NK cells secrete HMGB1 that induces DC maturation. (A) Western blot analysis of HMGB1 in supernatants (sups, upper panel) and cell lysates (CL, lower panel) from 4-hour cultures of IL-2-activated NK cells (lane 1), rNK cells (lane 2), coculture of iDC and IL-2-activated NK cells (lane 3), coculture of iDC and rNK cells (lane 4), iDCs (lane 5), and LPS-treated DCs (mDCs; lanes 6). One representative experiment of 4 performed is shown. (B) CD86⁺ expression on iDCs (▦, percentage; □, MFI) after 24 hours of culture without (iDCs) or with 1 μg/mL purified HMGB1, or with supernatants from iDCs, rNK cells or aNK cells, or from iDC/aNK cocultures, as indicated. One representative experiment of 4 performed is shown. (C) Confocal immunofluorescence analysis of a DC/NK conjugate stained for HMGB1 (left) and perforin (right). In DCs most HMGB1 is nuclear, whereas in NK cells it is almost equally distributed between nucleus and cytoplasm. Arrows point to the cytoplasmic staining of HMGB1 and perforin in NK cell cytoplasm. (D) Western blot analysis of HMGB1 in cytoplasmic fractions from iDCs (lane 1), LPS-treated DCs (mDCs, lane 2), rNK cells (lane 3), and aNK cells (lane 4). (E) Aliquots of iDCs (upper panel) and IL-2-activated NK cells (lower panel) were lysed and 50 μg total protein extract was loaded onto 2-dimensional gels, blotted, and hybridized with anti-HMGB1 antibody. Note the presence of more acidic HMGB1 spots in NK cells.