Hepatitis C virus (HCV) determines an acute hepatitis evolving to persistent infection in 50–80% of patients.1 Different mechanisms have been proposed to explain disease evolution, including viral escape, failure of the T helper immune network, and host genetic factors.2 Among CD4+ T cell subsets, lymphocytes expressing constitutively CD25 (interleukin 2 receptor alpha chain), namely T regulatory cells (Treg), appear to play a critical role in controlling chronic evolution of HCV mediated liver diseases.3 Here we investigate the frequency and functional activity of CD3+/CD4+/CD25+ Tregs in acute HCV infection in relation to its evolution over time.
The study was approved by the local ethics committee and included 16 consecutive patients with acute hepatitis C (AHC), 15 consecutive patients with acute hepatitis A (AHA), and 10 healthy donors (table 1). Criteria for diagnosis of acute hepatitis C were in accordance with previous reports.4 At the start of the study, our hospital did not offer interferon α treatment to acute patients, and even when treatment became available, none of the patients signed the written informed consent for treatment but agreed to be followed up. No patient had hepatitis B virus, cytomegalovirus, Epstein‐Barr virus, or human immunodeficiency virus infection markers, a history of alcohol abuse, or evidence of autoimmune liver disease (antinuclear antibody, antimitochondrial antibody, anti‐liver/kidney microsomal antibodies).
Table 1 Patient characteristics.
| Parameter | HCV‐T0 | HCV‐T6 | HAV | HD |
|---|---|---|---|---|
| Age (y) (mean (SD)) | 42 (3) | 40 (2) | 32 (5) | 35 (6) |
| Sex (M/F) | 10/6 | 12/12 | 11/4 | 7/3 |
| ALT level (U/l <40) | 1425 (539) | 100 (50) | 1418 (543) | <40 |
| AST level (U/l <40) | 892 (305) | 84 (24) | 851 (221) | <40 |
| HCV‐RNA (IU/ml (mean (SD)) | 310 (232)×103 | Positive | na | na |
| Genotype | ||||
| 1a/1b | 12/16 | 11/12 | 0 | |
| Non‐1 | 4/16 | 1/12 | ||
| Mode of infection | ||||
| Needlestick | 1/16 | 0 | ||
| Surgery procedure | 2/16 | 0 | ||
| Alimentary | 0 | 15/15 | ||
| Unclear | 13/16 | 0 | ||
| Immunological parameters | ||||
| CD3+ CD4+ | 956 (297)†** | 892 (103) | 725 (200)** | 772 (297)** |
| CD3+ CD4+ CD25+ | 130 (50)†** | 111 (12) | 46 (26)** | 46 (13)** |
| CD3+ CD4+ CD25+ | 13.5 (3)‡** | 11.2 (2) | 6.3 (1)** | 6 (1)** |
| T regulatory functional assay¶ | ||||
| Ratio 1:2¶ | 100§** | 92 | 80 | 79 |
| Ratio 1:20 | 99** | 85** | 70 | 55 |
HCV‐T0, patients with hepatitis C virus at time 0; HCV‐T6, patients hepatitis C virus after six months; HAV, patients with acute hepatitis A; HD, healthy donors; na, not applicable; ALT, alanine aminotransferase; AST, aspartate aminotransferase.
**Statistically significant (Mann‐Whitney U test, p<0.01).
†Total count number (cells/mm3; values are mean (SD)).
‡Percentage.
¶Purified CD3+ CD4+ CD25bright (Treg) were cocultured at different ratios (1:2 and 1:20) with autologous CD3+ CD4+ CD25− lymphocytes in the presence of CD3/CD28 conjugated beads (Dynal Biotech, Oslo, Norway).
§Percentage suppression was calculated assuming 0% counts per minutes (cpm) incorporated by 20 000 CD4+ CD25− purified T cells stimulated with CD3/CD28 coated beads (Dynal) in the absence of Treg. Mean (SD) of the absolute cpm found in healthy controls, HCV, and HAV was 25 000 (5000), 24 000 (6000), and 22 000 (4000), respectively.
