Mouse Ly-49A interrupts early signaling events in natural killer cell cytotoxicity and functionally associates with the SHP-1 tyrosine phosphatase

Abstract

The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2Dd. In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2Dd inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1-deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.

Figure 1

Inhibition of lysis of P388D1 (H-2D^d) targets correlates with the level of expression of mouse Ly-49A on RNK-16 cells. The various levels of Ly-49A expression are shown in FACS^® histograms (A–D). Cells were incubated with anti–Ly-49A (solid line) with FITC–goat anti–mouse Ab (FITC–GAM) or FITC-GAM alone (dotted line). Standard 4-h cytotoxicity assays were performed with either P388D1 cells (E–H) or YAC-1 targets (I–L). Effectors were wild-type RNK-16 cells (closed symbols) or RNK-16 transfected with Ly-49A (open symbols). Effector cells used were RNK-16 (A, E, and I) and clones of RNK-16 expressing Ly-49A: low expression, RNK-mLy-49A.2 (B, F, J); intermediate expression, RNKmLy-49A.8 (C, G, K); and high expression, RNK-mLy-49A.9 (D, H, L). Assays were carried out in the absence of antibody (squares), or in the presence of anti-Ly-49A F(ab′)₂ (diamonds) and control F(ab′)₂ (antiNK1.1) (circles). The dotted line in F–H is the killing curve for wild-type RNK-16 without mAb (from E for comparison).

Figure 2

Ly-49A inhibits phosphoinositide turnover in response to H-2D^d target cells. RNK-mLy-49A.9 cells fail to generate InsP₃ upon stimulation with P388D1 targets. [³H]myoinositol-labeled RNK-16 and RNK-mLy-49A.9 effectors (5 × 10⁶ cells) were stimulated with 10⁷ targets in a total volume of 1 ml at 37°C. Soluble InsP₃ was resolved by ion exchange chromatography. A brisk rise in InsP₃ was seen in RNK-16 cells (left) in response to either YAC-1 (open squares) or P388D1 (H-2D^d) (closed circles). Phosphoinositide turnover in RNK-mLy-49A.9 (right) was stimulated by YAC-1, but not by P388D1.

Figure 3

Ly-49A inhibits an early rise in tyrosine phosphorylation induced by target cell stimulation. Tyrosine phosphorylation of proteins in RNK-16 cells stimulated with P388D1 cells is shown on the left side of the figure. RNK-mLy-49A.9 cells stimulated with P388D1 cells are in the center, and RNK-mLy-49A.9 cells stimulated with YAC-1 cells are on the right. Target cell stimulation time points were 0, 0.5, 1.0, and 5.0 min. RNK-16 and RNKmLy-49A.9 effector cells were metabolically labeled with ³²Porthophosphate. After washing, 10⁷ labeled effector cells and 10⁷ unlabeled target cells were stimulated in a total volume of 1 ml complete phosphate-free RPMI with a brief 50 g contact spin, followed by incubation at 37°C for the indicated time. Cells were then immediately lysed in cold HNTG buffer with 1% Triton X-100. Clarifed precleared cell lysates were immunoprecipitated with APT (4G10), washed, and resolved by reducing 8% SDS-PAGE. Gels were dried and developed by autoradiography.

Figure 4

The tyrosine phosphatase SHP-1 associates with mouse Ly-49A in pervanadatestimulated RNK-16 cells. 1.5 × 10⁷ unstimulated or pervanadatestimulated RNK-16 cells or RNK-mLy-49A.9 cells were incubated in complete RPMI for 5 min at 37°C, washed, and lysed in cold HNTG lysis buffer containing 1% Triton X-100. Clarified precleared lysates were immunoprecipitated with anti–Ly-49A (A1, lanes 5–8) or isotypematched control mAb (NK1.1, PK136, lanes 1–4), washed, and resolved by 8% SDS-PAGE under nonreducing conditions. Proteins were transferred to PVDF membranes, immunoblotted with anti-SHP-1 antiserum, and developed with ¹²⁵I–protein A followed by autoradiography.

