Structural variation of the mouse natural killer gene complex.

The natural killer gene complex (NKC) on chromosome 6 contains clusters of genes that encode both activation and inhibitory receptors expressed on mouse natural killer (NK) cells. NKC genes, particularly belonging to the Nkrp1 and Ly49 gene families, display haplotype differences between different mouse strains and allelic polymorphisms of individual genes, as previously revealed by conventional analysis in a small number of inbred mouse strains. Herein we used array-based comparative genomic hybridization (aCGH) to efficiently compare the NKC in 21 mouse strains to the reference C57BL/6 strain. By using unsupervised clustering methods, we could sort these variations into the same groups as determined by previous RFLP analyses of Nkrp1 and Ly49 genes. Prospective analyses of aCGH and RFLP data validated these relationships. Moreover, aCGH data predicted monoclonal antibody reactivity with an allospecific determinant on molecules expressed by NK cells. Taken together, these data demonstrate the structural variation in the NKC between mouse strains as well as the usefulness of aCGH in analysis of complex, polymorphic gene clusters.

Cmv1 and natural killer cell responses to murine cytomegalovirus infection.

The dissection of genetic resistance to murine cytomegalovirus infection in inbred laboratory mouse strains led to the identification of a natural killer cell activation receptor that recognizes a virus-encoded protein. Herein, we summarize the genetic approach and findings that have provided novel insights into innate immune control of virus infections.

Extending missing-self? Functional interactions between lectin-like NKrp1 receptors on NK cells with lectin-like ligands.

The functions of natural killer (NK) cells are clearly regulated by major histocompatibility complex (MHC) class I molecules on their cellular targets. In mice, this is due to the action of MHC-specific inhibitory receptors belonging to the Ly49 family oflectin-like molecules. The Ly49 receptors are encoded in the NK gene complex (NKC) that contains clusters of genes for other lectin-like receptors on NK cells and other hematopoietic cells. Interestingly, recent studies have shown that some of these lectin-like receptors, belonging to the Nkrpl family, can recognize other lectin-like molecules, termed Clr, also encoded in the NKC. These genetically linked loci for receptor-ligand pairs suggest a genetic strategy to preserve this interaction and show several other contrasts with Ly49-MHC interactions. In this review, we discuss these issues and summarize recent developments concerning this non-MHC-dependent regulation of NK cell function.

Will pharmacogenetics allow better prediction of methotrexate toxicity and efficacy in patients with rheumatoid arthritis?

Methotrexate (MTX) remains the most commonly used disease modifying antirheumatic drug in rheumatoid arthritis (RA) because of its cost and experience in its use, despite the availability of new treatments such as leflunomide and the biological agents. However, a significant number of patients with RA either do not benefit from the drug or are unable to tolerate it. Pharmacogenetic approaches may help optimise treatment with MTX, and also other agents, in RA.

Pharmacogenetics in rheumatology: the prospects and limitations of an emerging field.

OBJECTIVES: To review the fundamental concepts of pharmacogenetics and analyse how the broad principles of this rapidly emerging field may influence the treatment of rheumatic disease in future. METHODS: The names of common rheumatic drugs and the terms 'pharmacogenetics', 'pharmacogenomics' and 'genetic polymorphism' were used as keywords to search the Medline and Current Contents databases. General review articles on pharmacogenetics were also examined. RESULTS: Pharmacogenetics is the study of how genetic differences influence the variability in drug toxicity and efficacy. Although the principles of pharmacogenetics have been known for several decades, recent technological advances have hastened the possibility of direct clinical applications. Most studies so far have been phenotypic analyses, but genotyping is now readily available for many polymorphisms. There are several examples pertinent to rheumatology that illustrate the important principles and foretell the usefulness of pharmacogenetics in individualizing therapy. However, further studies are needed. CONCLUSIONS: Because traditional pharmacotherapy in rheumatology has been empirical and because of the slow acting nature of many anti-rheumatic medications, the risk of significant side-effects and the increasing armamentarium of drugs available, pharmacogenetics is particularly relevant to rheumatology. There are many scientific and non-scientific concerns that should be addressed in future studies.

