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

Chantal Depatie, Anick Chalifour1, Catherine Paré1, Seung-Hwan Lee2, Silvia M. Vidal2 and Suzanne Lemieux3

Department of Biochemistry, McGill University, Montreal, H3G 1Y6 Québec, Canada
1 Centre de Recherche en Santè Humaine, INRS-Institut Armand-Frappier, Université du Québec, Laval, Québec H7V 1B7, Canada
2 Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa,Ontario K1H 8M5, Canada

Correspondence to: S. M. Vidal


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
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/Ly49g/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.

Keywords: Cmv1, Ly49 receptors, NK cells


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
In the mouse, natural resistance to murine cytomegalovirus (MCMV) infection is controlled by a dominant gene on chromosome 6 named Cmv1 (1). Common inbred strains present two allelic forms for Cmv1: a dominant resistant allele, Cmv1r, expressed in mice of the C57BL background, and a recessive susceptible allele, Cmv1s, expressed in BALB/c, A/J and DBA/2 strains. Phenotypically, resistant mice display splenic viral titers 103–104 times lower than those found in susceptible mice from day 2 post-infection when inoculated with a sublethal dose of virus. Studies in radiation chimeras following bone marrow transplantation together with in vivo depletion experiments using the mAb PK136 indicated that the effect of Cmv1 is mediated by NK cells (2,3). PK136 binds to NK1.1, a receptor encoded by the Nk1 gene that is expressed on the surface of almost all NK cells and a small population of T cells in C57BL mice and a few strains (4,5). The NK cell population is an important player in the innate response towards a variety of viral infections through lysis of infected target cells and production of an array of cytokines (reviewed in 6).

We and others (79) positioned Cmv1 to the NK gene complex (NKC), a chromosomal segment bearing numerous NK cell receptor genes, including Nk1, that stand as potential candidates for Cmv1 (10,11). By segregation analysis of two informative backcrosses totaling over 1967 meioses, we identified a 0.7 cM interval for Cmv1 (8) excluding the Nk1 gene. Segregation of Nk1 from Cmv1 was confirmed by Forbes et al. (9) shortly after. However, many other candidates remain, such as members of the Ly49 multigene family (12,13), and the Cd94 (14) and Nkg2 (15,16) genes.

Murine Ly49 genes encode NK cell receptors that form disulfide-linked dimeric type II integral membrane proteins and belong to the C-type lectin superfamily. The Ly49 family is composed of at least 14 members, most of which still await characterization (12,13). Allelic polymorphisms were established for some Ly49 genes expressed in BALB/c (Cmv1s) and C57BL/6 (Cmv1r) mice (1719). Genes encoding several homologous Ly49 molecules were found in rats and localized on chromosome 4 (20). However, only one Ly49 family member (Ly49L) has been detected in humans thus far. As expected, the Ly49L locus was localized in the NKC on syntenic chromosome 12p12–p13 (21). To date, no Ly49 receptor has been shown to have a ubiquitous NK cell surface expression. Rather, Ly49 receptors are expressed on overlapping cell subsets of variable size (18,19,2224). These receptors are involved in activation or inhibition of NK cell functions upon cross-linking with specific mAb or by interacting with MHC class I ligands (18,22,2428). Ly49 molecules with inhibitory properties, such as Ly49A, have immunoreceptor tyrosine-based inhibitory motifs (ITIM) in their cytoplasmic tail capable of recruiting cytoplasmic phosphatases (29). Ly49 receptors lacking cytoplasmic ITIM motifs transduce activating signals leading to target cell lysis and cytokine secretion (30).

Human and mouse CD94/NKG2 receptors are type II heterodimeric proteins that also belong to the C-type lectin superfamily and are expressed on NK cells. Unlike Ly49 receptors, they interact with non-classical MHC class I molecules bound to peptide (16,31). Signal transduction after engagement of human activating or inhibitory CD94/NKG2 receptors proceeds as for Ly49 molecules (32,33). Whether this will also be the case for CD94/NKG2 murine receptors has yet to be elucidated.

Numerous allelic variations in NKC genes complicate the task of assessing candidate mutations potentially underlying the Cmv1 phenotype (1419). Therefore, to address the candidacy of individual NKC members, (i) we studied the segregation of Cd94, Nkg2d, and the well characterized Ly49a, Ly49c and Ly49g genes with respect to Cmv1 in two informative backcrosses. These Ly49 genes were chosen based on the availability of receptor-specific mAb for in vivo analysis as a means to assess functionally their candidacy for Cmv1. Using mAb, (ii) we monitored the cell surface expression of Ly49A, Ly49C and Ly49G2 in splenic Cmv1r NK cell populations during the course of infection. Finally, (iii) we analyzed the role of specific NK-cell subsets in MCMV resistance by assaying MCMV spleen and liver titers after in vivo cell depletion with individual or combined anti-Ly49 mAb.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Inbred mouse strains A/J, BALB/cJ, C57BL/6J and B10.D2/nSnJ (6–10 weeks old) were purchased from the Jackson Laboratory (Bar Harbour, ME). We have previously described the breeding and maintenance of (A/JxC57BL/6)F1xA/J and (BALB/cxC57BL/6)F1xBALB/c segregating backcross mice (8).

