Elevated Frequency and Function of Regulatory T Cells in Patients

Abstract Background Regulatory T cells (Tregs) play a pivotal role in the persistence of hepatitis C virus infection. The aim of this study was to evaluate the frequency and function of Tregs in patients with chronic hepatitis C (CHC). Methods We enrolled 44 CHC patients with elevated alanine aminotransferase (ALT) levels (CH group), 13 CHC patients with persistent normal ALT levels (PNALT group), and 14 age-matched healthy subjects (HS group; controls). Tregs were identi? ed as CD4? , CD25? , and forkhead box P3 (Foxp3)? T lymphocytes, using threecolor ? uorescence-activated cell sorting (FACS).

The frequency of Tregs was determined by calculating the percentage of CD4? CD25high T cells among CD4 T cells. CD127 and CD45RA were also analyzed for subsets of Tregs. The levels of serum transforming growth factor (TGF)-b and interleukin (IL)-10 in immunosuppressive assays were detected by enzyme-linked immunosorbent assay (ELISA). The immunosuppressive abilities of Tregs were evaluated by measuring their ability to inhibit the proliferation of effector cells. Results Higher proportions of Tregs were found in the CH and PNALT groups compared with the HS group.

The populations of CD127 low/negative and CD45RA negative cells were higher in the CH group than in the PNALT group. The expressions of IL-10 and TGF-b in the CH and PNALT groups were signi? cantly higher than those in the HS group. In addition, the immunosuppressive ability of Tregs from the CH group was increased relative to that in the PNALT and the HS group. Conclusions CHC patients, irrespective of liver function, had higher frequencies of Tregs than healthy subjects; however, only CHC patients with in? ammation showed enhanced immunosuppressive function of Tregs.

Keywords Hepatitis C A Immunosuppression A Regulatory T cells Abbreviations HCV Hepatitis C virus CHC Chronic hepatitis C ALT Alanine aminotransferase HCC Hepatocellular carcinoma NK Natural killer Treg Regulatory T cell Foxp3 Forkhead box P3 TGF Transforming growth factor IL-10 Interleukin-10 K. -C. Tseng A Y. -H. Hsieh A N. -S. Lai Department of Internal Medicine, Buddhist Dalin Tzu Chi General Hospital, Chia-Yi, Taiwan K. -C. Tseng A Y. -H. Hsieh A N. -S. Lai School of Medicine, Tzuchi University, Hualien, Taiwan Y. -C. Ho A C. Li A S. -F.

Wu (&) Department of Life Science, Institute of Molecular Biology, National Chung-Cheng University, No. 168, University Rd. , Min-Hsiung, Chia-Yi 62102, Taiwan e-mail: [email protected] edu. tw Z. -H. Wen Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Kaohsiung, Taiwan Introduction Hepatitis C virus (HCV) is a small, enveloped, singlestranded, positive-sense RNA virus that belongs to the 123 J Gastroenterol family ? aviviridae and genus hepacivirus [1]. After HCV infection, the immune systems of 55–85% of infected patients cannot eliminate the virus, and in these cases, chronic hepatitis C (CHC) may develop [1].

Chronic hepatitis C is often asymptomatic, with persistently normal serum alanine aminotransferase (ALT) levels in 20–30% of patients, although it is usually associated with ? uctuating or persistently elevated ALT levels [2, 3]. Major long-term complications of CHC include cirrhosis, end-stage liver disease, and hepatocellular carcinoma (HCC) [1]. Retrospective and prospective studies on the long-term natural history of HCV infection have shown that 15–20% of CHC patients develop cirrhosis within 30 years [4].

Once cirrhosis occurs, the annual risk of HCC, hepatic decompensation, and liver-related death is approximately 1–4, 5, and 2–4%, respectively [4–7]. After HCV infection, interactions between innate and adaptive immune responses play pivotal roles in the perpetuation or clearance of HCV. Natural killer (NK) cells are specialized lymphocytes that provide one of the ? rst lines of defense during HCV infection. An overall reduction in NK cell activity may cause a predisposition to chronic HCV infection or, alternatively, HCV gene products may downregulate NK cell activity [8].

