[Show abstract][Hide abstract]ABSTRACT: The outcome of mouse CMV (MCMV) infection varies among different inbred mouse strains depending on NK cell effector functions governed through recognition receptor triggering. NK cells from different mouse strains possess diverse repertoires of activating or inhibitory Ly49 receptors, which share some of their polymorphic MHC class I (MHC-I) ligands. By examining the NK cell response to MCMV infection in novel BALB substrains congenic for different MHC (or H-2 in mice) haplotypes, we show that recognition of viral MHC-I-like protein m157 by inhibitory Ly49C receptor allows escape from NK cell control of viral replication. Dominant inhibition by Ly49C bound to self-H-2(b) encoded MHC-I molecules masks this effect, which only becomes apparent in distinct H-2 haplotypes, such as H-2(f). The recognition of m157-expressing cells by Ly49C resulted in both decreased NK cell killing in vitro and reduced rejection in vivo. Further, control of infection with m157-deletant (Δm157) MCMV was improved in mice carrying H-2 molecules unrecognized by Ly49C but allowing expansion of NK cell effectors expressing activating Ly49L receptors. Hence, our study is the first, to our knowledge, to demonstrate that MHC-I mimicry strategies used by MCMV to avoid NK cell control are biologically relevant during in vivo viral infection. Of value for human studies is that only a few genetic assortments conditional on the repertoires of viral MHC-I-like proteins/host NK receptors/MHC haplotypes should allow efficient protection against CMV infection.
Full-text available · Article · Nov 2014 · The Journal of Immunology
[Show abstract][Hide abstract]ABSTRACT: Herpes simplex encephalitis (HSE) is a lethal neurological disease resulting from infection with Herpes Simplex Virus 1 (HSV-1). Loss-of-function mutations in the UNC93B1, TLR3, TRIF, TRAF3, and TBK1 genes have been associated with a human genetic predisposition to HSE, demonstrating the UNC93B-TLR3-type I IFN pathway as critical in protective immunity to HSV-1. However, the TLR3, UNC93B1, and TRIF mutations exhibit incomplete penetrance and represent only a minority of HSE cases, perhaps reflecting the effects of additional host genetic factors. In order to identify new host genes, proteins and signaling pathways involved in HSV-1 and HSE susceptibility, we have implemented the first genome-wide mutagenesis screen in an in vivo HSV-1 infectious model. One pedigree (named P43) segregated a susceptible trait with a fully penetrant phenotype. Genetic mapping and whole exome sequencing led to the identification of the causative nonsense mutation L3X in the Receptor-type tyrosine-protein phosphatase C gene (Ptprc(L3X) ), which encodes for the tyrosine phosphatase CD45. Expression of MCP1, IL-6, MMP3, MMP8, and the ICP4 viral gene were significantly increased in the brain stems of infected Ptprc(L3X) mice accounting for hyper-inflammation and pathological damages caused by viral replication. Ptprc(L3X) mutation drastically affects the early stages of thymocytes development but also the final stage of B cell maturation. Transfer of total splenocytes from heterozygous littermates into Ptprc (L3X) mice resulted in a complete HSV-1 protective effect. Furthermore, T cells were the only cell population to fully restore resistance to HSV-1 in the mutants, an effect that required both the CD4(+) and CD8(+) T cells and could be attributed to function of CD4(+) T helper 1 (Th1) cells in CD8(+) T cell recruitment to the site of infection. Altogether, these results revealed the CD45-mediated T cell function as potentially critical for infection and viral spread to the brain, and also for subsequent HSE development.
Full-text available · Article · Sep 2013 · PLoS Pathogens
[Show abstract][Hide abstract]ABSTRACT: A large gap in our understanding of infant immunity is why natural killer (NK) cell responses are deficient, which makes infants more prone to viral infection. Here we demonstrate that transforming growth factor-β (TGF-β) was responsible for NK cell immaturity during infancy. We found more fully mature NK cells in CD11c(dnR) mice, whose NK cells lack TGF-β receptor (TGF-βR) signaling. Ontogenic maturation of NK cells progressed faster in the absence of TGF-β signaling, which results in the formation of a mature NK cell pool early in life. As a consequence, infant CD11c(dnR) mice efficiently controlled viral infections. These data thus demonstrate an unprecedented role for TGF-β in ontogeny that can explain why NK cell responses are deficient early in life.
Full-text available · Article · Aug 2012 · Nature Immunology
[Show abstract][Hide abstract]ABSTRACT: The association of Natural Killer (NK) cell deficiencies with disease susceptibility has established a central role for NK cells in host defence. In this context, genetic approaches have been pivotal in elucidating and characterizing the molecular mechanisms underlying NK cell function. To this end, homozygosity mapping and linkage analysis in humans have identified mutations that impact NK cell function and cause life-threatening diseases. However, several critical restrictions accompany genetic studies in humans. Studying NK cell pathophysiology in a mouse model has therefore proven a useful tool. The relevance of the mouse model is underscored by the similarities that exist between cell-structure-sensing receptors and the downstream signaling that leads to NK cell activation. In this review, we provide an overview of how human and mouse quantitative trait locis (QTLs) have facilitated the identification of genes that modulate NK cell development, recognition, and killing of target cells.
