Generation and cytokine responses of Spi-B–deficient mice. (A) Schematic representation of the gene targeting. Filled and open boxes represent coding and 3′-untranslated regions of Spib gene, respectively. Hatched boxes represent the coding region for neomycin resistance gene. B indicates BamH I; S, Sph I; and E, EcoR I. (B) Southern blot analysis of offspring from the heterozygote intercrosses. Genomic DNA was digested with BamH I, electrophoresed, and hybridized with the probe indicated in panel A. (C) For Northern blot analysis, Spi-B cDNA fragments corresponding to the 5′ region (nondeleted region, probe A) or to the deleted region (probe B) were used as probes. The same membrane was also hybridized with a β-actin cDNA fragment as a control. (D) FACS profiles of BM, spleen, iLN, and PB cells from WT or Spi-B–deficient mice. Dot plots of CD11c versus BST2 in BM, spleen, and iLN cells are shown. Numbers indicate percentages of gated cells among total live cells. For PB cells, dot plots of CD11c versus BST2 in CD19⁻CD11c⁺B220⁺ cells are shown. Numbers indicate the percentages of gated cells among CD19⁻CD11c⁺B220⁺ cells. Data are representative of at least 3 independent experiments. (E) Mice were injected intravenously with polyuridylic acid (polyU, a TLR7 agonist) or ODN1668 (a TLR9 agonist) or injected intraperitoneally with poly(I:C) (a TLR3/MDA5 agonist) and serum cytokine levels were measured at the indicated time points. Each symbol represents the data from 1 mouse and the bars indicate the mean. *P < .05; **P < .01