Epistasis between mouse Klra and major histocompatibility complex class I loci is associated with a new mechanism of natural killer cell-mediated innate resistance to cytomegalovirus infection

Abstract

Experimental infection with mouse cytomegalovirus (MCMV) has been used to elucidate the intricate host-pathogen mechanisms that determine innate resistance to infection. Linkage analyses in F(2) progeny from MCMV-resistant MA/My (H2 (k)) and MCMV-susceptible BALB/c (H2 (d)) and BALB.K (H2 (k)) mouse strains indicated that only the combination of alleles encoded by a gene in the Klra (also called Ly49) cluster on chromosome 6, and one in the major histocompatibility complex (H2) on chromosome 17, is associated with virus resistance. We found that natural killer cell-activating receptor Ly49P specifically recognized MCMV-infected cells, dependent on the presence of the H2 (k) haplotype. This binding was blocked using antibodies to H-2D(k) but not antibodies to H-2K(k). These results are suggestive of a new natural killer cell mechanism implicated in MCMV resistance, which depends on the functional interaction of the Ly49P receptor and the major histocompatibility complex class I molecule H-2D(k) on MCMV-infected cells.

Figure 1

Haplotype mapping on chromosome 6 in the vicinity of Cmv1. (a) Physical distance of markers used for haplotype mapping and genetic analysis. Markers in bold were arbitrarily positioned between well-defined markers. (b) Haplotype map of 30 polymorphic markers. Numbers indicate the relative size of PCR products (1 being the shortest) for microsatellite markers; letters indicate PCR–restriction-fragment length polymorphism markers. NP, no product.

Figure 2

Genetic analysis of MCMV resistance in MA/My mice. (a) MCMV viral titers in the spleen of MA/My (n = 16), BALB.K (n = 25), BALB/c (n = 15), (MA/My × BALB.K) F₁ (n = 6) and (MA/My × BALB/c) F₁ (n = 10) mice. Bars show standard deviation. (b) Empirical density (black line) of phenotypes of 226 (MA/My × BALB.K) F₂ mice (left) and 119 (MA/My × BALB/c) F₂ mice (right). Colored lines indicate the empirical density by genotypes at Klra5. In blue, distribution of homozygous BALB.K (n = 55) or BALB/c (n = 30) genotypes; in pink, distribution of the heterozygous genotype (MA/My × BALB.K, n = 118, or MA/My × BALB/c, n = 58); in brown, distribution of the homozygous MA/My genotypes (left, n = 53; right, n = 31) (c) Box plots of log₁₀ PFU counts of H2-Klra5 genotypes showing the combined effects of H2 (MA/My, H2^k; BALB/c, H2^d) and NKC (MA/My, Klra5^m; BALB/c, Klra5^c) loci on spleen viral titers of 119 (MA/My × BALB/c) F₂ progeny. The median and interquartile range are shown. Whiskers extend to the most extreme value, which is less than 1.5 times the interquartile range. Solid dots denote outliers.

Figure 3

Activation of Ly49P reporter cells by MCMV-infected cells. NFAT-GFP expression by reporter cells carrying individual Ly49 receptors was measured and GFP expression was analyzed by flow cytometry following culture of reporter cells: (a) with uninfected (filled histograms) or MCMV-infected (hollow histograms) MA/My MEF cells; (b) with Ba/F3 cells (filled histograms) or Ba/F3 cells expressing the MCMV protein, m157 (hollow histograms); (c) on plastic plates coated with monoclonal antibodies (mAb) to Ly49H-Ly49U, Ly49P and Ly49R.

Figure 4

Ly49P recognition of MCMV-infected cells is target MHC-dependent. NFAT-GFP stimulation by Ly49P and Ly49H reporter cells was determined after culture with MEF cells from MA/My (H2^k), BALB.K (H2^k), BALB/c (H2^d) and FVB (H2^q) inbred mouse strains and TpnT fibroblasts (H2^b) in the presence (hollow histograms) and absence (filled histograms) of MCMV infection. Ly49H reporter cells, which directly recognize the m157 MCMV protein even in the total absence of H2 molecules on the MCMV-infected cells, were equally activated when cultured with infected target cells from all of the above strains.

Figure 5

Characterization of Ly49P interaction with an infected cell. (a) Ly49P activation is MCMV-specific. Ly49P reporter cell activation after culture with target MEF cells from BALB.K mice. Target BALB.K MEFs were either treated with IFNβ or infected with MCMV or mouse herpesvirus 68 (MHV68). (b) Ly49P recognition of MCMV-infected cells is blocked by antibodies to H-2D^k and Ly49P (YE1/48) but not by antibodies to H-2K^k. The antibody to Ly49P was used to block recognition of the infected cells by the Ly49P receptor to ensure its proper expression. NFAT-GFP stimulation of Ly49P reporter cells was measured after culture with MCMV-infected BALB.K MEF cells in the absence of blocking antibody or in the presence of isotype control antibody or antibodies to H-2D^k, H-2K^k or Ly49P.