Residual MHCII expression in human MHCII deficiency. The RFX5-deficient human B-cell lines RO (A) and SJO (B) were cultured overnight with 10 μg/mL LPS, stained for CD19 and HLA-DR, and analyzed by flow cytometry. PBLs were used as healthy control cells. Only live cells are shown in all cases. RT-PCR analysis of HLA-DR expression by SJO cells (C). The cells were FACS sorted into HLA-DR⁺ and HLA-DR^- fractions. Cells (10⁵) were used for RT-PCR with primers for HLA-DRα and β. Primers were placed into highly homologous regions according to aligned HLA-DR alleles at the IMGT HLA database.⁵⁶  The α-chain PCR was intron spanning, the β-chain PCR was not. For the latter control, reactions lacking reverse transcriptase (RT) were included. PBLs (10⁵) from a healthy donor were used as positive controls. A PCR for β-actin served as control for the RT reaction. (D-G) MHCII expression on PBLs derived from 2 patients previously diagnosed to be MHCII deficient was analyzed after overnight culture in the presence of 10 μg/mL LPS and 0.5 μM CpG 2006 (patient 1) or 10 μg/mL LPS (patient 2). The patients were 1 year (F-G) and 4 years (D-E) of age, and the molecular defects were located in the RFX-ANK gene. After culture, the cells were stained for CD19 and HLA-DR (D,F) or CD3 and HLA-DQ (E,G) or CD3 and HLA-DP (data not shown) and analyzed by flow cytometry. Antibodies of irrelevant specificities were used as isotype controls for the HLA-DQ, HLA-DR, and HLA-DP stainings. Dot plots show cells in lymphocyte- and live-cell gates. In all experiments, similar results were obtained without treatment or treatment with 0.5 μM CpG, 50 μg/mL pI/pC, or CD40L.