CD94 surface density identifies a functional intermediary between the CD56bright and CD56dim human NK-cell subsets

Abstract

Human CD56(bright) natural killer (NK) cells possess little or no killer immunoglobulin-like receptors (KIRs), high interferon-gamma (IFN-gamma) production, but little cytotoxicity. CD56(dim) NK cells have high KIR expression, produce little IFN-gamma, yet display high cytotoxicity. We hypothesized that, if human NK maturation progresses from a CD56(bright) to a CD56(dim) phenotype, an intermediary NK cell must exist, which demonstrates more functional overlap than these 2 subsets, and we used CD94 expression to test our hypothesis. CD94(high)CD56(dim) NK cells express CD62L, CD2, and KIR at levels between CD56(bright) and CD94(low)CD56(dim) NK cells. CD94(high)CD56(dim) NK cells produce less monokine-induced IFN-gamma than CD56(bright) NK cells but much more than CD94(low)CD56(dim) NK cells because of differential interleukin-12-mediated STAT4 phosphorylation. CD94(high)CD56(dim) NK cells possess a higher level of granzyme B and perforin expression and CD94-mediated redirected killing than CD56(bright) NK cells but lower than CD94(low)CD56(dim) NK cells. Collectively, our data suggest that the density of CD94 surface expression on CD56(dim) NK cells identifies a functional and likely developmental intermediary between CD56(bright) and CD94(low)CD56(dim) NK cells. This supports the notion that, in vivo, human CD56(bright) NK cells progress through a continuum of differentiation that ends with a CD94(low)CD56(dim) phenotype.

Figure 1

Dissection of human CD56⁺CD3⁻ NK-cell subsets by CD94 surface expression. (A) Mononuclear cells freshly isolated from peripheral blood were stained with anti-CD3, anti-CD56, and anti-CD94 fluorescently labeled mAb. CD56⁺CD3⁻ NK cells were then gated for quantification of CD94 surface density. The dot plot shown is for a representative donor, and the numbers in the plot are averaged from 14 healthy donors and represent the percentage of total NK cells ± SD belonging to each subset of CD94^highCD56^bright, CD94^highCD56^dim and CD94^lowCD56^dim. (B) Comparison of CD94-PE antibody staining (gray) and its isotype control IgG1-PE staining (black) of NK cells gated on CD56⁺CD3⁻.

Figure 2

Quantification of surface antigen expression on human NK subsets defined by CD56 and CD94. Peripheral blood NK cells were enriched to purity 50% to 85% using RosetteSep cocktail and then stained with anti-CD3, anti-CD56, and anti-CD94–conjugated mAb, as well as a mAb directed against the surface antigens indicated in each histogram. Each of the 3 gated populations (CD94^highCD56^bright, CD94^highCD56^dim, and CD94^lowCD56^dim) was then assessed for positivity for surface expression of the fourth antigen as shown compared with its isotype control (not shown). The number in each histogram shows the percentage of cells that are positive for the indicated antigen(s), and is averaged from at least 3 different normal donors.

Figure 3

CD94 identifies functionally distinct human CD56^dim NK-cell subsets with regard to IFN-γ production. (A left panel) Enriched NK cells were costimulated with IL-12 and IL-18 for 24 hours followed by intracellular staining for IFN-γ. After gating on CD56^dimCD3⁻ cells, density of CD94 surface expression and intracellular IFN-γ production were simultaneously assessed as shown in the representative dot plot. (A right panel) Equal numbers of the 3 NK-cell subsets were stimulated with IL-12 plus IL-18, followed by measurement of secreted IFN-γ using ELISA. (B) Relative comparison of each of the 3 NK-cell subsets (left) costimulated by IL-12 and IL-18 for 24 hours in ability to produce IFN-γ using intracellular flow-cytometric analysis (right). Cells on the right side of the dashed vertical line are positive for IFN-γ. The figures are representative of at least 3 separate experiments. (C) Equal numbers of the 3 NK-cell subsets were stimulated with IL-12 plus IL-15 for 24 hours, followed by measurement of IFN-γ mRNA by real-time PCR (left) as well as secreted IFN-γ by ELISA (right). Bar graphs (A,C) represent mean ± SD for at least 3 experiments. Enriched NK cells under no monokine stimulation were used as a negative control for IFN-γ ELISA assay (A,C).

Figure 4

The CD94^highCD56^dim NK-cell subset shows an intermediate level of granzyme B and perforin 1 gene expression and CD94-mediated redirected killing compared with the CD94^highCD56^bright and the CD94^lowCD56^dim NK-cell subsets. (A-B) Real-time PCR analysis of GZMB (A) and PRF1 (B) using cDNA from the freshly sorted CD94^highCD56^bright, CD94^highCD56^dim, and CD94^lowCD56^dim NK-cell subsets. Data are summarized from 6 donors, and the error bars represent SD. (C) ⁵¹Cr cytotoxicity analysis of the aforementioned sorted 3 NK-cell subsets against the murine cell line P815 in the presence of the purified anti–human CD94 mouse mAb. Data are summarized from 3 donors, and the error bars represent SD.

Figure 5

The CD94^highCD56^dim NK-cell subset possesses higher STAT4 phosphorylation than the CD94^lowCD56^dim NK-cell subset. (A-B) Purified CD94^highCD56^dim (Hi) and CD94^lowCD56^dim (Lo) NK-cell subsets were cultured in media in the presence or absence of costimulation with rhIL-12 (10 ng/mL) and rhIL-15 (100 ng/mL) for 30 minutes. Lysates from these cells were used to determine the level of STAT5, and STAT3 (A) or STAT4 (B) phosphorylation by Western blotting. Assessment of β-actin or total STAT4 was included to control for protein loading. (C) CD94^highCD56^dim (Hi) and CD94^lowCD56^dim (Lo) NK-cell subsets were treated in media with or without rhIL-12 alone or costimulation by IL-12 and IL-18 for 30 minutes. Lysates from the harvested cells were used to detect STAT4 phosphorylation. β-Actin was included to control for protein loading. Data in panels A to C are representative of at least 3 experiments with similar results.