Figure 1. HMGN1 induces phenotypic and functional maturation of DCs. (A) Human MoDCs were incubated at 5 × 105/ml in the absence (sham) or presence of recombinant HMGN1 or HMGN2 (both at a final concentration of 1 µg/ml) for 48 h before they were immunostained and analyzed by flow cytometry for the expression of the indicated surface molecules (open area, isotype-matched control). Shown are the results of one experiment representative of five. (B) Human MoDCs were incubated at 5 × 105/ml in the absence (sham) or presence of recombinant HMGN1 or HMGN2 at the indicated doses for 24 h (left) or at 1 µg/ml for the indicated times (right), and cytokine levels in the culture supernatants were quantitated by cytokine array (mean ± SD; n = 3). (C) Human MoDCs cultured at 1 × 106/ml in the absence (sham) or presence of 1 µg/ml of recombinant HMGN1 for 24 h were suspended in chemotaxis medium, and their migration in response to 100 ng/ml CCL5, CCL21, or CXCL12 was measured by chemotaxis assay. The migration of DCs was enumerated by the mean (±SD) of six high-power fields (HPF) randomly chosen from triplicate wells. Shown are the results of one experiment representative of two. (D) Human MoDCs cultured in the absence or presence of 1 µg/ml HMGN1 or LPS for 48 h were irradiated at 3,000 rad and incubated in triplicate with allogeneic human peripheral blood T cells (105/well) in 0.2 ml of medium in wells of 96-well flat-bottomed plates at the indicated DC/T ratios for 5 d. The proliferation was measured as the mean (±SD) [3H]TdR incorporation of triplicate wells. Shown are the results of one experiment representative of three. (E and F) Human MoDCs were incubated at 1 × 106/ml in the absence (sham) or presence of synthetic HMGN1 N-terminal domain (N1ND, HMGN11–52) or C-terminal domain (N1CD, HMGN153–100) at 10 µg/ml for 48 h before measurement of DC surface marker expression by flow cytometry (E, gray area: sham-treated DCs) and cytokine production in the supernatants (F; mean ± SD; n = 3). (G) Human DCs were incubated at 5 × 105/ml in medium in the absence (sham) or presence of 1 mg/ml HMGN1 or mutated HMGN1 for 24 h before obtaining supernatants for measurement of the indicated cytokines. Shown is the mean (±SD) of triplicate wells of one experiment representative of two. *, P < 0.05 by Student’s t test when compared with the sham treatment.
Figure 1.

HMGN1 induces phenotypic and functional maturation of DCs. (A) Human MoDCs were incubated at 5 × 105/ml in the absence (sham) or presence of recombinant HMGN1 or HMGN2 (both at a final concentration of 1 µg/ml) for 48 h before they were immunostained and analyzed by flow cytometry for the expression of the indicated surface molecules (open area, isotype-matched control). Shown are the results of one experiment representative of five. (B) Human MoDCs were incubated at 5 × 105/ml in the absence (sham) or presence of recombinant HMGN1 or HMGN2 at the indicated doses for 24 h (left) or at 1 µg/ml for the indicated times (right), and cytokine levels in the culture supernatants were quantitated by cytokine array (mean ± SD; n = 3). (C) Human MoDCs cultured at 1 × 106/ml in the absence (sham) or presence of 1 µg/ml of recombinant HMGN1 for 24 h were suspended in chemotaxis medium, and their migration in response to 100 ng/ml CCL5, CCL21, or CXCL12 was measured by chemotaxis assay. The migration of DCs was enumerated by the mean (±SD) of six high-power fields (HPF) randomly chosen from triplicate wells. Shown are the results of one experiment representative of two. (D) Human MoDCs cultured in the absence or presence of 1 µg/ml HMGN1 or LPS for 48 h were irradiated at 3,000 rad and incubated in triplicate with allogeneic human peripheral blood T cells (105/well) in 0.2 ml of medium in wells of 96-well flat-bottomed plates at the indicated DC/T ratios for 5 d. The proliferation was measured as the mean (±SD) [3H]TdR incorporation of triplicate wells. Shown are the results of one experiment representative of three. (E and F) Human MoDCs were incubated at 1 × 106/ml in the absence (sham) or presence of synthetic HMGN1 N-terminal domain (N1ND, HMGN11–52) or C-terminal domain (N1CD, HMGN153–100) at 10 µg/ml for 48 h before measurement of DC surface marker expression by flow cytometry (E, gray area: sham-treated DCs) and cytokine production in the supernatants (F; mean ± SD; n = 3). (G) Human DCs were incubated at 5 × 105/ml in medium in the absence (sham) or presence of 1 mg/ml HMGN1 or mutated HMGN1 for 24 h before obtaining supernatants for measurement of the indicated cytokines. Shown is the mean (±SD) of triplicate wells of one experiment representative of two. *, P < 0.05 by Student’s t test when compared with the sham treatment.

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