Ly49h is necessary for genetic resistance to murine cytomegalovirus
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Natural killer (NK) cells play critical roles in antiviral immunity. While the importance of effector mechanisms such as interferons has been demonstrated through knockout mice, specific mechanisms of how viruses are recognized and controlled by NK cells are less well defined. Previous genetic studies have mapped the resistance genes for murine cytomegalovirus (MCMV), herpes simplex virus-1 (HSV-1), and ectromelia virus to the NK gene complex on murine chromosome 6, a region containing the polymorphic Ly49 and Nkrp1 families. Genetic resistance to MCMV in C57BL/6 has been attributed to Ly49H, an activation receptor, through susceptibility of the recombinant inbred strain BXD-8 that lacks Ly49h (also known as Klra8) but derived about half of its genome from its DBA/2 progenitor. However, it remained possible that epigenetic effects could account for the MCMV phenotype in BXD-8 mice. Herein, we report the generation of a novel congenic murine strain, B6.BXD8-Klra8Cmv1-del/Wum, on the C57BL/6 genetic background to evaluate the effect of deletion of a single NK activation receptor, Ly49H. Deletion of Ly49H rendered mice much more susceptible to MCMV infection. This increase in susceptibility did not appear to be a result of a difference in NK cell expansion or interferon-γ (IFN-γ) production between the C57BL/6 and the B6.BXD8 strains. On the other hand, the deletion of Ly49h did not otherwise affect NK cell maturation or Ly49D expression and had no effect on susceptibility to HSV-1 or ectromelia virus. In conclusion, Ly49h is necessary for genetic resistance to MCMV, but not HSV-1 or ectromelia virus.
KeywordsMCMV Ly49H Cytomegalovirus Cmv1 Natural killer cells
Natural killer (NK) cells play important roles in innate immunity, particularly to viruses (French and Yokoyama 2003; Gazit et al. 2006). This is best illustrated by the clinical course of patients with natural killer cell defects which are characteristically manifested as recurrent infections with herpesviruses, such as cytomegalovirus (CMV) and herpes simplex virus-1 (HSV-1; Biron et al. 1989). Thus, greater understanding of how natural killer cells specifically recognize and control viral infections will have important clinical applications.
Given that CMV viruses are species-specific and that murine CMV (MCMV) shares significant biologic and genomic similarity with human CMV, MCMV has been used extensively as an animal model for HCMV (Gonczol et al. 1985; Rawlinson et al. 1997). Several host factors, both major histocompatibility complex (MHC)-dependent and MHC-independent, account for MCMV resistance (Adam et al. 2006; Dighe et al. 2005; Grundy et al. 1981; Rodriguez et al. 2004; Scalzo et al. 1990). The first identified MHC-independent resistance gene, Cmv1, was mapped to distal mouse chromosome 6 in the vicinity of the NK gene complex (NKC) which encodes receptors expressed on NK cells (Scalzo et al. 1995). Identification of the Ly49H activation receptor as the product of the Cmv1 locus was aided by the BXD-8 recombinant inbred (RI) strain (Brown et al. 2001b; Dokun et al. 2001; Lee et al. 2001).
Ly49H is a member of the Ly49 family of C-type lectin-like receptors, which are encoded in the NKC region on distal mouse chromosome 6. The Ly49 family is extremely polymorphic, displaying haplotypes with variations in gene number and allelic polymorphism for each gene (Proteau et al. 2004; Wilhelm et al. 2002). The significant difference in viral susceptibility between the resistant C57BL/6 and susceptible BALB/c has been attributed to the expression of Ly49H on NK cells in C57BL/6, whereas BALB/c mice lack the Ly49h gene (Brown et al. 2001a; Lee et al. 2001, 2003). Interestingly, loci for genetic resistance to two other viruses, HSV-1 and ectromelia virus, an orthopoxvirus, have also been mapped to the NKC (Brown et al. 2001a; Delano and Brownstein 1995). Although antibody-blocking experiments do not support a role for Ly49H in resistance to these viruses (data not shown), Ly49H has not been extensively studied in resistance by genetic approaches.
