Objective To investigate the expression and significance of CD28,cytotoxic T-lymphocyte antigen-4 (CTLA-4),programmed death-1 (PD-1) and T cell immunoglobulin mucin-3 (Tim3) on T lymphocytes in chronic HBV-infected patients.Methods A total of 102 chronic HBV-infected patients,including 42 patients with chronic hepatitis B (CHB),30 patients with hepatitis B-induced liver cirrhosis (LC),and 30 patients with hepatocellular carcinoma (HCC),were enrolled from the First Affiliated Hospital to Soochow University during October 2012 and June 2013.Thirty healthy individuals were also enrolled as controls.Expression of CD28,CTLA-4,PD-1,Tim-3 on T lymphocytes in peripheral blood were determined by flow cytometry,and the differences among groups were analyzed using one-way ANOVA and LSD-t test.Spearman correlation test was performed to analyze the correlations of the expression of CD28,CTLA-4,PD-1,Tim-3 on T lymphocytes with HBV DNA loads,HBeAg and ALT.Results The expression of CD4 + CD28 +,CD8 + CD28 +,CD4 + CTLA-4 + in chronic HBV-infected patients were lower than those in healthy controls.CD4 + CD28 + expression in HCC group was lower than that in CHB group (t =2.373,P ＜ 0.05) ; CD8 + CD28 + expression in LC and HCC group was lower than that in CHB group (t =4.324 and 4.088,P ＜ 0.01) ; CD8 + PD-1 +,CD4 + Tim-3 + and CD8 + Tim-3 + expressions in CHB group were higher than those in LC,HCC group and healthy controls (t =3.051,3.130,3.121,3.254 and 3.723,P ＜0.01).CD8 + PD-1 + expression was positively correlated with ALT levels and HBV DNA loads (r =0.516 and 0.582,P ＜ 0.01) ; CD8 + Tim-3 + expression was also positively correlated with ALT levels andHBV DNA loads (r =0.578 and 0.556,P ＜0.01); PD-1 and Tim-3 expressions on CD8 T lymphocytes were positively correlated with each other (r =0.578,P ＜ 0.01).Conclusion The abnormal expression of the molecules on T lymphocytes in chronic HBV-infected patients is closely correlated with immune function disorder and the progression of the disease.

Objective To investigate the significance of changes in expression of co-stimulatory molecules on T lymphocytes in patients with chronic hepatitis B (CHB) infection.Methods In a casecontrol study,a total of 82 CHB cases including 50 male cases and 32 female cases (the mean age was 42.32 ±3.74) were enrolled in the First Affiliated Hospital to Soochow University from October 2012 to November 2013,together with 30 cases health control (15 male cases and 15 female cases,and the mean age was 42.32 ± 3.74).Patients were divided into three groups:40 cases of non-treated,30 cases of effective-treated and 12 cases of ineffective-treated with anti-viral drugs and immune-related therapy.The expression levels of CD28,CTLA-4,PD-1,Tim-3 on T cells subset from peripheral blood were determined by Flow Cytometry.Serum load of HBV DNA was detected by Real-time PCR and the serology markers such as HBeAg and ALT were detected by conventional methods The Kruskal-Wallis test was used to analysis groups comparison.Independent samples t-test and Mann-Whitney U test were used to two sample comparison.Spearman's rank correlation test was used to analyze the correlation.Results The expression levels of CD28,CTLA-4 and PD-1 on CD4 in health control group was the highest compared to ineffectivetreated group [404.65 (331.65-536.09) vs 277.15 (249.90-344.25) (H=29.81,P＜0.001);32.89 (29.69-39.69) vs 19.26 (11.90-20.56) (H =43.13,P ＜0.001),respectively].The expression level of PD-1 on CD8 in pre-treated group was the highest,while expression level in health control group was the lowest [15.47 (12.50-17.78) vs 3.05 (1.41-3.97) (H=56.60,P＜0.001)].Similarly,the expression level of Tim-3 on CD4 in ineffective-treated group was the highest,while Tim expression on CD4 in health control group was the lowest [199.62 (55.61-239.45) vs 70.62 (53.88-112.32) (H =41.03,P ＜ 0.001)].The expression level of Tim-3 on CD8 in pre-treated group was the highest,while it was the lowest in health control group [82.50 (78.69-84.58) vs 3.07 (1.56-6.87) (H=74.84,P ＜0.001)].Compared to the group with low viral load,the expression level of PD-1 on CD8 both in the pre-treated group with high viral load and the post-treated group was significantly increased [17.87 (13.38-20.94) (U=25.00,P＜0.001); 16.95 (5.39-18.27) (U=63.50,P＜0.001),respectively].Importantly,in the post-treated group,the PD-1 expression on CD4 T (r =0.689,P ＜0.001) and on CD8 T (r =0.751,P ＜ 0.001) was also positively correlated with ALT.Conclusions The abnormal expression of these co-stimulatory molecules may be present in the antiviral treatment process of CHB.It may provide new clues for the reasonable treatment of CHB.