The search for the missing 'missing-self' receptor on natural killer cells.

In this article, I outline the studies that led to the discovery of Ly49A as the first 'missing-self ' receptor on natural killer cells. It is a more detailed and formal version of my acceptance speech at the Novartis Award ceremony at the International Congress of Immunology in Stockholm. Yet, it is impossible to mention my gratitude to all of my previous mentors and colleagues or to credit all the scientists and immunologists who preceded us by making contributions, large and small, to knowledge upon which our findings were based. It is therefore humbling to accept the 2001 Novartis Award for Basic Immunology along with Professors Klas Kärre and Lorenzo Moretta.

Natural killer gene complex (Nkc) allelic variability in inbred mice: evidence for Nkc haplotypes.

Allelic variability for mouse Chromosome 6 Nkc loci was assessed in 22 common laboratory strains of mice using selected natural killer gene complex (Nkc)-linked sequence tagged site markers. Most Nkc markers distinguished three or more alleles for a particular locus in the assessed mouse strains. Nkc locus alleles were highly conserved among genealogically related inbred strains, whereas far less similarity was observed among unrelated strains. Concurrent strain-to-strain comparisons for all Nkc-linked loci revealed common and uncommon Nkc haplotypes, including some that were likely recombinant. Nkc allele and haplotype assignments in inbred mouse strains and correlation with phenotypic traits should facilitate positional gene cloning strategies for unknown Nkc-linked trait modification loci.

Ly49A allelic variation and MHC class I specificity.

The Ly49 family of natural killer (NK) cell receptors is encoded by a polygenic genetic locus. Allelic forms have been described and their expression appears to be regulated. The best-characterized Ly49 molecule, the C57BL/6 form of Ly49A, is an NK cell inhibitory receptor that binds H2Dd. To determine whether differences between Ly49a alleles may have functional consequences, allelic variants of Ly49a were cloned from several inbred mouse strains. Stable transfectants expressing each Ly49a allelic variant were generated and tested for reactivity with a panel of monoclonal antibodies (mAbs A1, JR9.318, YE1/32, and YE1/48) that recognize the C57BL/6 form of Ly49A. Binding to H2Dd was also assessed using fluorescently labeled H2Dd tetramers. Furthermore, cytotoxicity assays were performed using anti-Ly49A mAb-separated interleukin-2-activated NK cells. We show that despite binding to fluorescently labeled H2Dd tetramers, the Ly49A+ NK cells from representative mouse strains displayed significantly different degrees of inhibition with H2Dd targets. These results can be interpreted in the light of recent structural data on the Ly49A-H2Dd complex. Thus, the Ly49 family displays functionally significant allelic polymorphism which adds to the repertoire of NK cell receptors.

Analysis of a 1-Mb BAC contig overlapping the mouse Nkrp1 cluster of genes: cloning of three new Nkrp1 members, Nkrp1d, Nkrp1e, and Nkrp1f.

The murine Nkrp1 gene family encodes three previously identified activation and inhibitory receptors expressed on natural killer (NK) cells. This family includes the gene for NKR-P1C (NK1.1), the most specific serologic marker on C57BL/6-derived NK cells and is localized in a gene cluster in the NK gene complex (NKC). To further analyze the Nkrp1 family, we constructed and analyzed a bacterial artificial chromosome contig. A genomic organization of the Nkrp1 family was obtained and three new Nkrp1 genes were isolated from interleukin-2-activated NK cells. Thus, the Nkrp1 family adds to the repertoire of receptors expressed by NK cells.

Analysis of in situ NK cell responses during viral infection.

