Figure 6. Membrane-LT expression on B cells is not sufficient for induction of VCAM-1 expression on FDC in the absence of CD19.

(A) Splenic B cells from CD45.2⁺, CD19⁻, mLT-Tg (CD19^−/−, κLTα-Tg) mice alone, or in a 4:1 mixture with CD45.2⁺, CD19⁺, mLT⁻ (CD19^+/cre, LTβ^flox/flox) and CD19- mLT-Tg (CD19^−/− κLTα-Tg) were adoptively transferred into CD45.1⁺, CD19^−/− recipient mice and subsequently immunized i.p. with SRBC. Ten days post-immunization, FDC activation and GC formation were visualized in the spleen based on staining of tissue sections with biotinylated PNA (blue), anti-CD35 mAb (red), anti-VCAM-1 mAb (green) or anti-CD16/32 mAb (green). CD45.2⁺, donor B cells were visualized using anti-CD45.2 mAb (white). Scale bars are 100 μm. Images are representative of 4 independent experiments in which 3 mice were immunized per experimental group. (B) The MFI of VCAM-1 and CD16/32 expression was calculated on FDC from frozen spleen sections analyzed in Figures 5 and 6. The data represent the average MFI calculated from analyzing 3 spleen sections per mouse with 3 mice per experimental group over a series of 3 independent experiments. The student’s t-test was used to compare the MFI for VCAM-1 or CD16/32 expression on FDC in WT mice with that calculated on FDC from each of the other experimental groups. The data represent the mean ± standard error of the mean. *p<0.01 **p<0.001 (C) Analysis of Ab affinity maturation based on calculating the ratio of NP₈:NP₃₀ binding activity detected by ELISA in serum collected on days 7 and 14 after immunization of mice i.p. with NP-CGG in incomplete Freund’s adjuvant. The data represent the mean ratio calculated for 3 mice per experimental group for 3 independent experiments. (D) The relative change in affinity was calculated by dividing the mean for the ratio of NP₈:NP₃₀ binding activity on day 14 by the mean on day 7.