Objective To research the mechanism of the changes of T lymphocyte subtypes and provide reference for clinically prevention,diagnosis and treatment for NSCLC through analysis of the expression of Th1 ,Th2 in Non-small-cell carcinoma (NSCLC)patients.Methods Whole blood (EDTA anticoagulant treatment)from 60 NSCLC patients and 60 healthy sub-j ects were collected to detect of the expression of CD3 +T cells,CD4+T cells and CD8+T cells on T lymphocytes and the lev-els of Th1 and Th2 cells by flow cytometer (FCM),and the absolute value of T lymphocyte by hematology analyzer.Results Compared with normal control group,after surgery 1~3 days NSCLC groups,the percent of CD3 +T,CD4+T,CD8+T cells and the CD4+/CD8+ ratio in the NSCLC patients before surgery were significantly reduced 58.40±10.27 vs 66.58± 6.84,31.32±8.65 vs 39.40±6.43,34.23±8.00 vs 24.31±8.16,0.96±0.23 vs 1.58±0.23 (t=-6.726~14.916,P<0.05).The percent of CD3 +T,CD4+T,CD8+T cells and the CD4+/CD8+ ratio in the NSCLC patients after surgery 1~3 days were also significantly decreased 56.31±8.00 vs 66.58±6.84,27.72±7.55 vs 39.40±6.43,33.69±7.10 vs 24.31± 8.16,0.87±0.31 vs 1.58±0.23 (t=-6.720~14.367,P<0.05).The percent of CD4+T cells in the NSCLC patients af-ter surgery 4~7 days was increased 33.23±4.13 vs 39.40±6.43(t=6.257,P<0.05).Compared with the control group, within the helper T cell subsets,the cell content of Th1,Th2 cells (× 10/μl)and the Th1/Th2 ratio were significantly changed in different extent in the NSCLC group before surgery 6.79±1.34 vs 12.52±3.56,4.82±0.51 vs 2.32±0.82, 1.39±0.84 vs 5.36±1.42 (t=-20.087~18.630,P<0.05).The content of Th1 cells was lower in the NSCLC patients after 1~3 days and 4~7 days 8.86±1.52 vs 12.52±3.56,7.02±1.27 vs 12.52±3.56 (t=7.339~11.275,P<0.05). Conclusion The NSCLC patients presented immune dysfunction,like T lymphocytes and helper T cells decreased and Th2 cells were clearly in the ascendant.Also,the cytotoxic T cells increased by the stimulation of cancer cells,but they began to decrease after the surgery.

Objective: To investigate the specific cytotoxicities of the second and third generations of chimeric antigen receptor (CAR) -engineered T cells (CAR-T) on different lymphomas. Methods: CAR-Ts were prepared by lentivirus packaging and infection of T cells. CCK-8, ELISA and Lactate dehydrogenase cytotoxicity assay were applied to detect the proliferation capacity, the secretion level of inflammatory factor and the specific cytotoxicity. Flow cytometry assay showed the specific cytotoxicity and residual level of CAR-T in lymphomas of treated mice. Results: The results showed that the third generation CAR-T had greater capacity of the specific cytotoxicity and proliferation capacity than of the second generation CAR-T. But there was no prominent change of the secretion level of inflammatory factor. The specific cytotoxicity of the second generation CAR-T on highly aggressive lymphomas Raji was more prominent than in inert EHEB, but also could achieve satisfactory effect. The tumor burden in the mice injected with Raji was lower than in the mice injected with EHEB from nude mice experiment. But the residual level of CAR-T in the EHEB-injected mice was higher than in the Raji-injected ones. So the second generation CAR-T was more suitable for the treatment of indolent lymphoma. Conclusion The second generation CD19 CAR-T could treat aggressive lymphoma in a relatively short period, while the second generation CD19 CAR-T need a longer time in vivo to achieve satisfactory curative effect on the noble lymphoma.