Megan M. Tu

Ly49 Receptors: Innate and Adaptive Immune Paradigms

The Ly49 receptors are type II C-type lectin-like membrane glycoproteins encoded by a family of highly polymorphic and polygenic genes within the mouse natural killer (NK) gene complex. This gene family is designated Klra, and includes genes that encode both inhibitory and activating Ly49 receptors in mice. Ly49 receptors recognize class I major histocompatibility complex-I (MHC-I) and MHC-I-like proteins on normal as well as altered cells. Their functional homologs in humans are the killer cell immunoglobulin-like receptors, which recognize HLA class I molecules as ligands. Classically, Ly49 receptors are described as being expressed on both the developing and mature NK cells. The inhibitory Ly49 receptors are involved in NK cell education, a process in which NK cells acquire function and tolerance toward cells that express “self-MHC-I.” On the other hand, the activating Ly49 receptors recognize altered cells expressing activating ligands. New evidence shows a broader Ly49 expression pattern on both innate and adaptive immune cells. Ly49 receptors have been described on multiple NK cell subsets, such as uterine NK and memory NK cells, as well as NKT cells, dendritic cells, plasmacytoid dendritic cells, macrophages, neutrophils, and cells of the adaptive immune system, such as activated T cells and regulatory CD8+ T cells. In this review, we discuss the expression pattern and proposed functions of Ly49 receptors on various immune cells and their contribution to immunity.

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.

Mouse Nkrp1-Clr Gene Cluster Sequence and Expression Analyses Reveal Conservation of Tissue-Specific MHC-Independent Immunosurveillance

The Nkrp1 (Klrb1)-Clr (Clec2) genes encode a receptor-ligand system utilized by NK cells as an MHC-independent immunosurveillance strategy for innate immune responses. The related Ly49 family of MHC-I receptors displays extreme allelic polymorphism and haplotype plasticity. In contrast, previous BAC-mapping and aCGH studies in the mouse suggest the neighboring and related Nkrp1-Clr cluster is evolutionarily stable. To definitively compare the relative evolutionary rate of Nkrp1-Clr vs. Ly49 gene clusters, the Nkrp1-Clr gene clusters from two Ly49 haplotype-disparate inbred mouse strains, BALB/c and 129S6, were sequenced. Both Nkrp1-Clr gene cluster sequences are highly similar to the C57BL/6 reference sequence, displaying the same gene numbers and order, complete pseudogenes, and gene fragments. The Nkrp1-Clr clusters contain a strikingly dissimilar proportion of repetitive elements compared to the Ly49 clusters, suggesting that certain elements may be partly responsible for the highly disparate Ly49 vs. Nkrp1 evolutionary rate. Focused allelic polymorphisms were found within the Nkrp1b/d (Klrb1b), Nkrp1c (Klrb1c), and Clr-c (Clec2f) genes, suggestive of possible immune selection. Cell-type specific transcription of Nkrp1-Clr genes in a large panel of tissues/organs was determined. Clr-b (Clec2d) and Clr-g (Clec2i) showed wide expression, while other Clr genes showed more tissue-specific expression patterns. In situ hybridization revealed specific expression of various members of the Clr family in leukocytes/hematopoietic cells of immune organs, various tissue-restricted epithelial cells (including intestinal, kidney tubular, lung, and corneal progenitor epithelial cells), as well as myocytes. In summary, the Nkrp1-Clr gene cluster appears to evolve more slowly relative to the related Ly49 cluster, and likely regulates innate immunosurveillance in a tissue-specific manner.

Licensed and Unlicensed NK Cells: Differential Roles in Cancer and Viral Control

Natural killer (NK) cells are known for their well characterized ability to control viral infections and eliminate tumor cells. Through their repertoire of activating and inhibitory receptors, NK cells are able to survey different potential target cells for various surface markers, such as MHC-I – which signals to the NK cell that the target is healthy – as well as stress ligands or viral proteins, which alert the NK cell to the aberrant state of the target and initiate a response. According to the “licensing” hypothesis, interactions between self-specific MHC-I receptors – Ly49 in mice and KIR in humans – and self-MHC-I molecules during NK cell development is crucial for NK cell functionality. However, there also exists a large proportion of NK cells in mice and humans, which lack self-specific MHC-I receptors and are consequentially “unlicensed.” While the licensed NK cell subset plays a major role in the control of MHC-I-deficient tumors, this review will go on to highlight the important role of the unlicensed NK cell subset in the control of MHC-I-expressing tumors, as well as in viral control. Unlike the licensed NK cells, unlicensed NK cells seem to benefit from the lack of self-specific inhibitory receptors, which could otherwise be exploited by some aberrant cells for immunoevasion by upregulating the expression of ligands or mimic ligands for these receptors.