Evidence also suggests that the clearance and control of HCV infection is dependent on vigorous, multispeci? c immune responses by both CD8 and CD4 T lymphocytes [9]. An early and persistent Th1-dominant CD4 response appears to be critical for preventing chronic infection, whereas a weak or absent Th1 response with a more pronounced Th2 response is associated with the development and maintenance of chronic infection [10, 11]. Recently, regulatory T cells (Tregs) have been identi? ed as a specialized subset of T cells that can suppress autoreactive immune responses to maintain immunological tolerance and inhibit autoimmunity [12].

There are two general categories of Tregs. One Treg subset develops during the process of T-cell maturation in the thymus, resulting in the generation of a naturally occurring population of regulatory T cells. A second subset develops from ? na? ve CD4? CD25- T cells during immune responses in the periphery [13]. Although these Tregs are CD4? cells with high expression of CD25? , the most reliable marker for this subset is the transcription factor forkhead box P3 (Foxp3) [14]. The virus-speci? c induction of Tregs may have two different consequences.

First, it may be an important process that occurs to prevent excessive immuno-pathological damage. Second, it may enable the virus to establish viral persistence [15]. Many studies have investigated the relationship between Tregs and the outcome of CHC infection [16–23]. The majority of these studies showed that Tregs were present at higher frequencies in patients with persistent infections and elevated liver function than in patients who recovered and had normal liver function and in normal subjects. These ? ndings resulted from the Treg-related suppression of HCV-speci?

c T-cell responses [16–22]. However, Bolacchi et al. [23] demonstrated that CHC patients with normal ALT levels had higher levels of transforming growth factor (TGF)-b production by Tregs than CHC patients with elevated ALT levels. Of note, the majority of these studies only focused on the frequency but not the function of Tregs. Our aim in this study was to investigate the frequency and function of immune cells, including Tregs, in CHC patients with elevated liver function and to compare these patients with CHC patients who had persistently normal liver function and healthy subjects.

Patients and methods Study subjects A total of 71 subjects who had been followed at the Dalin Tzu Chi General Hospital in southern Taiwan during the period between November 2008 and June 2010 were recruited for this study. Among them, 44 were CHC patients with elevated ALT levels (CH group), 13 were CHC patients with persistent normal ALT levels (PNALT group), and 14 were healthy subjects (HS group; controls). The clinical backgrounds of these 3 groups were similar, except for their serum AST and ALT levels (Table 1).

The criteria for inclusion in the CH group were a positive anti-HCV antibody test for at least 6 months, a serum ALT level greater than the upper limit of the normal level, and detectable serum HCV RNA. The criteria for inclusion in the PNALT group were a positive anti-HCV antibody test for at least 6 months, but with a normal ALT level for more than 12 months. Exclusion criteria included malignant neoplasms, decompensated liver disease, acute hepatitis, autoimmune diseases, alcohol abuse, positive hepatitis B surface antigen, and HIV infection.

The study was approved by the Ethics Committee of the Dalin TzuChi General Hospital (approval number: B09604008-1). All patients signed written informed consents. HCV quanti? cation and genotyping Serum HCV RNA levels were measured using the COBAS TaqMan HCV assay (Roche Molecular Diagnostics, Basel, Switzerland), with a lower limit of quanti? cation of 25 IU per ml. HCV genotyping was performed using the LINEAR ARRAY Hepatitis C Virus Genotyping Test (Roche Molecular Diagnostics). 123

