Simon Bélanger

Evolution of the Ly49 and Nkrp1 recognition systems

The Ly49 and Nkrp1 loci encode structurally and functionally related cell surface proteins that positively or negatively regulate natural killer (NK) cell-mediated cytotoxicity and cytokine production. Yet despite their clear relatedness and genetic linkage within the NK gene complex (NKC), these two multi-gene families have adopted dissimilar evolutionary strategies. The Ly49 genes are extremely polymorphic and evolutionarily dynamic, with distinct gene numbers, remarkable allelic diversity, and varying MHC-I-ligand specificities and affinities among different murine haplotypes. In contrast, the Nkrp1 genes have opted for overall conservation of genomic organization, sequences, and ligand specificities, with only limited and focused allelic polymorphism. Possible selection pressures driving such varied evolution of the two gene families may include disequilibrium from ligand co-inheritance, pathogen immunoevasin strategies, flexibility in host counter-evolution mechanisms, and the prevalence and dynamics of inherent repetitive elements.

Ly49h-Deficient C57BL/6 Mice: A New Mouse Cytomegalovirus-Susceptible Model Remains Resistant to Unrelated Pathogens Controlled by the NK Gene Complex

Cmv1 was the first mouse cytomegalovirus (MCMV) resistance locus identified in C57BL/6 mice. It encodes Ly49H, a NK cell-activating receptor that specifically recognizes the m157 viral protein at the surface of MCMV-infected cells. To dissect the effect of the Ly49h gene in host-pathogen interactions, we generated C57BL/6 mice lacking the Ly49h region. We found that 36 h after MCMV infection, the lack of Ly49h resulted in high viral replication in the spleen and dramatically enhanced proinflammatory cytokine production in the serum and spleen. At later points in time, we observed that MCMV induced a drastic loss in CD8+ T cells in B6.Ly49h−/− mice, probably reflecting severe histological changes in the spleen. Overall, our results indicate that Ly49H+ NK cells contain a systemic production of cytokines that may contribute to the MCMV-induced pathology and play a central role in maintaining normal spleen cell microarchitecture. Finally, we tested the ability of B6.Ly49h−/− mice to control replication of Leishmania major and ectromelia virus. Resistance to these pathogens has been previously mapped within the NK gene complex. We found that the lack of Ly49H+ NK cells is not associated with an altered resistance to L. major. In contrast, absence of Ly49H+ NK cells seems to afford additional protection against ectromelia infection in C57BL/6 mice, suggesting that Ly49H may recognize ectromelia-infected cells with detrimental effects. Taken together, these results confirm the pivotal role of the Ly49H receptor during MCMV infection and open the way for further investigations in host-pathogen interactions.

Molecular and genetic basis for strain-dependent NK1.1 alloreactivity of mouse NK cells.

NK1.1 alloantigen expression can be used to define NK cells in certain mouse strains, such as B6 (NKR-P1C) and SJL (NKR-P1B). However, BALB/c NK cells do not react with the anti-NK1.1 mAb, PK136. To investigate the NK1.1− phenotype of BALB/c NK cells, we have undertaken NK1.1 epitope mapping and genomic analysis of the BALB/c Nkrp1 region. Bacterial artificial chromosome library analysis reveals that, unlike the Ly49 region, the Nkrp1-Ocil/Clr region displays limited genetic divergence between B6 and BALB/c mice. In fact, significant divergence is confined to the Nkrp1b and Nkrp1c genes. Strikingly, the B6 Nkrp1d gene appears to represent a divergent allele of the Nkrp1b gene in BALB/c mice and other strains. Importantly, BALB/c NK cells express abundant and functional Nkrp1 transcripts, and the BALB/c NKR-P1B receptor functionally binds Ocil/Clr-b ligand. However, the BALB/c NKR-P1B/C sequences differ from those of the known NK1.1 alloantigens, and epitope mapping demonstrates that directed mutation of a single amino acid in the NKR-P1BBALB protein confers NK1.1 reactivity. Thus, PK136 mAb recognizes, in part, a distal C-terminal epitope present in NKR-P1BSw/SJL and NKR-P1CB6, but absent in NKR-P1A/D/FB6 and NKR-P1B/CBALB. Allelic divergence of the Nkrp1b/c gene products and limited divergence of the BALB/c Nkrp1-Ocil/Clr region explain a longstanding confusion regarding the strain-specific NK1.1 alloantigen reactivity of mouse NK cells.

