Fig 1: TKO Tregs Display Marked Defects in Mobilization In Vitro and In Vivo(A) CD4+CD25− T cells from spleens of WT and TKO mice after 12 weeks of HFD were treated with anti-CD3 antibodies and TGF-β1 for differentiation into in vitro differentiated Tregs (iTregs). Percentage of WT and TKO CD4+CD25+Foxp3+ Tregs were measured by flow cytometry at the indicated time points (n = 6 per group). (B and C) CD4+CD25− T cells from spleens of WT and TKO mice after 12 weeks of HFD were activated by anti-CD3 antibodies for 24 h and subjected to qRT-PCR analysis (B) or ELISA from supernatants (C) for the indicated cytokines, chemokines, and growth factors (n = 5–9 per group).(D and E) Transwell migration study of CD4+CD25+ Tregs isolated from WT and TKO mice after 12 weeks of HFD. Cells were assessed for migration in the presence or absence of CCL19 (D) or CCL20 (E) (n = 3 per group).(F and G) Flow cytometry for CCR7 (F) or CCR6 (G) expression in WT and TKO Tregs (n = 6 per group).(H) Schematic of PKH26-labeled HFD WT and TKO Tregs adoptively transferred to HFD C57BL/6 mice. Flow cytometry shows percentage of PKH26-expressed cells in liver, VAT, and SAT of recipient mice (n = 6 per group).(I and J) Schematic of glucose uptake study of differentiated 3T3-L1 cells co-cultured with HFD WT and TKO iTreg supernatant (supe) (I). (J) Fluorescence intensity of 2-Deoxy-D-glucose (2-DG) uptake by differentiated 3T3-L1 cells co-cultured with supernatants of WT and TKO CD4+ Tregs in the presence or absence of insulin stimulation (n = 4 per group).(K and L) Schematic of glucose production study of mouse primary hepatocytes co-cultured with HFD WT and TKO iTreg supernatants (K). (L) Glucose production by mouse primary hepatocytes co-cultured with supernatants of HFD WT and TKO CD4+ Tregs (n = 6 per group).Statistical differences are indicated as *p<0.05, **p<0.01, and ***p<0.001. Results are reported as mean ± SEM.Related to Figures S3 and S4.
Fig 2: Optn deficiency hinders CII-induced DC migration. (A) qRT-PCR analyses of Optn in WT and Optn KO BMDCs. n = 3. (B) Scheme (left) and quantification (right) for Transwell analysis of CCL19/21-triggered migration of WT and Optn KO BMDCs after treating with CII (100 μg/mL) for 24 h. n = 3. (C) Scheme (left), representative images (middle) and bar graph (right) for the in vivo WT and Optn KO BMDC migration after labeling with CFSE and treating with CII (100 μg/mL) for 24 h. n = 3. (D) PCA analysis of transcriptome profiles of WT and Optn KO BMDCs after treating with CII (100 μg/mL) for 24 h. n = 3. (E) Volcano plot of transcriptome profiles of WT and Optn KO BMDCs after treating with CII (100 μg/mL) for 24 h (P < 0.05, fold-change >1.5). n = 3. (F) GSEA analyses of genes enriched in CII-pulsed WT or Optn KO BMDCs. n = 3. (G) Heatmap analyses of representative genes involved in cell migration in CII-pulsed WT or Optn KO BMDCs. n = 3. (H) qRT-PCR analyses of indicated genes in WT and Optn KO BMDCs after CII (100 μg/mL) treated for 24 h. n = 3. (I) Immunostaining of F-Actin (green) and membrane MHC-II (red) in WT and Optn KO BMDCs after CII (100 μg/mL) treatment for 24 h. Scale bar: 3 μm. n = 3. Data are presented as mean ± SD; ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.
Fig 3: CII establishes a characteristic gene profile to activate DC migration. (A) Scheme of the experimental approach for Affymetrix microarray gene chip analysis from the EMBL-EBI database (accession no. E-MTAB-2904). (B) Volcano plot of genes differentially expressed in human MoDCs after LPS or CII stimulation for 20 h (fold change >2; P < 0.05). n = 6. (C) Venn diagram of differentially up-regulated genes after LPS or CII stimulation for 20 h in human MoDCs. n = 6. (D) Heatmap and clustering analysis of differentially up-regulated genes after LPS or CII stimulation for 20 h in human MoDCs. n = 6. (E) Bar plots of gene ontology analysis of the up-regulated genes in different clusters of (D). n = 6. (F) Gene tree analysis based on gene expression levels after LPS or CII stimulation for 20 h in human MoDCs. n = 6. (G, H) GSEA enrichment scores for indicated gene sets in CII (G) or LPS (H) stimulated human MoDCs. n = 6. (I) Transwell analysis of CCL19/21-triggered migration of BMDCs upon LPS (50 ng/mL) or CII (100 μg/mL) treatment for 20 h. n = 3. Data are presented as mean ± SD; ∗∗∗P < 0.001.
