Heterogeneity among Ly-49C natural killer (NK) cells: characterization of highly related receptors with differing functions and expression patterns

Abstract

Ly-49C is a member of the polymorphic family of murine NK cell inhibitory receptors. The 5E6 antibody that defines a subset of NK cells responsible for the rejection of parental H-2d bone marrow by F1 mice has been shown previously to react with Ly-49C. Here, the 5E6 antibody was found to detect two Ly-49C-related molecules in B6 mice. Two cDNA clones were isolated from B6 NK cells, one identical to previously reported Ly-49CB6 and the other a novel cDNA. The deduced amino acid sequence of the latter differs from that of Ly-49CBALB at only 4 residues, whereas the previously reported Ly-49CB6 differs at 22 residues. Flow cytometric analyses of COS cells transfected with the two cDNAs showed that the 5E6 antibody binds to both Ly-49 molecules, while another anti-Ly-49C antibody, 4LO3311, binds to the newly described Ly-49C but not the previously reported Ly-49CB6. Two-color flow cytometric analysis detected 5E6+4LO3311- as well as 5E6+4LO3311+ subsets of NK cells from B6, but not BALB/c, mice. The level of Ly-49C expression on B6 NK cells detected by the 4LO3311 antibody was substantially lower than that on BALB/c NK cells. Binding specificity of the novel Ly-49CB6 was indistinguishable from that of Ly-49CBALB, whereas no binding was detectable with previously reported Ly-49CB6. These results demonstrate that the newly described Ly-49CB6, not the previously reported Ly-49CB6, is the probable B6 allelic form of Ly-49C. The previously reported Ly-49CB6 must be encoded by a separate gene and should be renamed Ly-49I. The implication of these results with respect to the role of Ly-49C in hybrid resistance is discussed.

Figure 1

(A) Cytoplasmic/transmembrane domain; (B) Stalk; (C) Carbohydrate recognition domain (CRD). Deduced amino acid sequences of two forms of Ly-49C from B6 NK cells. The amino acid sequences of Ly49C^B6 and Ly-49I^B6 are aligned with that of Ly-49C from BALB/c mice. The sequences are divided into those in the cytoplasmic/transmembrane, stalk, and CRD regions. The arrow indicates the begining of the region within the stalk region that previously has been shown to be important for the binding of Ly-49 to class I MHC (6). For the sequences of Ly-49C and I from B6 mice, only the residues different from those of BALB/c mice are shown. The CRD of Ly-49C was deduced from the previous studies on genomic clones of Ly-49, which indicates that the CRD is encoded by three exons (2). The nucleotide sequence of the Ly-49C^B6 cDNA is identical to an unpublished Ly-49C^B6 cDNA recently deposited in Genbank by Sentman and coworkers (these sequence data are available from EMBL/Genbank/DDBJ under accession number U56404).

Figure 2

Specificities of anti-Ly-49C antibodies. (A) Ly-49C^B6 and Ly-49I^B6 were expressed on COS cells and stained with 5E6 and 4LO3311. Solid histograms represent cells transfected with Ly-49C and empty histograms are cells transfected with vector alone. (B) COS cells were transfected with chimeric Ly-49A/C as indicated and stained with A1 (anti-Ly-49A), 5E6, or 4LO3311. Solid histograms represent cells transfected with chimeric Ly-49 and empty histograms are cells transfected with vector alone.

Figure 2

Specificities of anti-Ly-49C antibodies. (A) Ly-49C^B6 and Ly-49I^B6 were expressed on COS cells and stained with 5E6 and 4LO3311. Solid histograms represent cells transfected with Ly-49C and empty histograms are cells transfected with vector alone. (B) COS cells were transfected with chimeric Ly-49A/C as indicated and stained with A1 (anti-Ly-49A), 5E6, or 4LO3311. Solid histograms represent cells transfected with chimeric Ly-49 and empty histograms are cells transfected with vector alone.

Figure 3

Expression of Ly-49C on NK cells from BALB/c and B6 mice. (A) Splenic NK cells were first incubated with unlabeled anti-FcR (2.4G2) to block nonspecific binding of antibodies then stained with FITC-conjugated 5E6 or biotin-conjugated 4LO3311 and SA–PE. The x and y axes correspond to the log fluorescence intensity and cell count, respectively. Solid histogram shows anti-Ly-49C staining and dotted histogram shows autofluorescence of unstained cells. No difference was observed between unlabeled cells and cells incubated with FITC- or biotin-conjugated isotype-control mAbs (data not shown). (B) NK cells were stained for two-color analysis as described for A, except that SA–RED670^TM conjugate was used for the detection of the red fluorescence. The electronic compensations for the two-color analysis were set on samples incubated with each antibody alone.

Figure 4

Binding of Ly-49C to target cells. (A) COS cells were transfected with vector alone (a, b), Ly-49C^BALB (c, d), Ly-49C^B6 (e, f    ), or Ly-49I^B6 (g, h), and incubated with the murine lymphoid lines A20 (H-2^d) (a, c, e, g) and IC-21 (H-2^b) (b, d, f, h). After a 2 h coincubation, unbound cells were washed away, and plates were photographed. (B) The binding of IC-21 cells to COS cells transfected with Ly49C^B6 was tested as above in the absence (a) or presence (b) of the 4LO3311 antibody. IC-21 binding to cells transfected with vector alone is shown in the absence (c) or presence (d) of 4LO3311.

Figure 4

Binding of Ly-49C to target cells. (A) COS cells were transfected with vector alone (a, b), Ly-49C^BALB (c, d), Ly-49C^B6 (e, f    ), or Ly-49I^B6 (g, h), and incubated with the murine lymphoid lines A20 (H-2^d) (a, c, e, g) and IC-21 (H-2^b) (b, d, f, h). After a 2 h coincubation, unbound cells were washed away, and plates were photographed. (B) The binding of IC-21 cells to COS cells transfected with Ly49C^B6 was tested as above in the absence (a) or presence (b) of the 4LO3311 antibody. IC-21 binding to cells transfected with vector alone is shown in the absence (c) or presence (d) of 4LO3311.