LILRB1 Intron 1 Has a Polymorphic Regulatory Region That Enhances Transcription in NK Cells and Recruits YY1.

LILRB1 is a highly polymorphic receptor expressed by subsets of innate and adaptive immune cells associated with viral and autoimmune diseases and targeted by pathogens for immune evasion. LILRB1 expression on human NK cells is variegated, and the frequency of LILRB1+ cells differs among people. However, little is known about the processes and factors mediating LILRB1 transcription in NK cells. LILRB1 gene expression in lymphoid and myeloid cells arises from two distinct promoters that are separated by the first exon and intron. In this study, we identified a polymorphic 3-kb region within LILRB1 intron 1 that is epigenetically marked as an active enhancer in human lymphoid cells and not monocytes. This region possesses multiple YY1 sites, and complexes of the promoter/enhancer combination were isolated using anti-YY1 in chromatin immunoprecipitation-loop. CRISPR-mediated deletion of the 3-kb region lowers LILRB1 expression in human NKL cells. Together, these results indicate the enhancer in intron 1 binds YY1 and suggest YY1 provides a scaffold function enabling enhancer function in regulating LILRB1 gene transcription in human NK cells.

LILRB1 polymorphisms influence posttransplant HCMV susceptibility and ligand interactions.

UL18 is a human CMV (HCMV) MHC class I (MHCI) homolog that efficiently inhibits leukocyte immunoglobulin-like receptor subfamily B member 1 (LILRB1)+ NK cells. We found an association of LILRB1 polymorphisms in the regulatory regions and ligand-binding domains with control of HCMV in transplant patients. Naturally occurring LILRB1 variants expressed in model NK cells showed functional differences with UL18 and classical MHCI, but not with HLA-G. The altered functional recognition was recapitulated in binding assays with the binding domains of LILRB1. Each of 4 nonsynonymous substitutions in the first 2 LILRB1 immunoglobulin domains contributed to binding with UL18, classical MHCI, and HLA-G. One of the polymorphisms controlled addition of an N-linked glycan, and that mutation of the glycosylation site altered binding to all ligands tested, including enhancing binding to UL18. Together, these findings indicate that specific LILRB1 alleles that allow for superior immune evasion by HCMV are restricted by mutations that limit LILRB1 expression selectively on NK cells. The polymorphisms also maintained an appropriate interaction with HLA-G, fitting with a principal role of LILRB1 in fetal tolerance.

Vaccinia virus Western Reserve induces rapid surface expression of a host molecule detected by the antibody 4C7 that is distinct from CLEC2D.

In this study, the effect of active infection with vaccinia virus Western Reserve (VACV WR) on expression of C-type lectin domain family 2 (CLEC2D), a ligand of the human NK cell inhibitory receptor NKR-P1, was examined. As predicted, VACV infection led to a loss of CLEC2D mRNA in 221 cells, a B cell lymphoma line. Surprisingly, VACV infection of 221 cells caused a dramatic increase in cell surface staining for one CLEC2D-specific antibody, 4C7. There were no changes in other antibodies specific for CLEC2D and no indication that NK cells with NKR-P1A were inhibited, suggesting 4C7 detects a non-CLEC2D molecule following infection. The rapid increase in 4C7 signal requires virus attachment and is disrupted by UV treatment, but does not depend on new transcription or translation of either cellular or viral proteins. 4C7 does react with intracellular compartments, suggesting the molecule that is detected at the surface following infection is derived from an intracellular store. The phenomenon extends beyond lymphoid cells: it was observed in the non-human primate cell line Cos-7, but not with myxoma, a poxvirus distinct from VACV. To our knowledge, this is the first report of VACV or any poxvirus leading to rapid externalization of a host molecule. Among the VACV strains tested, the phenomenon was restricted to VACV WR and IHD-W, suggesting it has a virulence-, as opposed to a replication-related, function.

The Expanding Spectrum of Ligands for Leukocyte Ig-like Receptors.

The human leukocyte Ig-like receptor family is part of the paired receptor system. The receptors are widely expressed by various immune cells, and new functions continue to emerge. Understanding the range of functions of the receptors is of general interest because several types of pathogens exploit the receptors and genetic diversity of the receptors has been linked to various autoimmune diseases. Class I major histocompatibility molecules were the first ligands appreciated for these receptors, but the types of ligands identified over the last several years are quite diverse, including intact pathogens, immune-modulatory proteins, and molecules normally found within the CNS. This review focuses on the types of ligands described to date, how the individual receptors bind to several distinct types of ligands, and the known functional consequences of those interactions.

