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Subjects

Fresh heparinized peripheral blood samples were obtained from 30 patients diagnosed
with PBC and 30 unaffected age- and sex-matched controls 11]. All patients (Table 2) were female, and 90 % had readily detectable AMA; the diagnosis was made based on
internationally accepted criteria 11]. The mean age was 61 (43–78) years old (range years), and 100 % of them were taking
ursodiol. Four percent of the PBC patients included in this study were histologically
characterized as cirrhotics. Serum liver function tests were performed utilizing routine
laboratory methods. Subjects were excluded from the study if they had malignancies
or were using immunosuppressive drugs. Patients and controls were matched for age
and sex. After approval from appropriate institutional review board, all subjects
provided written informed consent prior to enrollment in the study. The study design
is illustrated in Fig. 1.

Table 2. Clinical, biochemical, and serological characteristics of PBC patients

CD14+, CD4+ T cell, and CD8+ T cell purification

Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation on a Ficoll-Hypaque
gradient for 30 min at 500g. First, CD14+ cells were isolated from PBMC by positive selection under endotoxin-free
conditions using anti-CD14 conjugated microbeads (Miltenyi Biotec, San Diego, CA,
USA). Thence, CD8+ cells were isolated from the eluate by positive selection using
anti-CD8 conjugated microbeads (Miltenyi Biotec, San Diego, CA, USA). Finally, the
CD4+ T cells were isolated by negative selection using a cocktail of antibodies against
CD8, CD14, CD16, CD19, CD36, CD56, CD123, TCR ?/?, and CD235a (Miltenyi Biotec, San
Diego, CA, USA). Aliquots of the CD14+, CD8+, and CD4+ T cells were subjected to viability
assays and flow cytometry analysis. The purity of these lymphocytic populations was
95 % and the viability 95 %. Genomic DNA from the isolated CD14+, CD8+, and CD4+
T cells was isolated using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA) 42].

Methylated DNA immunoprecipitation and methylation microarrays

DNA samples from PBC patients (?=?10) and controls (?=?10) were sonicated to generate fragments between 200 and 1000 base pairs (bp),
and then immunoprecipitated with a murine monoclonal antibody that specifically recognizes
5-methylcytidine (Diagenode, Liège, Belgium). The immunoprecipitated DNA fragments
were recovered using anti-mouse IgG magnetic beads. The MeDIP-enriched DNA was amplified
by PCR using universal primers and a limited number of cycles (GenomePlex® Complete
Whole Genome Amplification kit). The amplified DNA samples were then purified with
QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). One microgram of immunoprecipitated
and reference DNA was tagged respectively with Cyanine-5 (Cy5) and Cyanine-3 (Cy3)
labeled random 9-mers and then hybridized by the NimbleGen Array Hybridization Kit
(Roche NimbleGen, Madison, WI, USA). The labeled DNA was then purified by isopropanol/ethanol
precipitation. For array hybridization, Roche NimbleGen’s Human Promoter plus CpG
Island array was used, a 3 × 720k format array design containing 27,728 CpG islands
and all well-characterized promoter regions (from about ?2440 to +610 bp of the TSSs)
totally covered by ~720,000 probes.

Bisulfite sequencing

The CpG island DNA methylation status was determined by sequencing bisulfite-modified
genomic DNA 43] from PBC patients (?=?20) and controls (?=?20). One microgram of genomic DNA was bisulfite-converted using the EpiTect Bisulfite
Kit (Qiagen, Valencia, CA, USA). For each gene, primers were designed using the Methyl
Primer Express v1.0 program (Life Technologies-Applied Biosystems, Carlsbad, CA, USA)
corresponding to the region containing the oligonucleotide probe represented in the
DNA methylation bead array. Ten positive recombinant clones were selected randomly
and sequenced using ABI 3730 (Life Technologies-Applied Biosystems, Carlsbad, CA,
USA). The methylation level of gene is assessed according to the percentage of the
methylated CG sites in 10 clones. All primer sequences are listed in Additional file
1: Table S1.

Reverse transcription and quantitative polymerase chain reaction

Total RNA was extracted from all isolated samples (PBC ?=?30, controls ?=?30) using TRI Reagent (Sigma-Aldrich, St. Louis, MO, USA). cDNA was synthesized
from all different cell types using High Capacity cDNA Reverse Transcription Kits
(Life Technologies-Applied Biosystems, Carlsbad, CA, USA). One hundred nanograms of
cDNA in a total volume of 20 ?l was amplified for 40 cycles on an Applied Biosystems
7900 HT Sequence Detection System, using a TaqMan Gene Expression Assay specific for
CXCR3, UBE2A, FUNDC2, and IL1RAPL2 (Cat. #4331182, #4351372, Life Technologies-Applied
Biosystems, Carlsbad, CA, USA). TaqMan® Gene Expression Assays consist of a pair of
unlabeled PCR primers and a TaqMan® probe with a FAM™ dye label on the 5? end and
minor groove binder (MGB) nonfluorescent quencher (NFQ) on the 3? end. RNA from samples
of interest is reverse transcribed into cDNA, and the synthesized cDNA serves as the
template for real-time PCR. ROXâ„¢ dye is used as a passive reference to normalize real-time
PCR reactions. ROXâ„¢ dye provides an internal reference to which the FAMâ„¢ reporter
dye signal can be normalized, which is necessary to correct for fluorescent fluctuations
due to changes in concentration, volume, or light source intensity. Normalization
of the reporter dye signal results in increased data precision. All reactions were
run in duplicate. The relative mRNA expression level of each gene was determined by
normalizing its mRNA level to the internal control glyceraldehyde 3-phosphate dehydrogenase
(GAPDH).

Flow cytometry

Phenotypic analysis of CD4 T cells was assessed using fluorochrome-conjugated monoclonal
antibodies against cell surface markers on a FACSCantoII (BD Biosciences, San Diego,
CA, USA) using FACSDivaâ„¢ software (BD Biosciences, San Diego, CA, USA). Monoclonal
antibodies against human CD3, CD4, CD45RA, CD45RO, and CD183 (CXCR3) were purchased
from BioLegend (San Diego, CA, USA). Frozen cells were thawed and prepared for flow
cytometry as described 44]. Cells were stained for 15 min at room temperature with Aqua viability dye (Life
Technologies). Optimal concentrations of the mAbs were used throughout, and all assays
included positive and negative controls.

Statistical methods

NimbleScan software v2.5 (Roche NimbleGen, Madison, WI, USA) was utilized for DNA
methylation data analysis using a threshold p value of 0.05 equivalent to 1.31 based on the Gaussian distribution of data. Second,
exclusive elements corresponding to specific microarray probes were identified using
a one-sided Kolmogorov-Smirnov (KS) test in affected and healthy subjects; peaks found
only in either group were selected for further analysis. Peaks within 500 bp of each
other are merged. If several adjacent probes rise significantly above a set threshold,
the region is assigned to an enrichment peak (EP). NimbleScan detects peaks by searching
for at least two probes above a p value minimum cutoff (?log10) of 2. Third, elements of interest were inserted into
the UCSC Genome Browser to identify corresponding genes. The filter condition of different
methylated regions was set to be no less than six EPs in one group and not more than
three EPs in the other group. Based on GO analysis and potential involvement in the
pathogenesis of PBC, four genes were selected for validation using isolated CD4+,
CD8+, and CD14+ cells from 20 additional patients with PBC and 20 additional normal
controls. For RT-qPCR, the data were analyzed with Delta Delta Ct using global normalization.
Statistical differences between groups were identified using an unpaired t test taking into account transcripts with p value 0.05.