Dendritic cells support the in vivo development and maintenance of NK cells via IL-15 trans-presentation

Abstract

IL-15 is a key component that regulates the development and homeostasis of NK cells and is delivered through a mechanism termed trans-presentation. During development, multiple events must proceed to generate a functional mature population of NK cells that are vital for tumor and viral immunity. Nevertheless, how IL-15 regulates these various events and more importantly what cells provide IL-15 to NK cells to drive these events is unclear. It is known dendritic cells (DC) can activate NK cells via IL-15 trans-presentation; however, the ability of DC to use IL-15 trans-presentation to promote the development and homeostatic maintenance of NK cell has not been established. In this current study, we show that IL-15 trans-presentation solely by CD11c(+) cells assists the in vivo development and maintenance of NK cells. More specifically, DC-mediated IL-15 trans-presentation drove the differentiation of NK cells, which included the up-regulation of the activating and inhibitory Ly49 receptors. Although these cells did not harbor a mature CD11b(high) phenotype, they were capable of degranulating and producing IFN-gamma upon stimulation similar to wild-type NK cells. In addition, DC facilitated the survival of mature NK cells via IL-15 trans-presentation in the periphery. Thus, an additional role for NK-DC interactions has been identified whereby DC support the developmental and homeostatic niche of NK cells.

FIGURE 1

IL-15Rα⁺ DC influence in the generation of NK cells. A, NK1.1⁺CD3⁻ cells recovered in the BM, liver, and spleen of IL-15Rα^−/− (left column), CD11c/IL-15Rα Tg (middle column), and B6 (right column) mice were analyzed by flow cytometry. Data are representative of three independent experiments. B, Absolute numbers of NK cells (NK1.1⁺CD3⁻ cells) in the respective tissues of IL-15Rα^−/− (■), CD11c/IL-15Rα Tg ( ), and B6 (□) mice were calculated (average of three independent experiments, n = 7 mice/group). Error bars represent SE; *, p ≤ 0.05. Numbers above bars represent percentage of Wt levels.

FIGURE 2

Recovered developmental stages and tissue distribution of NK cells by DC-mediated IL-15 trans-presentation. A–D, Stages of NK cell development were identified by flow cytometric analysis of BM, spleen, and liver isolated from each group of mice (IL-15Rα^−/−, CD11c/IL-15Rα Tg, and Wt mice). Representative flow cytometry data from the BM are shown in A. NK precursors are characterized as CD122⁺NK1.1⁻ cells; immature are CD122⁺NK1.1⁺DX5⁻; early mature (M1) display CD122⁺NK1.1⁺DX5⁺CD11b^low phenotype; and late mature (M2) are a CD122⁺NK1.1⁺DX5⁺CD11b^high population. B, Absolute numbers of NK cells at the various developmental stages in the respective tissues (average of three independent experiments, n = 7 mice/group) in the indicated groups. C, Lymphocytes were isolated from the indicated tissues of IL-15Rα^−/− BM chimeras 8–12 wk after irradiation and BM reconstitution. Numbers of NK cells at the various developmental stages in the respective tissues of IL-15Rα^−/− BM chimeras are shown (average of three independent experiments, n = 8 mice/group). Error bars represent SE; *, p ≤ 0.05. D, NK cell subsets distinguished by their cell surface expression of CD27 and CD11b in the BM (top row) and spleen (bottom row) from each group of mice. Flow cytometric plots are representative of three independent experiments (n = 7 for each group of mice).

FIGURE 3

DC drive Ly49 expression of immature NK cells. A and B, Expression of Ly49 activating and inhibitory receptors on splenic NK cells as detected by flow cytometry in Wt (top row), CD11c/IL-15Rα Tg (middle row), and IL-15Rα^−/− (bottom row) mice. A, Histograms showing representative data from two independent experiments. B, Graph depicts the average percentage of the population that is positive for the indicated Ly49 molecule (n = 4/group). Error bar represent SE; *, p ≤ 0.05. C, In vitro acquisition of the Ly49 repertoire by BMDC. Ly49⁻ NK cells were sorted from spleens of Wt mice and cocultured with DC generated from BM of either IL-15Rα^−/− (left plot) or Wt (right plot) mice with GM-CSF. Plots show expression of Ly49 on NK1.1⁺CD3⁻ cells at the end of coculturing. Data are representative of two independent experiments. D, Cell surface expression of Ly49 activating and inhibitory receptors by NK cells derived from Wt (top row) and IL-15Rα^−/− (bottom row) BM donor cells. BM cells from Wt (CD45.2⁺) and IL-15Rα^−/− (CD45.1/CD45.2⁺) were injected in equal proportions into irradiated CD45.1⁺ Wt hosts. After reconstitution of hemopoietic cells (~8–10 wk later), the respective donor-derived NK1.1⁺CD3⁻ cells in the spleen were identified based on CD45 isoform expression. Plots are representative of three experiments (n = 5 mice/group).

FIGURE 4

DC assist in the expansion of NK cells in the BM. A, Representative histograms showing the in vivo BrdU incorporation [3 wk of BrdU (0.8 mg/ml) in drinking water] as detected by flow cytometry in mature NK cells (NK1.1⁺DX5+CD3⁻) found in the BM (top row), spleen (middle row), and liver (bottom row) in the three groups of mice. B, Graph depicts the average BrdU incorporation of mature NK cells from the individual groups in two independent experiments (n = 4 mice/group).

FIGURE 5

NK cells derived by DC-mediated IL-15 trans-presentation are functional but not activated under steady-state conditions. A, Effector function of NK cells. NK cells sorted from spleens of either Wt or CD11c/IL-15Rα Tg mice were incubated for 5 h in the absence (top row) or presence (bottom row) of plate-bound anti-NK1.1 Ab. Intracellular IFN-γ production and CD107 cell surface mobilization were measured via flow cytometry as indicators of effector function. Plot is representative of two independent experiments. B, Ex vivo comparison of the cell surface expression of the activation-induced marker, CD69, by splenic NK cells from Wt or CD11c/IL-15Rα Tg mice.

FIGURE 6

DC support the maintenance of peripheral NK cells. A, Graph shows the average numbers of donor NK cells (gated on CD45.1⁺NK1.1⁺DX5⁺CD11b⁺CD3⁻ cells) recovered in the BM, spleen, and liver 3 wk after the adoptive transfer into congenic CD45.2⁺IL-15Rα^−/− (■), CD11c/IL-15Rα Tg ( ), and Wt (□) mice. Error bars represent SE, and the numbers above bars represent percentage of Wt levels; *, p ≤ 0.05. B, Dilution of CFSE by donor cells found in the spleen of the three hosts. Plots are representative of two individual experiments (n = 4 for each group).