Assessment of Cmv1 candidates by genetic mapping and in vivo antibody depletion of NK cell subsets

Abstract

The mouse chromosome 6 locus Cmv1 controls resistance to infection with murine cytomegalovirus (MCMV). We have previously shown that Cmv1 is tightly linked to members of the NK gene complex (NKC) including the Ly49 gene family. To assess the candidacy of individual NKC members for the resistance locus, first we followed the co-segregation of Cd94, Nkg2d, and the well-characterized Ly49a, Ly49c and Ly49g genes with respect to Cmv1 in pre-existing panels of intraspecific backcross mice. Gene order and intergene distances (in cM) were: centromere-Cd94/Nkg2d-(0.05)-Ly49a/Ly49c/Ly49 g/Cmv1-(0. 3)-Prp/Kap/D6Mit13/111/219. This result excludes Cd94 and Nkg2d as candidates whereas it localizes the Ly49 genes within the minimal genetic interval for Cmv1. Second, we monitored the cell surface expression of individual Ly49 receptors in MCMV-infected mice over 2 weeks. The proportion of Ly49C(+) and Ly49C/I(+) cells decreased, the proportion of Ly49A(+) and Ly49G2(+) remained constant, and the cell surface density of Ly49G2 increased during infection, suggesting that NK cell subsets might have different roles in the regulation of MCMV infection. Third, we performed in vivo antibody depletion of specific NK cell subsets. Depletion with single antibodies did not affect the resistant phenotype suggesting that Ly49A(+), Ly49C(+), Ly49G2(+) and Ly49C/I(+) populations are not substantial players in MCMV resistance, and arguing for exclusion of the respective genes as candidates for Cmv1. In contrast, mice depleted with combined antibodies showed an intermediate phenotype. Whether residual NK cells, post-depletion, belong to a particular subset expressing another Ly49 receptor, or a molecule encoded by a yet to be identified gene of the NKC, is discussed.

Multiparametric Analysis of Host Response to Murine Cytomegalovirus in MHC Class I–Disparate Mice Reveals Primacy of Dk-Licensed Ly49G2+ NK Cells in Viral Control

“…Altogether the results suggested that self-licensed NK cells, possibly regulated by background genetic effects, contribute to highly efficient detection and elimination of viral targets. However, several reports have shown that Ly49G 2 + NK cells in B6 mice rapidly expanded in response to proliferation cues (36) or exposure to one of several different viruses (37–39). Moreover, licensed Ly49C/I+ NK cells were since shown to diminish viral control in MCMV-infected B6 mice (40).…”

Reduced MCMV Δm157 viral clearance in the absence of TSAd

“…Mice were infected with 2 * 10 6  PFU of Δm157 MCMV for 5 days. Viral titers of the stock virus or mouse organs (spleen and liver) were evaluated in vitro by standard plaque assays on a confluent BALB/c mouse embryonal fibroblast monolayer, as previously described40.…”

Genetic control of innate immune responses against cytomegalovirus: MCMV meets its match

“…Segregation analysis of polymorphic markers, including microsatellite markers, gene sequences and anonymous probes, with respect to Cmv1 lead to the localization of Cmv1 to a 0.35-cM interval in two independent genetic maps. [110][111][112] The map generated by our group revealed gene order and intergene distances (in cM) in the vicinity of Cmv1 as: (Figure 2a). A high-resolution physical map of the Cmv1 genetic interval based on long-range restriction mapping by PFGE and the assembly of a contig of YAC and BAC clones translated the genetic interval into 1.6 Mb of genomic DNA containing a minimum of 18 transcription units including the 14 Ly49 gene cluster (Figure 2b, c).…”

“…112 These results excluded Nk1.1, Cd94 and Nkg2d, while retaining the Ly49 gene cluster as candidates for Cmv1. [110][111][112] Interestingly, a similar mapping approach being performed by a second group initially excluded Ly49 genes as potential candidates by placing Cmv1 distal to Ly49b gene, raising the possibility that one or more NKC genes could be involved in the resistance/susceptibility phenotype. 115 Sequence comparison between Cmv1 r and Cmv1 s strains indicated allelic differences for transcripts analyzed in the Cmv1 interval 116,117 (Figure 2b, Figure 3), precluding assessment of their candidacy based upon mutation analysis.…”

“…All markers have been previously reported. 111,112,115,118,119 determined by the two previously described genetic maps 112,115 made it difficult to rule out a possible contribution of several linked loci to the susceptibility/ resistance phenotype.…”