The natural killer gene complex genetic locus Chok encodes Ly-49D, a target recognition receptor that activates natural killing

Abstract

Previously, we established that natural killer (NK) cells from C57BL/6 (B6), but not BALB/c, mice lysed Chinese hamster ovary (CHO) cells, and we mapped the locus that determines this differential CHO-killing capacity to the NK gene complex on chromosome 6. The localization of Chok in the NK gene complex suggested that it may encode either an activating or an inhibitory receptor. Here, results from a lectin-facilitated lysis assay predicted that Chok is an activating B6 NK receptor. Therefore, we immunized BALB/c mice with NK cells from BALB.B6-Cmv1(r) congenic mice and generated a mAb, designated 4E4, that blocked B6-mediated CHO lysis. mAb 4E4 also redirected lysis of Daudi targets, indicating its reactivity with an activating NK cell receptor. Furthermore, only the 4E4(+) B6 NK cell subset mediated CHO killing, and this lysis was abrogated by preincubation with mAb 4E4. Flow cytometric analysis indicated that mAb 4E4 specifically reacts with Ly-49D but not Ly-49A, B, C, E, G, H, or I transfectants. Finally, gene transfer of Ly-49DB6 into BALB/c NK cells conferred cytotoxic capacity against CHO cells, thus establishing that the Ly-49D receptor is sufficient to activate NK cells to lyse this target. Hence, Ly-49D is the Chok gene product and is a mouse NK cell receptor capable of directly triggering natural killing.

Figure 1

Lectin-facilitated lysis of CHO targets by BALB/c NK cells. Cytotoxicity assays against CHO cells in the presence or absence of ConA (8 μg/ml) were performed by using BALB/c and B6 IL-2-activated NK cells at effector:target (E:T) ratios of 10:1 and 20:1, respectively.

Figure 2

Establishment of anti-Chok mAb. (A) mAb 4E4 mediates dose-dependent inhibition of B6 NK-mediated CHO lysis. B6-derived IL-2-activated NK cells were assessed for their capacity to lyse CHO and YAC-1 targets in the presence of increasing concentrations of either mAb 4E4 or AF6–88.5.3, an isotype matched control (anti-H-2K^b), at an E:T ratio of 6.7:1. (B) mAb 4E4 blocks CHO killing by B6 and BALB.B6–Cmv1^r NK cells. CHO cell lysis by IL-2-activated NK cells derived from B6, BALB/c, and BALB.B6–Cmv1^r mouse strains was determined in medium alone or in the presence of 10 μg/ml of either mAb 4E4 or anti-H-2K^b control mAb (E:T of 6.7:1). (C) mAb 4E4 induces redirected lysis. mAbs 4E4, 2B4, and anti-H-2K^b control were evaluated for their capacity to influence B6 IL-2-activated NK cell-mediated lysis of FcR⁺ Daudi cells (E:T of 6.7:1).

Figure 3

mAb 4E4 identifies a subset of B6 NK cells that mediate CHO cell lysis. (A) mAb 4E4 reacts with a population of B6 but not BALB/c NK cells. Two-color flow cytometric analysis of naïve B6 and BALB/c splenocytes was performed with 4E4 and DX5 mAbs. (B) mAb 4E4-reactive cells mediate CHO lysis. Unseparated as well as FACS-sorted 4E4⁺ and 4E4⁻ IL-2-activated B6 NK cells were assessed for their capacity to lyse CHO and YAC-1 targets alone or in the presence of either mAb 4E4 or anti-H-2K^b control mAb (E:T of 5:1).

Figure 4

mAb 4E4 reacts with Ly-49D. (A) Flow cytometric analysis of 293T cells transfected with cDNA expression constructs for either Ly-49C or Ly-49D. (B) Two-color FACS analysis of B6 IL-2-activated NK cells by using 4E4 vs. 4E5 (anti-Ly-49D), A1 (anti-Ly-49A), 4D11 (anti-Ly-49G2/A), or 5E6 (anti-Ly-49C/I) mAbs.

Figure 5

Ly-49D expression in BALB/c NK cells confers cytotoxic capacity against CHO targets. (A) IL-2-activated BALB/c NK cells that were uninfected or infected with 50 pfu per cell of either wild-type vaccinia WR strain or a recombinant Ly-49D–vaccinia virus were analyzed for Ly-49D expression by using mAb 4E4. (B) The NK cell populations shown in A, as well as uninfected B6 NK cells, were assessed for their capacity to lyse CHO targets alone or in the presence of either 4E4 or an isotype-matched control mAb, mouse anti-rat Ig. YAC-l lysis is shown for comparison and was unaltered. Data shown represent lysis at E:T of 10:1.