Genetic control of natural killing and in vivo tumor elimination by the Chok locus

Abstract

The molecular mechanisms underlying target recognition during natural killing are not well understood. One approach to dissect the complexities of natural killer (NK) cell recognition is through exploitation of genetic differences among inbred mouse strains. In this study, we determined that interleukin 2-activated BALB/c-derived NK cells could not lyse Chinese hamster ovary (CHO) cells as efficiently as C57BL/6-derived NK cells, despite equivalent capacity to kill other targets. This strain-determined difference was also exhibited by freshly isolated NK cells, and was determined to be independent of host major histocompatibility haplotype. Furthermore, CHO killing did not correlate with expression of NK1.1 or 2B4 activation molecules. Genetic mapping studies revealed linkage between the locus influencing CHO killing, termed Chok, and loci encoded within the NK gene complex (NKC), suggesting that Chok encodes an NK cell receptor specific for CHO cells. In vivo assays recapitulated the in vitro data, and both studies determined that Chok regulates an NK perforin-dependent cytotoxic process. These results may have implications for the role of NK cells in xenograft rejection. Our genetic analysis suggests Chok is a single locus that affects NK cell-mediated cytotoxicity similar to other NKC loci that also regulate the complex activity of NK cells.

Figure 1

Strain-determined differences in CHO killing by NK cells. (A) Standard chromium-release assays were performed using B6 and BALB/c IL-2–activated NK cells (LAKs) against CHO and YAC-1 target cells at indicated E/T ratios. (B) Cytotoxicity assays against CHO or YAC-1 targets using freshly isolated NK cells from B6 and BALB/c.

Figure 2

Inheritance of strain difference in CHO cell killing and independence from host MHC haplotype. (A) Lytic capacity of IL-2–activated NK cells from the (BALB/c × C57BL/6)F₁ strain was compared with that of parental B6 and BALB/c IL-2–activated NK cells against the CHO and YAC-1 targets. (B) No association between H2 haplotype and capacity to lyse CHO. IL-2–activated NK cells derived from C57BL/10, BALB/c, and BALB.B were compared for their capacity to lyse CHO and YAC-1 targets.

Figure 3

No correlation between expression of 2B4 and NK1.1 activation antigens and capacity to lyse CHO cells. (A) IL-2– activated NK cells from B6, BALB/cJ, C57L/J, and NOD/LtJ were analyzed for their capacity to lyse CHO and YAC-1 targets. (B) Flow cytometric analysis of IL-2– activated NK cells from B6, BALB/cJ, C57L/J, and NOD/LtJ strains. NK cells from each of the strains were incubated with mAbs specific for FcγRII/III (a–d), NK1.1 (e–h), or 2B4 (i–l) followed by FITC-conjugated goat F(ab′)₂ anti–mouse Ig. Solid lines, specific staining; dotted lines, staining by secondary antibody alone. The 2.4G2 mAb is specific for both FcγRII and FcγRIII, even though mouse NK cells express only FcγRIII (reference 73).

Figure 4

The SDP of CHO killing among the CXB RI mouse strains demonstrates linkage to the NKC. (A) IL-2–activated NK cells derived from seven CXB RI strains as well as the progenitor strains, C57BL/6ByJ and BALB/cByJ, were assessed for their capacity to mediate CHO and YAC-1 lysis. The SDP is summarized using the symbols B and C to indicate the alleles inherited from the C57BL/6By and BALB/cBy progenitor strains, respectively. (B) Linkage of Chok to NKC-encoded loci on chromosome 6. The SDP observed for the Chok locus is compared with SDPs of other genes previously typed using the same CXB-RI panel (references , , , , , and 75). The symbols B and C represent alleles inherited from the progenitor C57BL/6By and BALB/cBy strains, respectively.

Figure 5

NK cells from BALB.B6-Cmv1^r as well as B6.BALB-Cmv1^s confirm Chok linkage to the NKC, and Chok regulates NK-mediated in vivo elimination of tumor targets through a perforin-dependent pathway. (A) Cytotoxicity assays against CHO and YAC-1 targets were performed using IL-2–activated NK cells derived from B6, BALB/c, and the NKC-congenic, BALB.B6-Cmv1^r, and B6.BALB-Cmv1^s mouse strains. (B) ¹²⁵I-radiolabeled CHO cells were injected into tail veins of perforin-deficient B6, BALB/c, or B6 mouse strains untreated or treated with anti-AGM1 antiserum 3 d before the assay. Each bar represents the mean percent retention of six mouse lungs except where ^# n = 5. (C) Lung clearance of [¹²⁵I]UdR-labeled YAC-1 or CHO targets was assessed in BALB/c and BALB.B6- Cmv1^r congenic mice, untreated or treated with 100 μg of either anti-NK1.1 mAb or an isotype-matched control anti-K^b mAb, intraperitoneally 2 d before the assay. Each bar represents the mean percent retention of four mouse lungs except where *n = 3. In all experiments, mice were killed and lungs were harvested 4 h after inoculation.

Figure 6

Antibodies against Ly-49D or Ly-49H do not block CHO lysis. (A) Cytotoxicity assays against CHO targets were performed using IL-2– activated NK cells derived from B6 and BALB/c in the presence or absence of anti–Ly-49D mAbs. (B) Neither mAb 3D10 (anti–Ly-49H) nor the isotype control had an effect on B6 NK–mediated CHO lysis.