NKG2A on CD8+ T lymphocytes from HCV-positive patients

466A

AASLD ABSTRACTS

633

ENHANCED EXPRESSION OF CD94/NKG2A ON CD8+ T LYMPHOCYTES FROM HCV-POSITIVE PATIENTS. Jacob

Nattermann, Hans Dieter Nischalke, Georg Feldmann, Tilman Sauerbruch, Ulrich Spengler, University of Bonn, Bonn, Germany Introduction: Impaired cytotoxic activity of CD8+ cytotoxic T lymphocytes (CTL) has been proposed to be involved in the establishment of chronic hepatitis C virus (HCV) infection. However, the mechanisms leading to suppressed cytolytic function of these ceils are only poorly understood. The C-type lectin heterodimer CD94-NKG2A, which specifically interacts with HLA-E, is an inhibitory natural killer ceil receptor (NKR), expressed on about 5% of peripheral h u m a n CD8+ T lymphocytes. Expression of this NKR on CD8+ lymphocytes has been proposed to be modulated by exposure to antigens and serum concentrations of IL-15 and TGF-/3, respectively. Although the functional role of CD94/NKG2A expression on CTL remains to be resolved, upregulation of CD94/NKG2A might act to sense antigen load, fine-tune CTL responses and control peripheral T-ceil tolerance. Therefore, we analyzed expression of CD94/ NKG2A on peripheral CD8+ T cells in HCV-infected patients. Furthermore, we measured serum levels of IL-15 and TGF-/3. METHODS: Thirty HCV mono-infected patients and 10 healthy, un-infected individuals were enrolled into this study. Expression of surface molecules were analyzed via flow-cytometry. Serum concentrations of IL-15 and TGF-/3 were measured by commercial ELISA. RESULTS: HCV-positive patients had a significantly higher proportion of CD94/NKG2A-positive CD8+ lymphocytes (113%) as compared to uninfected individuals (5.6%; p<0.05). Flow-cytometric analysis of T ceil receptor (TCR) expression revealed that NKG2Apositive T ceil from HCV(+) patients are composed of a higher proportion of c~/3 TCR-positive lymphocytes (14.1% vs 7.8%; p<0.04) but reduced numbers of y 3 TCR-positive lymphocytes (13.8% vs. 23.9%; p<0.04) as compared to uninfected patients. Most CD94/NKG2Apositive lymphocytes were negative for CCR7 (<20%) and CD28 (<35%), but positive for CD45RO (>80%), indicating a memory phenotype. No significant differences were found when serum concentrations of IL-15 and TGF-/3 were compared between HCV-infected and un-infected individuals. CONCLUSION: Infection with the hepatitis C virus results in up-regulation of NKG2A-expression on CD8+ T cells. This seems to be a result of exposure to viral antigens but not of elevated serum levels of IL-15 and TGF-/3 and might influence immune responses in HCV infection. Disclosures: Georg Feldmann - No relationships to disclose Jacob Nattermann - No relationships to disclose Hans Dieter Nischalke - No relationships to disclose Tilman Sauerbruch - No relationships to disclose Ulrich Spengler - No relationships to disclose

634

THE TANDEM-REPEAT POLYMORPHISM OF THE D C S I G N R GENE IN HCV INFECTION. Jacob Nattermann, Golo

Ahlenst~'el, Georg Feldmann, Hans Dieter Nischalke, Martin Vogel, University of Bonn, Bonn, Germany; Thomas Berg, HumboldtUniversity, Berlin, Germany; Jiirgen Rockstroh, Rainer Woitas, Tilman Sauerbruch, Ulrich Spengler, University of Bonn, Bonn, Germany Introduction: DC-SIGNR, a C-type lectin that functions as a transreceptor for HIV-1, has recently b e e n described as high affinity receptor for the hepatitis C virus envelope E2 protein. Exon 4 of the DC-SIGNR gene comprises a variable number of 69bp t a n d e m repeats, encoding for parts of the extracellular protein domain. Here, we analyzed the relevance of this gene polymorphism for the intraindividual replication of HCV. Methods: A cross-sectional comparison between HCV mono-infected patients (n-235), HCV/HIV-1 co-infected (n-119), and healthy, un-infected volunteers (n-100) was performed. DCSIGNR polymorphism was analyzed via PCR. For group comparisons of categorical data, Fisher's exact test was used. Differences of continuous data were analyzed for statistical significance with

