Fig. 2: CTA1-DD adjuvant treatment results in expansion and enhanced gene expression in targeted FDCs; a CD21^mT/mG mouse model for isolating GFP⁺ stromal cells.

a Representative sections of iLN from CD21^mT/mG naive mice (upper panel) or 12 days following a s.c. immunization with 10 μg CTA1-DD adjuvant (lower panel). Sequential tissue sections were stained for the FDC network (FDC-M2⁺) or GC (GL-7⁺); merged pictures show the overlap of CD21-GFP with FDC-M2. All images were taken with an inverted confocal microscope (LSM700) with x20 magnification. The size bars indicate 100 µm. These are representative sections from individual mice in groups of three mice showing similar results. b Intensity of GFP expression or FDC-M2 staining in tissue sections was measured using Fiji and is shown as color intensity per B-cell follicle for naive or CTA1-DD immunized mice (n = 3). c FACS analysis of CD45⁻ CD31⁻ LN cells from naive or CTA1-DD immunized CD21^mT/mG mice at 4 days (left panels) or the kinetics (right panel) of FDC responses after immunization. Representative panels of two experiments with n = 3 mice each and statistical significance (**p < 0.01) using one-way ANOVA of the effect of the adjuvant treatment on FDC frequencies compared to those observed in untreated mice (dotted line). d Representative sections of iLN from naive CD21^mT/mG mice (upper panel) or 12 days following a s.c. immunization with 10 μg CTA1-DD adjuvant (lower panel). Tissues from three mice per group were stained for FDC network (FDC-M2⁺) and cell proliferation was assessed by Ki67-staining; merged pictures show the overlap of CD21-GFP with FDC-M2⁺ and Ki67⁺ cells. e Validation of FDC status was done by Cr2 and Cxcl13 gene expression analysis of sorted GFP⁺ FDC-M2⁺, GFP⁺ gp38⁺ or GFP⁺ FDC-M2^- gp38^- (DN) cells from naive CD21^mT/mG mice. Data shown for n = 6 mice pooled from two experiments as relative gene expression to the reference genes Gapdh and Hprt. f Kinetics of the percentage and number of FDC-M2⁺ or Pdpn⁺ cells of GFP⁺ CD45^- CD31⁻ tdTomato⁻ cells in iLN following s.c. immunization with 10 μg CTA1-DD adjuvant of CD21^mT/mG mice (n = 6 pooled from two experiments). g Gene expression analysis using qPCR of mRNA from FDCs isolated from iLN 4 days after a s.c. immunization with 10 μg of CTA1-DD adjuvant. h Kinetics of gene expression in FDCs in iLN revealing early (day 1) and late (days 5/6) changes following s.c. immunization with 10 μg of CTA1-DD adjuvant as compared to gene expression in FDCs from naive control mice. For g and h, results are given as the mean fold-change ± SD of two pooled independent experiments (n = 3 in each group) relative to the mRNA levels of each gene observed in samples from unimmunized control mice (2^ddCt). The dotted line indicates a twofold change. Statistical significance relative to controls was assessed using repeated-measures ANOVA with Sidak correction for multiple measurements; *p < 0.033, **p < 0.002, ***p < 0.0002, ****p < 0.0001. i Chimeric mice were made through transplantation of WT BM to irradiated CR2^-/- mice (upper panel) or CR2^-/- BM transplantation to irradiated WT mice (lower panel), thus, reflecting an absence of CR2 in either FDCs (upper) or B cells (lower), respectively. The CR2^-/- chimeric mice were immunized s.c. with or without 10 μg CTA1-DD adjuvant and 4 days later FDCs were isolated and Cxcl13 gene expression was assessed using qPCR in CTA1-DD adjuvant-treated FDCs as compared to FDCs from naive control mice. Unless otherwise stated statistical significance was determined by one-way ANOVA followed by Tukey correction for multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.