of IFN-gama and IL-17, IL-21 and IL-4, which may be a substype of Tfh cell and play an important roles in antivirus.During the antivirus treatment, with the decreasing of hepatisis B e antigen and increasing of hepatisis B e antibody, the percent of circular Tfh cells, CCR+Tfh cells, plasma cells and the levels of serum IL-21 were increased, whereas the total B cells was constant in all subjects;In addition, the IL-21R and PD-1 molecules expressed on plasma cells was negatively associated with HBV-DNA and ALT,the IL-21R expressed on CCR7+Tfh cells was positively associated with GGT, consequently, we conclude that the Tfh cells and plasma cells were associated with immune mechanism of antivirus. CHAPTER TWO THE STUDY FOR THE INTERACTION BETWEEN TFH CELLS AND B LYMPHOCYTES IN VITROObjective To further study the interaction between Tfh cells and B lymphocytes under the stimulation of different cytokine the in vitro, and judge whether IL-21 could promote the production of HB e Ab.Methods and materials The subjects were consist of 10 CHB patients without therapy (HBEAG+), Heparinized peripheral venous blood were taken from all subjects. CXCR5+cells were further isolated by microbeads from the patients, and then cultured with the stimulus of IL-10, IL-21, IL-4 alone for one week. In some study, CXCR5+cell was stained with CFSE before culture to judge proliferation. Flow cytometry was used to detect the fluorescence intensity of CFSE, the expression and proportion of Tfh cells and B cells after 7 days'culture. ELISA was used to the level of secreted HBeAg in culture supernant. The All data were analyzed using SPSS for Windows software (version 17.0; SPSS). All data are provided as means and SDs for normal distributions. One-way anal-ysis of variance (ANOVA) was used to test the differences in means between various groups. Nonparametric statistical analysis was performed using the Kruskal-Wallis H test. For all tests, two-sided P< 0.05 was considered statistically significant. Results1 Detection the purity and composition of CXCR+cells by FCM The CXCR5+cells were isolated from PBMC in 10 patients infected with HBV, cultured with antibody stained with fluorescence, Detected the purity and composition of CXCR+cells by FCM,the results showed that the purity of CXCR+cells, which were composed of CD4+cell(55.25%±17.82%) and CD19+cells(36.98%±8.25%), was 68.82%±12.23%.2 Detection the proliferation of B cells stained with CFSE The CXCR5+cells were stained with CFSE, and then cultured under the stimulus of cytokines for 7days in vitro, we detected the percent of B cells and the dilution degrees of CFSE absorbed in B cells before and after culture, the result showed that the percent of B cells was 32.21%±6.82% before culture, after 7days'culture, the percent of B cells in the groups which stimulated by IL-21(54.35±6.38%) was significantly higher than IL-4 (48.22%±9.17%), IL-10 (40.21%±6.62%), and blank controls (37.71%±8.21%), and IL-4 groups which showed statistically significant different from IL-10 and blank controls (P < 0.05).3 Detection the quantity of HB e Ab in Supernatant by ELISA After 7days'culture under the stimulus of different cytokines in vitro, the quantity of HB e Ab in Supernatant was detected by ELISA, the results showed that the groups stimulated by IL-21(0.729±0.132 pg/ml) were significantly different from IL-4 (0.429±0.128 pg/ml), IL-10 (0.468±0.135 pg/ml), and blank controls (0.429±0.128 pg/ml),whereas the IL-4,IL-10 groups didn't show statistically significant different from blank controls.Conclusionwe could isolate the CXCR5+cells from PBMC in CHB patents by Magnetic beads methods, and establish a co-culture system between Tfh cells and B cells, which benefit to judge the interaction between the two kinds of cells, under the signal background of CD40L and icos molecules expressed on Tfh cells, the IL-21 and other cytokines could stimulate the proliferation of B cells and the production of immunoglobulin, as our study showed, the IL-21 perform well than other cytokines, especially, we found that the IL-21 could promote the proliferation of B cell isolated from CHB patients and production of hepatisis B antibody-Ig G, which may function in hepatisis B e antigen serum conversion.