NKp44, a novel triggering surface molecule specifically expressed by activated natural killer cells, is involved in non-major histocompatibility complex-restricted tumor cell lysis

Abstract

After culture in interleukin (IL)-2, natural killer (NK) cells acquire an increased capability of mediating non-major histocompatibility complex (MHC)-restricted tumor cell lysis. This may reflect, at least in part, the de novo expression by NK cells of triggering receptors involved in cytolysis. In this study we identified a novel 44-kD surface molecule (NKp44) that is absent in freshly isolated peripheral blood lymphocytes but is progressively expressed by all NK cells in vitro after culture in IL-2. Different from other markers of cell activation such as CD69 or VLA.2, NKp44 is absent in activated T lymphocytes or T cell clones. Since NKp44 was not detected in any of the other cell lineages analyzed, it appears as the first marker specific for activated human NK cells. Monoclonal antibody (mAb)-mediated cross-linking of NKp44 in cloned NK cells resulted in strong activation of target cell lysis in a redirected killing assay. This data indicated that NKp44 can mediate triggering of NK cell cytotoxicity. mAb-mediated masking of NKp44 resulted in partial inhibition of cytolytic activity against certain (FcgammaR-negative) NK-susceptible target cells. This inhibition was greatly increased by the simultaneous masking of p46, another recently identified NK-specific triggering surface molecule. These data strongly suggest that NKp44 functions as a triggering receptor selectively expressed by activated NK cells that, together with p46, may be involved in the process of non-MHC-restricted lysis. Finally, we show that p46 and NKp44 are coupled to the intracytoplasmic transduction machinery via the association with CD3zeta or KARAP/DAP12, respectively; these associated molecules are tyrosine phosphorylated upon NK cell stimulation.

Figure 1

Functional and molecular characteristics of NKp44 surface molecule. (a) Triggering of cytolytic activity. Four representative NK clones, derived from two donors, were analyzed in a redirected killing assay against the FcγR⁺ P815 target cells either in the absence (□) or presence of Z231 (▪), anti-CD16 ( ), anti-p46 ( ), anti-CD56 ( ), anti-CD69 ( ), or anti-CD40L ( ) mAbs at a 5:1 E/T ratio. All these mAbs are IgG1. Clones MA4 and MA5 were CD40L⁻, whereas clones MX340 and MX310 were CD40L⁺. (b) Biochemical characteristics. A ¹²⁵I-surface labeled polyclonal NK population, derived from donor LM, was immunoprecipitated with Z231 (lanes A and E), anti-CD69 (lanes B and F), anti-CD16 (lanes C and G) or BAB281 (lanes D and H) mAbs. Samples were analyzed in an 11% SDS-PAGE under nonreducing (lanes A–D) or reducing (lanes E–H) conditions. (c) N-glycosidase F digestion. NKp44 (lanes A and B), CD69 (lanes C and D), CD16 (lanes E and F), or p46 (lanes G and H) molecules isolated from the surface-labeled NK cell population shown in b were treated or not with N-glycosidase F. Samples were run in an 11% SDS-PAGE under reducing conditions.

Figure 1

Functional and molecular characteristics of NKp44 surface molecule. (a) Triggering of cytolytic activity. Four representative NK clones, derived from two donors, were analyzed in a redirected killing assay against the FcγR⁺ P815 target cells either in the absence (□) or presence of Z231 (▪), anti-CD16 ( ), anti-p46 ( ), anti-CD56 ( ), anti-CD69 ( ), or anti-CD40L ( ) mAbs at a 5:1 E/T ratio. All these mAbs are IgG1. Clones MA4 and MA5 were CD40L⁻, whereas clones MX340 and MX310 were CD40L⁺. (b) Biochemical characteristics. A ¹²⁵I-surface labeled polyclonal NK population, derived from donor LM, was immunoprecipitated with Z231 (lanes A and E), anti-CD69 (lanes B and F), anti-CD16 (lanes C and G) or BAB281 (lanes D and H) mAbs. Samples were analyzed in an 11% SDS-PAGE under nonreducing (lanes A–D) or reducing (lanes E–H) conditions. (c) N-glycosidase F digestion. NKp44 (lanes A and B), CD69 (lanes C and D), CD16 (lanes E and F), or p46 (lanes G and H) molecules isolated from the surface-labeled NK cell population shown in b were treated or not with N-glycosidase F. Samples were run in an 11% SDS-PAGE under reducing conditions.

Figure 1

Functional and molecular characteristics of NKp44 surface molecule. (a) Triggering of cytolytic activity. Four representative NK clones, derived from two donors, were analyzed in a redirected killing assay against the FcγR⁺ P815 target cells either in the absence (□) or presence of Z231 (▪), anti-CD16 ( ), anti-p46 ( ), anti-CD56 ( ), anti-CD69 ( ), or anti-CD40L ( ) mAbs at a 5:1 E/T ratio. All these mAbs are IgG1. Clones MA4 and MA5 were CD40L⁻, whereas clones MX340 and MX310 were CD40L⁺. (b) Biochemical characteristics. A ¹²⁵I-surface labeled polyclonal NK population, derived from donor LM, was immunoprecipitated with Z231 (lanes A and E), anti-CD69 (lanes B and F), anti-CD16 (lanes C and G) or BAB281 (lanes D and H) mAbs. Samples were analyzed in an 11% SDS-PAGE under nonreducing (lanes A–D) or reducing (lanes E–H) conditions. (c) N-glycosidase F digestion. NKp44 (lanes A and B), CD69 (lanes C and D), CD16 (lanes E and F), or p46 (lanes G and H) molecules isolated from the surface-labeled NK cell population shown in b were treated or not with N-glycosidase F. Samples were run in an 11% SDS-PAGE under reducing conditions.