The AP-1 transcription factor JunD activates the leukocyte immunoglobulin-like receptor 1 distal promoter.

Leukocyte immunoglobulin-like receptor 1 (LILRB1) is an inhibitory receptor that binds classical and non-classical MHC-I as well as UL18, a viral MHC-I homolog. LILRB1 is encoded within the leukocyte receptor complex and is widely expressed on immune cells. Two distinct promoters used differentially by lymphoid and myeloid cells were previously identified, but little is known regarding molecular regulation of each promoter or cell-type-specific usage. Here, we have investigated the transcriptional regulation of human LILRB1 focusing on elements that drive expression in NK cells. We found that while both the distal and proximal promoter regions are active in reporter plasmids in lymphoid and myeloid cells, the proximal promoter is used minimally to transcribe LILRB1 in NK cells compared with monocytes. We defined a 120-bp core region of transcriptional activity in the distal promoter that can bind several factors in NK cell nuclear extracts. Within this region, we investigated overlapping putative AP-1 sites. An inhibitor of JNK decreased LILRB1 transcript in a LILRB1⁺ NK cell line. Upon examining binding of specific AP-1 factors, we found JunD associated with the LILRB1 distal promoter. Finally, depletion of JunD led to a decrease in distal promoter transcript, indicating an activating role for JunD in regulation of LILRB1 transcription. This study presents the first description of regions/factors required for activity of the LILRB1 distal promoter, the first description of a role for JunD in NK cells and suggests a potential mechanism for dynamic regulation of LILRB1 by cytokines.

NK cells and poxvirus infection.

In recent years, our understanding of the role of natural killer (NK) cells in the response to viral infection has grown rapidly. Not only do we realize viruses have many immune-evasion strategies to escape NK cell responses, but that stimulation of NK cell subsets during an antiviral response occurs through receptors seemingly geared directly at viral products and that NK cells can provide a memory response to viral pathogens. Tremendous knowledge has been gained in this area through the study of herpes viruses, but appreciation for the significance of NK cells in the response to other types of viral infections is growing. The function of NK cells in defense against poxviruses has emerged over several decades beginning with the early seminal studies showing the role of NK cells and the NK gene complex in susceptibility of mouse strains to ectromelia, a poxvirus pathogen of mice. More recently, greater understanding has emerged of the molecular details of the response. Given that human diseases caused by poxviruses can be as lethal as smallpox or as benign as Molluscum contagiosum, and that vaccinia virus, the prototypic member of the pox family, persists as a mainstay of vaccine design and has potential as an oncolytic virus for tumor therapy, further research in this area remains important. This review focuses on recent advances in understanding the role of NK cells in the immune response to poxviruses, the receptors involved in activation of NK cells during poxvirus infection, and the viral evasion strategies poxviruses employ to avoid the NK response.

Cis association of leukocyte Ig-like receptor 1 with MHC class I modulates accessibility to antibodies and HCMV UL18.

Leukocyte Ig-like receptor (LIR) 1 (CD85j/ILT2/LILRB1) is an inhibitory receptor with broad specificity for MHC class I (MHC-I) and the human CMV MHC-I homologue UL18. LIR-1 can inhibit NK cells through the conventional interaction with MHC-I expressed on a target cell (in trans) but the nature and the effects of LIR-1 interactions with MHC-I in cis are not well understood. Here we show that MHC-I expressed in cis has an impact on the detection of LIR-1 with various antibodies. We found the cis interaction alters recognition by only one of two antibodies known to block functional trans recognition by LIR-1 on NK cells. Specifically, we observed an enhancement of recognition with GHI/75 in the presence of various MHC-I alleles on 721.221 cells. We found that blocking the LIR-1 contact site with anti-MHC-I antibodies decreased detection of LIR-1 with GHI/75. We also observed a decrease in GHI/75 following acid denaturation of MHC-I. Finally, disruption of LIR-1 cis interactions with MHC-I significantly enhanced UL18-Fc binding to NK92 cells and enhanced the relative inhibition of NK92 cells by HLA-G. These results have implications for LIR-1 function in scenarios such as infection when MHC-I levels on effector cells may be increased by IFNs.

Poxvirus infection-associated downregulation of C-type lectin-related-b prevents NK cell inhibition by NK receptor protein-1B.

