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: The CD153-CD30 axis regulates Cxcl13 and Ccl19 expression in Tnfrsf8-positive cells within lymphocytic foci of pSjD model mice.a In situ hybridization (ISH) of SG tissue sections from pSjD model mice, showing DAPI (white), Tnfrsf8 (red), Cxcl13 (blue), and Ccl19 (orange). The blue, orange, and magenta boxes indicate the corresponding regions in (b). b High-magnification images showing representative Tnfrsf8+ cells expressing Cxcl13 and/or Ccl19. Merged and single-channel images are shown. c The number of spots estimated for Cxcl13 (upper) and Ccl19 (lower) transcripts per Tnfrsf8+ (red) and Tnfrsf8− (blue) cells. ISH signals in SG sections from pSjD model mice (a, b) were quantified using QuPath. d ISH of SG tissue sections from Ctrl Ab- and anti-CD153 Ab-treated pSjD model mice, showing DAPI (white), Tnfrsf8 (red), Cxcl13 (blue), and Ccl19 (orange). e Representative high magnification images corresponding to (d). Merged images and individual color channel images for both the Ctrl Ab (upper)- and anti-CD153 Ab (lower)-treated groups are shown. f The numbers of spots estimated for Cxcl13 (left) and Ccl19 (right) transcripts per Tnfrsf8+ cell in Ctrl Ab- (black) and αCD153 Ab- (red) treated pSjD model mice. Data are presented as mean ± SEM; n = 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. Exact P values are provided in the Source Data. Scale bars = 50 μm (a, d) and 10 μm (b, e).
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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