J Gastroenterol Table 1 Characteristics of the study subjects Characteristics Age (years)a Gender (M/F) AST a CH group (n = 44) 56. 0 ± 10. 7 18/26 93. 6 ± 54. 4 PNALT group (n = 13) 57. 9 ± 7. 0 3/10 23. 5 ± 5. 1 HS group (n = 14) 54. 9 ± 11. 6 8/6 19. 4 ± 3. 3 P value 0. 749c 0. 198b CH vs. PNALT \0. 001d CH vs. HS \0. 001d PNALT vs. HS 0. 02d ALT a 137. 8 ± 82. 8 22. 4 ± 7. 2 20. 1 ± 4. 3 CH vs. PNALT \0. 001d CH vs. HS \0. 001d PNALT vs. HS 0. 319d HCV RNA (106 IU/ml)a,f Genotype 1 Non-1 f 2. 84 ± 3. 71 27 17 2. 64 ± 4. 02 5 2

NA NA CH vs. PNALT 0. 077e CH vs. PNALT: 0. 699b AST aspartate aminotransferase, ALT alanine aminotransferase, HCV hepatitis C virus, CH group chronic hepatitis C patients with elevated alanine aminotransferase, PNALT chronic hepatitis C patients with persistent normal alanine aminotransferase, HS healthy subjects, NA not applicable a b c d e f Values are expressed as means ± SD v2 test or Fisher’s exact test Analysis of variance (ANOVA) Student’s t-test Mann–Whitney U-test HCV RNA was undetectable for six subjects in the PNALT group

Preparation of peripheral blood mononuclear cells (PBMCs) Heparinized venous blood was obtained (10 ml), and cell surface markers were analyzed immediately after collection. PBMCs were isolated by Ficoll/sodium isophthalamide density-gradient centrifugation (relative density = 1. 077 g/ml) at 400g for 30 min at room temperature. Cells at the interphase were collected, washed, and resuspended in RPMI-1640 medium supplemented with 10% fetal calf serum (FCS), 5 mg/ml L-glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin [24].

Surface and intracellular antibody staining The monoclonal antibodies used to detect cell surface markers in direct immuno? uorescence assays were anti-CD3 (clone HIT31, or UCHT1), anti-CD4 (clone RPA-T4,), antiCD25 (clone M-A251s), anti-CD45RA (clone HI100), antiCD56 (clone MEM188, or B159), and anti-CD127 (clone HIL-7R-M21); all the antibodies were obtained from BD Pharmingen (Franklin Lakes, NJ, USA). Negative control samples were stained with isotype-matched control monoclonal antibodies (mAbs). After staining, the cells were washed extensively and analyzed by ? ow cytometry.

To analyze the intracellular expression levels of Foxp3, freshly isolated PBMCs were ? xed with 1% paraformaldehyde and permeabilized with 0. 5% Triton X-100, followed by detection using anti-Foxp3 (eBioscience, San Diego, CA, USA). Flow cytometry was performed with a Becton Dickinson (Franklin Lakes, NJ, USA) FACScalibur cytometer, and the data were analyzed using CellQuest software (BD Biosciences, San Jose, CA, USA). Isolation of different cell populations PBMC single-cell suspensions were prepared from whole blood and depleted of erythrocytes by ammonium chloride lysis.

To purify Tregs, the cell suspensions were incubated with a human Treg separation cocktail containing biotinconjugated anti-CD8, CD11b, CD16, CD19, CD36, CD41a, CD56, CD123, CD235a, and cd TCR, and APC-conjugated anti-CD25 mAbs (BD Biosciences, San Jose, CA, USA). Streptavidin-conjugated beads (BD Biosciences) were added, and the bound cells were isolated using a BD IMagnet. Bead-bound cells were used as supporting cells. Suspensions of these cells were incubated with anti-APC coated beads (BD Biosciences), and then puri? ed again using a BD IMagnet. After this second round of cell puri?

cation, the suspended cells were used as responder cells and the beadbound cells were the puri? ed Tregs. For the coculture assays, 3 types of cells from the same donor were used. 123 J Gastroenterol Detection of cytokines by enzyme-linked immunosorbent assay (ELISA) To measure plasma cytokine concentrations, samples were obtained from each individual’s peripheral blood. The resulting plasma or the supernatant of cocultured medium was transferred to a microtube and stored at -80°C. The sample was thawed at room temperature at the time of cytokine measurement.