Innate immune recognition of virus-infected cells includes NK cell detection of changes to endogenous cell-surface proteins through inhibitory receptors. One such receptor system is the NK cell receptor protein-1B (NKR-P1B) and its ligand C-type lectin-related-b (Clr-b). NKR-P1B and Clr-b are encoded within the NK cell gene complex, a locus that has been linked to strain-dependent differences in susceptibility to infection by poxviruses. In this study, we report the impact of vaccinia virus (VV) and ectromelia virus infection on expression of Clr-b and Clr-b-mediated protection from NK cells. We observed a loss of Clr-b cell-surface protein upon VV and ectromelia virus infection of murine cell lines and bone marrow-derived macrophages. The reduction of Clr-b is more rapid than MHC class I, the prototypic ligand of NK cell inhibitory receptors. Reduction of Clr-b requires active viral infection but not expression of late viral genes, and loss of mRNA appears to lag behind loss of Clr-b surface protein. Clr-b-mediated protection from NK cells is lost following VV infection. Together, these results provide the second example of Clr-b modulation during viral infection and suggest reductions of Clr-b may be involved in sensitizing poxvirus-infected cells to NK cells.

Channel catfish leukocyte immune-type receptor mediated inhibition of cellular cytotoxicity is facilitated by SHP-1-dependent and -independent mechanisms.

Channel catfish (Ictalurus punctatus) leukocyte immune-type receptors (IpLITRs) are immunoregulatory proteins belonging to the immunoglobulin superfamily that likely play an important role in the regulation of teleost immune cell effector responses. IpLITRs are expressed by myeloid and lymphoid subsets and based on their structural features can be classified as either putative stimulatory or inhibitory forms. We have recently demonstrated at the biochemical and functional levels that stimulatory IpLITR-types induced intracellular signaling cascades resulting in immune cell activation. Alternatively, we have shown that putative inhibitory IpLITRs may abrogate immune cell responses by recruiting teleost Src homology 2 (SH2) domain-containing cytoplasmic phosphatases (SHP) to their tyrosine-containing cytoplasmic tails. In the present study, we used vaccinia virus to express recombinant chimeric proteins encoding the extracellular and transmembrane regions of human KIR2DL3 fused with the cytoplasmic tails of two putative inhibitory IpLITRs (i.e. IpLITR1.2a and IpLITR1.1b) in mouse spleen-derived cytotoxic lymphocytes. This approach allowed us to study the specific effects of IpLITR-induced signaling on lymphocyte killing of B cell targets (e.g. 721.221 cells) using a standard chromium release assay. Our results suggest that both IpLITR1.2a and IpLITR1.1b are potent inhibitors of lymphocyte-mediated cellular cytotoxicity. Furthermore, using a catalytically inactive SHP-1 mutant in combination with site-directed mutagenesis and co-immunoprecipitations, we also demonstrate that the IpLITR1.2a-mediated functional inhibitory response is SHP-1-dependent. Alternatively, IpLITR1.1b-mediated inhibition of cellular cytotoxicity is facilitated by both SHP-1-dependent and independent mechanisms, possibly involving the C-terminal Src kinase (Csk). The involvement of this inhibitory kinase requires binding to a tyrosine residue encoded in the unique membrane proximal cytoplasmic tail region of IpLITR1.1b. Overall, this represents the first functional information for inhibitory IpLITR-types and reveals that catfish LITRs engage SHP-dependent and -independent inhibitory signaling pathways to abrogate lymphocyte-mediated killing.

The first Ig domain of KIR3DL1 contacts MHC class I at a secondary site.

KIR3DL1 is a highly polymorphic inhibitory killer cell Ig-like receptor (KIR) implicated in resistance to viral diseases such as AIDS. KIR3DL1 contains three Ig domains and is specific for MHC class I (MHC-I) molecules belonging to the HLA-Bw4 serogroup. The receptor's second and third Ig domains confer the Bw4 specificity, but the role of the first Ig domain (D0) in ligand recognition has remained enigmatic. We found that KIR3DL1 expressed in YTS cells and as a soluble receptor can weakly recognize additional MHC-I molecules including HLA-B*0702 and HLA-G. This interaction is highly sensitive to blocking with Abs to the MHC-I α3-domain and the anti-KIR3DL1 Ab Z27, but not the canonical blocking Ab DX9. Using chimeric receptors between KIR3DL1 and KIR2DL1 expressed on YTS cells and as soluble Fc-fusion proteins, we show that the D0 domain confers the broad functional recognition and binding as well as the reactivity with Z27. These results suggest that the presence of a second and independent site of interaction between D0 and MHC-I and that MHC-I could bridge KIR3DL1 molecules together in a manner that facilitates signaling.

