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: 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 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: CD153+ PD-1+ CD4+ T cells and CD30+ fibroblasts increased in patients with pSjD.In situ hybridization (ISH) of labial salivary glands (LSG) from patients with non-SjD sicca and pSjD, showing TNFSF8 expression (left) and the number of cases with TNFSF8+ cells ( > 10 cells per focus) in lymphocytic foci (right; n = 9 non-SjD sicca, n = 13 pSjD). b Flow cytometric analysis of CD153 and PD-1 expression in CD4+ T cells in LSG from patients with non-SjD sicca and pSjD (left). The frequency of CD153+PD-1+ among CD4+ T cells in LSG from patients with non-SjD sicca (black) and pSjD (red) (right). c Immunohistochemistry of CD30 in LSG from patients with non-SjD sicca and pSjD (left). The number of CD30+ cells in LSG from patients with non-SjD sicca (black) and pSjD (red) (right). d ISH of LSG from patients with pSjD, showing DAPI (white), TNFRSF8 (red), and COL1A1 (blue). The orange box indicates the corresponding region shown in (e). e High magnification images of TNFRSF8⁺ COL1A1+ cells. The merged and individual channel images for each color are shown (DAPI, white; TNFRSF8, red; COL1A1, blue). f The number of spots estimated for COL1A1 transcript per TNFRSF8+ (red) and TNFRSF8− (blue) cells. g Immunofluorescence of LSG from patients with pSjD, showing DAPI (white), CD30 (red), and Ki-67 (blue). h ISH of LSG from a patient with pSjD, showing DAPI (white), COL1A1 (red), CXCL13 (blue), and CCL19 (orange). i ISH of LSG from a patient with pSjD, showing DAPI (white), TNFSF8 (orange), CD4 (blue), and TNFRSF8 (red), and. j Multiplex immunofluorescence of LSG from patients with pSjD. k Spatial analysis. Data are presented as mean ± SEM; n = 6–20 individuals per group; each point represents one individual. 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, c), 50 μm (d, g, h), 10 μm e, 20 μm (g, i), and 280 μm (j).