Each cytokine was measured according to the manufacturer’s instructions for each relevant assay kit. The concentration of IL-10 was measured with a commercial ELISA kit (BD OptiEIA ELISA kits). The detection limit was 2 pg/ml. The concentration of TGF-b was measured with a commercial ELISA kit (Quantikine human TGF-b1 immunoassay; R & D Systems, Minneapolis, MN, USA). The total TGF-b1 concentration was measured according to the manufacturer’s instructions: 0. 1 ml of 1 N HCl was added to dilute plasma, and the sample was mixed to activate TGF-b1 and then incubated for 1 h at room temperature, after which the mixture was neutralized with 0.

1 ml of 1 N NaOH. The detection limit was 4. 61 pg/ml. Measurement of the immunosuppressive function of Tregs To evaluate the immunosuppressive function of Tregs, supporting cells were resuspended in T-cell medium at a concentration of 5 9 107 cells/ml and incubated with 20 lg/ml mitomycin C (Sigma-Aldrich, Saint Louis, MO, USA) at 37°C for 40 min. The cells were then washed twice and resuspended in T-cell medium. Responder cells were resuspended in T-cell medium at 106 cells/ml and incubated with 10 lM carboxy?

uorescein diacetate succinimidyl ester (CFSE) (Invitrogen, Grand Island, NY, USA) at 37°C for 10 min. After a fourfold volume of prechilled T-cell medium was added, the cells were washed twice and resuspended in T-cell medium. Supporting cells and responder T cells were cocultured at a ratio of 5:1 in the presence or absence of activating anti-CD3 antibodies (BD Biosciences) at 1 lg/ml. Immunosuppressive ability was assessed by adding additional Tregs at a ratio of 1/5:1 or 1/2:1 to the effector T cells. After 4. 5 days of culture, the cells were evaluated by ? ow cytometry.

The percent suppression of proliferation by Tregs was calculated as proliferation with Tregs follows [25]: 1 A proliferation without Tregs A 100. Statistical analysis For the baseline variables, analysis of variance (ANOVA), Student’s t-test, and the Mann–Whitney U-test were used to compare the means of continuous variables. The v2test was used for the comparisons between categorical variables. Fisher’s exact test was used instead of the v2test when the numbers were small. The percentages of cell populations and cytokine expression were compared using Student’s t-test. A P value of\0.

05 was considered signi? cant. Results Lymphocyte populations were altered in the peripheral blood of the PNALT and CH groups To investigate whether CHC patients had signi? cant alterations in lymphocyte subpopulations in their peripheral blood, we primarily focused on NK cells, CD4? T cells, and CD8? T cells. The PBMCs of 44 CHC patients with elevated liver function (CH group), 13 CHC patients with normal liver function (PNALT group), and 14 age-matched healthy subjects (HS group) were stained with anti-CD3, anti-CD4, anti-CD8, anti-CD25, and anti-CD56 antibodies and analyzed by ? ow cytometry. CD4?

T cells were identi? ed as the CD3? CD4? population (Fig. 1a). The percentage of CD4? T cells was increased in the CH and PNALT groups compared with that in the HS group (Fig. 1c; CH vs. HS: 34. 158 ± 10. 174 vs. 26. 815 ± 8. 362%, P = 0. 014; PNALT vs. HS: 35. 822 ± 10. 400 vs. 26. 815 ± 8. 362%, P = 0. 015). However, the percentage of CD4? T cells did not signi? cantly differ between the CH and PNALT groups. NK cells were identi? ed as the CD3-CD56? population (Fig. 1b). The percentage of NK cells was signi? cantly decreased in the CH and PNALT groups compared to that in the HS group (Fig. 1c; CH vs.