CXCL13 is a plasma biomarker of germinal center activity

Significance A major challenge for vaccine science is that there is no way to measure germinal center activity in humans. This challenge is particularly acute for human clinical trials of candidate vaccines (and most nonhuman primate studies of candidate vaccines), because germinal centers are the engines of Ab affinity maturation, and generation of highly affinity-matured Ab responses is the goal of all Ab-eliciting vaccines. Here, we report that we have identified the chemokine CXCL13 [chemokine (C-X-C motif) ligand 13] as a biomarker of germinal center activity. We show explicit relationships between plasma CXCL13 concentrations and germinal center frequencies in lymph nodes in a series of different conditions, including licensed and experimental vaccines, and in humans, nonhuman primates, and mice. Significantly higher levels of plasma CXCL13 [chemokine (C-X-C motif) ligand 13] were associated with the generation of broadly neutralizing antibodies (bnAbs) against HIV in a large longitudinal cohort of HIV-infected individuals. Germinal centers (GCs) perform the remarkable task of optimizing B-cell Ab responses. GCs are required for almost all B-cell receptor affinity maturation and will be a critical parameter to monitor if HIV bnAbs are to be induced by vaccination. However, lymphoid tissue is rarely available from immunized humans, making the monitoring of GC activity by direct assessment of GC B cells and germinal center CD4+ T follicular helper (GC Tfh) cells problematic. The CXCL13–CXCR5 [chemokine (C-X-C motif) receptor 5] chemokine axis plays a central role in organizing both B-cell follicles and GCs. Because GC Tfh cells can produce CXCL13, we explored the potential use of CXCL13 as a blood biomarker to indicate GC activity. In a series of studies, we found that plasma CXCL13 levels correlated with GC activity in draining lymph nodes of immunized mice, immunized macaques, and HIV-infected humans. Furthermore, plasma CXCL13 levels in immunized humans correlated with the magnitude of Ab responses and the frequency of ICOS+ (inducible T-cell costimulator) Tfh-like cells in blood. Together, these findings support the potential use of CXCL13 as a plasma biomarker of GC activity in human vaccine trials and other clinical settings.

Preventing Postoperative Metastatic Disease by Inhibiting Surgery-Induced Dysfunction in Natural Killer Cells

Natural killer (NK) cell clearance of tumor cell emboli following surgery is thought to be vital in preventing postoperative metastases. Using a mouse model of surgical stress, we transferred surgically stressed NK cells into NK-deficient mice and observed enhanced lung metastases in tumor-bearing mice as compared with mice that received untreated NK cells. These results establish that NK cells play a crucial role in mediating tumor clearance following surgery. Surgery markedly reduced NK cell total numbers in the spleen and affected NK cell migration. Ex vivo and in vivo tumor cell killing by NK cells were significantly reduced in surgically stressed mice. Furthermore, secreted tissue signals and myeloid-derived suppressor cell populations were altered in surgically stressed mice. Significantly, perioperative administration of oncolytic parapoxvirus ovis (ORFV) and vaccinia virus can reverse NK cell suppression, which correlates with a reduction in the postoperative formation of metastases. In human studies, postoperative cancer surgery patients had reduced NK cell cytotoxicity, and we show for the first time that oncolytic vaccinia virus markedly increases NK cell activity in patients with cancer. These data provide direct in vivo evidence that surgical stress impairs global NK cell function. Perioperative therapies aimed at enhancing NK cell function will reduce metastatic recurrence and improve survival in surgical cancer patients.

Positive regulation of plasmacytoid dendritic cell function via Ly49Q recognition of class I MHC.

Plasmacytoid dendritic cells (pDCs) are an important source of type I interferon (IFN) during initial immune responses to viral infections. In mice, pDCs are uniquely characterized by high-level expression of Ly49Q, a C-type lectin-like receptor specific for class I major histocompatibility complex (MHC) molecules. Despite having a cytoplasmic immunoreceptor tyrosine-based inhibitory motif, Ly49Q was found to enhance pDC function in vitro, as pDC cytokine production in response to the Toll-like receptor (TLR) 9 agonist CpG-oligonucleotide (ODN) could be blocked using soluble monoclonal antibody (mAb) to Ly49Q or H-2Kb. Conversely, CpG-ODN–dependent IFN-α production by pDCs was greatly augmented upon receptor cross-linking using immobilized anti-Ly49Q mAb or recombinant H-2Kb ligand. Accordingly, Ly49Q-deficient pDCs displayed a severely reduced capacity to produce cytokines in response to TLR7 and TLR9 stimulation both in vitro and in vivo. Finally, TLR9-dependent antiviral responses were compromised in Ly49Q-null mice infected with mouse cytomegalovirus. Thus, class I MHC recognition by Ly49Q on pDCs is necessary for optimal activation of innate immune responses in vivo.

Ly49 cluster sequence analysis in a mouse model of diabetes: an expanded repertoire of activating receptors in the NOD genome

The mouse Ly49 and human killer cell immunoglobulin-like receptors (KIR) gene clusters encode activating and inhibitory class I MHC receptors on natural killer (NK) cells. A direct correlation between the presence of multiple activating KIR and various human autoimmune diseases including diabetes has been shown. Previous studies have implicated NK cell receptors in the development of diabetes in the non-obese diabetic (NOD) inbred mouse strain. To assess the contribution of Ly49 to NOD disease acceleration the Ly49 gene cluster of these mice was sequenced. Remarkably, the NOD Ly49 haplotype encodes the largest haplotype and the most functional activating Ly49 of any known mouse strain. These activating Ly49 include three Ly49p-related and two Ly49h-related genes. The NOD cluster contains large regions highly homologous to both C57BL/6 and 129 haplotypes, suggesting unequal crossing over as a mechanism of Ly49 haplotype evolution. Interestingly, the 129-like region has duplicated in the NOD genome. Thus, the NOD Ly49 cluster is a unique mix of elements seen in previously characterized Ly49 haplotypes resulting in a disproportionately large number of functional activating Ly49 genes. Finally, the functionality of activating Ly49 in NOD mice was confirmed in cytotoxicity assays.