Blood samples were collected at admission to hospital (T0), which occurred at least one week after the first clinical symptoms, and re‐tested six months later (T6). HCV‐RNA was assayed qualitatively and quantitatively using Amplicor Roche system 2.5 with cut off points of 50 and 600 IU/ml, respectively. T lymphocytes subsets from peripheral blood were counted by multicolour flow cytometry.5,6 For functional studies, Tregs were isolated by positive selection using antibody coated magnetic beads (Dynal Treg Kit; Dynal, Oslo, Norway). Tregs suppressor activity was measured as described previously.7 The Mann‐Whitney U test (two tailed) and Spearman's test were used for statistical analysis.
An increase in Treg frequency and suppressor activity was observed in 12 (all genotype 1) of 16 AHC patients and persisted for six months after diagnosis (T6), by which time HCV‐RNA was positive, assessed both qualitatively quantitatively, and transaminase levels exceeded twice the upper limit of normal. No significant increase in Tregs was observed in AHA patients (table 1, fig 1). Four of 16 HCV infected patients who showed no significant increase in Treg frequency or function at the time of admission to hospital (patient 1 8% and 80 cells/mm3 genotype 2a/2c; patient 2 7% and 72 cells/mm3 genotype 1b; patient 3 8.5% and 82 cells/mm3 genotype 3a/3c; patient 4 9% and 85 cells/mm3 with genotype 2a/2c) went on to clear the virus (negative HCV‐RNA) and had normal transaminase levels six months after acute hepatitis.
Figure 1 CD4+ CD25+ Treg levels in representative patients with acute hepatitis C virus (HCV) and controls. CD4+ CD25+ Treg levels were higher in acutely HCV infected patients (A) who underwent chronic evolution in comparison with acutely HCV infected patient who resolved the infection (B). (C) Patient with hepatitis A virus (HAV). (D) Healthy donor.
CD4+/CD25+ are known to be increased in patients with chronic hepatitis C but not in those who resolve the infection.3 Our findings indicate that an increase in Treg number and function, if present in the acute hepatitis phase of the infection, predicts a chronic evolution and persists over time. Interestingly, all patients with elevated Tregs and chronic evolution harboured genotype 1 virus, suggesting a possible influence of that subset on chronic evolution,8 perhaps through Treg triggering. Whether the elevation in regulatory T cells reported here is just a predictor of chronic evolution of HCV infection or a promoter of HCV infection chronicity, remains to be investigated.
Footnotes
Conflict of interest: None declared.
References
- 1.Davis G L, Albright J E, Cook S F.et al Projecting future complications of chronic hepatitis C in the United States. Liver Transpl 20039331–338. [DOI] [PubMed] [Google Scholar]
- 2.Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol 20055215–229. [DOI] [PubMed] [Google Scholar]
- 3.Cabrera R, Tu Z, Xu Y.et al An immunomodulatory role for CD4(+)CD25(+) regulatory T lymphocytes in hepatitis C virus infection. Hepatology 2004401062–1071. [DOI] [PubMed] [Google Scholar]
- 4.Nomura H, Sou S, Tanimoto H.et al Short‐term interferon‐alfa therapy for acute hepatitis C: a randomized controlled trial. Hepatology 2004391213–1219. [DOI] [PubMed] [Google Scholar]
- 5.Perrella A, Atripaldi L, Bellopede P.et al Flow cytometry assay of myeloid dendritic cells (mDCs) in peripheral blood during acute hepatitis C: possible pathogenetic mechanisms. World J Gastroenterol 2006121105–1109. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Longhi M S, Ma Y, Bogdanos D P.et al Impairment of CD4(+)CD25(+) regulatory T‐cells in autoimmune liver disease. J Hepatol 20044131–37. [DOI] [PubMed] [Google Scholar]
- 7.Longhi M S, Ma Y, Mitry R R.et al Effect of CD4+ CD25+ regulatory T‐cells on CD8 T‐cell function in patients with autoimmune hepatitis. J Autoimmun 20052563–71. [DOI] [PubMed] [Google Scholar]
- 8.Yoo T W, Donfield S, Lail A.et al Hemophilia Growth and Development Study. Effect of hepatitis C virus (HCV) genotype on HCV and HIV‐1 disease. J Infect Dis 20051914–10. [DOI] [PubMed] [Google Scholar]