Virus, infection and virus titration
The Smith strain of MCMV, obtained from the ATCC (Rockville, MD), was propagated by salivary gland passages in 3-week-old BALB/c mice as previously described (8). Otherwise, mice were infected i.p. with 2x103 p.f.u. of MCMV. Spleens harvested at indicated days were used for NK cell enrichment and flow cytometry (FCM) analysis, and for virus titration by plaque assay on monolayers of mouse embryo fibroblasts, as described (8). To ensure that every mouse was properly infected, liver viral load, which is not controlled by Cmv1, was also determined. Viral titers are expressed as log10 of MCMV p.f.u. per organ.

Detection of polymorphisms and linkage analysis
Oligonucleotide sequences are presented in Table 1Go. Ly49c was mapped by allele-specific oligonucleotide (ASO) hybridization of a 144 bp PCR product within Ly49c exon 4, amplified with Ly49c-L/R oligonucleotides. The PCR reaction was performed using 20 ng of genomic DNA in a 20 µl volume reaction for 30 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 30 s. A 5 µl aliquot of the PCR reaction was fractionated by electrophoresis on a 1.5% agarose gel containing ethidium bromide to verify successful amplification. The remaining PCR products were denatured in a solution of 0.4 N NaOH and 10 mM EDTA, and boiled for 5 min prior to transfer to a nylon membrane (Hybond-N; Amersham, Little Chalfont, UK) with a DOT-Blot apparatus (BioRad, Hercules, CA). The filter was then hybridized with [{gamma}-32P]ATP-labeled Ly49c-3 oligonucleotide specific for the C57BL/6 allele. Washing conditions were 56°C with 0.5xSSC (1xSSC is 0.15 M sodium chloride/0.015 M sodium citrate) and 0.1% SDS for 40 min. Hybridization signals were detected only from PCR products of the C57BL/6 allele. The mapping of Ly49a and Ly49g was accomplished by single sequence length polymorphism (SSLP) using sequence-specific primers flanking novel (CA)n repeats closely linked to each of the genes. For this, total genomic DNA from YAC 109B6 (Whitehead I mouse) (Research Genetics, Huntsville, AL) containing the Ly49a and Ly49g genes (data not shown) was used to create a cosmid library of mouse-specific clones as previously described (34). A total of 120 clones were screened by oligonucleotide hybridization using oligonucleotide Ly49a-3, corresponding to the promoter region of Ly49a (35), and oligonucleotide Ly49g-3, corresponding to unique exon 3 sequences of Ly49g (36). The same oligonucleotides were used to confirm the presence of the respective genes by cosmid DNA sequencing. To create small insert libraries, cosmid DNA was digested with MboI and subcloned into the BamHI site of plasmid pBluescript II KS+ (Stratagene, Burlingame, CA). Inserts containing (CA)n repeated sequences were identified as previously described (37), and used to derive Ly49a/D6Ott11 and Ly49g/D6Ott22 primer pairs. SSLP were identified by PCR amplification using 20 ng of genomic DNA as described (8). Cd94 was mapped by following the segregation of two transversions (T666C and C670T), which distinguish BALB/c and A/J from C57BL/6 strains (14), by direct DNA sequencing of a 244 bp PCR product corresponding to the 3' untranslated region. The PCR product, obtained from genomic DNA with oligonucleotides Cd94-L/R, was gel purified and sequenced using dye terminator chemistry (Amersham). Nkg2d was mapped by following the segregation of an XbaI restriction fragment length polymorphism (RFLP) identified within a 700 bp PCR product corresponding to intron 11 and amplified with oligonucleotides Nkg2d-L/R. For genetic mapping we followed the segregation of these polymorphisms on a panel of 981 (A/JxC57BL/6)F1xA/J and 920 (BALB/cxC57BL/6)F1xBALB/c segregating backcross mice described previously (8). Genetic linkage was determined by segregation analysis. Gene order was deduced by minimizing the number of crossovers between different loci within the linkage group (38).


