Cytokine-induced memory-like natural killer cells

Abstract

The mammalian immune response to infection is mediated by 2 broad arms, the innate and adaptive immune systems. Innate immune cells are a first-line defense against pathogens and are thought to respond consistently to infection, regardless of previous exposure, i.e., they do not exhibit memory of prior activation. By contrast, adaptive immune cells display immunologic memory that has 2 basic characteristics, antigen specificity and an amplified response upon subsequent exposure. Whereas adaptive immune cells have rearranged receptor genes to recognize the universe of antigens, natural killer (NK) cells are innate immune lymphocytes with a limited repertoire of germ-line encoded receptors for target recognition. NK cells also produce cytokines such as IFN-gamma (IFN-gamma) to protect the host during the innate response to infection. Herein, we show that cytokine-activated NK cells transferred into naïve hosts can be specifically detected 7-22 days later when they are phenotypically similar to naïve cells and are not constitutively producing IFN-gamma. However, they produce significantly more IFN-gamma when restimulated. This memory-like property is intrinsic to the NK cell. By contrast, memory-like NK cells do not express granzyme B protein and kill targets similarly to naïve NK cells. Thus, these experiments identify an ability of innate immune cells to retain an intrinsic memory of prior activation, a function until now attributed only to antigen-specific adaptive immune cells.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Adoptively transferred, preactivated, and control NK cells can be detected after 1–3 weeks and are phenotypically similar. (A) CFSE-labeled preactivated (IL-12 + IL-18 with 10 ng/mL IL-15) or control-treated (10 ng/mL IL-15) NK cells were transferred into Rag1−/− hosts and could be detected 7 (Upper) to 22 (Lower) days later in the spleen, identified as CFSE+NK1.1+. Percentages represent transferred NK cells, gated on live lymphocytes. (B) Preactivated (solid black line) and control (dashed line) NK cells express similar levels of NK activation markers (CD69 and CD11b), CD27, and B220. (C) Cytokine receptor expression 7 days after adoptive transfer (isotype control, shaded). Results are representative of 2–5 independent experiments.

Fig. 2.

Preactivated NK cells proliferate in vivo and do not constitutively make IFN-γ but produce abundant IFN-γ upon restimulation. (A) NK cells preactivated with IL-12 + IL-18 (plus 10 ng/mL IL-15) proliferate in vivo significantly more than control-treated (10 ng/mL IL-15) cells (40.5% v. 6.3%;*, P < 0.0001; n = 7). (B) Preactivated (Left) donor-derived (CFSE+) and host (CFSE−) NK cells do not constitutively produce IFN-γ 7 days after adoptive transfer. (C) Representative FACS plot of CFSE+ donor NK cell (Upper) and CFSE− host NK cell (Lower) IFN-γ production (x axis) after restimulation with IL-12 + IL-15 for 4 h. All plots are gated on NK1.1+ NK cells. The numbers indicate the percentage of CSFE+ (donor) cells (Upper) or CSFE− (host) cells (Lower) in the corresponding gates, demonstrating significantly more preactivated NK cells produce IFN-γ upon restimulation. (D) Percentage of IFN-γ positive CFSE+ (donor) and CFSE− (host) NK cells after stimulation with IL-12 + IL-15 (**, P < 0.01, n = 4). (E and F) Engagement of Ly49H (E) or NK1.1 (F) by culture with plate-bound monoclonal antibody for 8 h (***, P < 0.0001; ****, P = 0.004). IFN-γ production with anti-Ly49H is shown as a ratio of % IFN-γ positive donor NK cells (CFSE+NK1.1+) to % IFN-γ positive host NK cells (CFSE−NK1.1+) to account for well-to-well variability. Error bars indicate SEM.

Fig. 3.

Memory-like NK cells lack granzyme B protein and kill target cells similar to control NK cells. (A) Preactivated, memory-like NK cells (solid black line) do not express granzyme B protein 7 days after adoptive transfer into Rag1−/− hosts as compared with a positive control, NK cells activated with high-dose (100 ng/mL) IL-15 for 2 days. (Dashed line, control-treated NK cells; shaded, negative control; gray line, positive control). (B) Donor and host NK cells were sorted from recipients that had received activated or control NK cells 7 days prior and were used as effectors in a cytotoxicity assay with Yac-1 targets at the indicated effector to target (E:T) ratios. Data represent the mean of duplicate wells and results are representative of 2 independent experiments.

Fig. 4.

Similar IFN-γ production by memory-like NK cells regardless of proliferation. (A) Resting NK cells did not produce IFN-γ protein. (B) After restimulation with IL-12 + IL-15, parental (generation 0) and 3 daughter generations of preactivated memory-like NK cells were identified based on CFSE dilution and IFN-γ production in each generation measured 7 days after adoptive transfer. (C and D) There was no significant difference in IFN-γ production in generations 0–3 after restimulation with either IL-12 + IL-15 (C) or anti-Ly49H plate-bound antibody (D). Results represent the mean of 3 independent experiments. Error bars indicate SEM.