Effect of Ly49 Haplotype Variance on NK Cell Function and Education

The class I MHC-specific receptors expressed by murine NK cells exhibit remarkable variation. Specific activating killer Ig-related receptor/Ly49 have major effects on autoimmune and infectious disease induction and outcome in humans and mice. However, these studies are greatly affected by individual background genetics. Furthermore, the educational impact of variable inhibitory KIR/Ly49 gene numbers on NK cell development and the subsequent ability to survey for MHC class I (MHC-I) expression remain unknown. To address these questions, Ly49 congenic mice were generated that maintain a 129-derived Ly49 gene cluster on a C57BL/6 genetic background (B6.Ly49129 mice), and the in vitro and in vivo NK cell function of these mice was compared with their inbred parental 129S1 and C57BL/6 counterparts. Notably, target cell recognition directed by activating Ly49 receptors was profoundly affected by allelic variation in B6.Ly49129 congenic cells versus C57BL/6 NK cells. Furthermore, when assessing NK cell function based on education and subsequent recognition of the C57BL/6 MHC-I haplotype by inhibitory Ly49 receptors, B6.Ly49129 congenic mice exhibited robust NK cell activity, demonstrating efficient NK cell education by the 129S1 Ly49 cluster during development. The responsiveness of NK cells expressing 129S1 Ly49 was shown to be mediated by subsets expressing one or more self-MHC receptors, including Ly49I, Ly49O, Ly49V, and NKG2A. These findings demonstrate that the genetically segregating and diverse MHC-I and Ly49 loci in mice exhibit independent and epistatic effects on NK cell education that can be uncoupled during the intercrossing of inbred strains.

In Vivo RNA Interference Screens Identify Regulators of Antiviral CD4+ and CD8+ T Cell Differentiation

Classical genetic approaches to examine the requirements of genes for T cell differentiation during infection are time consuming. Here we developed a pooled approach to screen 30-100+ genes individually in separate antigen-specific T cells during infection using short hairpin RNAs in a microRNA context (shRNAmir). Independent screens using T cell receptor (TCR)-transgenic CD4(+) and CD8(+) T cells responding to lymphocytic choriomeningitis virus (LCMV) identified multiple genes that regulated development of follicular helper (Tfh) and T helper 1 (Th1) cells, and short-lived effector and memory precursor cytotoxic T lymphocytes (CTLs). Both screens revealed roles for the positive transcription elongation factor (P-TEFb) component Cyclin T1 (Ccnt1). Inhibiting expression of Cyclin T1, or its catalytic partner Cdk9, impaired development of Th1 cells and protective short-lived effector CTL and enhanced Tfh cell and memory precursor CTL formation in vivo. This pooled shRNA screening approach should have utility in numerous immunological studies.

Ly49 family receptors are required for cancer immunosurveillance mediated by natural killer cells

According to the missing-self hypothesis, natural killer (NK) cells survey for target cells that lack MHC-I molecules. The Ly49 receptor family recognizes loss of MHC-I and is critical for educating NK cells, conferring the ability to eliminate transformed or infected cells. In this study, we evaluated their requirement in innate immune surveillance of cancer cells using genetically manipulated mice with attenuated expression of Ly49 receptors (NKC(KD)) in several models of carcinoma and metastasis. We found that NKC(KD) mice exhibited uncontrolled tumor growth and metastases. Expression of two MHC-I alleles, H-2K(b) and H-2D(b), was decreased in tumors from NKC(KD) mice in support of the likelihood of NK-mediated tumor immunoediting. These tumor cells exhibited directed alterations to their cell surface expression in response to the genetically altered immune environment to evade host recognition. Immunoediting in NKC(KD) mice was restricted to MHC-I molecules, which are ligands for Ly49 receptors, while expression of Rae-1 and Mult1, ligands for another NK cell receptor, NKG2D, were unaffected. Restoring NK cell education in NKC(KD) mice with a transgene for the inhibitory self-MHC-I receptor Ly49I restored suppression of cancer onset and growth. Interestingly, immune surveillance mediated by activating Ly49 receptors remained intact in NKC(KD) mice, as demonstrated by the ability to stimulate the NKG2D receptor with tumor cells or splenocytes expressing Rae-1. Together, our results genetically establish the integral role of Ly49 in NK cell-mediated control of carcinogenesis through MHC-I-dependent missing-self recognition.