1b shows H and E-stained tissue sections of NALT from normal BALB/c mice before and after teasing. NALT cells were readily isolated, Fulvestrant purchase yielding approximately 2.5 × 105 viable cells per palate. Because we had exsanguinated the mice from the inferior vena cava, we noted few erythrocytes; thus more than 96% of the cells were the following immune cells: CD3+ cells (53.5

± 3.8%; mean ± SD; n =3); CD4+ cells (38.6 ± 2.6%; mean ± SD; n =3); CD8+ cells (17.5 ± 2.5%; mean ± SD; n =3); B220+ cells (40.0 ± 3.7%; mean ± SD; n = 3); Mac-1+ cells (1.5 ± 0.4%; mean ± SD; n =3); CD11c+ cells (0.6 ± 0.0%; mean ± SD; n =3); and Ly-6G+ cells (0.3 ± 0.1%; mean ± SD; n =3). The cell yield from NALT and their phenotypic composition were essentially the same as those reported previously (17, 18), showing that they had been accurately prepared. Figure 2 shows the time-dependent Wnt antagonist changes in the total number of cells in NALT or submandibular lymph nodes of BALB/c mice after one i.n. injection of cedar pollen. The total number of NALT cells did not change significantly from days 0–14 after

i.n. injection of the allergen (Fig. 2a); and the percentages of B220+, CD3+, Mac-1+, CD11c+, and Ly-6C+ cells were also unchanged (data not shown). In contrast, the total number of submandibular lymph node cells started to increase on day 3 after i.n. injection of the allergen, reached a peak (≈ threefold that of the PBS-injected C-X-C chemokine receptor type 7 (CXCR-7) control) on day 10, and declined to the basal level by day 14 (Fig. 2b). Of particular interest, the percentage of B220+ cells on day 0 (≈ 36%) started to increase from day 3 (≈ 49%), reached a plateau on days 5–10 (54–55%), and decreased to the basal level by day 14 (≈ 42%). In contrast, those of CD3+ cells, Mac-1+, CD11c+, and Ly-6C+ cells decreased time-dependently and returned to the basal level by day 14 (data not shown), suggesting that B220+ cells (e.g., B or pre-B cells) in the submandibular lymph nodes might be the cells that respond to i.n. injections of allergen. Bulk cells from submandibular lymph

nodes from mice that had been treated once i.n. with allergen produced a significant amount of IgE Ab on day 7 (mean ± SE, 3.8 ± 1.0 ng/mL; n= 30) with a peak on day 10 (7.8 ± 1.6 ng/mL; n =30). The concentrations then decreased to the control level by day 14 (0.1 ± 0.1 ng/mL; mean ± SEM; n= 30), demonstrating time-dependent changes in the amount of IgE Ab similar to the changes in total cell numbers. In contrast, the bulk cells from the NALT from mice that had been treated once i.n. with allergen did not produce significant amounts of IgE (n =12) on days 0–14. The bulk cells of the axillary lymph nodes, Peyer’s patches, inguinal lymph nodes, and mesenteric lymph nodes produced 1.8 ± 0.3 (mean ± SEM; n =15), 1.3 ± 1.4 (mean ± SD; n =9), 0.5 ± 0.3 (mean ± SD; n =9), 0.1 ± 0.3 (mean ± SD; n =9) ng/mL IgE on day 10, respectively (data not shown).