The BXD-8 RI strain arose from the consanguineous mating between C57BL/6 and DBA/2 progenitor strains for 20 generations. Consequently, half of the genome of this recombinant inbred strain is derived from the resistant C57BL/6 progenitor strain, while the other half from the susceptible DBA/2. BXD-8 mice carried C57BL/6 markers for distal chromosome 6 flanking the NKC region, but demonstrated increased susceptibility to MCMV compared to the C57BL/6 strain. This increased susceptibility has previously been shown to correlate with the absence of Ly49H expression by staining with the monoclonal antibody 3D10 on BXD-8 NK cells and a selective deletion in Ly49h by Southern analysis (Brown et al. 2001a). However, since approximately half of the BXD-8 genome is derived from the DBA/2 strain, it was not clear whether any DBA/2 alleles had an epistatic genetic effect on its increased susceptibility. Transgenic expression of a bacterial artificial chromosome containing the C57BL/6 allele of Ly49h into the susceptible BALB/c genetic background also conferred resistance to MCMV (Lee et al. 2003), but it remained possible that other background BALB/c alleles may modify the Ly49h effect.
To further analyze the role of Ly49h in NK cell function, particularly in infections, we backcrossed the BXD-8 RI strain onto C57BL/6 strain by marker-assisted backcrossing (speed congenics). We termed this novel strain B6.BXD8-Klra8Cmv1-del/Wum, referred to by the shorthand of B6.BXD8 in this paper, and characterized the NK cell function of this strain.
Materials and methods
C57BL/6NCR mice were purchased from National Cancer Institute. The RI strain BXD-8 was purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and was backcrossed to C57BL/6 for nine generations to generate the novel strain B6.BXD8-Klra8Cmv1-del/Wum. At each successive generation, heterozygous pups carrying the deleted Ly49h allele were identified by flow cytometry. Microsatellite genotyping was performed by the Speed Congenics Core of the Rheumatic Diseases Core Center at Washington University to screen for C57BL/6 markers for the initial seven generations. Subsequently, two more generations were backcrossed onto C57BL/6 while following the heterozygous expression of Ly49H phenotype by flow cytometry. All animals were maintained in a specific pathogen-free facility at Washington University School of Medicine, and all experiments were approved by the Animal Studies Committee.
Genomic DNA for BXD-8 was purchased from Jackson Laboratory. Splenic genomic DNA for C57BL/6 and B6.BXD8 were extracted using Gene Pure following manufacturer’s protocol. Digested DNA was separated on a 1% agarose gel and transferred onto a nitrocellulose membrane. Membrane was subsequently hybridized with the BglII–PstI fragment of Ly49H complementary DNA (cDNA) labeled with 32αP-dCTP using Rediprime II System (Amersham, UK) following the manufacturer’s protocol and image acquired using the Typhoon system (Amersham).
Viral titers were quantified using methods previously published (French et al. 2004). Briefly, whole spleens were harvested and frozen in 1 ml aliquots of D10 [Dulbecco’s modified Eagle’s medium supplemented with 10% newborn calf serum (Hyclone), 100 U/ml penicillin, 100 μg/ml streptomycin and 2 mM glutamine]. Liver sections were weighed in 1 ml aliquots of D10 medium. Tissues were subsequently thawed, homogenized via dounce homogenizers, and used to infect NIH-3T12 fibroblast monolayers in triplicates in six-well plates. Titers were calculated as log(PFU/spleen) or log(PFU/100 mg of liver).
