Effects of MHC class I alleles on licensing of Ly49A+ NK cells

Abstract

NK cells are innate immune lymphocytes that can react to cells lacking self-MHC class I. However, NK cells that cannot engage self-MHC through an inhibitory receptor are resistant to stimulation through their activation receptors. To become licensed (i.e., functionally competent to be triggered through its activation receptors), an NK cell must engage host MHC class I via a MHC class I-specific inhibitory receptor, such as a member of the murine Ly49 family. To explore potential determinants of NK cell licensing on a single Ly49 receptor, we have investigated the relative licensing impacts of the b, d, k, q, r, and s H2 haplotypes on Ly49A(+) NK cells. The results indicate that licensing is essentially analog but is saturated by moderate-binding MHC class I ligands. Interestingly, licensing exhibited a strong inverse correlation with a measure of cis engagement of Ly49A. Finally, licensing of Ly49A(+) NK cells was found to be less sensitive to MHC class I engagement than Ly49A-mediated effector inhibition, suggesting that licensing establishes a margin of safety against NK cell autoreactivity.

Figure 1. IFNγ production by Ly49A-monopositive NK cells varies with MHC haplotype

(A) Representative gating scheme for flow cytometric analysis of intracellular IFNγ production by Ly49-Amonopositive NK cells (Ly49A⁺ NK1.1⁺ CD3⁻ CD19⁻ NKG2A⁻ Ly49C⁻ Ly49F⁻ Ly49G2⁻ Ly49I⁻). The number in the final dot plot represents the percent IFNγ⁺ cells among the Ly49A⁺ population. (B) Average frequency ± SD of IFNγ production by naïve Ly49A-monopositive NK cells (Ly49A⁺ NK1.1⁺ CD3⁻ CD19⁻ NKG2A⁻ Ly49C⁻ Ly49F⁻ Ly49I⁻; Ly49G2⁺ NK cells were also gated out in two of three experiments) incubated with plate-bound anti-NK1.1 antibody (5 µg PK136). The dotted line indicates the mean IFNγ production frequency of Ly49A⁺ NK cells of the H2^d haplotype, which is known to strongly license Ly49A⁺ NK cells. N=6 or 7 per group, pooled results from three independent experiments. See Materials and Methods for antibody clones used. For each MHC haplotype, the data are shown in the same rank order in Figs. 1, 3, 5, and supplemental Figs 1 and 2, according to the MFI of anti-Ly49A staining depicted in Fig. 3B. Numerical values for the data shown in these figures is displayed in the Table. *: p<0.05; **: p<0.01.

Figure 2. IFNγ production by NK cells from B10.RIII mice is not influenced by a known region of genetic contamination

IFNγ production by Ly49A⁺ NK cells from mice expressing RIII- or C57BL/6 (B6)-derived regions on chromosome 10 or at the MHC locus. B10.RIII mice were crossed with B6 mice, and the F2 pups were typed by microsatellite analysis to select for mice homozygous for the RIII- or B6-derived segments on chromosome 10 and chromosome 17, as indicated. Bars represent average frequency ± SD of IFNγ production by naïve Ly49A⁺ NK cells (NK1.1⁺ CD3⁻ CD19⁻) incubated with plate-bound anti-NK1.1 antibody (2 µg PK136). N=9 per group. Pooled results from three independent experiments. *: p<0.05; ***: p<0.001

Figure 3. Correlations between licensing, soluble Ly49A tetramer binding, and putative cis binding of Ly49A

