Human lymph node NK cells produce IFN-γ upon interaction with autologous DCs. (A) Lymph node mononuclear cells were cultured for 6 h alone (Medium), in the presence of 100 ng/ml LPS (LPS), or in the presence of immature (DC) or LPS-matured (DC + LPS) autologous monocyte-derived DCs. One of five experiments is shown. (B) Lymph node mononuclear cells were cultured for 6 h alone, with LPS and in the presence of spleen DCs, which were matured (DC + LPS) or not (DC) for 24 h in the presence of 100 ng/ml LPS. IFN-γ production was analyzed on CD3^– cells; numbers inside the dot plots indicate the percentage of NK cells producing IFN-γ in these representative experiments. One of three experiments is shown.

DCs present IL-15 on their surface upon maturation. DCs derived from monocytes by culture in the presence of GM-CSF and IL-4 (GM-CSF + IL-4) (A) and DCs directly isolated from spleen (B) present IL-15 on their surface upon maturation with LPS, proinflammatory cytokines (IL-1β, IL-6, TNF-α, and prostaglandin E₂) or live bacillus Calmette–Guérin. Monocyte/macrophages cultured without cytokines (Medium) or in the presence of GM-CSF alone (GM-CSF) and then activated by the same stimuli used for DC maturation failed to express IL-15 on their surfaces. Addition of CD40L-transfected cells to DC cultures (CD40L) in the last 48 h was sufficient, even in the absence of inflammatory stimuli, to induce surface IL-15 expression in DCs but not in monocyte/macrophages (data not shown). Dotted lines represent isotype controls. One of three experiments is shown. (C) NK cells purified from peripheral blood were labeled with CFSE and cultured in the presence of autologous DCs matured by different stimuli. At day 6 of the cultures, cells were harvested, and CFSE dilution was evaluated (LPS, DCs matured by 100 ng/ml LPS; CD40L, DCs matured by CD40L-transfected cells; Cytokines, DCs matured by proinflammatory cytokines). One of three experiments is shown. (D) Western blot analysis of IL-15 at the indicated time points of DC differentiation from peripheral blood monocytes and with (mDC) and without (iDC) maturation by proinflammatory cytokines for 2 days. One of three experiments is shown. (E) Flow cytometric analysis of CD83 (Center) and IL-15Rα (Right) on monocytes (Mono), immature DCs (iDC; day 8) and mature DCs (mDC; day 8 and last 2 days incubated with proinflammatory cytokines). As a control for the polyclonal anti-IL-15Rα goat antibody a goat anti-mouse polyclonal antibody was used (control). One of three experiments is shown.

NK cells and DCs colocalize in T cell areas of normal human lymph node. Sequential cryosections of human lymph node show that DEC-205-positive DCs (A) and CD3^–CD56⁺ NK cells (B) are in close proximity in the T cell area (T) of the tissue, but not in B cell follicles (B). (C) Arrowheads indicate NK cells expressing CD56 but not CD3 in the T cell area of lymph nodes. (Magnification: ×10 in A and B and ×40 in C.)

DCs stimulate IFN-γ secretion and proliferation mainly by CD56^bright NK cells. (A) Splenic CD56⁺CD3^– NK cells were analyzed for their CD56^brightCD16^– and CD56^dimCD16⁺ subsets. (B) IFN-γ secretion by purified splenic CD56⁺CD3^– NK cells upon monocyte-derived DC stimulation was analyzed. (C) Proliferation of purified splenic CD56⁺CD3^– NK cells upon monocyte-derived DC stimulation. The numbers in the blots indicate the percentages of the responding NK populations. One of two experiments is shown.

IL-12 and IL-15 are crucial for DC-mediated IFN-γ secretion and proliferation by NK cells, respectively. (A Upper) Lymph node mononuclear cells were cultured for 6 h in the presence of mature DCs isolated from autologous spleen in the presence of the indicated blocking antibodies. Analyses were performed on CD3^– cells. Similar results were obtained when autologous monocyte-derived DCs were used to activate lymph node NK cells (data not shown). (A Lower) NK cells purified from peripheral blood were cultured in the presence of autologous monocyte-derived DCs. One of three experiments is shown. (B) IFN-γ production by the EBNA1-specific CD4⁺ T cell clone A4.E116 in response to autologous monocyte-derived DCs, which had been pulsed with the cognate EBNA1_514-527 peptide, in the presence of blocking antibodies. One of three experiments is shown. NK cells purified from human lymph nodes (C) or from peripheral blood (D) were labeled with CFSE and cultured for 6 days in the presence of autologous DCs alone (Medium) and, where indicated, with blocking antibodies (IgG1, isotype control; αIL-2, anti-IL-2; αIL-12, anti-IL-12; αIL-15, anti-IL-15) or separated by transwell (Trans). Numbers inside dot plots indicate the percentage of cycling NK cells. One of two experiments is shown for C, and one of three experiments is shown for D.