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Table 1. Summary of PCR primers used for genetic mapping
 
Antibodies
Hybridomas producing anti-HSA (rat IgG2b, clone M1/69), anti-CD8 (rat IgG2a, clone 53-6.72), anti-Fc{gamma}RIII (rat IgG2b, clone 2.4G2) and anti-NK1.1 (mouse IgG2a, clone PK136) mAb were purchased from ATCC (Rockville, MD). Hybridomas producing anti-CD4 (rat IgG2b, clone MT4) and anti-Ly49A (mouse IgG2a, clone A1) mAb were kindly provided by Dr E. F. Potworowski (INRS-Institut Armand-Frappier, Laval) and Dr J. P. Allison (UCA, Berkeley, CA) respectively. 4LO3311 and 4LO439 mouse IgG3 mAb have been described (39). To derive 5GA5, a novel mAb recognizing Ly49C and Ly49I, Armenian hamsters (obtained from Dr G. Yerganian, Cytogen Research & Development, West Roxbury, MA) were immunized 4 times at 3 week intervals with BALB/c splenic LAK cells. Cell fusion and cloning of hybridomas were as described (39). Screening of hybridoma supernatants was done by dot-blot on Ly49C antigen purified from BALB/c LAK cells by affinity chromatography over 4LO3311 mAb-coated Sepharose 4B beads (Pharmacia, Baie d'Urfée, France). The 5GA5 mAb detects an epitope, common to Ly49C and Ly49I receptors, localized within the carbohydrate recognition domain (data not shown). Two-color FCM analysis of C57BL/6 NK-enriched spleen cells with 5GA5 and 4LO3311 mAb showed that both antibodies recognized the same Ly49C+ population, and that the Ly49C+ and Ly49I+ populations are of comparable size (data not shown). Similar studies in BALB/c cells showed that 5GA5 and 4LO3311 mAb detect the same percentage of cells, indicating that 5GA5 does not detect an Ly49I+ population in this strain. This finding, consistent with data obtained with another anti-Ly49C/I mAb, SW5E6 (19), suggests that Ly49I is not expressed in BALB/c mice or, alternatively, that the antibody detects a polymorphic determinant that affects binding. Cold competition assays in BALB/c NK-enriched spleen cells showed that 5GA5 and 4LO3311 do not compete for binding (data not shown). Characteristics and reactivity of all antibodies used in this study are summarized in Table 2Go. With the exception of mAb PK136 and 4LO439, which were purified from ascitic fluid, all other mAb were purified from hybridoma supernatants using Protein G–Sepharose 4 Fast Flow (Pharmacia). The mAb were conjugated to FITC or biotin (both from Sigma) using standard procedures. Isotype control mAb were purchased from PharMingen (San Diego, CA).


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Table 2. Reactivity of anti-NK antibodies used for FCM analyses and NK cell depletions
 
Depletion of NK cell subsets
At 48 h before infection with MCMV, C57BL/6 or B10.D2 mice were inoculated i.p. with 1 mg of purified anti-Ly49 mAb of a given specificity, a mixture of three or four mAb (1 mg of each) or 200 µl of a 1:4 dilution of ascitic fluid containing the anti-NK1.1 mAb PK136. At day 2 post-infection, the extent of depletion was assessed by FCM analysis using the corresponding biotin-conjugated antibody.

Enrichment of splenic NK cells
Splenic NK cells were prepared by a negative selection procedure. Briefly, nylon wool non-adherent cells were incubated for 45 min on ice with a mixture of rat anti-HSA, anti-CD4 and anti-CD8 mAb, and then reactive cells were eliminated with sheep anti-rat IgG-coated magnetic beads (Dynal, Great Neck, NY). When applied to C57BL/6 spleen cells, this procedure yields a population containing 70–85% NK1.1+ cells.

FCM analysis
Splenic NK cells (2–3x105/sample) suspended in PBS containing 1% BSA and 0.02% sodium azide were first incubated for 20 min at room temperature with mAb 2.4G2 to block FcR. Cells were then incubated for 30 min on ice with optimal concentrations (30 ng to 2 µg/sample) of FITC- or biotin-conjugated mAb or both, added in sequence. Phycoerythrin (PE)–labeled streptavidin (SA–PE) and Red670-labeled streptavidin (SA–Red670) conjugates (Canadian Life Sciences, Burlington, Ontario, Canada) were used for the detection of the red fluorescence in one- and two-color analyses respectively. After washing, stained cells were analyzed on a Epics XL-MCL flow cytometer (Coulter, Hialeah, FL) calibrated with FLOWCHECK Fluorospheres. Results are expressed as the percentage of lymphocytes gated with forward and side scatters that reacted with mAb. The mean fluorescence intensity (MFI) was established on a logarithmic scale. Data analysis using XL software, version 1.5, was based on the collection of 5000–10,000 events per sample.

