DL4 expression on Ccl19-Cre–expressing follicular fibroblasts regulates Notch-mediated T_FH and GC differentiation. (A) LN sections of R26-EYFP^ΔCcl19 mice 5 d after NP-CGG immunization stained for EYFP, CD21/35, and Pdpn expression. EYFP staining reveals Cre recombinase activity through the deletion of the lox-stop-lox cassette in the R26 gene locus. Strong CD21/35 staining in the B zone (BZ) is indicative for activated FDCs and weaker staining for B cells, whereas Pdpn stains FRCs in the T zone (TZ) and activated FDCs in the BZ. White boxes indicate areas of the BZ and TZ, respectively, which are also shown in higher magnification on the right side. (B, left) Representative FACS profiles of LN stromal cells of DL4^Δ/ΔCcl19 mice 5 d after NP-CGG immunization, gated on Aqua⁻CD45⁻Ter119⁻cells, and further classified as CD35⁺Pdpn⁺CD31⁻ (FDC), CD35⁻Pdpn⁻CD31⁻ (DN), CD35⁻Pdpn⁺CD31⁺ (LEC), or CD35⁻Pdpn⁻CD31⁺ (BEC) stromal cells. Percentages indicate the fraction present in each quadrant or gate. (right) Representative histograms showing DL4 surface expression on FDCs, FRCs, LECs, and BECs, derived from LNs of DL4^lox/lox and DL4^Δ/ΔCcl19 mice. Percentages indicate the proportion of cells expressing DL4 in DL4^lox/lox mice (red) versus DL4^Δ/ΔCcl19 mice (blue). (C) Representative histograms showing CD157 (BP-3) and CD21/35 surface expression on FDC (CD35⁺Pdpn⁺CD31⁻), FRC (CD35⁻Pdpn⁺CD31⁻), and B cells (CD19⁺TCRβ⁻). (D) DL4 mRNA expression was assessed for stromal cell subsets (sorted as shown in B) from 6 LNs per mouse derived from DL4^lox/lox and DL4^Δ/ΔCcl19 mice (n = 2) 5 d after NP-CGG immunization. The numbers indicate the fold enrichment relative to the mRNA expression level observed for total LN from DL4^lox/lox mice. (E) PCR for the deleted (Δ) versus undeleted (loxP) DL4 gene was performed on DNA from stromal cell subsets (sorted as shown in B) of LNs 5 d after NP-CGG immunization from control and DL4^Δ/ΔCcl19 mice. (F) LN sections of DL4^lox/lox mice 6 d after NP-CGG immunization were stained for DL4, CD21/35, and desmin, as indicated. Arrows indicate different cell types and sites of DL4 expression with BZ and TZ shown on the right in higher magnification. (G) LN sections of R26-EYFP^ΔCcl19 mice 5 d after NP-CGG immunization were stained for DL4, EYFP, and CD21/35, as indicated, with higher magnifications shown on the right. (H) LN sections of DL4^lox/lox and DL4^Δ/ΔCcl19 mice 6 d after NP-CGG immunization were stained for DL4, CD21/35, and desmin, as indicated. Note the loss of DL4 staining in activated CD21/35⁺ FDCs of DL4^Δ/ΔCcl19 mice. FDC, follicular DCs; MRCs; BRCs; FRC, T zone fibroblastic reticular cells; HEV, high endothelial venules. Size bars as indicated. Experiments shown are representative of two independent experiments and at least two mice of each genotype.