A total of 27 patients were enrolled at the Unit of Infectious Diseases and Hepatology of the Azienda Ospedaliero-Universitaria of Parma, Parma, Italy. Twenty patients displayed clinical, biochemical, and virological evidence of anti-HBe-positive chronic hepatitis B (HBsAg positive, anti-HBc positive, HBe negative, anti-HBe positive), with fluctuating levels of ALT (ranging from 22 to 1,372 U/liters) and HBV DNA (ranging from 0.049 ×105 to 1,220 ×105 copies/ml). Nine patients were monitored for at least 3 years; all other patients were monitored for at least 12 months. Patients C8 to C20 were HLA-A2 positive. Screening for HLA-A2 was performed by staining peripheral blood mononuclear cells (PBMC) with a fluorescent anti-HLA-A2.01 antibody (Serotec).
Seven patients had clinical, biochemical, and virological evidence of acute HBV infection (aminotransferase levels at least 10 times the upper limit of the normal range of levels and HBsAg and immunoglobulin M anti-HBc antibodies detected in the serum). All patients were negative for anti-hepatitis C virus, delta virus, human immunodeficiency virus type 1 (HIV-1), and HIV-2 antibodies and for other markers of viral or autoimmune hepatitis. The study was approved by the Ethical Committee of the Azienda Ospedaliero-Universitaria of Parma, and all subjects gave written, informed consent.
The presence or absence of HBsAg, anti-HBsAg, total and immunoglobulin M anti-HBc, HBe, anti-HBe, anti-hepatitis D virus, anti-hepatitis C virus, and anti-human immunodeficiency virus types 1 and 2 was determined by using commercial enzyme immunoassay kits (Abbott Labs, IL; Ortho-Clinical Diagnostic, Johnson & Johnson, Raritan, NJ; DiaSorin, Vercelli, Italy). Levels of HBV DNA in serum samples were quantified by PCR (Cobas Amplicor test; Roche Diagnostics, Basel, Switzerland). HBV genotyping was performed by detecting type-specific sequences in the HBV pol gene domains B and C (INNO-LIPA HBV genotyping line probe assay; Innogenetics, Alpharetta, GA).
Synthetic peptides, peptide-HLA class I tetramers, and antibodies.
A panel of 315 15-mer peptides overlapping by 10 residues and covering the overall protein sequence of HBV genotype D were purchased from Chiron Mimotopes (Victoria, Australia). 15-mer peptides were pooled in 16 mixtures as indicated in Table . Synthetic peptides and the corresponding phycoerythrin (PE)-labeled tetrameric peptide-HLA class I complexes representing the HLA-A2-restricted epitopes on the core peptide spanning amino acids 18 to 27 (FLPSDFFPSV), the envelope peptides spanning amino acids 183 to 191 (FLLTRILTI), 335 to 343 (WLSLLVPFV), and 348 to 357 (GLSPTVWLSV), and the polymerase peptides spanning amino acids 575 to 583 (FLLSLGIHL) and 816 to 824 (SLYADSPSV) of HBV genotype D; the HLA-A2 restricted epitope cytomegalovirus (CMV) pp65 (NLVPMVATV); and the influenza virus epitope matrix (GILGFVFTL) were purchased from Proimmune (Oxford, United Kingdom) and from Beckman-Coulter, Inc. (Fullerton, CA). Anti-CD8 (peridinin chlorophyll protein [PerCP]- or allophycocyanin [APC]-conjugated), anti-CD3 (PerCP-conjugated), anti-CD4 (PE-conjugated), and anti-interleukin-2 (anti-IL-2; APC-conjugated) antibodies were purchased from Becton Dickinson Immunocytometry Systems (San Jose, CA). Anti-gamma interferon (anti-IFN-γ)-fluorescein isothiocyanate (FITC) was purchased from Sigma-Aldrich (St. Louis, MO). Anti-perforin (FITC-conjugated), anti-PD-1 (FITC-conjugated), and anti-CD127 antibodies and goat anti-mouse antibody conjugated with FITC or APC were purchased from BD Biosciences-Pharmingen (San Jose, CA). Anti-CCR7 FITC-conjugated antibody was purchased from R&D Systems (Minneapolis, MN).
HBV amino acid coordinates of 15-mer peptide pools
Isolation of PBMC and in vitro expansion of HBV-specific CD8 cell population.
PBMC were isolated from fresh heparinized blood by Ficoll-Hypaque density gradient centrifugation and resuspended in RPMI 1640 supplemented with 25 mM HEPES, 2 mM l-glutamine, 50 μg/ml of gentamicin, and 8% human serum (complete medium). For CD8 expansion, PBMC were resuspended in complete medium supplemented with IL-7 (5 ng/ml) and IL-12 (100 pg/ml) at a concentration of 2 × 106/ml, seeded at 200 μl/well in 96-well plates, and stimulated with HLA-A2-restricted HBV peptides at a 1 μM final concentration. Recombinant IL-2 (50 IU/ml) was added on day 4 of culture, and the immunological assays were performed on day 10. For PBMC stimulation with overlapping 15-mer peptides covering the overall HBV protein sequence, only IL-2 was added on day 4.
Cell surface and intracellular staining. (i) Staining with tetramers and other surface markers.