Statistical analysis
Significance of the differences observed in reference to control groups was assessed using the two-tailed Student's t-test. Only P values <0.01 (*) and <0.001 (**) are indicated.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Linkage analysis
We previously reported the generation of a genetic linkage map, comprised of 45 DNA markers corresponding to either cloned genes or microsatellites, which has improved the resolution of the genetic localization of the murine host resistance locus Cmv1 on mouse chromosome 6 (8). Segregation analysis of the above markers with respect to Cmv1 in 1967 backcross animals defined a minimal genetic interval for Cmv1 of 0.7 cM with the following gene order and intergene distances: centromere–Nk1/D6Mit61/135/257/289/ 338–0.4–Ly49a/D6Mit370/Cmv1–0.3–Tel/D6Mit374/290/220/196/195/110. Ly49a (36) was the only gene to co-segregate with Cmv1 in 1967 meioses. This locus was mapped using a diagnostic RFLP detected by an Ly49a cDNA probe. As such, it was not possible to determine if this RFLP corresponded uniquely to Ly49a or to another member of the Ly49 gene family as multiple bands were seen on Southern blots of digested genomic DNA hybridized with the cDNA probe.

The testing for possible candidates for Cmv1 initially required improving the resolution of our genetic linkage map to precisely localize individual Ly49 family members in addition to the newly identified NKC members, Nkg2d and Cd94. To this end, we developed novel polymorphic markers in the vicinity of Cmv1, and used them for linkage analysis in our reported intraspecific panels of 981 (A/JxC57BL/6)F1x A/J mice and 920 (BALB/cxC57BL/6)F1xBALB/c mice (8). Ly49c was mapped by exploiting the existence of two proximal nucleotide transversions between BALB/c, A/J and C57BL/6 strains. Using ASO hybridization, we followed the segregation of these transversions (T442G and A444C: A/J, BALB/c -> C57BL/6) using a C57BL/6-specific primer (19). Allelic variants have also been reported for Ly49a and Ly49g in the above strains (17,18). At present, however, it is difficult to determine if these variants correspond to homolog or paralog genes. To ascertain the genetic localization, simple sequence repeats (SSR) in the vicinity of the Ly49a and Ly49g genes were cloned and mapped by SSLP. The nomenclature and characteristics of the two SSR, D6Ott11 and D6Ott22, together with the polymorphisms used to map Cd94 and Nkg2d, are listed in Table 1Go. The SSR markers were isolated from YAC-derived cosmid clones containing either Ly49a or Ly49g. D6Ott11, which lies within 35 kb of the Ly49a promoter region (35), generated polymorphic fragments for the strains analyzed, whereas D6Ott22, which lies within 45 kb of Ly49g exon 3, amplified a C57BL/6 specific fragment.

The segregation of the SSR, as well as the other novel markers in this study, was followed on a subset of animals from our backcross panels (8). Thirty-seven animals that presented a recombination event between the anchor loci D6Mit216 and D6Mit25 were typed. As these panels have been phenotyped for Cmv1, this approach provided direct information regarding Cmv1 linkage. To determine gene order, the individual haplotypes of the 37 informative meioses were established, and the location of the new markers and genes was integrated with the established 45 markers (Fig. 1Go). Assuming that there were no double crossover events, a single crossover was detected between Cd94/Nkg2d and Cmv1, and no recombination was observed between Cd94 and Nkg2d. This result positions Cd94/Nkg2d proximal to Cmv1 at an estimated recombinational distance of 0.05 cM and decreased our genetic interval from 0.7 to 0.35 cM. However, no crossovers were detected between Cmv1 and Ly49a/D6Ott11, and Ly49c and Ly49g/D6Ott22, identifying these loci as attractive candidates for the host resistance locus. Combined pedigree analysis for the 7.9 cM segment encompassing Cmv1 produced the following locus order and interlocus distance (cM): D6Mit216–(5.1)–D6Mit52 (0.5)–D6Mit94–(0.2)–Nk1/D6Mit61/135/257/289/338–(0.35)–Nkg2d/Cd94–(0.05)–Cmv1/Ly49a(D6Ott11)/Ly49c/Ly49g(D6Ott22)/D6Mit370–(0.3)–Prp/Kap/D6Mit13/11/219–(0.3)–Tel/D6Mit374/290/220/196/195/110. These results delineate a minimal genetic interval for Cmv1 of 0.35 cM that is defined by 12 tightly linked markers.



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Fig. 1. Segregation analysis and linkage map in the vicinity of the Cmv1 host resistance locus. (A) Each column represents a chromosomal haplotype identified in the backcross progeny (BALB/cxC57BL/6)F1xBALB/c. Solid boxes: C57BL/6 alleles. Open boxes: BALB/c alleles. The number of the progeny with each haplotype is shown at the bottom of each column. Haplotype analysis of the (A/JxC57BL/6)F1xA/J is not shown. (B) Schematic representation of the composite map position of Cmv1 on mouse chromosome 6. The gene order and the mapped loci were determined by pedigree analysis, and the intergene distances are given as estimates of recombination frequencies within backcross animals. The centromere of the chromosome is shown as a black circle. Recombination frequencies are shown to the right of the chromosome.

