High frequency of functional anti-YMDD and -mutant cytotoxic T lymphocytes after in vitro expansion correlates with successful response to lamivudine therapy for chronic hepatitis B

Abstract

Background: Many determinants for a sustained response to lamivudine therapy have been reported but the role of T cell responsiveness remains unclear. The finding that tyrosine-methionine-aspartate-aspartate (YMDD) motif of the reverse transcriptase domain of hepatitis B virus (HBV) DNA polymerase carries a HLA-A2 restricted cytotoxic T lymphocyte (CTL) epitope makes quantitative measurement of the numbers of peptide specific CTLs feasible using MHC tetramer-peptide complex staining. Aim: To investigate the correlation between anti-YMDD motif CTL activity and the efficacy of lamivudine therapy in HLA-A2 positive patients with chronic hepatitis B (CH-B). Methods: The function and phenotype of peptide and interleukin 2 expanded peripheral blood mononuclear cells were quantified by cell lytic assay and immunocytochemical analysis by staining with HLA-A2-peptide tetramer complexes. Results: After in vitro expansion, sustained responders had more potent CTL responses against YMDD, YVDD, and YIDD, as well as other epitopes on HBV antigens than non-responders. The frequency of YMDD/YVDD/YIDD motif specific CTLs increased significantly with an effective cell lytic function during and after therapy in sustained responders but not in non-responders. YMDD specific CTLs cross reacted with YIDD and YVDD mutant epitopes, and shared T cell receptor gene usages with YIDD and YVDD specific CTLs. Conclusions: Sustained responders, at least HLA-A2 patients, elicited a more potent CTL immunity against YMDD and its mutants. YMDD specific CTLs are cross reactive with YVDD and YIDD mutant epitopes, which may further contribute to immune clearance of the mutant viruses and a successful response to lamivudine therapy in CH-B patients.

HBcAg-specific CD4+CD25+regulatory T cells modulate immune tolerance and acute exacerbation on the natural history of chronic hepatitis B virus infection

“…Likewise, Figure 1b shows the prediction of 15-mer peptides of continuous amino acid sequences on HBcAg 1-30 using the SYFPEITHI scoring system on DRB1*0401 molecule correlates proliferation assays of CD4 + CD25 + T reg cells from DRB1*0401 patients in terms of [H 3 ] thymidine incorporation. The proliferation in response to peptide P16 (HBcAg [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] , with an SYFPEITHI score = 20 on DRB1*0401 molecule) was comparable to that of rHBcAg, thus this peptide (as P2 in Supplementary Table 1) was selected for the construction of DRB1*0401 tetramer in this study. These data of proliferation are consistent with reports that MHC class II tetramers may identify peptide-specific human CD4 + T cells proliferating to viral antigens [27,34], and with that T reg cell population could be induced and expanded by foreign antigen [35].…”

“…Amino acids that are regarded as having a negative effect on the binding ability are given values between )1 and )3. The 17 peptides with 15-mer amino acids used in this study were arbitrarily designed (their SYFPEITHI scores not shown, Supplementary Table 1), of which, P2, LSFLPSDFFP SVRDL (HBcAg [16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] ) and P13, PPAYRPPNAPILSTL (HBcAg 129-143 ) had the highest SYFPEITHI scores, i.e., 20 and 28, for DRB1*0401 and DRB1*0101 molecules, respectively. They were used for the construction of each of MHC class II tetramers.…”

“…Preparation of HLA-A2-HBcAg 18-27 tetrameric complexes HLA-A2-restricted epitope peptide HBcAg [18][19][20][21][22][23][24][25][26][27] FLPSDFFPSV, an immunodominant cytotoxic T lymphocyte (CTL) epitope on HBcAg, is currently the most widely used epitope for assaying CTL activities in HBV patients [2,[17][18][19][20][21]. We used HLA-A2-HBcAg 18-27 tetrameric complexes in this study to monitor CTL activities for the correlation with T reg f during AEs in HLA-A2 patients.…”

“…The culture procedures using the 15-mer peptide P2 or P13 combined with recombinant HBcAg (rHBcAg) to stimulate LITs were carried out as described [20,22]. Generally, the CRI-p culture method gives 5-to 15-fold increase in total T reg cell numbers compared to that obtained directly from fresh LITs or PBMCs ex vivo [20,22].…”

Clinical and virological features of non-breakthrough and severe exacerbation due to lamivudine-resistant hepatitis B virus mutants

“…This difference from our study may suggest that our patients were predominately Genotype C, in contrast to the European study, which mainly involved those of Genotype A or D. Another possibility may be the presence of fewer substitutions in the rt region at commencement of therapy, either alone or with emergence of YMDD mutant, as discussed below. A recent report showed that sustained lamivudine responders with HLA-A2 elicited more potent cytotoxic Tlymphocyte (CTL) immunity against YMDD and its mutant (YIDD and YVDD) [Lin et al, 2005]. Although we do not have HLA type data for the patients in our study, anti-mutant CTLs such as those described above may contribute in suppressing the elevation of mutant virus loads.…”