Fig 4: CD30 deficiency attenuated autoimmune pathology by limiting fibroblast proliferation and chemokine production in salivary glands of pSjD model mice.a Hematoxylin and eosin (H&E) staining of salivary glands (SG) from pSjD-WT and pSjD-Tnfrsf8 knockout (KO) mice and quantification of lymphocytic foci per unilateral lobe of SG (right). b The numbers of immune cell populations in the SG of pSjD-WT and pSjD-Tnfrsf8 KO mice was determined by flow cytometric analysis. CD45+ cells: 7AAD−CD45+, CD4+ T cells: 7AAD−CD45+ CD4+CD8α−, CD8+ T cells: 7AAD−CD45+ CD4−CD8α−, B cells: 7AAD−CD45+CD19+, CD153+CD4+ T cells: 7AAD−CD45+CD153+PD-1+CD4+CD8α−. The cell numbers were calculated by [total viable cell count × frequency]. c Immunofluorescence of SG sections from pSjD-WT and pSjD-Tnfrsf8 KO mice, showing DAPI (white), podoplanin (blue), Ki-67 (red), and CD45 (yellow). The number of podoplanin+ Ki-67+ cells in 5 foci was counted and then averaged. d In situ hybridization of SG sections from pSjD-WT and pSjD-Tnfrsf8 KO mice (left), showing DAPI (white), Col1a1 (red), Cxcl13 (blue), and Ccl19 (orange). The numbers of spots estimated for Cxcl13 (middle) and Ccl19 (right) transcripts per Col1a1+ cell in pSjD-WT (black) and pSjD-Tnfrsf8 KO (red) mice. Data are presented as mean ± SEM; n = 3–4 mice per group; each point represents one mouse. Sample sizes are provided in the Source Data. Statistical significance was determined using an unpaired two-tailed Student’s t-test. **P < 0.01, *P < 0.05. Exact P values are provided in the Source Data. Scale bars = 100 μm a and 50 μm (c, d).
Fig 5: Ccl19 and Cxcl13 promoted CD4+ T-cell and B cell migration to salivary glands, driving autoimmune pathology in pSjD model mice.a The proportions of Ccr7+ among CD4+ T, B, and CD8+ T cells in salivary glands (SG) of pSjD model mice. b The proportions of Cxcr5+ cells among CD4+ T, B, and CD8+ T cells in SG of pSjD model mice. c Ccr7 expression in CD4+ T-cell subsets (PD-1−CD153−, PD-1+CD153−, and PD-1+CD153+) shown as histograms (left) and geometric mean fluorescence intensity (gMFI; right). d Cxcr5 expression in CD4+ T-cell subsets (PD-1−CD153−, PD-1+CD153−, and PD-1+CD153+) shown as histograms (left) and gMFI (right). e Experimental design: Anti-Ccl19 and Cxcl13 antibodies (αCcl19 + Cxcl13 Ab) or isotype control antibody (Ctrl Ab) was intraperitoneally injected into pSjD model mice from 6 weeks of age. f Hematoxylin and eosin (H&E) staining of SG from αCcl19 + Cxcl13 Ab- and Ctrl Ab-treated mice (left) and quantification of lymphocytic foci per unilateral lobe of SG (right). g The numbers of immune cell populations in SG from αCcl19 + Cxcl13 Ab- and Ctrl Ab-treated pSjD model mice was determined by flow cytometric analysis. CD45+ cells: 7AAD−CD45+, CD4+ T cells: 7AAD−CD45+CD4+CD8α−, CD8+ T cells: 7AAD−CD45+CD4−CD8α+, B cells: 7AAD−CD45+CD19+, CD153+CD4+ T cells: 7AAD−CD45+CD153+PD-1+CD4+CD8α−. The cell numbers were calculated by [total viable cell count × frequency]. Data are presented as mean ± SEM; n = 3–4 mice per group; each point represents one mouse. Sample sizes are provided in the Source Data. Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparison test (a–d) or unpaired two-tailed Student’s t-test (f, g). **** P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. Exact P values are provided in the Source Data. Scale bar = 100 μm (f).
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