 
Cell surface expression of Ly49A, Ly49C and Ly49G2 receptors during early MCMV infection
The anti-Ly49 mAb used in this study for FCM analysis and in vivo depletion were either allele specific [A1: anti-Ly49A (40); 4LO439: anti-Ly49G2 (39 and unpublished observations)] or receptor specific [4LO3311: anti-Ly49C (19,39,41)] (Table 2Go). 5GA5 recognizes both Ly49C and Ly49I, and is the only mAb that has dual specificity in this study (see Methods for a description of 5GA5). However, it is possible that some of the mAb used react with the Ly49j-n gene products for which no cDNA has been cloned so far (13). The relative proportion of NK cells which expresses a given Ly49 molecule in normal C57BL/6 mice is as follows: Ly49G2+ > Ly49C+ = Ly49I+ > Ly49A+. Two-color FCM analysis of C57BL/6 NK-enriched spleen cells revealed that, collectively, the subsets of NK cells stained by A1, 4LO3311, 4LO439 and 5GA5 constitute 85% of the NK1.1+ cell population (Fig. 2Go).



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Fig. 2. FCM analysis of Ly49+ NK cell subsets. Co-expression of NK1.1 and Ly49 receptors was assessed by incubating NK-enriched spleen cells from C57BL/6 mice with FITC-conjugated anti-NK1.1 mAb PK136, and a mixture of the biotinylated mAb A1 (anti-Ly49A), 4LO3311 (anti-Ly49C), 5GA5 (anti-Ly49C/I) and 4LO439 (anti-Ly49G2). The red fluorescence was detected with SA–Red670 conjugate. Numbers indicate the percentage of cells in each quadrant.

 
The cell surface expression of Ly49 receptors was monitored by FCM analysis of NK-enriched spleen cells over a 2 week period after Cmv1r C57BL/6 mice were infected with a sublethal dose of MCMV. At no time point was there significant variation in the mean number of spleen cells harvested from infected mice. However, whereas in normal mice the yield of splenic NK cells recovered after the enrichment procedure was 1.52 ± 0.38% (0.7–1.5x106 cells per spleen), this value dropped significantly in infected mice, reaching a maximal 2.3-fold reduction at day 3 post-infection. To control for variability within the entire NK cell population during the course of infection, NK1.1 cell surface expression was followed using the PK136 mAb (4). From 2 to 14 days post-infection, the percentage of NK1.1+ cells was reduced (Fig. 3Go). Maximal effect was seen at 3 days post-infection when NK1.1+ represent 60% of control values (P < 0.01). The cell surface expression of the Ly49+ cell subsets displays different variation patterns during the course of infection. The proportion of Ly49C+ and Ly49C/I+ cells decreased to 60 and 46% of the control values respectively 3 days post-infection (P < 0.001); the proportion of cells expressing either Ly49A or Ly49G2 at their cell surface remained relatively constant over the infection period. Interestingly, the cell surface density of Ly49G2 increased 2-fold 3 and 4 days post-infection (P < 0.001). Except for a minor variation in the level of surface expression of the NK1.1 receptor 2 days after infection, the levels of all other receptors remained unchanged.



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Fig. 3. Modulation of cell surface expression of NK cell receptors in C57BL/6 (Cmv1r) mice infected with MCMV. Surface expression of NK1.1, Ly49A, Ly49C, Ly49C/I and Ly49G2 receptors in MCMV-infected mice was followed during 2 weeks post-infection by FCM analysis of NK-enriched spleen cells stained with biotinylated mAb and SA–PE conjugate. The relative size of each cell subset and the density of expression (MFI) of the corresponding receptor are illustrated. Except for the control group which included 10 mice individually tested, each bar corresponds to the mean ± SD of three to seven determinations. Due to the decrease in spleen cell recovery in infected mice, NK-enriched cells were prepared occasionally from pooled spleen cells of two to three infected mice. Statistically significant differences in comparison with uninfected mice at P values <0.01 (*) and <0.001 (**) are indicated.