NK cells are required for early control of murine CMV (MCMV) infection, but the distribution of murine NK cells in situ has not been clearly defined. We tested the reactivity of all available NK cell receptor-specific mAbs by immunohistochemistry. Only one mAb, 4D11 (anti-Ly-49G2), was reactive with C57BL/6 tissue sections. mAb 4D11-reactive cells expressed the nuclear morphology and flow cytometric profile of NK cells. In lymphoid organs, NK cells were distributed primarily in the splenic red pulp, between adjacent lobes in lymph node and randomly in the cortex and medulla of the thymus. No NK cells were detected in normal liver sections. Two days following MCMV infection, most splenic NK cells were associated with the lymphoid follicles and marginal zone. By day 3 following infection, the number of liver NK cells had increased significantly and the cells were detected within inflammatory foci. These changes were independent of IL-12, IFN-gamma, and TNF-alpha, as assessed in mice with targeted mutations. Concurrent immunostaining for NK cells and viral Ags revealed close association of NK cells and MCMV-infected cells in the spleen and liver. Similar results were obtained in CD1(-/-) and recombination activation gene-1(-/-) mice lacking NK T or T and B cells, respectively, indicating specificity of staining for NK cells. Thus, following MCMV infection, NK cells accumulate at sites of viral replication in an IL-12-, IFN-gamma-, and TNF-alpha-independent manner.

Specific and nonspecific NK cell activation during virus infection.

The natural killer (NK) cell activation receptor Ly49H is required for resistance to murine cytomegalovirus (MCMV). We show here that NK cell proliferation and production of interferon-gamma (IFN-gamma) was not dependent on Ly49H expression during early MCMV infection. During a later phase of infection, however, Ly49H+ NK cells selectively proliferated and this expansion was blocked by anti-Ly49H administration. With vaccinia virus infection, neither the early nor late phase of NK cell proliferation was selective for Ly49H+ NK cells. These findings indicated that Ly49H+ NK cells were specifically activated by MCMV and that MCMV infection was characterized by nonspecific and specific phases of NK cell activation in vivo.

CD47 (integrin-associated protein) engagement of dendritic cell and macrophage counterreceptors is required to prevent the clearance of donor lymphohematopoietic cells.

Integrin-associated protein (CD47) is a broadly expressed protein that costimulates T cells, facilitates leukocyte migration, and inhibits macrophage scavenger function. To determine the role of CD47 in regulating alloresponses, CD47(+/+) or CD47(-/-) T cells were infused into irradiated or nonconditioned major histocompatibility complex disparate recipients. Graft-versus-host disease lethality was markedly reduced with CD47(-/-) T cells. Donor CD47(-/-) T cells failed to engraft in immunodeficient allogeneic recipients. CD47(-/-) marrow was unable to reconstitute heavily irradiated allogeneic or congenic immune-deficient CD47(+/+) recipients. These data suggested that CD47(-/-) T cells and marrow cells were cleared by the innate immune system. To address this hypothesis, dye-labeled CD47(-/-) and CD47(+/+) lymphocytes or marrow cells were infused in vivo and clearance was followed. Dye-labeled CD47(-/-) cells were engulfed by splenic dendritic cells and macrophages resulting in the clearance of virtually all CD47(-/-) lymphohematopoietic cells within 1 day after infusion. Host phagocyte-depleted CD47(+/+) recipients partially accepted allogeneic CD47(-/-) T cells. Thus, dendritic cells and macrophages clear lymphohematopoietic cells that have downregulated CD47 density. CD47 expression may be a critical indicator for determining whether lymphohematopoietic cells will survive or be cleared.

Murine natural killer cell activation receptors.

Natural killer (NK) cells express two types of receptors involved in target recognition: inhibitory receptors for target cell MHC class I molecules and activation receptors. While there has been significant progress in understanding the inhibitory receptors, less is known about the activation receptors. Detailed analysis of several mouse NK-cell activation receptors provides insight into the physiologic relevance of these receptors in the innate immune response.

Cloning of Clr, a new family of lectin-like genes localized between mouse Nkrp1a and Cd69.

We report the identification of a novel family of genes, named Clr, encoding C-type lectin-like molecules, which maps in the natural killer (NK) gene complex (NKC) on mouse Chromosome 6. Genomic sequence analysis indicates the presence of at least seven members between Nkrpla and Cd69. By RT-PCR, at least three members of the family are expressed on interleukin-2-activated NK cells. Sequence analysis revealed complete open reading frames of 203-205 amino acids, with a carboxyl-terminal C-type lectin-like carbohydrate recognition domain (CRD). The CRDs of the Clr proteins exhibit a significant degree of homology with the known NKC-encoded NK-cell receptors. However, a key cysteine usually present in the CRD is missing in the Clr proteins, suggesting that their ligands and functions are distinct from other molecules encoded in the NKC.