Flow cytometric analysis of natural killer cells
Single-cell suspensions were prepared from spleens as previously described (French et al. 2004). Splenocytes were stained with APC-PK136 (NK1.1), PerCP-Cy5.5-2C11 (CD3), and PerCP-Cy5.5-1D3 (CD19) in supernatant produced by 2.4G2 hybridoma to block FcRII/III. The following antibodies were used to identify respective cell surface markers: PE-eBio12AB (Ly49A/D), PE-5E6 (Ly49C/I), FITC-4E4 (Ly49D), FITC-3D10 (Ly49H), PE-16a11 (NKG2AB6), and PE-CX5 (NKG2D). Except for the Ly49H antibody, which was made in-house, the other antibodies were purchased from BD Biosciences (San Jose, CA, USA). For intracellular IFN-γ staining, cells were permeabilized with Cytofix/Cytoperm (PharMingen, La Jolla, CA, USA) per manufacturer’s protocol and stained with PE-conjugated anti-IFN-γ (clone XMG1.2) purchased from BD Biosciences. To evaluate BrdU incorporation, each mouse was injected with 2 mg of BrdU intraperitoneally 3 h prior to spleen harvest. Splenocytes were stained with surface markers and then permeabilized using Cytofix/Cytoperm following the manufacturer’s protocol. Intracellular BrdU was determined by staining with FITC-anti-BrdU antibody (BD Biosciences) as previously described (Dokun et al. 2001).
Total RNA was extracted from spleens using Trizol (Invitrogen, Carlsbad, CA, USA). cDNA was subsequently generated using oligo-dT primers while following the protocol for Superscript III First Strand Synthesis (Invitrogen). PCR primers for Ly49D were: forward 5′-TTCAGGGTTGCAGAACGAGATGAG-3′; reverse 5′-AGGATCCCGAGAGCTATCACAATG-3′. The internal probe sequence was 5′-AAAGCTCGCCTCAGAGTTCCCTGGCA-3′. The primers for Ly49H were: forward 5′-CACAAGTCTTCAGGGTTGAACAGC-3′; reverse 5′-CAACAATTACCAGCCGAAGGGAAC-3′; internal probe 5′-TGGCCTAAGAGTCCCTTGGCAGCTCATTGT-3′. Messenger RNA (mRNA) expression was normalized against β-actin expression and then compared to levels expressed in C57BL/6 splenocytes.
Further characterization of NK cells from B6.BXD8 mice demonstrated comparable levels of the maturation marker, Mac-1, as NK cells from C57BL/6 (Fig. 2b, middle panel). Inasmuch as Mac-1 is expressed on functionally mature NK cells, these data suggest full functional maturation of B6.BXD8 NK cells. Indeed, both B6.BXD8 and C57BL/6 NK cells produced similar levels of IFN-γ after NK1.1 activation (Fig. 3b). Thus, B6.BXD8 NK cells do not have any gross defect in IFN-γ production.
During MCMV infection, we previously reported that NK cells are both non-specifically and specifically activated when considering Ly49H expression (Dokun et al. 2001). However, it was not clear if specific activation of NK cells through Ly49H contributed to non-specific activation. When we infected B6.BXD8 mice with MCMV, we found that B6.BXD8 NK cells express IFN-γ at similar levels as those of C57BL/6 NK cells at 38 hours post-MCMV infection (Fig. 4b). This finding indicated that the early, non-specific IFN-γ production occurs normally during MCMV infection in the absence of Ly49h. Another non-specific effect is the stimulation of NK cell proliferation early during MCMV infection. We observed no difference in the incorporation of BrdU in C57BL/6 versus B6.BXD8 splenocytes at 38 hours after MCMV infection (Fig. 4c). Thus, the “non-specific” (with respect to Ly49H expression) activation of NK cells does not require triggering through Ly49H itself (Dokun et al. 2001).
Natural killer cells play important roles in controlling viral infections, and how NK cells are able to recognize and control diverse viral pathogens with a limited repertoire of innate receptors is still poorly defined. In the case of C57BL/6 mice and control of MCMV viremia, we show here that the deletion of a single NK cell receptor, namely Ly49h, in the C57BL/6 genetic background greatly increased host susceptibility. This current paper further extends previous findings that Ly49H, a single NK activation receptor, plays a critical role in the control of MCMV infection in C57BL/6 mice because the present data exclude any epistatic genetic effect of DBA/2 alleles in the phenotype of BXD-8 mice. Taken together with previous work (Brown et al. 2001b; Lee et al. 2001, 2003), these data provide strong genetic evidence that Ly49h plays a major role in the ability of C57BL/6 mice to resist MCMV infection.
