Analysis of LN development in Lta^−/− mice. (A) FACS analysis of CD45⁻ stromal cell from E15 and E16 wild-type and Lta^−/− iLNs with mAbs for VCAM-1 and ICAM-1. Percentages of CD45⁻ cells according to their expression of VCAM-1 and ICAM-1 are shown in each quadrant. (B) FACS analysis of cell suspensions of E15 and E16 wild-type and Lta^−/− iLNs with mAbs for CD4 and CD45. Numbers on each quadrant correspond to percentages of CD3⁻ cells expressing CD4 and CD45. (C) Immunofluorescence staining of E15 wild-type and Lta^−/− iLN sections with ERTR7 (red) and DAPI (blue) (10×/1.4 NA). (D) A statistically significant reduction in total cell numbers in iLN of Lta^−/− E16 embryos compared with their wild-type counterparts. Results shown are wild-type: 7780 cells SEM 657, Ltα^−/−: 5339 cells SEM 778, unpaired t test P < .03. (E) Statistically significant decrease in percentages of cycling CD45⁻ stromal cells from E16 Lta^−/− iLNs with respect to their wild-type counterparts as measured by Ki67 staining. Results shown are Ki67⁺ cells wild-type: 38% SEM 3, Ltα^−/−: 19.6% SEM 2.9, unpaired t test P < .009. (F) Annexin V staining of CD45⁻ stromal cell from E15 and E16 Lta^−/− and wild-type iLNs. Note the increased frequency of cells undergoing apoptosis (Annexin V⁺) in the Ltα^−/− cells compared to their wild-type counterparts. Numbers above brackets correspond to percentages of Annexin V⁺ cells. (G) The addition of wild-type LTic's rescued the defect of E15 Lta^−/− reaggregate iLNs to form a lymphoid aggregate upon kidney capsule grafting that recruited CD19⁺ B cells and CD3⁺ T cells. Numbers on the quadrants correspond to percentages of either CD3⁺ T cells, CD19⁺ B cells, or double negative cells. Results shown here are from 1 representative assay out of 5 independent experiments. Error bars in panels D,E represent SEM.