HS: 14. 843 ± 9. 120 vs. 30. 430 ± 13. 242%, P \ 0. 001; PNALT vs. HS: 18. 774 ± 11. 543 vs. 30. 430 ± 13. 242%, P = 0. 020). However, the percentage of NK cells did not signi? cantly differ between the CH and PNALT groups. The CD3? CD8? population, which consisted of cytotoxic anti-virus immune cells other than NK cells, also showed no differences between these groups (Fig. 1c). Regulatory T-cell populations were increased in the PNALT and CH groups As described above, the population of CD4? T cells was increased in the CHC patients. CD4 T-helper cells include a variety of subtypes, including Tregs.

Human peripheral blood contains heterogeneous populations of CD4? CD25? cells with either high expression of CD25, which is associated with regulatory functions, or moderate expression of CD25 [16, 26]. We de? ned a threshold for CD25 expression such that those cells that showed greater expression than CD4-CD25? T cells were considered to be CD4? CD25high Treg cells (Fig. 2b). As shown in Fig. 2, Tregs were identi? ed by CD3? CD4? CD25high 123 J Gastroenterol Fig. 1 Expression of CD4, CD8 and CD56 on peripheral blood mononuclear cells (PBMCs) in the HS, PNALT, and CH groups.

PBMCs were isolated from the whole blood of healthy subjects (HS, n = 14), chronic hepatitis C patients with persistent normal alanine aminotransferase (PNALT, n = 13), and chronic hepatitis C patients with elevated alanine aminotransferase (CH, n = 44). a CD4? T-cell analysis of PBMCs was performed by anti-CD3 and anti-CD4 staining. b Natural killer cell analysis of PBMCs was performed by anti-CD3 and anti-CD56 staining. c The mean percentages and standard deviations of CD3? CD8? cells, CD3? CD4? cells, and CD3-CD56? cells in the PBMCs were determined. CD3? CD4?

cells were statistically significantly increased in the PNALT (P = 0. 015) and CH (P = 0. 014) groups compared with the HS group (HS 26. 815 ± 8. 362%, PNALT 35. 822 ± 10. 400%, CH 34. 158 ± 10. 174%); however, CD3-CD56? cells were statistically signi? cantly decreased in the PNALT (P = 0. 020) and CH (P \ 0. 001) groups compared with the HS group (HS 30. 430 ± 13. 242%, PNALT 18. 774 ± 11. 543%, CH 14. 843 ± 9. 120%) staining (Fig. 2a, b). Tregs were signi? cantly increased in both the CH and PNALT groups compared to the HS group (Fig. 2c; CH vs. HS: 2. 860 ± 1. 459 vs. 1. 521 ± 0.

363%, P = 0. 001; PNALT vs. HS: 2. 832 ± 1. 416 vs. 1. 521 ± 0. 363%, P = 0. 002). CD127 and CD45RA expression in the PNALT and CH groups The above data showed that the percentages of CD4? CD25high were signi? cantly increased in both the PNALT and CH groups (Fig. 2c). It has been indicated that low expression levels of CD127, the receptor alpha chain of IL-7, identify CD4? CD25? cells that express a high level of Foxp3 and have suppressive activities. To characterize the CD cell populations in CHC patients, CD4? T cells were examined for the expression of CD25 and CD127. As shown in Fig.

3a, the population was de? ned by CD127 low/- and CD25?. We compared the percentages of CD4? CD25? CD127 low/- cells between the PNALT and CH groups. The percentage was signi? cantly higher in the CH group compared to the PNALT group (Fig. 3b; CH vs. PNALT: 18. 016 ± 5. 200 vs. 9. 230 ± 2. 789%, P = 0. 018). We also detected the expression of CD45RA in the PNALT and CH groups. As shown in Fig. 3c, the expression of CD25? populations was mostly distributed in the CD45RA- cells. We compared the percentages of CD4? CD25? CD45RA- cells between the PNALT and CH groups. The percentage was signi?

cantly higher in the CH group compared to the PNALT group (Fig. 3d; CH vs. PNALT: 12. 512 ± 3. 999 vs 3. 976 ± 1. 107%, P = 0. 003). Increased Treg Foxp3 expression in the PNALT and CH groups Foxp3 is the most important transcription factor expressed by Tregs that in? uences their survival, differentiation, and function. To investigate the expression of Foxp3 in Tregs from the three studied subject groups, freshly isolated PBMCs were evaluated after intracellular staining with antiFoxp3 (Fig. 4a). The percentage of Foxp3-expressing Tregs was increased in the CH and PNALT groups compared to the HS group (Fig.

