Interleukin-21 differentially affects human natural killer cell subsets

Abstract

Interleukin-21 (IL-21) is a cytokine with pleiotropic effects on various cell types including dendritic cells, B cells, T cells and natural killer (NK) cells. To evaluate if IL-21 affects human NK cell subpopulations in a similar fashion, functional studies were performed on CD56(dim) and CD56(bright) NK cells, both bearing IL-21 receptors at identical densities. Stimulation with IL-21 strongly induced proliferation of CD56(bright) NK cells and cytotoxicity against K562 target cells was preferentially augmented in CD56(dim) NK cells. In contrast, stimulation with IL-2 and IL-21 alone or in combination failed to induce interferon-gamma and tumour necrosis factor-alpha production in the two NK cell subsets. Intracellular analysis of signal transducer and activator of transcription (STAT) proteins revealed that IL-21 by itself induces phosphorylation of STAT1 and STAT3 in CD56(dim) NK cells, and to an even higher degree in CD56(bright) NK cells. In this CD56(bright) NK cell population alone, IL-2 weakly phosphorylated STAT1 and STAT3, which was further increased when cells were treated with the combination of both cytokines. In contrast, STAT5 was strongly phosphorylated only in CD56(bright) NK cells by low-dose IL-2, while IL-21 did not affect STAT5 at all. In summary, we present data indicating that the NK-cell-directed cytokines IL-2 and IL-21 not only affect functions in NK cell subpopulations differently but can also act additively.

Figure 1

Expression of IL-21R on NK cell subsets. (a) Affymetrix array analysis revealed positive signals for IL-21R-specific mRNA in resting and activated CD56^dim (open blocks) and CD56^bright (solid blocks) NK cells using Probe Set 219971_at. Expression data are publicly accessible at EMBO-EBI ArrayExpress database (URL: http://www.ebi.ac.uk/aerep/entry?) under accession number: E-MEXP-380. Flow cytometry revealed a low surface expression of IL-21R on NK cells. Histograms of CD56^dim (b) and CD56^bright (c) subsets document the expression of IL-21R on resting (grey-filled) and activated (solid line) NK cells as compared to the isotype controls (dotted line).

Figure 2

Regulation of activation markers by IL-21 and/or IL-2. PBMCs of healthy donors (n = 7) were stimulated with IL-21 and/or IL-2 and activation markers CD69 (a) and CD25 (b) were analysed using flow cytometry. The percentage of receptor-bearing CD56^dim (open blocks) and CD56^bright (solid blocks) NK cells are shown. Solid connecting lines represent significant changes as compared to a medium control, whereas dotted lines depict significant differences between CD56^dim and CD56^bright NK cells following activation with the respective stimulus. For comparisons between the two populations, values were adjusted for medium levels ***P < 0·001; **P < 0·01; *P < 0·05; ±SEM.

Figure 3

Patterns of IL-21- and/or IL-2-induced proliferation of NK cell subsets. Proliferation of sorted NK cell subsets in response to activation with IL-21 and/or IL-2 was investigated by [³H]thymidine assay. Mean values of seven assays ±SEM are shown. Asterisks above solid lines represent significant increases as compared to the medium control. Asterisks above dotted lines illustrate significant differences between CD56^dim (open blocks) and CD56^bright (solid blocks) NK cells activated with the same mediator. For comparisons between the two populations values were adjusted for medium levels. ***P < 0·001; **P < 0·01; *P < 0·05; ± SEM.

Figure 4

IL-21 and/or IL-2 enhance cytotoxicity and conjugate formation. (a) Sorted NK cell subsets were activated with IL-21 and/or IL-2 for 20 hr and cytotoxicity against K562 was determined in standard 4-hr ⁵¹Cr-release assays; lytic units (LU₂₀/10⁷ cells) are depicted. (b) The impact of IL-21 and/or IL-2 on conjugate forming ability of CD56^dim (open blocks) and CD56^bright (solid blocks) NK cells complexed to target cells was measured using flow cytometry and gating on respective NK subsets. Mean values of NK cells bound to targets are given in percentage ± SEM (n = 6).

Figure 5

Intracellular staining of pSTAT proteins. PBMCs were stimulated with cytokines as indicated and analysed for phosphorylation of STAT1 (a), STAT3 (b) and STAT5 (c) by flow cytometry. Grey-filled histograms represent medium controls and open histograms represent the respective stimulus.

Figure 6

FACS analyses of phosphorylated STATs in NK cell populations. PBMCs were stimulated with cytokines as indicated (1: IL-2, 2: IL-21, 3: IL-2 + IL-21, filled grey area: medium control) and analysed for phosphorylation of STAT1 (a), STAT3 (b), and STAT5 (c) by gating on CD56^dim (left panel) and CD56^bright (right panel) NK cells. Bar diagrams depict MFIs of pSTAT1 (d), pSTAT3 (e), and pSTAT5 (f) as assessed for CD56^dim (open blocks) and CD56^bright (solid blocks) NK cells (n = 7). Asterisks above solid connecting lines represent significant enhancement compared to medium control. Asterisks above dotted lines illustrate significant differences between CD56^dim and CD56^bright NK cells activated with the same stimulus. For comparisons between the two populations values were adjusted for medium levels; ***P < 0·001; **P < 0·01; *P < 0·05; ± SEM.