Modulation of the inhibitory receptor leukocyte Ig-like receptor 1 on human natural killer cells.

Leukocyte Ig-like receptor 1 (LIR-1) is an inhibitory Ig superfamily receptor with broad specificity for MHC-I expressed on leukocytes including natural killer (NK) and T cells. The extent of LIR-1 expression on NK cells is quite disparate between donors but the regulation of LIR-1 in NK cells is poorly understood. We examined expression profiles of LIR-1 on NK and T lymphocytes in 11 healthy donors over 1 year. Four of the 11 donors demonstrated substantial increases in LIR-1⁺ NK cells. High levels of LIR-1 expression were not correlated with exposure to human cytomegalovirus or the fraction of CD57⁺ NK cells in the donor. LIR-1 levels on ex vivo NK and CD56⁺ T cells were increased in vitro by short term exposure to IL-2 or IL-15 compared to control but not with various other cytokines tested. Sorted CD56(bright) NK cells also increased LIR-1 expression when cultured in IL-2. Maintenance of LIR-1 on longer term NK cells was also dependent on continuous stimulation by IL-15 or IL-2. While we could not detect increases in total LIR-1 mRNA in response to cytokine treatment by qPCR, we observed a shift in activity of LIR-1 promoter reporter constructs in the presence of IL-2 favoring the more translationally active transcript from the proximal promoter. Together these results show LIR-1 on NK cells is under the control of cytokines known to drive NK cell maturation and activation and suggest availability of such cytokines may alter the NK repertoire in vivo as we observed in several donors with fluctuating levels of LIR-1 on their NK cells.

LILRB1 polymorphism and surface phenotypes of natural killer cells.

Leukocyte Ig-like receptor (LIR)-1 is an inhibitory receptor that binds a broad range of class I HLA molecules and is encoded by the LILRB1 gene within the leukocyte receptor complex. In contrast to uniform expression on monocytes and B cells, LIR-1 expression on natural killer (NK) cells varies considerably between individuals. To investigate how polymorphism is related to the observed patterns of expression, we analyzed the LILRB1 gene and its transcriptional activity in a group of individuals with various levels of expression on NK cells. We found that LILRB1 transcription is correlated with surface protein expression on NK cells. In a cohort of 24 donors, we found high expression on NK cells to be associated with three linked SNPs (AGG verses GAA) within the putative regulatory region. We also identified several new protein variants and observed variants with P, T, T, and I at positions 68, 95, 142, and 155, respectively, more frequently in donors with low expression on NK cells. These results suggest that there is a significant degree of diversity within the LILRB1 locus and that it influences expression patterns on NK cells. These genetic differences may underpin variation in individual immune responses involving LIR-1 on NK cells.

Natural killer cell conjugate assay using two-color flow cytometry.

This flow cytometry-based method is a quick way to detect adhesion of NK cells to target cells. The two cell types are labeled with distinct fluorescent dyes and following co-incubation, the number of NK cells firmly adhered to target cells is quantified using two-color flow cytometry.

Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.

Analysis of the linker for activation of T cells and the linker for activation of B cells in natural killer cells reveals a novel signaling cassette, dual usage in ITAM signaling, and influence on development of the Ly49 repertoire.

The linker for activation of T cells (LAT) and the linker for activation of B cells (LAB/NTAL/LAT2) are integral proteins in receptor coupling to downstream events. Both proteins are expressed in natural killer (NK) cells and LAT is phosphorylated during target cell interactions or ligation of the immunoreceptor tyrosine-based activation motif (ITAM)-coupled CD16. Regardless, Lat(-/-) mice exhibit normal natural and antibody-mediated killing. Here we place both LAT and LAB in the DAP12 pathway of NK cells. Moreover, we unveil a LAT-independent pathway that requires expression of Syk. Mice lacking either LAT or LAB have a skewed Ly49 repertoire, and activated NK cells from Lat(-/-) mice have reduced responses to the ITAM-coupled receptor NK1.1. In contrast, resting Lat(-/-) NK cells show intact NK1.1 responses, whereas NK cells without LAB are hyperactive. Elimination of both adaptors severely reduces NK1.1 signaling under both conditions. Together these data show that NK ITAMs preferentially use a signaling cassette regulated by interplay between LAT and LAB. Activation by interleukin-2 causes a shift to greater dependency on LAT due to suppression of Syk signaling. The overlapping use of multiple adaptors permits fine-tuning of NK-cell ITAM responses over the course of an immune response.

