Regulation of natural cytotoxicity receptors by heparan sulfate proteoglycans in -cis: A lesson from NKp44

Abstract

NKp44 (NCR2) is a distinct member of natural cytotoxicity receptors (NCRs) family that can induce cytokine production and cytolytic activity in human NK cells. Heparan sulfate proteoglycans (HSPGs) are differentially expressed in various normal and cancerous tissues. HSPGs were reported to serve as ligands/co-ligands for NKp44 and other NCRs. However, HSPG expression is not restricted to either group and can be found also in NK cells. Our current study reveals that NKp44 function can be modulated through interactions with HSPGs on NK cells themselves in -cis rather than on target cells in -trans. The intimate interaction of NKp44 and the NK cell-associated HSPG syndecan-4 (SDC4) in -cis can directly regulate membrane distribution of NKp44 and constitutively dampens the triggering of the receptor. We further demonstrate, that the disruption of NKp44 and SDC4 interaction releases the receptor to engage with its ligands in -trans and therefore enhances NKp44 activation potential and NK cell functional response.

Keywords: Heparan sulfate; Heparan sulfate proteoglycans; Immune regulation; NCR2; NK cell activation; NKp44; Natural cytotoxicity receptors; Syndecan 4.

Conflict of interest statement

Conflict of interest

The authors declare no financial or commercial conflict of interest.

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Figures

Figure 1. Characterization of HS binding site mutation and its effect on NKp44-mediated IFN-γ production by NK cells

A,C. FACS analysis of NKp44 (NKp44-mCherry fusion protein; as gated on corresponding mCherry) expression by NK92 cell lines: (A) NK92-44wt, (B) NK92-44mut, (C) NK92 parental cell line. D. FACS analysis of NKp44 expression by NK92-44wt and NK92-44mut cell lines as assayed by combined staining with anti-NKp44 mAb (clone 3.43.13) and secondary allophycocyanin-conjugated anti-mouse IgG1 Ab staining; NK92-44wt (dark solid line), NK92-44mut (grey solid line), secondary allophycocyanin-conjugated anti-mouse IgG1 Ab single staining (control) of NK92-44wt (dark dotted line; tinted) andNK92-44mut (grey dotted line; tinted). E-H. Representative images of FACS analysis: IFN-γ production by NK92-44wt and NK92-44mut cell lines as performed by combined intracellular staining with biotin-conjugated anti-IFN-γ mAb and SA- allophycocyanin: NK92-44wt (E, F) or NK92-44mut (G, H) cell lines were stimulated overnight with plate-bound anti-NKp44 mAb (clone 3.43.13; F, H) or Mock control (anti-2B4 mAb; E, G). I,J. Summary of FACS analysis: I (E-F; NK92-44wt) and J (G–H; NK92-44mut). Data represent mean ± SD of n=3 biological replicas for each treatment. (A–J) Data are representative of 3 independent experiments. *p ≤ 0.01 (also NK92wt (I) to NKp44mut (J)); t-test.

Figure 2. Impact of HS binding site mutation on NKp44-mediated functional response of NK cells

A–B. Impact of HS binding site mutation on NKp44 mediated cytotoxicity. NK92 effector cell lines were co-incubated with K562 target cell line: summary of specific degranulation of NK92-44wt and NK92-44mut cell lines as assayed by anti-CD107a mAb (A); NKp44 mediated cytotoxicity: summary of specific lysis of K562 cell line by NK92-44wt and NK92-44mut cell lines as assayed by multiparametric FACS analysis (B). C-D. Impact of soluble HS on NKp44 mediated IFN-γ secretion: NK92-44wt or NK92-44mut cells were activated overnight by plate-bound anti-NKp44 mAb in the presence of 5µg/ml of either soluble HS or CS or assay medium alone (NT). IFN-γ in culture supernatant was assayed by ELISA: IFN-γ concentration in the sup ranged from 0.1ng/ml (Ctr. Ab) to 4ng/ml (anti-NKp44 mAb). A-D. Data represents mean ± SD of two to three independent experiments in n=6 biological replicas for each treatment. *p ≤ 0.01, **p ≤ 0.05; t-test.

Figure 3. NKp44 and SDC4 co-distribution in non-activated NK cells

(A–F) NK92-44wt and NK92-44mut SDC4-GFP co-expressing cells were complemented with standard assay medium (NT) or with assay medium containing 10µg/ml of either HS or CS. ImageStream analysis of NKp44-mCherry and SDC4-GFP (A, B) polarization and (C, D) co-localization: polarization and co-localization coefficient correspond to the proportion of positive cells. Data represents mean ± SD of n=3 independent experiments and indicated number of biological replicas (n = 3,000 for each treatment). *p

Figure 4. NKp44 and SDC4 co-localization in activated NK cells

NK92-44wt and NK92-44mut SDC4-GFP co-expressing cells were complemented with standard assay medium (NT) or with assay medium containing 10µg/ml of either HS or CS. Confocal microscope image analysis of (A) non-treated (NT) and (B) HS- or CS-treated cells: NKp44-mCherry and SDC4-GFP co-localization coefficient (M2; see Materials and Methods) corresponds to the proportion of NKp44-mCherry co-localized with SDC4-GFP. Data represents mean ± SD of n=5 independent experiments and indicated number of biological replicas (N = 100 for each treatment*p ≤ 0.01; **p ≤ 0.05; t-test. C,D. Representative images of non-treated (NT) and HS- or CS-treated cells are shown. Images are representative of 5 independent experiments.

Figure 5. NKp44 and SDC4 proximal interaction in activated NK cells

NK92-44wt and NK92-44mut SDC4-GFP co-expressing cells were complemented with standard assay medium (NT) or with assay medium containing 10µg/ml of either HS or CS. Confocal microscope images analysis of non-treated (NT) (A) and HS- or CS-treated (B) cells: FRET efficiency is shown for SDC4-GFP (donor) and NKp44-mCherry (acceptor; FRET channel). Data represents mean ± SD of n=3 independent experiments and indicated number of biological replicas (N = 100 for each treatment). P-values were calculated using T-test: *p-value ≤ 0.01. C,D. Representative images of non-treated (NT) and HS- or CS-treated cells are shown (FRET channel diagram and FRET efficiency scale are shown). Images are representative of 3 independent experiments.

Figure 6. NKp44 and SDC4 co-distribution and function in activated primary NK cells

IL-2 activated primary NK cells were complemented with standard assay medium (NT) or with assay medium containing 10µg/ml of either HS or CS (A,B,D). Primary NK cells were stained with specific anti-NKp44 mAb (FITC channel) and goat anti-SDC4 (APC channel) antibodies (see Materials and methods). ImageStream analysis of NKp44 and SDC4 (A) polarization and (B co-localization): polarization and co-localization coefficient correspond to the proportion of positive cells. (A and B) Data represents mean ± SD of two independent experiments and indicated number of biological replicas (N = 15,000 for each treatment). C. Impact of soluble HS on NKp44 mediated IFN-γ secretion: IL-2 activated primary NK cells were stimulated overnight by plate-bound anti-NKp44 mAb in the presence of 5µg/ml of either soluble HS or CS or assay medium alone (NT). IFN-γ in culture supernatant was assayed by ELISA: IFN-γ concentration in the sup ranged from 0ng/ml (not detected; Ctr. Ab) to 0.8ng/ml (anti-NKp44 mAb). Data represent mean ± SD of two independent experiments in n=6 biological replicas for each treatment. *p ≤ 0.01; t-test. D. Representative images of non-treated (NT) and HS- or CS-treated cells are shown. Images are representative of two independent experiments.