A total of 1 × 106 to 4 × 106 PBMC, either freshly isolated or obtained following in vitro expansion of CD8 cell populations for 10 days, were incubated for 30 min at room temperature with PE-labeled tetramers in RPMI 1640 and 8% human serum. After a wash with phosphate-buffered saline-0.1% fetal calf serum, staining was performed for 15 min in the dark by using FITC-, PerCP-, or APC-conjugated antibodies. The cells were then washed and analyzed immediately on a BD Biosciences flow cytometer (FACSCalibur) by using the CellQuest software. Tetramer-positive responses are reported as the percentage of tetramer-positive T cells among the total CD8 population. Frequencies of tetramer-positive cells exceeding 0.02% of CD8+ cells were considered to indicate positive responses. The expansion (n-fold) was calculated as the ratio between the percentage of tetramer-positive T cells after 10 days of in vitro stimulation and the percentage of tetramer-positive T cells detected ex vivo.
(ii) IFN-γ staining.
Tetramer-stained cells were incubated in medium alone (control) or with viral peptides (1 μM) for 1 h; brefeldin A (10 μg/ml) was added for an additional 18 h of incubation. After washing, the cells were stained with anti-CD8 PerCP-conjugated monocolonal antibody for 20 min at 4°C and then fixed and permeabilized as described above. Cells were finally stained with FITC-conjugated anti-IFN-γ for 15 min at room temperature, washed again, and analyzed by flow cytometry.
Enzyme-linked immunospot (ELISPOT) assays were performed using the panel of 315 15-mer peptides pooled in 16 mixtures as described above. HBV-specific T-cell responses were analyzed after overnight incubation with individual peptide mixtures of either PBMC (ex vivo analysis) or short-term polyclonal T-cell lines previously expanded in vitro by 10 days of stimulation with the same peptide mixtures used for the ELISPOT assays (in vitro analysis). Briefly, 96-well plates (Multiscreen-IP; Millipore S.A.S., Malshelm, France) were coated overnight at 4°C as recommended by the manufacturer with 5 μg/ml capture mouse anti-human IFN-γ monoclonal antibody (1DIK; Mabtech, Sweden). Plates were then washed seven times with phosphate-buffered saline-0.05% Tween 20 and blocked with RPMI 1640-10% fetal calf serum for 2 h at 37°C. PBMC (2 × 105/well) or T cells from short-term polyclonal T-cell lines (5 × 104/ well) were seeded in duplicate for each individual peptide mixture. Plates were incubated for 18 h at 37°C in the presence or absence of peptides. After a wash with phosphate-buffered saline-0.05% Tween 20, 50 μl of 1 μg/ml biotinylated secondary mouse anti-human IFN-γ monoclonal antibody (7B6-1; Mabtech, Sweden) was added. After 3 h of incubation at room temperature, plates were washed four times, 100 μl of goat alkaline phosphatase anti-biotin antibody (Vector Laboratories Inc., Burlingame, CA) was added to the wells, and the plates were incubated for a further 2 h at room temperature. Plates were then washed four times, and 75 μl of alkaline phosphatase-conjugated substrate (5-bromo-4-chloro-3-indolyl phosphate; Bio-Rad Laboratories, Hercules, CA) was added. After 4 to 7 min, the colorimetric reaction was stopped by washing with distilled water. Plates were air dried, and spots were counted using an automated ELISPOT reader (AID ELISPOT reader system; Autoimmune Diagnostika). Numbers of IFN-γ-secreting cells were expressed as the numbers of spot-forming cells per 2 × 105 cells. The number of specific IFN-γ-secreting cells was calculated by subtracting the value for the unstimulated control from the value for the stimulated sample. While unstimulated controls for ex vivo analysis were the same for all stimulated PBMC samples, distinct controls were set up for each individual polyclonal T-cell line derived from 10 days of PBMC stimulation with individual peptide mixtures. Positive controls consisted of PBMC stimulated with phytohemagglutinin. Wells were considered positive if they had values at least two times the background value and the number of spots was more than 10. To avoid the possibility of missing weakly positive wells, all in vitro experimental sample sets with a ratio of spot-forming cells in peptide-stimulated and unstimulated wells of more than 1.5 were retested by ICS. ICS was also applied to every positive sample to determine the T-cell subset (CD8+ or CD4+) responsible for IFN-γ production.
Adherent mononuclear cells isolated from PBMC of patients with chronic HBV infection were incubated for 45 min at 37°C with anti-PD-L1 (10 μg/ml) or isotype control antibody (e-Bioscience, Boston, MA). Cells were then washed and coincubated for 10 days with nonadherent cells and specific peptides (the HBV core peptide spanning amino acids 18 to 27 or pools of overlapping 15-mer peptides covering the overall HBV protein sequence) at the final concentration of 1 μM (IL-2 was added on day 4). On day 10, cells were stained with surface antibodies, tetramers, anti-IFN-γ, and anti-IL-2 and analyzed by flow cytometry.
Levels of IFN-γ production in groups with different viremia levels were compared by using the Mann-Whitney test. The correlation between HBV DNA log10 values and frequencies of HBV-specific T cells was evaluated by using linear regression analysis and Spearman's correlation coefficient. The percentages of CD127+ cells and PD-1-positive cells among tetramer-positive CD8 lymphocytes in patients with acute and chronic HBV infections were compared by using the Mann-Whitney test. Frequencies of tetramer-positive CD8 cells and IFN-γ- and IL-2-producing CD8 T cells in samples from chronic patients upon peptide stimulation in the presence or absence of anti-PD-L1 antibody were compared by using the Wilcoxon paired test.