4b; CH vs. HS: 2. 263 ± 1. 122 vs. 0. 787 ± 0. 751%, P = 0. 007; PNALT vs. HS: 2. 350 ± 1. 292 vs. 0. 787 ± 0. 751%, P = 0. 018); however, there was no difference between the CH and PNALT groups. Increased IL-10 expression in regulatory T-cell inhibition assay and TGF-b expression in PBMCs in the PNALT and CH groups To estimate the cytokine expression in the immunosuppressive assay of Tregs in the three studied subject groups, we detected the IL-10 and TGF-b expression levels.

The supernatants of Tregs cocultured with effector T cells (1:2) were collected from the HS, PNALT, and CH groups. 123

J Gastroenterol Fig. 2 Analysis of regulatory T cells among the groups. a PBMCs were gated on total CD3? T cells and analyzed for co-expression of CD4 and CD25 within the T-cell gate. b CD4? CD25high T cells are shown in the rectangle drawn in the upper right region of the density plot illustrating CD4? CD25high Treg cells. The threshold for CD25high expression was set at a level that was greater than that of the CD4-CD25? cells (dotted line); CD4? CD25low and CD4? CD25- populations are also shown. c The mean percentages and standard deviations of CD4?

CD25high cells in the CD4 T cells were calculated (HS 1.521 ± 0. 363%, PNALT 2. 832 ± 1. 416%, CH 2. 860 ± 1. 459%), and these results were compared between the HS and CH groups (P = 0. 001) and between the HS and PNALT groups (P = 0. 002) ELISA was performed. The expression of IL-10 in the CH and PNALT groups was signi? cantly higher than that in the HS group (Fig. 5a; CH vs. HS: 64. 812 pg/ml ± 9. 724 vs. 29. 657 pg/ml ± 5. 933, P \ 0. 001; PNALT vs. HS: 72. 818 pg/ml ± 11. 989 vs. 29. 657 pg/ml ± 5. 933, P \ 0. 001). The IL-10 production was dependent on the ratio of cocultured effector T cells and Tregs (E:T ratio). As shown in Fig.

5b, the IL-10 concentration was highest at an E:T ratio of 1:1, then it gradually decreased. TGF-b is important in the suppressive function of Tregs. However, we could not detect any differences between the three subject groups (Fig. 5c), probably due to the culture medium containing a high level of TGF-b in serum. But we also evaluated the TGF-b concentration in the serum of the three studied groups. The TGF-b concentration was much higher in the CH and PNALT groups than that in the HS group (Fig. 5d; CH vs. HS: 898. 213 pg/ ml ± 113. 897 vs. 5. 005 pg/ml ± 0. 114, P \ 0.

001; PNALT vs. HS: 837. 413 pg/ml ± 192. 182 vs. 5. 005 pg/ml ± 0. 114, P \ 0. 001). Enhanced immunosuppressive ability of circulating Tregs in CH patients We next evaluated the immunosuppressive abilities of Tregs. The ability of Tregs to mediate immunosuppression was analyzed by coculturing Tregs with CFSE-labeled, autologous CD4? CD25- responder T cells at different ratios. After 4. 5 days of culture, the cells were evaluated by ? ow cytometry. As a result of this stimulation, the mean ? uorescence intensity (MFI) of the dividing cells containing CFSE was decreased, as shown in Fig.

6a. The decrease in the MFI of the positive control sample without Tregs was the most apparent. When Tregs were added to the coculture system, the proliferation of the effector cells was inhibited to varying extents. The histogram of one of the CH patients is shown in Fig. 6a. The immunosuppressive ability of the Tregs was determined by calculating the ratio of the MFI from the effector cells cocultured with Tregs to the MFI of 123 J Gastroenterol Fig. 3 CD127 and CD45RA expression in the PNALT and CH groups. a Expression of CD127 and CD25 in CD4?

