Figure 2.

AA subjects show reduced CD28 mRNA expression but similar frequencies of CD4⁺CD28⁻ T cells with GG and AG. (A) RNA from freshly isolated peripheral blood mononuclear cells (PBMCs) was extracted, transcribed into cDNA and tested for CD28 mRNA expression in healthy individuals homozygous (AA) or heterozygous (AG) for the PSC risk variant and homozygous for the protective allele (GG) (n = 14 per group). Data are expressed as 2^−ΔCt relative to beta actin. Lines indicate median values. **p = 0.002 and **p = 0.003 with Mann Whitney U test after exclusion of the outlier detected in the AA group by Grubbs test. (B) CD28 mRNA expression in total PBMCs in CMV seronegative donors. Data are expressed as 2^−ΔCt relative to beta actin. AA and AG healthy individuals homozygotes and heterozygotes for the PSC risk variant and GG homozygotes for the protective allele. Lines indicate median values. *p = 0.02 and *p = 0.03 with Mann Whitney U test after exclusion of the outlier detected in the AA group by Grubbs test. (C) Representative flow cytometry plots showing the gating strategy to define CD4⁺CD28⁻ T cells in PBMCs. CD3⁺CD4⁺ cells were selected after duplet exclusion and CD28 expression was studied in the CD4⁺ population. (D) Data show the proportion of CD4⁺ T cells that have lost CD28 expression. Line indicates median with interquartile range. (E) CD28 median fluorescence intensity (MFI) across genotypes. Line indicates median value.