IFNγ stimulation of monoallelic PSMB10 deficiency primary skin fibroblasts

Immunoproteasomes are specialized multiprotein proteases, that degrade intracellular proteins thereby generating antigenic peptides for HLA class I antigen presentation. Mutations in immunoproteasomal genes are associated with systemic autoinflammatory diseases. We identified a newborn with severe T lymphopenia and found a de novo heterozygous PSMB10 p.Gly209Arg variant. Molecular dynamics analysis predicted and biochemical studies confirmed the variant to impact assembly and function of the immunoproteasome resulting in impaired cytokine responses to interferons. Skin fibroblasts obtained from the patient (PSMB10G209R) and a healthy individual of comparative age (PSMB10WT) were treated with or without 75U/ml recombinant Human IFN-γ. 5 different cell passages for independent treatments to assess significance of alterations. RNA was isolated from cells to investigate the alteration in IFN response between PSMB10WT and PSMB10G209R. Skin fibroblasts obtained from the patient (PSMB10G209R) and a healthy individual of comparative age (PSMB10WT) were treated with or without 75U/ml recombinant Human IFN-γ. 5 different cell passages for independent treatments to assess significance of alterations. RNA was isolated from cells to investigate the difference of IFN response between PSMB10WT and PSMB10G209R. 5 passages(7, 8, 9, 10, 12) of PSMB10G209R skin fibroblasts and 5 passages(7, 8, 9, 10, 12) of PSMB10WT skin fibroblasts were used as biological replicates for hIFN-γ treatment. Cells were planted in 6 well plates 16 hours before treatment. Medium was replaced with or without 75U/ml recombinant human IFN-γ for 24 hours. Total RNA isolated from cells were isolated and subjected to Agilent microarray analysis.