Full-text available · Article · Jan 2012 · Frontiers in Immunology
[Show abstract][Hide abstract]ABSTRACT: Natural killer (NK) cells and CD8+ T cells play a prominent role in the clearance of mouse cytomegalovirus (MCMV) infection. The role of NK cells in modulating
the CD8+ T-cell response to MCMV infection is still the subject of intensive research. For analyzing the impact of NK cells on mounting
of a CD8+ T-cell response and the contribution of these cells to virus control during the first days postinfection (p.i.), we used
C57BL/6 mice in which NK cells are specifically activated through the Ly49H receptor engaged by the MCMV-encoded ligand m157.
Our results indicate that the requirement for CD8+ T cells in early MCMV control inversely correlates with the engagement of Ly49H. While depletion of CD8+ T cells has only a minor effect on the early control of wild-type MCMV, CD8+ T cells are essential in the control of Δm157 virus. The frequencies of virus epitope-specific CD8+ T cells and their activation status were higher in mice infected with Δm157 virus. In addition, these mice showed elevated levels of alpha interferon (IFN-α) and several other proinflammatory cytokines
as early as 1.5 days p.i. Although the numbers of conventional dendritic cells (cDCs) were reduced later during infection,
particularly in Δm157-infected mice, they were not significantly affected at the peak of the cytokine response. Altogether, we concluded that increased
antigen load, preservation of early cDCs' function, and higher levels of innate cytokines collectively account for an enhanced
CD8+ T-cell response in C57BL/6 mice infected with a virus unable to activate NK cells via the Ly49H–m157 interaction.
[Show abstract][Hide abstract]ABSTRACT: Ly49 receptor expression on NK cells from FVB H2-Dk transgenic and
nontransgenic mice. (A) The indicated Ly49 specific monoclonal antibodies
(black peak) or isotype controls (red peak) were gated on NKp46+
splenic NK cells from
mice and analyzed by FACS. The proportion of Ly49 receptor expression is
indicated in each histogram. (B) Quantification of expression frequency of
the indicated NK receptors in
(1.81 MB EPS)
[Show abstract][Hide abstract]ABSTRACT: Frequencies of Ly49+ and KLRG1+NK cells in double congenic mice.
Quantification of expression frequency of indicated NK receptors in the
parental MA/My strain and
mice. Data are presented as mean ± SEM and P values
of significant differences between groups are indicated.
(0.89 MB EPS)
[Show abstract][Hide abstract]ABSTRACT: Binding of Ly49-specific monoclonal antibody to MA/My activating receptors.
cDNAs encoding MA/My Ly49P, Ly49R and Ly49U receptors were expressed in
NFAT-GFP 2B4 T-cell hybridomas . Expression of the
three receptors was detected by the anti-Flag M2 monoclonal antibody.
Binding of the isotype control monoclonal antibody (red histogram) or the
indicated Ly49-specific monoclonal antibody (black histogram) to Ly49P,
Ly49R, and Ly49U receptors was assessed by flow cytometry and analyzed using
Flowjo software. The percentage of binding is indicated in each
(1.19 MB EPS)
[Show abstract][Hide abstract]ABSTRACT: Ly49P+2B4 reporter cell stimulation by MCMV-infected MEF cells produced
from FVB-Tg(Dk)+ mice. (A) Stimulation of Ly49P
reporter cells by co-culture with MEF cells from the indicated backgrounds
that were uninfected (black histograms) or MCMV infected at an MOI of 1 for
24 h (grey histograms). Ly49P-specific activation was detected by NFAT-GFP
expression using flow cytometry. (B) Stimulation of Ly49P or Ly49H reporter
cells by co-culture with
MEF cells that were uninfected (left) or infected with
Δm157 (middle) or Δm04 (right)
MCMV deletion mutants. Reporter cell stimulation was detected by monitoring
expression of GFP by flow cytometry. The percentage of positive cells in
each gated population is indicated.
(0.87 MB EPS)
[Show abstract][Hide abstract]ABSTRACT: NK cell–dependent MCMV infection control in congenic and F3 mice. (A)
Viral loads in spleens (top) and livers (bottom) 3 d post-infection in
MCMV-resistant MA/My progenitor,
transgenic mice that were NK cell depleted (white squares) or not (black
circles) with anti-asialo GM1 antibody. (B) Number of NK cells per spleen
(top) and BrdU incorporation (bottom) at 7 d post-MCMV infection in
MCMV-resistant mice of the indicated genotypes. For the number of NK cells,
data are presented as mean ± SEM and statistically significant
differences between groups are indicated. For BrdU incorporation data are
represented by fold increase between noninfected and infected animals.
