Natural killer cell education in mice with single or multiple major histocompatibility complex class I molecules

Abstract

The ability of murine NK cells to reject cells lacking self MHC class I expression results from an in vivo education process. To study the impact of individual MHC class I alleles on this process, we generated mice expressing single MHC class I alleles (K(b), D(b), D(d), or L(d)) or combinations of two or more alleles. All single MHC class I mice rejected MHC class I-deficient cells in an NK cell-dependent way. Expression of K(b) or D(d) conveyed strong rejection of MHC class I-deficient cells, whereas the expression of D(b) or L(d) resulted in weaker responses. The educating impact of weak ligands (D(b) and L(d)) was further attenuated by the introduction of additional MHC class I alleles, whereas strong ligands (K(b) and D(d)) maintained their educating impact under such conditions. An analysis of activating and inhibitory receptors in single MHC class I mice suggested that the educating impact of a given MHC class I molecule was controlled both by the number of NK cells affected and by the strength of each MHC class I-Ly49 receptor interaction, indicating that NK cell education may be regulated by a combination of qualitative and quantitative events.

Figure 1.

All single MHC class I mice reject MHC class I⁻ grafts. MHC⁻ spleen cells were mixed with spleen cells syngeneic to the recipient and coinjected i.v. into tilorone-treated recipients expressing either D^b, D^d, K^b, or L^d alone. Control and test cells were labeled with different doses of CFSE, allowing their subsequent distinction using FACS analysis. Eighteen hours later, the fraction of surviving MHC⁻ cells in spleen was determined in each host by comparing the ratio between CFSE^high and CFSE^low cells in the recipient spleen. White bars show recipients in which NK cells were depleted before the transfer. All groups contained four mice except the NK-depleted MHC⁻ and NK-depleted D^d groups, which contained two mice each. The relative survival of MHC⁻ cells in D^b, D^d, K^b, and L^d mice was in all cases statistically different from that in MHC⁻ mice (P < 0.01 for D^b; P < 0.0001 for D^d, K^b, and L^d). The survival was also significantly lower in D^d, K^b, and L^d mice compared with D^b mice and in K^b and D^d mice compared with L^d mice (P < 0.05). Results show averages of two experiments. Error bars show SDs. Graft, MHC class I phenotype on the test graft; missing, MHC lacking in this test graft in relation to the syngeneic control cells that were cotransferred.

Figure 2.

Influence on the educating impact by coexpressed MHC class I molecules. Syngeneic cells (K^bD^b) together with cells expressing either D^b or K^b were injected into K^bD^b mice as in Fig. 1. (A) K^b-negative grafts were more efficiently rejected than D^b-negative grafts in K^bD^b (B6) mice. The relative survival of target cells missing only K^b was significantly lower than the survival of cells missing D^b (P < 0.001). Results show averages of two experiments with four mice per group. Error bars show the standard deviations. (B) The lack of either K^b or D^d, but not D^b, on the grafted cells mediated strong rejection in K^bD^bD^d (D8) mice. The relative survival of target cells missing D^b was significantly higher than the survival of cells missing D^d or K^b (P < 0.05). Results show averages of two experiments with three or four mice per group. Error bars show standard deviations.

Figure 3.

Missing K^b on the graft induces strong rejection in K^bD^bL^d mice, whereas lack of either D^b or L^d results in a weaker response. Averages of two or three experiments with a total of four to six mice per group are shown. The survival of target cells missing K^b was significantly lower than the survival of cells missing L^d (P < 0.05) or D^b (P < 0.0001). Error bars show SDs.

Figure 4.

The lack of L^d evokes rejection of different strengths depending on the MHC background. Syngeneic cells and cells differing from the recipient only with regard to lack of L^d were coinjected into K^bD^bL^d, D^bL^d, K^bL^d, or L^d mice. The survival of cells missing only L^d was significantly better in K^bD^bL^d mice than in D^bL^d or K^bL^d mice (P < 0.01). The bars show averages of two to four experiments with a total of three to seven mice in each group. The single L^d group includes also data from Fig. 1. Error bars show SDs.

Figure 5.

Unique expression patterns of Ly49 receptors in mice with single MHC class I molecules. The surface levels of Ly49G2, -I, -A, -F, and -C were measured on NK1.1⁺CD3⁻ cells from mice expressing no MHC class I (MHC⁻), D^b, K^b, L^d, or D^d. The expression levels are expressed as percent of median fluorescence intensity (MFI) compared with MHC⁻ mice. The bars show averages of four to seven experiments with one to three mice in each experiment. Error bars show SD. *, P < 0.05; **, P < 0.01.

Figure 6.

Summary of the educating impact of the four tested MHC class I molecules in relation to the number of additional MHC class I molecules that are coexpressed. Missing K^b and missing D^b were both tested in two different hosts with three MHC class I molecules (K^bD^bD^d and K^bD^bL^d). The two symbols in the K^b and D^b plots indicate the different hosts with three MHC class I molecules. Similarly, missing L^d was tested in K^bL^d and D^bL^d mice, indicated by the two squares in the L^d plot with two MHC class I molecules.