Decreased Jumonji Domain-Containing 3 at the Promoter Downregulates Hematopoietic Progenitor Kinase 1 Expression and Cytoactivity of T Follicular Helper Cells from Systemic Lupus Erythematosus Patients

Figure 4

Effects of JMJD3 upregulation on Tfh cells from 3 SLE patients. (a, b) Relative JMJD3 and HPK1 protein levels in SLE Tfh cells transfected with pcDNA3.1 blank plasmid or pcDNA3.1-JMJD3-expressing plasmid were measured by western blot analysis after stimulation. β-Actin was used as endogenous control. (c–f) Relative JMJD3 (c), H3K27me3 (d), H3K4me3 (e), MLL1, MLL2, MLL3, and MLL4 (f) enrichments at the HPK1 promoter in SLE Tfh cells transfected with pcDNA3.1 blank plasmid or pcDNA3.1-JMJD3-expressing plasmid were quantified by combining ChIP and qPCR assays after stimulation. Results were normalized to input DNA (total chromatin). (g) The proliferation activity of SLE Tfh cells transfected with pcDNA3.1 blank plasmid or pcDNA3.1-JMJD3-expressing plasmid was assessed by MTT assays after stimulation. (h) The relative concentrations of IL-21, BAFF, IFNγ, and IL-17A in the supernatants of SLE Tfh cells transfected with pcDNA3.1 blank plasmid or pcDNA3.1-JMJD3-expressing plasmid were examined using ELISA after stimulation. (i) After stimulation, some SLE Tfh cells transfected with pcDNA3.1 blank plasmid or pcDNA3.1-JMJD3-expressing plasmid were cocultured with autologous B cells for 8 days, whereafter the concentrations of IgM, IgG1, IgG2, and IgG3 in the supernatants were detected by ELISA. The data from every pcDNA3.1 blank plasmid group member were set as 1, and the multiple of every pcDNA3.1-JMJD3-expressing plasmid group member relative to its homologous pcDNA3.1 blank plasmid group member was calculated.

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