Results shown are representative of 1–2 experiments.
(1.56 MB EPS)
[Show abstract][Hide abstract]ABSTRACT: Lack of NKG2A/C/E and CD94 antibody staining on NK cells from MA/My mice. (A)
NKG2A/C/E and CD94 expression on NKp46+ NK cells from MA/My, FVB/N, and
DBA/2J (as they carry a NKG2/CD94 deficiency ) mice was determined by
flow cytometry using the indicated monoclonal antibodies. (B) CD94 and NKG2A
RNA expression in enriched NK cells from the indicated mice strains was
analyzed by RT-PCR. β-actin was used as an internal control.
(0.87 MB EPS)
[Show abstract][Hide abstract]ABSTRACT: Ly49 receptor and MHC class I expression on NK cells from F3 mice. The
indicated Ly49-specific monoclonal antibodies were gated on NKp46+
splenic NK cells from F3 mice and the proportion of expression is indicated
in each histogram. Right panels: expression of MHC-I
H2-Dq and H2-Dk
molecules on total splenocytes was determined. 2–3 mice per genotype
were analyzed. We found that the expression of the activating and the
inhibitory receptors were almost comparable between strains for the
exception of Ly49G which was barely detectable in the
mice using both anti- Ly49G antibodies (LGL-1 and AT8 (data not shown)).
This receptor is perfectly expressed in the
B6.H20 parental strain (data not shown) and
doesn't seem to be correlated with a defect of NK maturation since the
Killer cell lectin-like receptor G1 (KLRG1) is equally expressed between the
(1.37 MB EPS)
[Show abstract][Hide abstract]ABSTRACT: Effect of Ly49-specifc monoclonal antibody depletion in MA/My mice during the
course of MCMV infection. MCMV viral load was assessed in untreated MA/My
mice (mock) or treated with the indicated monoclonal antibody prior to MCMV
infection. Viral load in spleen was determined by plaque assay after 3 d of
(1.47 MB EPS)
[Show abstract][Hide abstract]ABSTRACT: Co-expression of H2-Dq and H2-Dk in
FVB-Tg(Dk)+ mice. Endogenous
H2-Dq (bottom) and transgenic
H2-Dk (top) expression in splenic T and B
transgenic (black peak) or nontransgenic (black peak) littermates was
determined by flow cytometry.
(0.57 MB EPS)
[Show abstract][Hide abstract]ABSTRACT: The cytomegalovirus resistance locus Cmv3 has been linked to an epistatic interaction between two loci: a Natural Killer (NK) cell receptor gene and the major histocompatibility complex class I (MHC-I) locus. To demonstrate the interaction between Cmv3 and
H2k, we generated double congenic mice between MA/My and BALB.K mice and an F2 cross between FVB/N (H-2q) and BALB.K
(H2k) mice, two strains susceptible to mouse cytomegalovirus (MCMV). Only mice expressing H2k in conjunction with Cmv3MA/My or
Cmv3FVB were resistant to MCMV infection. Subsequently, an F3 cross was carried out between transgenic FVB/H2-Dk and MHC-I deficient mice in which only the progeny expressing Cmv3FVB and a single H2-Dk class-I molecule completely controlled MCMV viral loads. This phenotype was shown to be NK cell–dependent and associated with subsequent NK cell proliferation. Finally, we demonstrated that a number of H2q alleles influence the expression level of H2q molecules, but not intrinsic functional properties of NK cells; viral loads, however, were quantitatively proportional to the number of H2q alleles. Our results support a model in which H-2q molecules convey Ly49-dependent inhibitory signals that interfere with the action of H2-Dk on NK cell activation against MCMV infection. Thus, the integration of activating and inhibitory signals emanating from various MHC-I/NK cell receptor interactions regulates NK cell–mediated control of viral load.
Full-text available · Article · Apr 2011 · PLoS Genetics
[Show abstract][Hide abstract]ABSTRACT: Cytomegaloviruses (CMV) are ubiquitous, opportunistic DNA viruses that have mastered the art of immune evasion through their ability to mimic host proteins or to inhibit antiviral responses. The study of the host response against CMV infection has illuminated many facets of the complex interaction between host and pathogen. Here, we review evidence derived from the animal models and human studies that supports the central role played by innate immune receptors in the recognition of virus infection and their participation in the many layers of defense.
[Show abstract][Hide abstract]ABSTRACT: Genetically distinct inbred strains of mice that differ in their susceptibility to mouse cytomegalovirus (MCMV) are invaluable for dissecting complex host-pathogen interactions. Their study has allowed the identification of host-resistance loci, including several activating NK cell receptors of major histocompatibility complex (MHC) class I. In this chapter, we provide a practical guide to the genetic mapping and functional characterization of NK cell receptors that control innate immunity against MCMV via specific recognition of infected cells.
Article · Jan 2010 · Methods in molecular biology (Clifton, N.J.)