HEPATOLOGY, October 2003

the Mann Whitney U test. A p-value <0.05 was considered to be statistically significant. Results: No significant differences were detected with regard to the distribution of DC-SIGNR alleles/genotypes in all study groups. Moreover, we found no association between single DCSIGNR allele and the genotype of the infecting HCV strains. However, we found higher average HC viral loads in HCV monoinfected individuals with 5- (14.8x106 -+4.1 copies/ml), 6- (15.5x106 _+5.1 copies/ml), and 7-tandem repeat alleles (13.7x106 -+2.7 copies/ ml) than in subjects with 4- (8.6x106 -+6.6 copies/ml) and 9-tandem repeats (5.5x106 -+ 5.3 copies/ml; p<0.02). These differences in average viral levels were also seen, w h e n patients were compared according to their DC-SIGNR genotypes. Of note, HIV/HCV co-infected patients had significantly higher levels of serum HCV-RNA as compared to HCV mono-infected subjects (4.8 vs. 13.5 x 106 copies/ml; p<0.001), and no association was observered between the DC-SIGNR polymorphism and hepatitis C viraemia within the cohort of HIV/HCV co-infected subjects. Conclusion: Taken together, our study demonstrates a relation between length of DC-SIGNR and viral load in hepatitis C monoinfected patients, supporting a role of DC-SIGNR for modulating the course of hepatitis C. Disclosures: Golo Ahlenstiel - No relationships to disclose Thomas Berg - No relationships to disclose Georg Feldmann - No relationships to disclose Jacob Nattermann - No relationships to disclose Hans Dieter Nischalke - No relationships to disclose Jiirgen Rockstroh - No relationships to disclose Tilman Sauerbruch - No relationships to disclose Ulrich Spengler - No relationships to disclose Martin Vogel - No relationships to disclose Rainer Woitas - No relationships to disclose

635 CD4+CD25+

T-REGULATORY LYMPHOCYTES CONTROL EXPANSION OF HEPATITIS VIRUS SPECIFIC CD8+ T CELLS. Simon M Rushbrook, Cambridge University, Cambridge, UK;

Scoff Ward, Nuffield Department of Medicine, Oxford, UK;~ sther Unitt, Cambridge University, Cambridge, UK; Paul Klenerman, Oxford University, Oxford, UK; Graeme J M Alexander, Cambridge University, Cambridge, UK Background: Persistent infection with HCV is associated with poor expansion of HCV specific CD8+ T ceils in contrast to those who eliminate the virus. We demonstrated previously that T-regulatory lymphocytes (T-regs) in patients with chronic HCV infection inhibit HCV specific Thl responses. Activated T-regs are characterised by membranous expression of CD4, CD25, CD45RO, TGF-beta; and intraceilular CTLA-4. We propose that persistent HCV infection is favoured by the generation of virus peptide specific T-regs that inhibit expansion of HCV specific CD8+ cytolytic T ceils. Methods: Peripheral blood was collected from 20 HLA-A2 positive patients with chronic HCV infection. Function of T-regs was assessed by inhibition of anti-CD3 induced proliferation using flow cytometry and proliferation assays. Prior to stimulation with HLA-A2 matched peptides from EBV, CMV or HCV lymphocytes were (a) unfractionated (b) depleted of T-regs using anti-CD25 or (c) depleted of T-regs using anti-CD25 and then reconstituted with T-regs. After 14 days, the proportion of tetramer-specific ceils was determined by flow cytometry. Results: Circulating T-regs suppressed proliferation of CD8+T ceils (n - 6, p - <0.05). CD4+CD25+ T ceils expressed membranous TGF-beta; with anti-CD3 stimulation. Tetramer-bound CD8+ T ceils (HCV-antigen specific) expressed high level TGFbeta Receptor-II in contrast to u n b o u n d CD8+ T ceils. Following depletion of T-regs significant expansion of antigen specific T ceils was noted in 10/20 patients for at least one of the 3 HCV peptides studied (median increase 43-fold, range 1-5163, p-<0.001). Similarly following depletion, 16/20 patients had a 3-fold expansion of CD8+ T ceils for the EBV peptide (p - <0.01) and 5/20 patients had a 7-fold expansion to the CMV peptide (p - 0.063). Re-