CD57 defines a functionally distinct population of mature NK cells in the human CD56dimCD16+ NK-cell subset

Abstract

Natural killer (NK) cells are innate immune lymphocytes that express a heterogeneous repertoire of germline-encoded receptors and undergo a distinct pattern of maturation. CD57 is a marker of terminal differentiation on human CD8(+) T cells. Very few newborn or fetal NK cells express CD57; however, the frequency of CD57-bearing NK cells increases with age. We assessed the transcriptional, phenotypic, and functional differences between CD57(+) and CD57(-) NK cells within the CD56(dim) mature NK subset. CD57(+) NK cells express a repertoire of NK-cell receptors, suggestive of a more mature phenotype, and proliferate less when stimulated with target cells and/or cytokines. By contrast, a higher frequency of CD57(+) NK cells produced interferon-γ and demonstrated more potent lytic activity when these cells were stimulated through the activating receptor CD16; however, they are less responsive to stimulation by interleukin-12 and interleukin-18. Finally, CD57 expression is induced on CD57(-)CD56(dim) NK cells after activation by interleukin-2. A combination of a mature phenotype, a higher cytotoxic capacity, a higher sensitivity to stimulation via CD16, with a decreased responsiveness to cytokines, and a decreased capacity to proliferate suggest that CD57(+) NK cells are highly mature and might be terminally differentiated.

Figure 1

CD57⁻CD56^dimCD16⁺ NK cells are phenotypically less mature than CD57⁺ NK cells. (A) Flow cytometry was performed on total PBMCs, and CD3⁻CD56⁺ NK cells were gated (as shown in supplemental Figure 1) and analyzed for expression of CD57 and CD56. Two representative donors are shown. (B-E) Flow cytometry was performed on total PBMCs, and CD56^dimCD16⁺ NK cells were gated on CD57⁺ and CD57⁻ cells that were assessed for expression of different markers. Representative histograms for each marker are shown (shaded area represents isotype-matched control Ig; thin line, CD57⁺; and bold line, CD57⁻). The percentage of cells expressing CD62L and CD27 (B) and KLRG1 (D) was determined on each subset. The gMFI of NKp46, NKp30, NKG2D (C) and CD16 is shown (E). ● represents CD57⁺; and ◇, CD57⁻. ***P < .0005. (B-E) P values were determined comparing CD57⁺ and CD57⁻ NK cells (n = 23, for KLRG1 n = 18 and for NKp30 n = 14).

Figure 2

More CD56^dimCD16⁺CD57⁺ NK cells express KIR and LIR-1 than CD56^dimCD16⁺CD57⁻ NK cells. CD56^dimCD16⁺ NK cells that are CD57⁺ or CD57⁻ were assessed for the expression of LIR-1 and KIRs. (A) Representative histogram of LIR-1 expression (shaded area represents isotype-matched control Ig; thin line, CD57⁺; and bold line, CD57⁻). The percentage of cells expressing LIR-1 was determined in each subset. (B) The percentage of cells expressing each KIR was determined in each subset. ● represents CD57⁺; and ◇, CD57⁻. *P < .05. ***P < .0005. (A-B) P values were determined comparing CD57⁺ and CD57⁻ NK cells (n = 23).

Figure 3

CD57 is acquired after activation of CD57⁻CD56^dim NK cells. Sorted CD57⁻CD56^dim NK cells were cultured with 500 IU/mL or 1000 IU/mL IL-2 for 5 days. Representative histograms of CD57 expression by the NK cells before sort, after sort, and after stimulation are shown (shaded area represents isotype-matched control Ig; and bold line, CD57 stained cells).

Figure 4

More CD57⁺CD56^dimCD16⁺ NK cells produce IFN-γ in response to activation by CD16, but they are less responsive to IL-12 and IL-18. Enriched NK cells were activated for 6 hours with different cytokines (A), with anti-CD16 at 10 μg/mL (B), with anti-CD16 at different concentrations (0.5, 1, or 10 μg/mL) and 200 IU/mL IL-2 (C), with plate-bound antibodies at 10 μg/mL and 200 IU/mL IL-2 (D), or with K562 or 721.221 target cells and 200 IU/mL IL-2 (E). CD56^dimCD16⁺ NK cells were gated on CD57⁺ and CD57⁻ cells, and the percentages of cells expressing IFN-γ were determined in each subset (A-E). The gMFI of IFN-γ staining of each subset is shown (A-C). Only minimal amounts of IFN-γ were detected in NK cells cultured in either IL-12 or IL-18 alone (not shown), similar to the amount detected in NK cells cultured in IL-2 alone (A). Black bars represent CD57⁺; and white bars, CD57⁻. **P < .005. ***P < .0005. P values were determined between CD57⁺ and CD57⁻ NK cells (n = 8).

Figure 5

CD57⁺CD56^dimCD16⁺ NK cells proliferate less than CD57⁻CD56^dimCD16⁺ NK cells in vitro but are not more sensitive to AICD. (A) CD57⁺ and CD57⁻ sorted NK cells were CFSE-labeled and cultured in 100 IU/mL IL-2 with K562 cells expressing membrane-tethered IL-15 and 4-1BBL. Cells were analyzed by flow cytometry at different time points. Representative histograms of CFSE dilution over time (thin line represents CD57⁺; and bold line, CD57⁻; n = 7 for A, n = 3 for B). (B) Total PBMCs were activated with plate-bound antibodies (10 μg/mL) in 200 IU/mL IL-2 for 6 hours. Flow cytometry was performed, and CD56^dimCD16⁺ NK cells were gated on CD57⁺ and CD57⁻ cells. The percentages of cells expressing annexin V were determined for each subset (black bars represent CD57⁺; and white bars, CD57⁻). *P < .05. **P < .005. P values were determined between CD57⁺ NK cells and CD57⁻ NK cells (n = 5).

Figure 6

CD57⁺CD56^dimCD16⁺ NK cells have a higher cytotoxic potential and mediate better CD16-induced cytotoxicity. (A) Percentages of NK cells that express perforin, granzyme B, or both cytotoxic molecules were assessed on CD57⁺CD56^dimCD16⁺ NK cells and CD57⁻CD56^dimCD16⁺ NK cells (● represents CD57⁺; and ◇, CD57⁻). ***P < .0005. P values were determined between CD57⁺ and CD57⁻ NK cells (n = 23). (B-C) Low buoyant density lymphocytes were activated with K562 or 721.221 target cells (B) or with plate-bound antibodies (C) in media with 200 IU/mL IL-2. CD56^dimCD16⁺ NK cells were gated on CD57⁺ and CD57⁻ cells, and the percentages of CD107⁺ cells were determined on each subset (black bars represent CD57⁺; and white bars, CD57⁻). P values were determined between CD57⁺ and CD57⁻ NK cells (n = 8). (D) Sorted CD57⁺ and CD57⁻ CD56^dimCD16⁺ NK cells were activated with P815 target cells coated with anti-CD56 or anti-CD16. *P < .05. P value was determined between the percentage of specific lysis of P815 plus anti-CD16 by CD57⁺ and CD57⁻ NK cells (n = 6, in triplicate).