Figure 5

Ly-49A function is impaired in me^v/me^v LAK cells. 9-d Ly-49A⁺ and Ly-49A⁻ LAK cells were tested in 4-h cytotoxicity assays against D12 (C1498.D^d) targets against Ly-49A⁺ effector cells (A–C) or Ly-49A⁻ effector cells (D–F) from +/+ (A and D), +/me^v (B and E), or me^v/me^v mice (C and F). Assays were done in the absence of antibody (open squares), or in the presence of anti–Ly-49A (A1, closed diamonds), or isotype-matched control antibody (anti-gp42, 3G7, open circles).

Figure 6

Ly-49A expression on Ly-49A⁺ and Ly-49A⁻ LAK cells isolated from +/+, +/me, me/me mice. 6-d LAK cells were separated into Ly-49A⁺ and Ly-49A⁻ populations by panning with anti–Ly-49A Ab. Ly-49A⁻ cells were additionally treated with rabbit anti–mouse Ab and complement depletion. FACS^® analysis was performed on day 9 LAK cells using FITC–anti-Ly-49A (A1). Staining was performed in the presence of unlabeled blocking antibodies (IgG2a mouse myeloma protein, 1 μg/10⁶ cells in 0.1 ml 2.4G2 supernatant). Ly-49A expression is shown in FACS^® histograms (A–F). Dotted lines represent cells incubated with saline; solid lines represent FITC–anti-Ly-49A staining.

Figure 7

Ly-49A function is impaired in me/me LAK cells. 9-d Ly-49A⁺ and Ly-49A⁻ LAK cells were tested in 4-h cytotoxicity assays against D12 (C1498.D^d) and C1498 (H-2^b) targets. D12 targets (A–F) and C1498 targets (G–L) were tested against Ly-49A⁺ effector cells (A–C and G–I) or Ly-49A⁻ effector cells (D–F and J–L) from +/+ (A, D, G, J), +/me (B, E, H, K), or me/me mice (C, F, I, L). Assays were done in the absence of antibody (open squares), or in the presence of anti–Ly-49A (A1, closed diamonds), or isotype-matched control antibody (anti-gp42, 3G7, open circles).

Figure 8

Lysis of P388D1 (H-2D^d) cells is not altered in RNK-mLy49A/Y8F transfectants. Ly-49A expression on RNK transfectants was assessed by staining cells with either saline (dotted line) or FITC–anti-Ly-49A (solid line). FACS^® histograms show Ly-49A expression in wild-type RNK-16 (B), RNK-mLy-49A.9 (D), RNK-mLy-49A/Y8F.1 (F) and RNK-mLy-49A/Y8F.4 (H). Standard 4-h cytotoxicity assays were performed using P388D1 (H-2D^d) as targets. Effector cells were wild-type RNK-16 (A), RNK-mLy-49A.9 (C), RNK-mLy-49A/Y8F.1 (E), or RNK-mLy-49A/Y8F.4 (G). Effectors were preincubated with either media alone (open squares), anti-Ly-49A (closed circles), or isotype-matched control antibody (anti-NK1.1, PK136, closed triangles), before addition of targets.

Figure 9

Cold-target competition of unlabeled P388D1 and B-16S in the lysis of labeled B-16S targets. 4-h cytotoxicity assays were performed with RNK-16 (left) or RNK-mLy-49A.9 cells (right) as effectors. B-16S (H-2^b) target cells were labeled with ⁵¹Cr and tested at an effector to labeled target ratio of 10:1. Cold targets were either unlabeled B-16S cells (H-2^b) or P388D1 cells (H-2D^d). Cold targets were added in 10-fold excess to labeled targets. 10⁵ cold targets and 10⁴ labeled targets were added at the same time to 10⁵ effectors in a total volume of 0.2 ml. Effectors were preincubated with no antibody, F(ab′)₂ anti–Ly-49A or F(ab′)₂ antiNK1.1. Results are expressed as percent inhibition = (1 − [percent cytotoxicity with cold target/percent cytotoxicity without cold target]) × 100.