 
Splenic MCMV replication in mice depleted of selected NK cell subsets
In vivo depletion of NK cells with PK136 prior to MCMV infection converted resistant C57BL/6 and C57BL/6 -> BALB.B bone marrow chimeric mice to the susceptible phenotype (2,3). We therefore investigated the phenotypic effect of selectively depleting cell populations which express specific Ly49 receptors in MCMV-infected resistant mice. Two days prior to infection, C57BL/6 mice were inoculated with anti-Ly49 mAb or PK136. The extent of depletion was assessed by staining NK-enriched spleen cells with biotinylated mAb and SA–PE conjugate at 2 days post-infection. Due to the high quantum yield of PE it was possible to detect residual cells expressing low levels of targeted receptors which are missed when using FITC-conjugated mAb (data not shown). To rule out the possibility that Ly49+ receptors are masked by the respective antibodies, NK-enriched spleen cells from treated mice were assayed for surface IgG and showed no positive staining. Furthermore, as expected, the recoveries of NK-enriched spleen cells from treated mice and their content of NK1.1+ were reduced according to depletion efficiencies of the targeted cell populations (data not shown).

A 79–89% reduction in the proportion of NK cells expressing Ly49A or Ly49G2 was obtained after depletion with either anti-Ly49A or anti-Ly49G2 allele-specific mAb but all mice remained resistant (Fig. 4AGo, upper panel). Depletion of the Ly49C+ cell population of >50%, using either the receptor-specific mAb 4LO3311 or the Ly49C/I cross-specific mAb 5GA5, could not be achieved, even with higher doses of antibody (data not shown). As Ly49C cell surface expression is markedly down-regulated in the presence of the H-2Kb ligand (41,42), we envisaged the possibility that the efficiency of depletion was adversely affected by the low receptor density in the C57BL/6 strain. Therefore, we attempted to deplete Ly49C+ cells in the B10.D2 (Cmv1r, non-H-2Kb) strain which displays a higher cell surface expression level of Ly49C than C57BL/6 mice (41). The proportion of Ly49C+ and Ly49C/I+ cells decreased by 69 and 87% in B10.D2 mice inoculated with 4LO3311 and 5GA5 respectively (Fig. 4AGo, lower panel). If Ly49C+ cells are a major component in mediating resistance to MCMV, the level of depletion should arguably be sufficient to detect variations in splenic viral load. However, depletion of Ly49C+ cells and all other Ly49+ cell subsets did not alter the resistance phenotype of C57BL/6 and B10.D2 MCMV-infected mice. No positive cells were detected when NK-enriched spleen cells from mAb-depleted mice were stained with biotinylated anti-mouse IgG antibodies and SA–PE conjugate, showing that Ly49+ cell subsets were really depleted and not Ly49 receptors only masked by their respective antibodies.



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Fig. 4. Effect of Ly49 cell subset depletion on splenic MCMV replication. (A) C57BL/6 (upper panel) or B10.D2 (lower panel) (Cmv1r) mice were depleted of selected Ly49 cell subsets as described in Methods and then infected with MCMV. At day 2 post-infection, spleen and liver were harvested and used for virus load determination and depletion control. An asterisk indicates <2.39 log10 p.f.u. Left panels display mean viral loads ± SD as log10 p.f.u./organ from two to seven mice individually tested. BALB/c (Cmv1s), C57BL/6 and B10.D2 (Cmv1r) infected mice, either not depleted or depleted of total NK cells with the anti-NK1.1 mAb PK136, were used as controls. Profiles of residual spleen cells reacting with a given FITC- or biotin-conjugated mAb (solid line) are shown in right panels as overlays on those obtained from non-depleted infected mice of the same strain (dotted line). (B) B10.D2 mice inoculated with 3-mAb (A1, 4LO3311 and 4LO439) or 4-mAb (A1, 4LO3311, 5GA5 and 4LO439) mixtures were infected and tested 2 days later as described above. Histograms in the right panel illustrate the extent of depletion achieved with the 3-mAb (upper panel) and 4-mAb (lower panel) mixtures on the NK1.1+ cell population (stained with PK136) and on the Ly49+ populations (stained with mixed biotinylated mAb). Data illustrated are representative of two to four identical experiments. Numbers indicated correspond to the mean percentage of depletion in reference to cell subset size in non-depleted infected mice.

