Fig. 7: Histological analysis, immune characterization and gene expression quantification in vehicle and 3C7 mAb-injected murine SGs.
a Workflow of the mouse inducible ELS formation, following SGs cannulation. The experimental timeline shows that the AV was injected into the SG on day 0; on days 4 and 10, the 3C7 monoclonal anti-Slc5a12 antibody was administered. On day 14, SG tissues were collected for subsequent analyses. Post collection, tissues were processed for immunofluorescence and qPCR to assess relevant biomarkers and gene expression levels. b, Representative images of H&E staining of the whole SGs from vehicle-cannulated and 3C7 mAb-cannulated mice. Black arrows point to inflammatory foci (peri-ductal leukocytic infiltrates with more than 50 lymphocytes). c,d, Comparison between vehicle-cannulated and 3C7 mAb-cannulated mice (n = 5 or 6 per group) of focus score calculated on H&E images (c) and aggregate area fraction (% of SG area occupied by the inflammatory infiltrate), calculated on immunofluorescence images (d). e, Representative immunofluorescence staining of SG tissue sections from vehicle and 3C7 mAb-injected mice. CD3 (green) is used as a marker for T cells, B220 (red) is used as a marker for B cells and DAPI (blue) is used as a marker for nuclei. The images reveal the distribution and localization of T cells and B cells in the tissue and their segregation. Scale bars, 100 µm. f, Comparison between vehicle and 3C7 mAb-cannulated mice of the positive area for CD3 (T cells), B220 (B cells) and both (T cell–B cell intersection), respectively, as calculated from e. Data are presented as means; error bars, s.d. (n = 4 biological replicates per group). Statistical analysis was performed using an unpaired t-test. In c, d and f, box and whisker plots show the 75th and 25th percentiles of the data, and minimum and maximum values. Statistical significance was determined using a Mann–Whitney U-test (two-sided). g, Prevalence of segregated and non-segregated aggregates over the total number in vehicle and 3C7 mAb-cannulated mice, as calculated from e. h, qPCR analysis of gene expression levels for Cxcr5, Cxcl13, Ccr7, Ccl19, Ltb, Ltbr, Il17, Il21 and Il21r in vehicle and 3C7 mAb-injected mice. Gene expression levels are normalized to a housekeeping gene and presented as relative expression levels. Data are expressed as means from n = 8–10 mice per group; error bars, s.d. Statistical significance was determined using a Mann–Whitney U-test and outliers were excluded by Grubb’s test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).