Although we were not able to pinpoint the genetic lesion in B6.BXD8, our novel strain demonstrated a specific deficiency of Ly49H at the protein level but intact expression of other examined receptors in the NKC region. Ly49n and Ly49k, two loci juxtaposing Ly49h, were previously identified as pseudogenes (McQueen et al. 1999). Their presence or absence is thus unlikely to account for the increased susceptibility of B6.BXD8 to MCMV, as compared to C57BL/6. Furthermore, while it is theoretically plausible that DBA/2 genes could have recombined into the Ly49h region in B6.BXD8 during the recombinant inbreeding process, this is very unlikely because a double crossover event would be needed in a very tight region. Regardless, B6.BXD8 has a specific Ly49h deletion on the C57BL/6 genetic background.
Interestingly, we previously detected non-stochastic expression of Ly49H with Ly49D on NK cells in normal B6 mice (Smith et al. 2000). These activation receptors both signal through the DAP12 signaling chain. One possible explanation for their co-expression is their interdependent transcriptional regulation. However, our current studies indicate that the absence of Ly49h did not appear to affect Ly49D expression, indicating that Ly49D expression is independent of Ly49H expression, raising the possibility that their tendency to be expressed with each other in normal mice may be related to their common requirement for DAP12.
Our current study found no gross defect with NK cell maturation markers, expression of other NKC-encoded receptors, or NK cell function in B6.BXD8 mice, indicating that Ly49H is not necessary for NK cell development. Importantly, unlike other Ly49 members which interact with host MHC class I molecules and modulate NK activation, Ly49H has no known endogenous ligand. Whether Ly49H plays roles other than antiviral protection against MCMV remains to be determined, and it is possible that Ly49H enables NK cells to recognize pathogens other than MCMV. However, in our current study, we found that B6.BXD8 and C57BL/6 mice are equally susceptible to HSV-1 and ectromelia virus, two pathogens whose resistance is encoded in the NKC region.
Our previous studies indicated that NK cells appear to undergo two phases of activation during MCMV infection, an early (days 1–2) “non-specific” (independent of Ly49H expression) and a later (days 3–6) “specific” phase whereby Ly49H+ NK cells undergo selective proliferation (Dokun et al. 2001). However, it was not clear if triggering through Ly49H itself contributed to the non-specific phase. Herein, we found that NK cells in B6.BXD8 mice have no defect in their non-specific responses, indicating that Ly49h is dispensable for early non-specific NK cell stimulation during MCMV infections.
Given the importance of Ly49 family in modulating NK cell functions, the novel strain B6.BXD8 may serve as a useful tool for further delineation of NK activation receptors. Previously, functional analysis of Ly49 activation receptors were derived from examining mice deficient or defective in the adaptor molecule DAP12, since activation Ly49 receptors do not contain intrinsic signaling motifs and signal downstream by association with the DAP12 adaptor molecule (Bakker et al. 2000; Kaifu et al. 2003; Smith et al. 1998). However, DAP12 can also associate with other activation receptors and is expressed in several different cell types (Bouchon et al. 2001), indicating that DAP12-deficient mice have broader defects. Therefore, B6.BXD8 will allow the evaluation of the endogenous function of Ly49H and indirectly other activation receptors with more precision.
This work was supported by NIH grant R01AI051345 to W.M.Y. Microsatellite typing was supported by the Rheumatic Diseases Core Center grant P30AR048335. W.M.Y. is an investigator of the Howard Hughes Medical Institute. T.P.C. is supported by the Abbott Research Scholars fellowship.
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Note added in proof:
The B6.BXD8 strain has been accepted for repository at The Jackson Laboratory and will be available through their facility.