healthmedicinet

Human Subjects

Twenty-nine chronic hepatitis B (CHB) patients, 28 chronic HBV-infected patients in
the immune tolerant (IT) stage, 33 in the HBeAg-negative inactive carrier (IC) and
22 healthy controls (HC) were enrolled in the cross-sectional study. CHB patients
as well as IT and IC were diagnosed according to the described criteria 39]. Subjects with previous antiviral therapy, co-infection of HIV or other hepatitis
virus, diabetes, severe systemic illness, regular alcohol over-consumption and hepatocellular
carcinoma were excluded. Basic characteristics of the enrolled subjects were summarized
in Table 1.

Six CHB patients from a phase IV, multi-center, open-label clinical trial of telbivudine
(600 mg/d, n?=?21, trial number CLDT600ACN07T) were enrolled for longitudinal study,
and heparinized blood was taken at baseline, 12 and 24 weeks of telbivudine treatment
(Table 2), respectively. The study protocol was approved by the Medical Ethical Committee
of Nanfang Hospital of Southern Medical University. Prior to the collection of blood
sample, the informed consent for participation in the study was obtained from each
participant or parent if the participant is under 18 years of age.

Serological assays and HBV-DNA assays

Presence of HBsAg, HBeAg, anti-HBs, anti-HBc, anti-HBe, anti-HCV, and anti-HDV was
determined using commercial AxSYM MEI kits (Abbott Laboratories, North Chicago, IL,
USA). The HBV-DNA level was quantified using the Roche Diagnostics Cobas Taqman 48
(Meylan, France). The biochemical analyses were conducted with an OLYMPUS AU5400 Full
Automatic Biochemical Analyzer (Olympus Corp., Tokyo, Japan).

ELISA Analysis of Soluble SRA

Soluble SRA levels in human serum samples were measured in duplicate using a commercial
ELISA kit purchased from Uscn Life Sciences (Wuhan, China).

Protein preparation

Recombinant extracellular domain of human SRA was expressed in Escherichia coli using the pET expression system (Novagen, Madison, WI, USA) and purified by nickel-chelating
resins (GE Healthcare, Piscataway, NJ, USA) according to the protocols. The expressed
protein was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE) analysis and western blot analysis using anti-human SRA antibody (Abcam,
Cambridge, MA, USA).

Cell surface and intracellular staining

Whole blood was incubated with FCM Lysing Solution (Mutisciences, Hangzhou, China)
for removal of red blood cells, followed by staining with SRA-ECD protein biotinylated
with Sulfo-NHS-LC-Biotin (Pierce, Rockford, IL, USA) and anti-CD3-APC (BD Biosciences,
San Jose, CA, USA) for flow cytometric analysis of binding of SRA-ECD protein with
T cells.

Intracellular cytokine staining (ICS) was performed as previously described 40],41]. Briefly, freshly isolated PBMCs from CHB patients and HLA-A2(+) PBMCs were selected
for stimulation by HBV core18-27 peptide (FLPSDFFPSV) and 18-mer overlapping peptide
pool covered C open reading frame (1 ?g/mL, GL Biochem, Shanghai, China) plus anti-CD49d
(1 ?g/mL, BD Biosciences) and anti-CD28 (1 ?g/mL) for 10 days. Then the cells were
re-stimulated with 3 ?g/mL of core18-27 and the 18-mer peptide pool for 1 hour, followed
by incubated with 1 mg/mL of brefeldin A (BD Biosciences) for an additional 5 hours.
After surface staining with anti-CD8-APC (BD Biosciences), cells were fixed, permeabilized,
and stained with anti-IFN-?-PE and anti-TNF-?-PECy7 (BD Biosciences) respectively
according to the manufacturer’ s instructions. In this study, positive response was
defined as greater production of either IFN-? or TNF-? by core peptide-stimulated
CD8⁺ T cells compared with either culture medium-stimulated or HIV gag peptide-stimulated
CD8⁺ T cells. Patients with a positive CD8⁺ T cell response were chosen for the T-cell suppression assays. The percent inhibition
was calculated by the following formula: percentage of inhibition?=?[(normal activity
– inhibited activity)/(normal activity)] ×100%.

In Vitro T-Cell Stimulation

CellTrace 5(6)-carboxyfluorescein diacetate succinimidyl ester (CFSE) (Molecular Probes,
Eugene, OR, USA) labeled human T cells were plated at 2?×?10⁵ cells/well in 1 ?g/mL of anti-CD3 Abs (eBioscience, San Diego, CA, USA)-coated plates
in the presence of 0.5 ?g/mL of anti-CD28 Abs (eBioscience) with or without indicated
concentrations of recombinant SRA-ECD protein. In some case, 100 U/ml of IL-2 was
added in the incubation. Proliferation was analyzed using FACS based on the dilution
of fluorescence intensity. Supernatants were collected at 48 hours after the stimulation
for cytokine assays using commercial ELISA kit (eBioscience).

Western blot

Purified T cells were serum starved overnight in complete medium supplemented with
0.5% FCS, and subsequently activated on anti-CD3 antibodies (1 ?g/ml) coated plates
in the presence of anti-CD28 antibodies (1 ?g/ml) with or without SRA-ECD protein
(10 ?g/ml). Activated cells were lysed using RIPA buffer (50 mM Tris, 150 mM NaCl,
and 1% Nonidet P-40, pH 7.4) and protein lysates were subjected to western blot analysis
with antibodies against phospho-ZAP70, ZAP-70 (Cell Signaling Technology, Danvers,
MA, USA) or GAPDH (Thermo scientific Inc., Waltham, MA, USA).

Statistical analysis

Continuous data are expressed as either the median (10th–90th percentile) or the mean?±?SEM.
Independent samples t test, Mann–Whitney test or Chi square test were used in group
comparisons. Spearman’s rank order correlation coefficient was used for correlation
analysis. All statistical analyses were based on two-tailed hypothesis tests with
a significance level of p??0.05.