T cells in the PNALT and CH groups. Plots were gated for CD4? T cells. CD127 low/CD25? cells were gated in the box R15. b The percentages of CD127 low/- CD25? cells in the PNALT and CH groups were calculated (PNALT 9. 230 ± 2. 789%, CH 18. 016 ± 5. 200%, P = 0. 018). c Expression of CD45RA and CD25 in CD4? T cells in the PNALT and CH groups. CD45RA and CD25 were analyzed in CD4? T cells. The major population expressing CD25? was CD45RA-. d The percentages of CD45RA-CD25? cells in the PNALT and CH groups were calculated (PNALT 3. 976 ± 1. 107%, CH 12. 512 ± 3. 999%, P = 0.

003) Fig. 4 Forkhead box P3 (Foxp3) expression of PBMCs in the HS, PNALT, and CH groups. a Foxp3 expression in CD4? T cells was detected by intracellular staining. b The mean percentages and standard deviations of the Foxp3? cells were determined for the HS, PNALT, and CH groups (HS 0. 787 ± 0. 751%, PNALT 2. 350 ± 1. 292%, CH 2. 263 ± 1. 122%). Foxp3? cells were statistically signi? cantly increased in the PNALT (P = 0. 018) and CH (P = 0. 007) groups compared with the HS group the positive controls (effector cells only), as described in the ‘‘Patients and methods’’ section.

When cocultured at an effector:Treg (E:T) ratio of 1:1/2 or 1:1/5, the suppressive percentages of Treg were calculated to be, for 1:1/2: HS 14. 73%, PNALT 13. 386%, CH 45. 02%; for 1:1/5: HS 3. 208%, PNALT 4. 686%, CH 26. 82%. The immunosuppressive abilities of the Tregs were signi? cantly increased in the CH group compared with the PNALT and HS groups (CH vs. HS: 1:1/2, P = 0. 010; 1:1/5, P = 0. 033; CH vs. PNALT: 1:1/2, P = 0. 013; 1:1/5, P = 0. 055), but the immunosuppressive ability was not signi? cantly different in the PNALT and HS groups (Fig. 6b).

This indicated that the Tregs derived from the CH group exhibited a stronger ability to suppress effector T-cell functions than the Tregs from the PNALT and the HS groups. 123 J Gastroenterol Fig. 5 Cytokine expressions of regulatory T cells (Tregs) in the HS, PNALT, and CH groups. a The supernatants of effector T cells cocultured with Tregs (Teff/Treg = 2:1) were collected from the HS, PNALT, and CH groups. Enzyme-linked immunosorbent assay (ELISA) was performed to detect interleukin (IL)-10 (CH vs. HS, P \ 0. 001; PNALT vs. HS, P \ 0. 001). b The Teff:Treg ratios from one of the patients were 1:1, 2:1, and 5:1, and IL-10 was detected.

c The supernatants of effector T cells cocultured with Tregs (Teff/ Treg = 2:1) were collected from the HS, PNALT, and CH groups. ELISA was performed to detect transforming growth factor (TGF)-b. d Serum was collected from the HS, PNALT, and CH groups. ELISA was performed to detect TGF-b (CH vs. HS, P \ 0. 001; PNALT vs. HS, P \ 0. 001) Discussion In this study, we evaluated the frequency and function of Tregs and the associated T-cell responses in PBMCs from CHC patients with elevated ALT levels and compared these data with the data for both CHC patients with persistent normal ALT levels and healthy subjects.

We demonstrated that the frequencies of Tregs in the CH and PNALT groups were higher than that in the HS group. Furthermore, we also demonstrated that the functional immunosuppression mediated by Tregs from the CH group was stronger than that mediated by Tregs from the PNALT and HS groups. There are two plausible mechanisms for cytotoxic T lymphocyte (CTL)-mediated HCV clearance from the liver after HCV infection: (1) the induction of apoptosis in infected hepatocytes (cytolytic mechanism) or (2) the release of interferon (IFN)-c to suppress HCV replication (non-cytolytic mechanism) [27].