Membranous structures transfer cell surface proteins across NK cell immune synapses.

Intercellular transfer of cell surface proteins is widespread and facilitates several recently discovered means for immune cell communication. Here, we examined the molecular mechanism for intercellular exchange of the natural killer (NK) cell receptor KIR2DL1 and HLA-C, prototypical proteins that swap between NK cells and target cells. Transfer was contact dependent and enhanced for cells expressing cognate receptor/ligand pairs but did not depend on KIR2DL1 signaling. To a lesser extent, proteins transferred independent from specific recognition. Intracellular domains of transferred proteins were not exposed to the extracellular environment and transferred proteins were removed by brief exposure to low pH. By fluorescence microscopy, transferred proteins localized to discrete regions on the recipient cell surface. Higher resolution scanning electron micrographs revealed that transferred proteins were located within specific membranous structures. Transmission electron microscopy of the immune synapse revealed that membrane protrusions from one cell interacted with the apposing cell surface within the synaptic cleft. These data, coupled with previous observations, lead us to propose that intercellular protein transfer is mediated by membrane protrusions within and surrounding the immunological synapse.

Activating Ly-49 receptors regulate LFA-1-mediated adhesion by NK cells.

NK cells are important for innate resistance to tumors and viruses. Engagement of activating Ly-49 receptors expressed by NK cells leads to rapid NK cell activation resulting in target cell lysis and cytokine production. The ITAM-containing DAP12 adapter protein stably associates with activating Ly-49 receptors, and couples receptor recognition with generation of NK responses. Activating Ly-49s are potent stimulators of murine NK cell functions, yet how they mediate such activities is not well understood. We demonstrate that these receptors trigger LFA-1-dependent tight conjugation between NK cells and target cells. Furthermore, we show that activating Ly-49 receptor engagement leads to rapid DAP12-dependent up-regulation of NK cell LFA-1 adhesiveness to ICAM-1 that is also dependent on tyrosine kinases of the Syk and Src families. These results indicate for the first time that activating Ly-49s control adhesive properties of LFA-1, and by DAP12-dependent inside-out signaling. Ly-49-driven mobilization of LFA-1 adhesive function may represent a fundamental proximal event during NK cell interactions with target cells involving activating Ly-49 receptors, leading to target cell death.

Regulation of natural killer cell activity.

Information regarding the role of natural killer (NK) cells in the response to viruses, intracellular bacteria and parasites continues to emerge. NK cells can directly lyse infected cells, secrete cytokines and interact with dendritic cells to drive the adaptive immune response. There are a large number of activating and inhibitory receptors that govern NK cell activity. Recent studies have revealed how signals are transmitted and integrated from the variety of receptors, how particular receptors influence NK development and functional status, and how NK cells access lymph nodes and sites of infection. The potential for NK cells to exhibit specific and memory-like responses has begun to blur the 'innate' definition of NK cells.

Microclusters of inhibitory killer immunoglobulin-like receptor signaling at natural killer cell immunological synapses.

We report the supramolecular organization of killer Ig-like receptor (KIR) phosphorylation using a technique applicable to imaging phosphorylation of any green fluorescent protein-tagged receptor at an intercellular contact or immune synapse. Specifically, we use fluorescence lifetime imaging (FLIM) to report Förster resonance energy transfer (FRET) between GFP-tagged KIR2DL1 and a Cy3-tagged generic anti-phosphotyrosine monoclonal antibody. Visualization of KIR phosphorylation in natural killer (NK) cells contacting target cells expressing cognate major histocompatibility complex class I proteins revealed that inhibitory signaling is spatially restricted to the immune synapse. This explains how NK cells respond appropriately when simultaneously surveying susceptible and resistant target cells. More surprising, phosphorylated KIR was confined to microclusters within the aggregate of KIR, contrary to an expected homogeneous distribution of KIR signaling across the immune synapse. Also, yellow fluorescent protein-tagged Lck, a kinase important for KIR phosphorylation, accumulated in a multifocal distribution at inhibitory synapses. Spatial confinement of receptor phosphorylation within the immune synapse may be critical to how activating and inhibitory signals are integrated in NK cells.