Involvement of HMGN1 generated by nonleukocytes at the site of immunization in the induction of immune response. (A) HMGN1 KO (Hmgn1^−/−) and littermate-matched WT (Hmgn1^+/+) mice (n = 4) were i.p. immunized with OVA (50 µg/mouse) in the presence or absence of LPS (1 µg/mouse). Serum cytokines were measured at 24 and 96 h after immunization. Shown is the mean (±SD) serum cytokine concentration of each group, which is representative of three independent experiments. (B) HMGN1 KO (Hmgn1^−/−) and littermate-matched WT (Hmgn1^+/+) mice (n = 3) were immunized i.p. with PBS, OVA (50 µg/mouse), or OVA + LPS (1 µg/mouse). After 4 h, peritoneal leukocytes were enumerated for total leukocytes (top) or DCs (bottom; defined as CD11c and I-A/E double-positive cells). Shown is the mean (±SD) cell number of individual group of one experiment representative of two. (C) C57BL/6 mice (n = 3) were injected with OVA (50 µg/mouse) alone or OVA + LPS (1 µg/mouse). HMGN1 expression in the peritoneal cavity 4 or 22 h after injection was assessed by Western blot with anti-HMGN1 antibody. Shown are the results of one experiment representative of two. (D and E) Bone marrow chimeric mice were generated by reconstituting lethally irradiated Hmgn1^−/− mice with Hmgn1^+/+ bone marrow mononuclear cells (WT→KO) or vice versa (KO→WT). The chimeric mice (n = 3) were immunized i.p. with PBS containing OVA (50 µg/mouse) or OVA in the presence of LPS (1 µg/mouse) on day 1 and boosted on day 14. On day 10 and 20, OVA-specific IgG antibody responses were measured by ELISA (D). On day 21, splenocytes were cultured in a 48-well plate (2.5 × 10⁶/0.5 ml/well) in the presence of 100 µg/ml OVA for 48 h. The cytokines in the supernatants were measured and are shown as the mean concentration (±SD) of each group (E). Shown are the results of one experiment representative of two. *, P < 0.05.