Fig 1: PPP2R2D deficiency in T cells mitigates imiquimod-induced lupus-like pathology in mice.Topical imiquimod was applied to the ear skin of R2Dfl/fl and LckCreR2Dfl/fl mice (n = 7/group) for 8 weeks. (A) The representative picture and cumulative data of the weight of spleens. FACS analysis of the percentage of CD3+CD4+IFN-?+ (B), CD3+CD4+IL-17A+ (C), CD3+CD4+IL-2+ (D), and CD3+CD4+FoxP3+ (E) cells in spleens. (F and G) The expression levels of Treg markers CTLA-4 (F) and GITR (G) in splenic Tregs (CD3+CD4+FoxP3+) were determined by FACS. (H and I) The anti-dsDNA IgG level in serum (H) and the levels of albumin and creatinine in urine (I) were measured by ELISA. (J and K) The deposition of complement 3 (C3) and IgG in glomeruli was determined by immunofluorescence staining. Representative figures (J) and cumulative data (K) depicting numbers of glomeruli with C3 and IgG double deposition in coronal sections of kidney. Scale bar: 50 µm. (L) Representative H&E staining of kidney tissues. Scale bar: 20 µm. (M) Cumulative data elucidating the histopathologic scores for glomerulonephritis. (N) Kidney-infiltrating lymphocytes including total T cells (Thy1.2), CD4+ T cells, and CD8+ T cells were counted and analyzed by FACS. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.001 using unpaired t test.
Fig 2: PPP2R2D expression increases and negatively regulates IL-2 production in human T cells following stimulation.Human T cells derived from PBMCs of healthy subjects were stimulated with CD3 (OKT3) and CD28 antibodies. (A) The mRNA expression of the different regulatory subunits of PP2A in T cells at 0.5 and 24 hours after stimulation (n = 4). Dashed line represents the expression level of each subunit in T cells without stimulation. (B) The mRNA expression of PPP2R2D and IL-2 in T cells at 0, 0.5, 2, 6, 12, and 24 hours after stimulation (n = 6–7). All the expression levels were normalized to the samples without stimulation. (C) Western blot analysis of protein expression levels of PPP2R2D, p-CREB, CREB, and ß-actin in T cells at 0, 6, 12, and 24 hours after stimulation. (D) Cumulative data (n = 4) for quantification of the levels of PPP2R2D and p-CREB in the blots shown in C. (E and F) Intracellular staining of IL-2 production in T cells at 0, 6, 12, and 24 hours after stimulation was analyzed by FACS. Cells were subjected to silencing of PPP2R2D (E) or to transfecting with PPP2R2D plasmid (F), and they were rested overnight before stimulation for indicated times. n = 3. (B and D) *P < 0.05, ****P < 0.001, when compared with corresponding 0 hour using 2-way ANOVA with Holm-Šidák multiple-comparisons test. (E and F) **P < 0.01, ***P < 0.001, ****P < 0.001 using multiple t test.
Fig 3: PPP2R2D expression is increased in T cells from patients with SLE.(A) The mRNA expression of PPP2R2D and in T cells from healthy subjects (n = 7) or from patients with SLE (n = 19). HC, healthy control. (B) Western blot analysis of the protein expression levels of PPP2R2D and ß-actin in T cells from patients with SLE or matched healthy donors. Healthy donor 1 (H1) is age-, sex-, and ethnicity-matched with lupus patient 1 (L1); H2 is age-, sex-, and ethnicity-matched with L2, and so on. (C) Cumulative data for quantification of the level of PPP2R2D in the blots shown in B. Healthy controls (HC), n = 11; SLE patients, n = 11. (D) Pearson correlation analysis showing the relationship between PPP2R2D expression and Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). *P < 0.05 using unpaired t test (A) or paired t test (C).
Fig 4: Chromatin accessibility profiles of Tconv cells with or without PPP2R2D expression using ATAC-seq analysis.CD4+ Tconv cells were sorted out from spleens of R2Dfl/fl or LckCreR2Dfl/fl mice (n = 2 mice/group) and ex vivo stimulated by plate-bound CD3 and CD28 antibodies for 4 hours before being subjected to ATAC-seq. (A) Correlation heatmap indicating cross-correlation between each replicate or each group. (B) Histogram showing the distance from the nearest transcription start site (TSS) for all ATAC-seq peaks. (C) Volcano plot showing differential chromatin accessibility in CD4+ Tconv cells isolated from R2Dfl/fl (WT) and LckCreR2Dfl/fl (KO) mice. Fold change (FC) is calculated as log2 (LckCreR2Dfl/fl/R2Dfl/fl). Red indicates sites that were significantly different (adjusted P = 0.01, FC = 4). (D) Transcription factor (TF) family binding motifs enriched in loci more accessible in LckCreR2Dfl/fl or R2Dfl/fl Tconv cells; the x axis shows the enrichment factor (ratio of the percentage of differential sites with motifs to the percentage of nondifferential sites with motifs), and the y axis shows the significance level of enrichment. TF families are indicated by color. (E) Accessibility tracks for selected gene loci (IL-2, Fos, Jun, Nfatc1, Nfkb1, and Rela) in R2Dfl/fl (up) and LckCreR2Dfl/fl (down) Tconv cells were plotted using the integrative genomics viewer (IGV). ATAC-seq data are average of 2 biological replicates at each cell type.
Fig 5: Loss of PPP2R2D expression in T cells enhances the suppressive capacity of Tregs.In vitro suppression of R2Dfl/fl or LckCreR2Dfl/fl CD4+ Tconv or CD8+ Tconv cells by R2Dfl/fl or LckCreR2Dfl/fl Tregs after incubation together at various ratios. (A–D) Proliferation index of CD4+ Tconv (A and B) or CD8+ Tconv (C and D) cells is shown. Representative flow charts of the dilution of fluorescent dye CellTrace Violet (A and C) and cumulative data (B and D) are shown. n = 6 mice/group. (E–H) Expression of IFN-? in CD4+ Tconv (E and F) or CD8+ Tconv (G and H) cells is shown. Representative flow charts of the expression of IFN-? (E and G) and cumulative data (F and H) are shown. n = 5 mice/group. *P < 0.05, **P < 0.01 using multiple t test.
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