(A) Naïve splenocytes from MHC-congenic and K^b−/− D^b−/− mice were stained with soluble Ly49A tetramers. Mean fluorescence intensity (MFI) ± SEM is shown. N=3 for each group. Representative of three independent experiments. (B) Naïve splenocytes from MHC-congenic mice were stained for Ly49A (mAb A1), NK1.1, CD3, and CD19. MFI ± SEM of Ly49A staining of Ly49A⁺ NK cells (NK1.1⁺ CD3⁻ CD19⁻) is shown. N=3 for each group. Representative of two independent experiments. (C) Correlation analysis of frequency of IFNγ production by Ly49A-monopositive NK cells from and Ly49A tetramer staining of MHC-congenic and MHC class I-deficient mice. For details on data, see Figs. 1B and 3A. (D) Correlation analysis of frequency of IFNγ production by and anti-Ly49A staining MFI of Ly49A-monopositive NK cells from MHC-congenic mice. For details on data, see Figs. 1B and 3B. *: p<0.05; **: p<0.01; ***: p<0.001.

Figure 4. Licensing of Ly49A⁺ NK cells is not affected by haploinsufficiency of the MHC class I ligand

(A) H2D^d expression on splenocytes from mice homozygous (black), hemizygous (dark gray), or nullizygous (light gray) for a D^d transgene. Each histogram represents one mouse. Representative of four independent experiments. (B) Ly49A tetramer staining of splenocytes, histograms colored as in A, from a single experiment. (C) Frequency of IFNγ production by anti-NK1.1-stimulated (5 µg PK136) Ly49A⁺ NK cells from mice of the indicated D^d transgene genotype. Similar results were obtained when Ly49C, I, and G2⁺ cells were gated out of the analysis. Frequency was normalized to the average frequency of IFNγ production by D^{d tg/tg} Ly49A⁺ NK cells in each experiment. Each symbol represents one mouse, pooled from two independent experiments. (D) MFI ± SEM of anti-Ly49A staining (mAb JR9) on Ly49A⁺ NK cells from littermates of the indicated D^d transgene genotype. N=4 or 6 per group. Representative of three independent experiments. (E) H2D^k expression on splenocytes from mice homozygous (black), hemizygous (dark gray), or nullizygous (light gray) for the endogenous H2^k class I genes. Each histogram represents one mouse. Representative of three independent experiments. (F) Ly49A tetramer staining of splenocytes, histograms colored as in E, from a single experiment. (G) Frequency of IFNγ production by anti-NK1.1-stimulated (5 µg PK136) Ly49A⁺ NK cells from mice of the indicated MHC class I genotype. Frequency was normalized to the average frequency of IFNγ production by H2^k/k Ly49A⁺ NK cells in each experiment. Each symbol represents one mouse, pooled from three independent experiments. (H) MFI ± SEM of anti-Ly49A staining (mAb JR9) on Ly49A⁺ NK cells (NK1.1⁺ CD3⁻ CD19⁻) from mice of the indicated MHC class I genotype. N=5 and 6 for the H2^k/k and H2^k/− groups, respectively. Results from a K^b−/− D^b−/− mouse is shown for reference. Representative of three independent experiments.

Figure 5. Ly49A-mediated inhibition of effector function is more sensitive than NK cell licensing

(A) Aggregate results of three independent ⁵¹Cr-release assays of Ly49A⁺ LAK cells from H2D^d-transgenic K^b−/− D^b−/− mice killing Con A-activated MHC-congenic splenocytes. Each condition was tested in triplicate in each experiment. (B) Specific inhibition ± SEM of killing of MHC-congenic Con A blasts by Ly49A⁺ LAK cells at an E:T of 8:1. Specific inhibition = 100 × (Specific lysis of K^b−/− D^b−/− Con A blasts – Specific lysis of MHC-congenic Con A blasts) / Specific lysis of K^b−/− D^b−/− Con A blasts. Aggregate results from three independent experiments in which each condition was assayed in triplicate. (C) Specific lysis of Con A blasts homozygous, hemizygous, or nullizygous for a D^d transgene by Ly49A⁺ LAK cells. Representative of two independent experiments. (D) Specific lysis of Con A blasts homozygous, hemizygous, or nullizygous for endogenous H2K^k and H2D^k genes by Ly49A⁺ LAK cells. Representative of two independent experiments. *: p<0.05; ***: p<0.001.