Only the former mechanism leads to an elevation of ALT levels, due to hepatocyte destruction. Chronic infections are usually marked by reduced frequencies of virus-speci? c CD4 and CD8 T cells, suggesting that the appropriate immune response required for spontaneous recovery either fails to develop or is suppressed [28]. Approximately 15–30% of patients achieve spontaneous viral clearance after the initial HCV-speci? c CD4 and CD8 cell responses [9]. Tregs appear to play an important role in determining whether resolution or persistence occurs during acute HCV infection [26]. SmykPearson et al.

[26] demonstrated that patients in whom an acute hepatitis C infection had resolved showed a decrease in Treg functional suppression compared with the level of suppression seen with persistence. This phenomenon is similar with regard to the status of CHC infection. During chronic infection with HCV, HCV-speci? c CD4 and CD8 T cells respond to the HCV viremia, which causes hepatocyte destruction. Our hypothesis was that the more rigorous the T-cell response, the stronger the Treg response would be to compensate for the destructive immune responses. However, this stronger Treg response may also cause viral persistence.

Our study showed that the frequencies of Tregs in the CH and PNALT groups were higher than that in the HS group, a ? nding which was also demonstrated in previous studies [16–22]. In addition to the frequency of Tregs, we also investigated the immunosuppressive function of Tregs. Although there was no statistically signi? cant difference in the 123 J Gastroenterol Fig. 6 Immunosuppressive ability of Tregs in the HS, PNALT, and CH groups. Supporting cells, effector T cells, and Tregs were puri? ed from PBMCs with isolation kits using whole blood from the HS (n = 5), PNALT (n = 5), and CH (n = 11) groups.

a Immunosuppression was estimated using carboxy? uorescein diacetate succinimidyl ester (CFSE) dilution; the histogram from one sample from the CH group is shown here. Supporting cells and effector T cells were cocultured at a ratio of 5:1 and stimulated with anti-CD3 (1 lg/ml). The immunosuppressive ability of Tregs was assessed using different ratios of effector T cells to Tregs (1:1/5 or 1:1/2) and estimated by determining the mean ? uorescence intensity (MFI). b The suppressive percentages were calculated (1:1/2: HS 14. 73%, PNALT 13. 386%, CH 45. 02%; 1:1/5: HS 3. 208%, PNALT 4.

686%, CH 26. 82%), and these results were compared between the HS and CH groups (1:1/2, P = 0. 010; 1:1/5, P = 0. 033) and between the PNALT and CH groups (1:1/2, P = 0. 013; 1:1/5, P = 0. 055) frequency of Tregs between the CH and PNALT groups, there was a statistically signi? cant difference in the functional suppressive abilities of Tregs between these two groups. That is, Tregs had more potent suppressive abilities in CHC patients with in? ammation. This result was consistent with a previous study [19]. Furthermore, our study also demonstrated that there was a signi?

cant difference between the frequency of Tregs in the PNALT and HS groups, but there was no statistically signi? cant difference in the immunosuppressive function of Tregs between these two groups. Combined with the baseline characteristics, we concluded that CHC patients, irrespective of liver function, had higher frequencies of Tregs than the healthy subjects; however, the immunosuppressive function of Tregs was enhanced only in CHC patients with in? ammation (CH group). There have also been a few studies that investigated the functional suppression of Tregs in CHC patients with and without abnormal liver function [16, 18, 19].

However, these studies focused on changes in Th1 cytokine expression by CD4 T cells with or without Treg depletion. IL-10 and TGF-b are two important cytokines for the development and function of Tregs. The release of TGF-b and IL-10 by Tregs suppresses effector T-cell functions and immune responses. In our data, the IL-10 secretion determined from the Treg suppression assay was signi? cantly higher in CHC patients than in the healthy subjects; fur