Mature natural killer cells with phenotypic and functional alterations accumulate upon sustained stimulation with IL-15/IL-15Ralpha complexes

Abstract

Cytotoxic lymphocytes such as natural killer (NK) and CD8 T cells play important roles in immunosurveillance by killing virally infected or malignant cells. The homeostatic cytokine, IL-15, promotes the development, function, and survival of NK and CD8 T cells. IL-15 is normally presented in trans as a surface complex with IL-15 receptor-alpha-chain (IL-15Rα) by dendritic cells (DCs) and monocytes. Signaling by IL-15 occurs via the IL-2/IL-15 receptor β-chain (CD122) which is expressed primarily by NK1.1(+) cells and CD8 T cells. The use of preformed complexes of IL-15 with soluble IL-15Rα complexes to boost the effector function of CD122(+) cytolytic lymphocytes such as NK and CD8 T cells has recently gained considerable attention. Here we describe the impact of transient and prolonged in vivo stimulation by IL-15/IL-15Rα complexes on NK and CD8 T cells. Whereas transitory stimulation increased the number of activated NK cells and significantly enhanced their effector function, prolonged stimulation by IL-15/IL-15Rα complexes led to a marked accumulation of mature NK cells with considerably impaired activation, cytotoxicity, and proliferative activity, and an altered balance of activating and inhibitory receptors. In contrast to NK cells, CD8 T cells exhibited an activated phenotype and robust T cell receptor stimulation and effector function upon chronic stimulation with IL-15/IL-15Rα complexes. Thus, prolonged stimulation with the strong activating signal leads to a preferential accrual of mature NK cells with altered activation and diminished functional capacity. These findings point to a negative feedback mechanism to preferentially counterbalance excessive NK cell activity and may have important implications for cytokine immunotherapy.

Fig. 1.

Lowered activation status and altered balance of activating and inhibitory receptors on NK cells upon sustained in vivo stimulation with IL-15/IL-15Rα complexes. The first five histograms show representative histograms for expression of activation markers (B220, CD11c, CD44, CD43, and CD69) on splenic NK cells. Other histograms show expression of activating and inhibitory receptors (NKG2D, NKp46, 2B4, CD84, DNAM1, Ly-49H, Ly-49D, and NKG2A) on splenic NK cells. Dot plot shows expression of Ly-49I/C/F/H versus Ly-49H to determine percentage of Ly-49I/C/F⁺ NK cells. Numbers indicate percentage; italicized numbers indicate mean fluorescence intensity (MFI).

Fig. 2.

Accumulation of mature NK cells upon sustained in vivo stimulation with IL-15/IL-15Rα complexes. (A) Representative dot plots showing CD11b versus CD27 (Left) and KLRG1 (Right) expression on NK cells from spleens (Un, untreated; Tr, transient; Su, sustained). Numbers indicate percentage. (B) Percentage of KLRG1⁺ NK cells in spleen (black, n = 9), lung (dark gray, n = 3), liver (gray, n = 3), lymph nodes (light gray, n = 3), and bone marrow (white, n = 3). (C) Number of KLRG1⁺ NK cells in the spleen of untreated mice or upon transient and sustained treatments (n = 6). Significance: *P < 0.05, **P < 0.01; NS, not significant. Data are means ± SD.

Fig. 3.

Reduced proliferative capacity of NK cells upon sustained in vivo stimulation with IL-15/IL-15Rα complexes. (A) Percentage of proliferating NK cells in spleens of untreated mice or upon transient and sustained treatments determined by intracellular K_i-67 staining (n = 6). (B) Representative histograms showing K_i-67 staining in NK cells at different maturation stages, on the basis of CD11b and KLRG1 expression. Gray filled, isotype control; black line, KLRG1. Numbers indicate percentage. R1, CD11b⁺KLRG1⁻; R2, CD11b⁺KLRG1⁺; and R3, CD11b⁻KLRG1⁻. (C) Percentage of apoptotic KLRG1⁺ NK cells (gate R2) analyzed by FLICA assay (n = 3). Significance: *P < 0.05, **P < 0.01; NS, not significant. Data are means ± SD.

Fig. 4.

Impaired effector function of NK cells upon sustained in vivo stimulation with IL-15/IL-15Rα complexes. (A) Percentage of IFNγ⁺ NK cells in spleen (gated on CD49b⁺CD3ε⁻ cells) upon 5-h anti-NK1.1 stimulation (n = 5). White bars, anti-NK1.1 (PK136); black bars, isotype control. (B) IFNγ production by NK cells upon 5-h PMA/ionomycin stimulation. MFI of IFNγ⁺ NK cells shown for spleen (n = 11). (C) TNFα production by NK cells upon 5-h PMA/ionomycin stimulation. Percentage of TNFα⁺ NK cells shown for spleen (n = 3). (D) Degranulation of NK cells indicated by CD107a staining. Splenocytes were stimulated on anti-NK1.1–coated plates and intracellularly stained with anti-IFNγ. Percentage of CD107a⁺ IFNγ⁻ (black) and IFNγ⁺ (white) NK cells shown (n = 6–7). (E) Cytotoxicity of NK cells analyzed in vitro. NK cell-enriched splenocytes were cultured with a constant number (1 × 10⁴) of YAC1 cells at different effector:target ratios as indicated for 4 h and cytotoxicity was measured by lactate dehydrogenase release assay. Filled square, untreated; filled circle, transient; open circle, sustained (n = 3). (F) Cytotoxicity of NK cells analyzed in vivo by CFSE-based assay. Percentage of in vivo killing in untreated (n = 12) mice or upon transient (n = 12) and sustained (n = 10) treatments calculated as described in Materials and Methods. Each circle represents an individual mouse. Lines indicate the average value. Significance: *P < 0.05, **P < 0.01, ***P < 0.001; NS, not significant. Data are means ± SD.