A "chimeric" C57l-derived Ly49 inhibitory receptor resembling the Ly49D activation receptor.

Ly49D is a natural killer (NK) cell activation receptor that is responsible for differential mouse inbred strain-determined lysis of Chinese hamster ovary (CHO) cells. Whereas C57BL/6 NK cells kill CHO, BALB/c-derived NK cells cannot kill because they lack expression of Ly49D. Furthermore, the expression of Ly49D, as detected by monoclonal antibody 4E4, correlates well with CHO lysis by NK cells from different inbred strains. However, one discordant mouse strain was identified; C57L NK cells express the mAb 4E4 epitope but fail to lyse CHO cells. Herein we describe a Ly49 molecule isolated from C57L mice that is recognized by mAb 4E4 (anti-Ly49D). Interestingly, this molecule shares extensive similarity to Ly49D(B6) in its extracellular domain, but its cytoplasmic and transmembrane domains are identical to the inhibitory receptor Ly49A(B6), including a cytoplasmic ITIM. This molecule bears substantial overall homology to the previously cloned Ly49O molecule from 129 mice the serologic reactivity and function of which were undefined. Cytotoxicity experiments revealed that 4E4(+) LAK cells from C57L mice failed to lyse CHO cells and inhibited NK cell function in redirected inhibition assays. MHC class I tetramer staining revealed that the Ly49O(C57L)-bound H-2D(d) and lysis by 4E4(+) C57L LAK cells is inhibited by target H-2D(d). The structural basis for ligand binding was also examined in the context of the recent crystallization of a Ly49A-H-2D(d) complex. Therefore, this apparently "chimeric" Ly49 molecule serologically resembles an NK cell activation receptor but functions as an inhibitory receptor.

Vital involvement of a natural killer cell activation receptor in resistance to viral infection.

Natural killer (NK) cells are lymphocytes that can be distinguished from T and B cells through their involvement in innate immunity and their lack of rearranged antigen receptors. Although NK cells and their receptors were initially characterized in terms of tumor killing in vitro, we have determined that the NK cell activation receptor, Ly-49H, is critically involved in resistance to murine cytomegalovirus in vivo. Ly-49H requires an immunoreceptor tyrosine-based activation motif (ITAM)-containing transmembrane molecule for expression and signal transduction. Thus, NK cells use receptors functionally resembling ITAM-coupled T and B cell antigen receptors to provide vital innate host defense.

Development of intestinal intraepithelial lymphocytes, NK cells, and NK 1.1+ T cells in CD45-deficient mice.

The transmembrane protein tyrosine phosphatase CD45 is differentially required for the development and function of B, T, and NK cells, with mice partially deficient for CD45 having a significant inhibition of T cell, but not NK or B cell, development. CD45-mediated signaling has also been implicated in the development of intrathymic, but not extrathymic, intestinal intraepithelial T lymphocytes (iIELs) in the CD45ex6(-/-) mouse. As NK1.1(+) CD3(+) (NK-T) cells can also develop through extrathymic pathways, we have investigated the role of CD45 in NK-T cell development. In mice with a complete absence of CD45 expression (CD45ex9(-/-)) the NK-T cell population was maintained in the iIEL compartment, but not in the spleen. Functionally, CD45-deficient NK-T cells were unable to secrete IL-4 in response to TCR-mediated signals, a phenotype similar to that of CD45-deficient iIELs, in which in vitro cytokine production was dramatically reduced. Using the CD45ex9(-/-) mouse strain, we have also demonstrated that only one distinct population of NK-T cells (CD8(+)) appears to develop normally in the absence of CD45. Interestingly, although an increase in cytotoxic NK cells is seen in the absence of CD45, these NK calls are functionally unable to secrete IFN-gamma. In the absence of CD45, a significant population of extrathymically derived CD8alphaalpha(+) iIELs is also maintained. These results demonstrate that in contrast to conventional T cells, CD45 is not required during the development of CD8(+) NK-T cells, NK cells, or CD8alphaalpha(+) iIELs, but is essential for TCR-mediated function and cytokine production.