Figure 2

Expression of NKp44 molecules in freshly isolated or cultured polyclonal and clonal NK or T cell populations. (top) A polyclonal NK cell population (A), NK clones (B–E), a polyclonal T population (F), TCR-γ/δ⁺ (G), or TCR-α/β⁺ (H) T cell clones or TCR-γ/δ⁺ p58.2⁺(I) and TCR-α/β⁺ p70⁺ (L) T cell clones were analyzed by immunofluorescence and FACS^® analysis for surface expression of NKp44 molecules. (bottom) Freshly isolated PBMCs were cultured in the presence of the irradiated H9 cell line and rIL-2 and analyzed by two-color immunofluorescence and FACS^® analysis at various time intervals for the expression of NKp44, CD56, p46, and CD69 surface molecules in combination with CD3 molecules. Cells were stained with mAbs to the indicated molecules followed by FITC- or PE-conjugated isotype-specific goat anti–mouse second reagents. The contour plots were divided into quadrants representing unstained cells (lower left), cells with only red fluorescence (upper left), cells with red and green fluorescence (upper right), and cells with only green fluorescence (lower right).

Figure 3

Comparison between NKp44 and p46 molecules. (a) Preclearing experiments. The ¹²⁵I-surface labeled NK cell population derived from donor KK was immunoprecipitated three times with Z231 (lanes A–C) or BAB281 (lanes E–G). The NKp44 or p46 precleared lysates were then immunoprecipitated with BAB281 (lane D) or Z231 (lane H) mAbs, respectively. Samples were analyzed in an 11% SDS-PAGE under reducing conditions. (b) Peptide mapping analysis of NKp44 and p46 molecules. ¹²⁵I-labeled NKp44 (A) or p46 (B) molecules were purified from the polyclonal NK cell population KK. Proteins were digested with pepsin and peptides were analyzed by electrophoresis in the first dimension followed by chromatography in the second dimension.

Figure 3

Comparison between NKp44 and p46 molecules. (a) Preclearing experiments. The ¹²⁵I-surface labeled NK cell population derived from donor KK was immunoprecipitated three times with Z231 (lanes A–C) or BAB281 (lanes E–G). The NKp44 or p46 precleared lysates were then immunoprecipitated with BAB281 (lane D) or Z231 (lane H) mAbs, respectively. Samples were analyzed in an 11% SDS-PAGE under reducing conditions. (b) Peptide mapping analysis of NKp44 and p46 molecules. ¹²⁵I-labeled NKp44 (A) or p46 (B) molecules were purified from the polyclonal NK cell population KK. Proteins were digested with pepsin and peptides were analyzed by electrophoresis in the first dimension followed by chromatography in the second dimension.

Figure 4

Association of p46 and NKp44 with immunoreceptor tyrosine-based activation motif–bearing molecules. (a) Cell lysates derived from a polyclonal NK cell population were immunoprecipitated with mAbs to the indicated molecules. Samples were run in a 15% SDS-PAGE under reducing conditions, transferred to PVDF membranes, and probed with anti-ζ mAb. (b) Cell lysates derived from a polyclonal NK cell population unstimulated (NaV⁻) or stimulated (NaV⁺) with 100 μM Na-pervanadate, were immunoprecipitated with mAbs to the indicated molecules. Samples were analyzed in a 15% SDS-PAGE under reducing conditions, transferred to PVDF membranes, and probed with anti-PTyr mAb.

Figure 4

Association of p46 and NKp44 with immunoreceptor tyrosine-based activation motif–bearing molecules. (a) Cell lysates derived from a polyclonal NK cell population were immunoprecipitated with mAbs to the indicated molecules. Samples were run in a 15% SDS-PAGE under reducing conditions, transferred to PVDF membranes, and probed with anti-ζ mAb. (b) Cell lysates derived from a polyclonal NK cell population unstimulated (NaV⁻) or stimulated (NaV⁺) with 100 μM Na-pervanadate, were immunoprecipitated with mAbs to the indicated molecules. Samples were analyzed in a 15% SDS-PAGE under reducing conditions, transferred to PVDF membranes, and probed with anti-PTyr mAb.

Figure 5

Inhibition of non-MHC-restricted cytotoxicity by mAb-mediated masking of NKp44 and p46 molecules. The representative NK clone MID1 was analyzed for cytolytic activity against the indicated FcγR⁻ target cell lines either in the absence or presence of anti-p46, anti-NKp44, anti-CD69, or a combination of anti-p46 + anti-NKp44, anti-p46 + anti-CD69, or anti-NKp44 + anti-CD69 mAbs. Percentage of lysis ± SD at the 8:1 E/T ratio is shown. Each bar represents the mean of five different experiments.