 
Next we assessed the resistance/susceptibility phenotype of B10.D2 MCMV-infected mice inoculated with a combination of three (A1, 4LO3311 and 4LO439; `3-mAb') or four (A1, 4LO3311, 4LO439 and 5GA5; `4-mAb') anti-Ly49 mAb (Fig. 4BGo). The efficiency of these depletions was determined by staining with PK136 or 3-mAb or 4-mAb to detect residual cells. Injection of 3-mAb successfully depleted 88% of the targeted cells and 80% of NK1.1+ cells but, as expected, the residual population still reacted with 4-mAb, which detects Ly49I+ cells (Fig. 4BGo, upper right panel). After inoculation with 4-mAb, <10% of cells stained with 3-mAb or 4-mAb, corresponding to a depletion of 85% of the NK1.1+ cells (Fig. 4BGo, lower right panel). As expected, expression of Ly49A and Ly49G2 receptors was reduced in B10.D2 mice that express H-2Dd, a ligand for these two receptors (18,25). However, despite the low expression levels of Ly49A and, to a lower extent, of Ly49G2 in B10.D2 mice, 82% of Ly49A+ cells and 89% of Ly49G2+ cells were depleted in mice inoculated with 3-mAb and 4-mAb mixtures (data not shown). On the basis of the splenic viral load, the resistance phenotype of mice depleted with 3-mAb was unchanged. However, some mice depleted with 4-mAb exhibited up to a 50-fold increase in splenic titers, suggestive of a progression towards susceptibility (Fig. 4BGo: left panel).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The host resistance locus Cmv1 is expressed in the splenic NK cell population and sits in a region containing numerous NK cell-related genes with a high degree of polymorphism within families, such as the Ly49. Therefore, for our positional cloning approach, relying solely on sequence comparison is not sufficient to identify candidate genes. Thus, using methods complementary to genetic localization, which serves to eliminate potential genes and helps decrease the encompassing interval, has become imperative to assess candidacy. Previously published results segregated Ly49a and Ly49g from the Cmv1 locus (9). However, these genes could not be excluded as possible candidates as the mice that exhibited crossovers directly proximal to Cmv1 displayed intermediate phenotypes. Experimental error affecting phenotype assignment or, alternatively, contribution of other closely linked genes to control of viral replication could explain these observations. The results presented here show that Cd94 and Nkg2d are positioned 0.05 cM proximal to the Cmv1 locus, thereby excluding these two genes from the list of candidate genes. In contrast, we show that Ly49a, Ly49c and Ly49g do not segregate from Cmv1 in 1901 meiosis analyzed, and stand as Cmv1 candidates.

Modulation of Ly49 receptor expression with respect to that of MHC class I ligands has been reported but not in the context of viral infection (23,4145). Considering that MCMV causes a down-regulation of MHC class I molecules (4647), it was of interest to monitor the cell surface expression of Ly49 receptors during the course of infection. The receptor calibration model postulates that the level of receptor expression decreases in the presence of its cognate MHC class I ligand (48). This allows NK cells to increase their sensitivity in distinguishing between cells bearing normal amounts of MHC class I molecules and those expressing aberrant levels. Therefore, down-regulation of MHC class I surface expression, as in the case of MCMV-infected cells, should lead to NK cell-mediated lysis of these cells. The only Ly49 receptor known to have specificity for an MHC class I molecule of the H-2b haplotype expressed in C57BL/6 (Cmv1r) mice is Ly49C (26,27). In agreement with the receptor calibration model, the lowest density of Ly49C receptors is found in H-2b mice (41). In H-2 congenic mice on C57BL and BALB backgrounds, the expression level of Ly49C is 3–4 times lower in H-2b than in H-2d mice. Furthermore, in C57BL/6 TAP1/ß2-microglobulin 2m)–/– mutant mice, deficient in MHC class I expression, the level of Ly49C detected by the receptor-specific 4LO3311 is 8 times higher than in C57BL/6 mice (42). Since H-2Kb, which binds to Ly49C with high affinity, is down-regulated during MCMV infection (46), we would have predicted a selective increase in the cell surface expression of Ly49C in MCMV-infected C57BL/6 mice. However, no changes in Ly49C surface expression were observed. Two recent reports using MHC class I mosaic mouse models demonstrate that Ly49 expression is regulated by the NK cell's own MHC class I expression (49,50). This observation might explain the absence of any increase in Ly49C expression in MCMV-infected mice.

The only significant change concerning level of expression of Ly49 receptors during the course of infection was an up-regulation in cell surface density of Ly49G2 at days 3 and 4. Ly49G2, a splice variant of Ly49G, has been characterized as an inhibitory receptor which only binds to an H-2d MHC class I ligand (18). Despite the absence of an established ligand in H-2b mice, a moderate increase in the level of Ly49G2 cell surface expression was reported in ß2m–/– mice and a 25% increase in TAP1/ß2m–/– mice, both of which have C57BL background (23,44). The 2-fold increase of Ly49G2 detected in this study with the allele-specific anti-Ly49G2 4LO439 is significant and might point to a yet unidentified role for this receptor during MCMV infection. However, this role would be different from resistance to MCMV as it is observed only 3–4 days after infection, whereas control of splenic viral replication is seen earlier. Moreover, antibody depletion of the NK cell subset expressing the Ly49G2 receptor in Cmv1r mice did not abolish resistance.

