Expression analysis of the ligands for the Natural Killer cell receptors NKp30 and NKp44

Abstract

Background: The natural cytotoxicity receptors (NCR) are important to stimulate the activity of Natural Killer (NK) cells against transformed cells. Identification of NCR ligands and their level of expression on normal and neoplastic cells has important implications for the rational design of immunotherapy strategies for cancer.

Methodology/principal findings: Here we analyze the expression of NKp30 ligand and NKp44 ligand on 30 transformed or non-transformed cell lines of different origin. We find intracellular and surface expression of these two ligands on almost all cell lines tested. Expression of NKp30 and NKp44 ligands was variable and did not correlate with the origin of the cell line. Expression of NKp30 and NKp44 ligand correlated with NKp30 and NKp44-mediated NK cell lysis of tumor cells, respectively. The surface expression of NKp30 ligand and NKp44 ligand was sensitive to trypsin treatment and was reduced in cells arrested in G(2)/M phase.

Conclusion/significance: These data demonstrate the ubiquitous expression of the ligands for NKp30 and NKp44 and give an important insight into the regulation of these ligands.

Figure 1. Immunoperoxidase stainings of acetone-fixed tumor cells on cytospins.

Intracellular compartments of HT-29, 1106mel, Jurkat (A–C) and HeLa cells (D) were stained with NKp46-Ig, NKp44-Ig and NKp30-Ig fusion proteins as indicated. In (D), intracellular stainings of HeLa cells with the ER marker calnexin, the Golgi marker GM130, the early endosome marker EEA-1, and the late endosome marker LAMP-1 were performed in parallel for comparison with NCR-Ig staining pattern. A–C, ×150, D, ×250.

Figure 2. Detection of NKp30L and NKp44L on the surface of cells by FACS analysis.

(A) 293T cells were analyzed without treatment (none) or after incubation with medium or CS1-ILZ, followed by anti-ILZ mAb (ILZ-11) and PE-labeled goat anti-mouse IgG (GaM-PE) staining. (B, C) 293T cells were stained with CS1-ILZ, NKp30-ILZ (B) or NKp44-ILZ (C) as described in (A) and analyzed by FACS. Mean fluorescence intensities of the stainings are indicated in brackets.

Figure 3. Analysis of NKp30L and NKp44L expression on pancreatic carcinoma cell lines (A) and breast carcinoma cell lines (B).

The indicated cells were stained with CS1-ILZ, NKp30-ILZ or NKp44-ILZ and analyzed by FACS. The mean fluorescence intensities (MFI) of the NKp30-ILZ (black circles) and NKp44-ILZ (white circles) stainings were divided by the MFI of CS1-ILZ background staining. Each dot represents the result of an independent staining.

Figure 4. Analysis of NKp30L and NKp44L expression on solid tumor cell lines of different origin (A) and of cell lines hematopoietic origin (B).

The indicated cells were analyzed for NKp30L and NKp44L expression as described in figure 3.

Figure 5. Reduced expression of NKp30L in cells arrested in mitosis.

(A) 293T cells were treated with DMSO for 18 hours, 10 µM colchicine (col) for 1 hour, 100 ng/ml nocodazole (noc) or 0.1 µM colchicine for 18 hours and the cell cycle stage of the treated cells was analyzed by FACS. Numbers represent percentage of cells in G₁ phase (left) S phase (middle) and G₂/M phase (right). (B and C) the same cells as in (A) were analyzed for the expression of NKp30L as described in Fig. 2.

Figure 6. Correlation between NKp30L and NKp44L expression and NKp30 and NKp44-mediated lysis by NK cells.

(A) EM-3 and SD-1 cells were used as targets in a 4 h ⁵¹Cr release assay using IL2-activated NK cells from two different donors (NK1 and NK2) as effector cells at the indicated effector to target (E/T) ratios. (B) ⁵¹Cr release assay of EM-3 and SD-1 target cells in the presence of control antibodies (cIgG1) or blocking anti-NKp30 antibodies (αNKp30). (C–E) Lysis of K562 (C), HeLa (D) and Shep (E) cells by IL-2 activated NK cells in the presence of control antibodies (cIgG1) or blocking anti-NKp30 (αNKp30) and/or anti-NKp44 antibodies (αNKp44).

Figure 7. Surface expression of NKp30L and NKp44L is sensitive to Trypsin treatment.

EM-3 cells were left untreated (control) or treated with trypsin. (A) The expression of NKp30L and NKp44L was then analyzed as described in figure 3 or cells were used as targets in a ⁵¹Cr release assay (B).