Ly49 receptors activate angiogenic mouse DBA+ uterine natural killer cells

In humans, specific patterns of killer immunoglobulin-like receptors (KIRs) expressed by uterine natural killer (uNK) cells are linked through HLA-C with pregnancy complications (infertility, recurrent spontaneous abortion, intrauterine growth restriction and preeclampsia). To identify mechanisms underpinning the associations between NK cell activation and pregnancy success, pregnancies were studied in mice with genetic knockdown (KD) of the MHC-activated Ly49 receptor gene family. B6.Ly49KD pregnancies were compared to normal control B6.Ly49129 and C57BL/6 (B6) pregnancies. At mid-pregnancy (gestation day (gd9.5)), overall uNK cell (TCRβ−CD122+DBA+DX5− (DBA+DX5−)) and TCRβ−CD122+DBA−DX5+ (DBA−DX5+)) frequencies in pregnant uterus were similar between genotypes. Ly49KD lowered the normal frequencies of Ly49+ uNK cells from 90.3% to 47.8% in DBA−DX5+ and 78.8% to 6.3% in DBA+DX5− uNK cell subtypes. B6.Ly49KD matings frequently resulted in expanded blastocysts that did not implant (subfertility). B6.Ly49KD mice that established pregnancy had gestational lengths and litter sizes similar to controls. B6.Ly49KD neonates, however, were heavier than controls. B6.Ly49KD implantation sites lagged in early (gd6.5) decidual angiogenesis and were deficient in mid-pregnancy (gd10.5) spiral arterial remodelling. Ultrastructural analyses revealed that B6.Ly49KD uNK cells had impaired granulogenesis, while immunocytochemistry revealed deficient vascular endothelial cell growth factor (VEGFA) production. Perforin and IFNG expression were normal in B6.Ly49KD uNK cells. Thus, in normal mouse pregnancies, Ly49 receptor signaling must promote implantation, early decidual angiogenesis and mid-pregnancy vascular remodelling. Disturbances in these functions may underlie the reported genetic associations between human pregnancy complications and the inability of specific conceptus MHCs to engage activating KIR on uNK cells.

Genetic Investigation of MHC-Independent Missing-Self Recognition by Mouse NK Cells Using an In Vivo Bone Marrow Transplantation Model

MHC-I–specific receptors play a vital role in NK cell–mediated “missing-self” recognition, which contributes to NK cell activation. In contrast, MHC-independent NK recognition mechanisms are less well characterized. In this study, we investigated the role of NKR-P1B:Clr-b (Klrb1:Clec2d) interactions in determining the outcome of murine hematopoietic cell transplantation in vivo. Using a competitive transplant assay, we show that Clr-b−/− bone marrow (BM) cells were selectively rejected by wild-type B6 recipients, to a similar extent as H-2Db−/− MHC-I–deficient BM cells. Selective rejection of Clr-b−/− BM cells was mitigated by NK depletion of recipient mice. Competitive rejection of Clr-b−/− BM cells also occurred in allogeneic transplant recipients, where it was reversed by selective depletion of NKR-P1Bhi NK cells, leaving the remaining NKR-P1Blo NK subset and MHC-I–dependent missing-self recognition intact. Moreover, competitive rejection of Clr-b−/− hematopoietic cells was abrogated in Nkrp1b-deficient recipients, which lack the receptor for Clr-b. Of interest, similar to MHC-I–deficient NK cells, Clr-b−/− NK cells were hyporesponsive to both NK1.1 (NKR-P1C)–stimulated and IL-12/18 cytokine–primed IFN-γ production. These findings support a unique and nonredundant role for NKR-P1B:Clr-b interactions in missing-self recognition of normal hematopoietic cells and suggest that optimal BM transplant success relies on MHC-independent tolerance mechanisms. These findings provide a model for human NKR-P1A:LLT1 (KLRB1:CLEC2D) interactions in human hematopoietic cell transplants.

Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion

The immune response to influenza virus infection comprises both innate and adaptive defenses. NK cells play an early role in the destruction of tumors and virally-infected cells. NK cells express a variety of inhibitory receptors, including those of the Ly49 family, which are functional homologs of human killer-cell immunoglobulin-like receptors (KIR). Like human KIR, Ly49 receptors inhibit NK cell-mediated lysis by binding to major histocompatibility complex class I (MHC-I) molecules that are expressed on normal cells. During NK cell maturation, the interaction of NK cell inhibitory Ly49 receptors with their MHC-I ligands results in two types of NK cells: licensed (“functional”), or unlicensed (“hypofunctional”). Despite being completely dysfunctional with regard to rejecting MHC-I-deficient cells, unlicensed NK cells represent up to half of the mature NK cell pool in rodents and humans, suggesting an alternative role for these cells in host defense. Here, we demonstrate that after influenza infection, MHC-I expression on lung epithelial cells is upregulated, and mice bearing unlicensed NK cells (Ly49-deficient NKCKD and MHC-I-deficient B2m -/- mice) survive the infection better than WT mice. Importantly, transgenic expression of an inhibitory self-MHC-I-specific Ly49 receptor in NKCKD mice restores WT influenza susceptibility, confirming a direct role for Ly49. Conversely, F(ab’)2-mediated blockade of self-MHC-I-specific Ly49 inhibitory receptors protects WT mice from influenza virus infection. Mechanistically, perforin-deficient NKCKD mice succumb to influenza infection rapidly, indicating that direct cytotoxicity is necessary for unlicensed NK cell-mediated protection. Our findings demonstrate that Ly49:MHC-I interactions play a critical role in influenza virus pathogenesis. We suggest a similar role may be conserved in human KIR, and their blockade may be protective in humans.

Optimized Tetramer Analysis Reveals Ly49 Promiscuity for MHC Ligands

Murine Ly49 receptors, which are expressed mainly on NK and NKT cells, interact with MHC class I (MHC-I) molecules with varying specificity. Differing reports of Ly49/MHC binding affinities may be affected by multiple factors, including cis versus trans competition and species origin of the MHC-I L chain (β2-microglobulin). To determine the contribution of each of these factors, Ly49G, Ly49I, Ly49O, Ly49V, and Ly49Q receptors from the 129 mouse strain were expressed individually on human 293T cells or the mouse cell lines MHC-I–deficient C1498, H-2b–expressing MC57G, and H-2k–expressing L929. The capacity to bind to H-2Db– and H-2Kb–soluble MHC-I tetramers containing either human or murine β2-microglobulin L chains was tested for all five Ly49 receptors in all four cell lines. We found that most of these five inhibitory Ly49 receptors show binding for one or both self–MHC-I molecules in soluble tetramer binding assays when three conditions are fulfilled: 1) lack of competing cis interactions, 2) tetramer L chain is of mouse origin, and 3) Ly49 is expressed in mouse and not human cell lines. Furthermore, Ly49Q, the single known MHC-I receptor on plasmacytoid dendritic cells, was shown to bind H-2Db in addition to H-2Kb when the above conditions were met, suggesting that Ly49Q functions as a pan–MHC-Ia receptor on plasmacytoid dendritic cells. In this study, we have optimized the parameters for soluble tetramer binding analyses to enhance future Ly49 ligand identification and to better evaluate specific contributions by different Ly49/MHC-I pairs to NK cell education and function.

Critical role for the Ly49 family of class I MHC receptors in adaptive natural killer cell responses

Significance Finding that “innate” natural killer cells possessed T cell-like immune memory was an unprecedented discovery that rocked our understanding of the divide between innate and adaptive immunity. In this report we demonstrate that Ly49 receptors on natural killer cells, which engage the class I antigen-presenting platform on target cells, are critical components of adaptive natural killer cell memory. We also demonstrate the clinical potential of natural killer cell memory, using it to mediate a cancer vaccine against melanoma. These findings offer insights into how adaptive natural killer cell memory might function and what clinical applications this mysterious immune response might have. Adaptive natural killer (NK) cell memory represents a new frontier in immunology. Work over the last decade has discovered and confirmed the existence of NK cells with antigen-specific memories, which had previously been considered a unique property of T and B cells. These findings have shown that antigen-specific NK cells gain their specificity without the use of RAG proteins, representing a novel mechanism for generating antigen specificity, but the details of this mechanism have remained a mystery. We have discovered that members of the Ly49 family of surface receptors are critically involved in both the sensitization and the challenge phases of an NK cell memory response, as is antigen presentation from their binding partner, the class I MHC. Moreover, we demonstrate that the Ly49-interacting component of a presented antigen dictates the specificity of the NK cell memory response, implicating Ly49 receptors themselves in antigen-specific recognition. Finally, we demonstrate that adaptive NK cell memories can protect against an otherwise lethal melanoma without T cell or B cell support. These findings offer insight into the mechanism behind NK cell antigen specificity and demonstrate the clinical potential of this adaptive immune cell.

Correction: Influenza Virus Targets Class I MHC-Educated NK Cells for Immunoevasion

[This corrects the article DOI: 10.1371/journal.ppat.1005446.].