1) CD40L bisulfite sequencing data were obtained on a minimum of

1). CD40L bisulfite sequencing data were obtained on a minimum of seven clones prepared from each of CHIR-99021 cell line both CD4+ and CD8+ T cells isolated from the PBMCs of 20 PBC patients and 20 unrelated controls. The CD40L promoter sequences amplified from CD4+ T cells of PBC patients

showed significantly lower methylation, as compared to healthy controls. The methylation patterns of individually sequenced clones are shown for two representative subjects in Fig. 2A. Overall promoter methylation was determined as the percentage of methylated CpG sites of all possible CpG sites, which indicated a significant reduction in CD4+ T cells from patients, compared to healthy controls (0.54 versus 0.64; P < 0.001), to subjects with type I diabetes (0.54 in PBC versus 0.66; P < 0.001), and to psoriasis patients (0.54 in PBC versus 0.67; P < 0.001) (Fig. 2B). Similarly, site-specific methylation was calculated for each of the 10 CpG sites in the CD40L click here promoter region (Fig. 2C) and ranged from 0.33 to 0.61

in PBC patients versus 0.29-0.78 in healthy subjects, 0.29-0.78 in type I diabetes, and 0.32-0.75 in psoriasis patients (Fig. 2C). No detectable difference in the level of CD40L promoter methylation in isolated CD8+ T cells was observed between PBC patients and controls (data not shown). However, in general, the levels of CD40L promoter methylation were significantly downmethylated in CD4+ T cells, compared to CD8+ T cells from both PBC patients (Fig. 3A) and controls (data not shown), as confirmed by a significantly lower CD4+/CD8+ methylation ratio in PBC patients (0.72 versus 0.85; P = 0.0002) (Fig. 3B). Levels of CD40L mRNA were also evaluated in CD4+ T cells from both patients and healthy controls by reverse-transcriptase PCR. Levels of CD40L mRNA expression was increased in CD4+T cells from PBC patients, compared to controls (2−(ΔΔCt) = 2.53 versus 1.12; P = 0.0178) and inversely correlated with levels of CD40L promoter methylation (r2 = 0.2347, P = 0.0355; Fig. 4). Based on the fact that patients with

mutations of the X-linked CD40L gene exhibit high titers of serum IgM,18 we evaluated the potential correlation between levels of CD40L methylation and serum IgM levels. The sera from 16 of the 6-phosphogluconolactonase 20 PBC patients (80%) included in the study had high relative levels of IgM (Fig. 5A) and showed significantly lower levels of CD40L promoter methylation within CD4+ T cells, compared to their normal IgM counterparts (Fig. 5B). Interestingly, IgM levels inversely correlated with levels of CD40L promoter methylation (r2 = 0.5448, P = 0.0011; Fig. 5C). To determine the potential contribution of the presence of mutations of the CD40L gene that could influence IgM levels,17, 18 we sequenced CD40L in gDNA samples isolated from each of the PBC patients and analyzed them for the presence of mutations previously documented for the CD4L gene (Fig. 6).

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