The NK cell MHC class I receptor Ly49A detects mutations on H-2Dd inside and outside of the peptide binding groove.

The NK cell inhibitory receptor Ly49A recognizes the mouse MHC class I molecule H-2D(d) and participates in the recognition of missing self. Previous studies indicated that the determinant recognized by Ly49A exists in alpha1/alpha2 domain of H-2D(d). Here we have substituted polymorphic as well as conserved residues of H-2D(d) alpha1/alpha2 domain (when compared with H-2K(d), which does not interact with Ly49A). We then tested the ability of the H-2D(d) mutants to interact with Ly49A by soluble Ly49A tetramer binding and NK cell cytotoxicity inhibition assays. Individual introduction of mutations converting the H-2D(d) residue into the corresponding H-2K(d) residue (N30D, D77S, or A99F) in H-2D(d) partially abrogated the interaction between Ly49A and H-2D(d). Introduction of the three mutations into H-2D(d) completely abolished Ly49A recognition. Individual introduction of D29N or R35A mutation into the residues of H-2D(d) that are conserved among murine MHC class I severely impaired the interaction. The crystal structure of H-2D(d) reveals that D77 and A99 are located in the peptide binding groove and that N30, D29, and R35 are in the interface of the three structural domains of MHC class I: alpha1/alpha2, alpha3, and beta(2)-microglobulin. These data suggest that Ly49A can monitor mutations in MHC class I inside and outside of the peptide binding groove and imply that inhibitory MHC class I-specific receptors are sensitive to mutations in MHC class I as well as global loss of MHC class I. Our results also provide insight into the molecular basis of Ly49A to distinguish MHC class I polymorphism.

The functional binding site for the C-type lectin-like natural killer cell receptor Ly49A spans three domains of its major histocompatibility complex class I ligand.

Natural killer (NK) cells express receptors that recognize major histocompatibility complex (MHC) class I molecules and regulate cytotoxicity of target cells. In this study, we demonstrate that Ly49A, a prototypical C-type lectin-like receptor expressed on mouse NK cells, requires species-specific determinants on beta2-microglobulin (beta2m) to recognize its mouse MHC class I ligand, H-2D(d). The involvement of beta2m in the interaction between Ly49A and H-2D(d) is also demonstrated by the functional effects of a beta2m-specific antibody. We also define three residues in alpha1/alpha2 and alpha3 domains of H-2D(d) that are critical for the recognition of H-2D(d) on target cells by Ly49A. In the crystal structure of the Ly49A/H-2D(d) complex, these residues are involved in hydrogen bonding to Ly49A in one of the two potential Ly49A binding sites on H-2D(d). These data unambiguously indicate that the functional effect of Ly49A as an MHC class I-specific NK cell receptor is mediated by binding to a concave region formed by three structural domains of H-2D(d), which partially overlaps the CD8 binding site.

Uterine macrophages express the gp49B inhibitory receptor in midgestation.

Mouse gp49B is an immunoreceptor tyrosine-based inhibitory motif-bearing receptor identified on mast cells and NK cells. In this report, however, we show that this receptor is expressed on macrophages accumulating in the uterine metrial gland in midgestation, along with gp49A that has a very homologous extracellular domain with gp49B but has a short cytoplasmic domain without ITIM. Culture of bone marrow cells in the conditioned medium of the metrial gland resulted in the selective proliferation of macrophages expressing both Fcgamma-activating receptors and gp49B inhibitory receptor. Stimulation of macrophages with immobilized IgG, but not with anti-FcgammaRII/III, induced a considerable amount of TNF-alpha and IL-10 production, suggesting that the high-affinity receptor for IgG (FcgammaRI) can transmit activating signals in cytokine production of macrophages. Furthermore, coligation of gp49B with FcgammaRI resulted in the inhibition of TNF-alpha production. Thus, our data provide evidence that gp49B is an endogenous negative regulator of macrophage activation and may regulate the function of macrophages during pregnancy.