Fig 1: 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).
Fig 2: 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 3: 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).
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).
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