Concerning the variation in size of NK cell subpopulations, we observed a decrease in the proportion of the Ly49C+ cells starting at day 3 and no change in the Ly49A+ and Ly49G2+ populations. As we mentioned above, the Ly49C+ population is the only one expressing a receptor for which a known ligand is present in the resistant C57BL/6 strain. Therefore, it is possible to speculate that down-regulation of the ligand in response to MCMV infection results in activation of the cognate NK cell by cytokines in the microenvironment or other mechanism resulting in the regulation of the population by apoptosis. In contrast to our observations, Tay et al. (51) reported an increase in the number of Ly49A and Ly49G2 expressing cells at day 3 post-infection only. These discrepancies can be explained in part by the use of a different mAb for Ly49G2 detection. Our data were obtained using the allele-specific mAb 4LO439 which recognizes specifically Ly49g2 cDNA-transfected COS cells, whereas the 4D11 mAb used in the Tay study cross-reacts with cells transfected with Ly49g2 and Ly49a cDNAs (12,44). In addition, rather than using total spleen cell suspensions, our results are based on FCM analysis of NK-enriched splenic cells to reduce non-specific binding of antibodies, thereby increasing the sensitivity of our assay.

Previous reports have demonstrated the successful conversion of resistant mice towards susceptibility using antibodies to selectively deplete NK cells (2,3,52). To test the candidacy of Ly49 genes, we selectively depleted subpopulations of NK cells in Cmv1r mice expressing a specific receptor at their cell surface prior to infection and looked for susceptibility. Conversion would not provide absolute validation of candidacy as the depleted NK cell population expresses other molecules at its surface that might be responsible for resistance. However, non-conversion permits exclusion of possible candidates. Depletion of single Ly49 cell subsets did not alter the resistant phenotype, indicating that the targeted subsets do not play a significant role in resistance to MCMV. Although depletion levels ranged from 69 to 89%, we believe that depletions were sufficient to justify the experiments as removal of NK1.1+ cells with PK136 was also not 100% but conversion to susceptibility was still seen. Using a different set of mAb for depleting NK cell subsets in C57BL/6 mice, Tay et al. (51) recently reached similar conclusions.

The data pertaining to targeted depletion of multiple subsets might suggest a threshold amount of NK cells needed to restrict viral growth. With a depletion of 80% of NK1.1+ cells achieved with 3-mAb, a resistant phenotype persisted whereas mice inoculated with 4-mAb had 15% NK1.1+ cells left and the majority of these mice exhibited a significant increase in viral load. The Ly49I+ population depleted with 4-mAb (but not with 3-mAb) is unlikely to be responsible for the resistance as no change in phenotype was seen in B10.D2 mice inoculated with the anti-Ly49C/I mAb 5GA5 alone. This suggests that the threshold value for maintaining resistance is likely between 15 and 20% of NK1.1+ cells. This would indicate that the Cmv1 locus encodes a protein ubiquitously expressed on NK cells. Alternatively, Cmv1 may be expressed in a small subset which does not express Ly49A, Ly49C and Ly49G2, and therefore remains undepleted. NK1.1+ T cells expressing either {alpha}ß or {gamma}{delta} T cell receptors are unlikely to be involved as these cells are not eliminated in mice inoculated with anti-asialo GM1 antiserum, a treatment that abrogates MCMV resistance (52,53).

The Ly49 family includes, so far, 14 identified members, the majority of which have unknown cell subset distribution and functions. To better evaluate candidates, experiments are in progress to use transgenic technology to create animals expressing different allelic forms of Ly49 genes. The generation of a panel of transgenic animals with an overlapping set of genomic clones spanning the Cmv1 domain will also facilitate dissection of other NKC loci. This includes the Chok locus responsible for preferential target cell lysis of CHO cells by splenic cells of C57BL/6 mice (54) and the Rmp1 locus responsible for innate resistance to lethal ectromelia infection (55).


    Acknowledgments
 
The authors are grateful to Jack Brennan and Fumio Takei for their contribution in the determination of the 4LO439 and 5GA5 mAb specificity. We also acknowledge Yvette Lusignan and Marcel Desrosiers for their expert assistance in FCM analyses, and Line Laroche and Pierre Bèrubè for technical support. We also thank the Medical Research Council of Canada for their grant support. A. C and C. P. were supported by scholarships from NSERC, and S. M. V. is recipient of a MRC scholarship.


    Abbreviations
 
3-mAb A1, 4LO3311 and 4LO439
4-mAbA1, 4LO3311, 5GA5 and 4LO439
ß2mß2-microglobulin
ASOallele-specific oligonucleotide
FCMflow cytometry
ITIMimmunoreceptor tyrosine-based inhibitory motif
MCMVmurine cytomegalovirus
MFImean fluorescence intensity
NKCNK gene complex
PEphycoerythrin
SAstreptavidin
SSRsimple sequence repeat
SSLPsimple sequence length polymorphism

    Notes
 
The first two authors contributed equally to this work

Transmitting editor: H. R. MacDonald

Received 22 March 1999, accepted 31 May 1999.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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