Characterization of inhibitory and stimulatory forms of the murine natural killer cell receptor 2B4

Abstract

The receptor 2B4 belongs to the Ig superfamily and is found on the surface of all murine natural killer (NK) cells as well as T cells displaying non-MHC-restricted cytotoxicity. Previous studies have suggested that 2B4 is an activating molecule because cross-linking of this receptor results in increased cytotoxicity and gamma-interferon secretion as well as granule exocytosis. However, it was recently shown that the gene for 2B4 encodes two different products that arise by alternative splicing. These gene products differ solely in their cytoplasmic domains. One form has a cytoplasmic tail of 150 amino acids (2B4L) and the other has a tail of 93 amino acids (2B4S). To determine the function of each receptor, cDNAs for 2B4S and 2B4L were transfected into the rat NK cell line RNK-16. Interestingly, the two forms of 2B4 had opposing functions. 2B4S was able to mediate redirected lysis of P815 tumor targets, suggesting that this form represents an activating receptor. However, 2B4L expression led to an inhibition of redirected lysis of P815 targets when the mAb 3.2.3 (specific for rat NKRP1) was used. In addition, 2B4L constitutively inhibits lysis of YAC-1 tumor targets. 2B4L is a tyrosine phosphoprotein, and removal of domains containing these residues abrogates its inhibitory function. Like other inhibitory receptors, 2B4L associates with the tyrosine phosphatase SHP-2. Thus, 2B4L is an inhibitory receptor belonging to the Ig superfamily.

Figure 1

Expression of 2B4 isoforms in RNK-16 cells. (A) Diagram of the gene products (2B4L or 2B4S) that arise by alternative splicing of the murine 2B4 gene. The two forms are identical up to amino acid 308, at which point sequences unique to each form are represented (░⃞ or ▧). The potential tyrosine phosphorylation sites are represented by (Y). cDNAs for each form were cloned into expression vectors and transfected into the rat NK cell line RNK-16. (B) The staining profiles of stable transfectants of RNK-16 cells are shown. Cells were transfected with expression constructs as indicated and stained with FITC-conjugated isotype control antibodies (■) or FITC-conjugated anti-2B4 (□).

Figure 2

Redirected lysis of P815 targets by 2B4L or 2B4S transfectants. RNK-16 cells transfected with empty vector (RNK-16) or with 2B4 expression constructs (2B4L or 2B4S) were used as effectors in a standard chromium release assay against the murine FcR+ target P815. The effectors used are shown on the top of the figure. Effectors were preincubated with 25 μg/ml of isotype control antibody (cAb) or with anti-2B4 antibody before the addition of targets as indicated.

Figure 3

Expression of 2B4 deletion constructs in RNK-16 cells. (A) Various 2B4 deletion constructs used to map functional domains of 2B4 are depicted. Residues unique to 2B4L are represented by shaded regions. Stop codons were added at the desired positions by PCR mutagenesis, and the resulting products were placed into expression vectors and used to transfect RNK-16 cells. (B) The staining profiles of stable transfectants of RNK-16 cells are shown. Transfected cell lines were stained with FITC-conjugated isotype control antibody (■) or with FITC-conjugated anti-2B4 antibody (□).

Figure 4

Redirected lysis of P815 targets by RNK-16 clones expressing the 2B4 deletion mutants. Various transfected RNK-16 cell lines were used as effectors (indicated on x axis) at an effector-to-target ratio of 100:1 in a standard chromium release assay. As indicated, effectors were preincubated with control antibody (cAb) or with anti-2B4 as described for Fig. 2.

Figure 5

2B4L expression inhibits tumor lysis by RNK-16. A typical chromium release assay using YAC-1 tumor targets is shown. Various RNK-16 transfectants (described on top of the figure) were used as effectors at the indicated effector-to-target (E:T) ratios. Where indicated, effectors were preincubated with anti-2B4 antibody as described for Fig. 2.

Figure 6

2B4L expression inhibits redirected lysis by NKR-P1A but not antibody-dependent cellular cytotoxicity in RNK-16 cells. (A) A redirected lysis assay using P815 targets is shown. Various RNK-16 transfectants were used at an effector-to-target ratio of 100:1. Where indicated, effectors were preincubated with anti-rat NKR-P1A (3.2.3 antibody) or with anti-2B4 antibody as described in Fig. 2. As shown for Fig. 2, baseline lysis of P815 targets by all transfectants was consistently <2%. (B) An antibody-dependent cellular cytotoxicity assay using P815 targets is shown. Various RNK-16 transfectants were used as effectors as described for Fig. 6. Where indicated, effectors were preincubated with anti-P815 (rabbit polyclonal antisera) or anti-2B4 antibody as described for Fig. 2. As shown for Fig. 2, baseline lysis of P815 targets by all transfectants was consistently <2%.

Figure 7

2B4L tyrosine phosphorylation and SHP-2 association. (A) Approximately 2 × 10⁷ cells of various RNK-16 transfectants or C57/Bl6 LAKs were incubated at 37°C for 10 min in the presence or absence of 1 mM pervanadate. Cells were lysed and immunoprecipitated with 5 μg of anti-2B4 antibody or control antibody (cAb) as indicated. Immunoprecipitates were resolved by nonreducing SDS/PAGE and immunoblotted with horseradish peroxidase-conjugated anti-phosphotyrosine antibody and analyzed by enhanced chemiluminescence. Bands corresponding to 2B4 are located near the 66-kDa marker. (B) Approximately 1 × 10⁸ cells of 2B4L expressing RNK-16 cells were incubated with or without pervanadate as described above. Cells were then lysed and subjected to immunoprecipitation with anti-2B4 antibody. Immunoprecipitates were then resolved by nondenaturing SDS/PAGE and subjected to immunoblotting with anti-SHP-2 antibody followed by horseradish peroxidase-conjugated secondary antibody and analyzed by enhanced chemiluminescence. The lane marked total lysate represents 1 × 10⁶ cell equivalents loaded onto the gel.