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.

The vitronectin receptor serves as an accessory molecule for the activation of a subset of gamma/delta T cells.

Constitutive production of cytokines was observed in 3 of 12 gamma/delta T cell lines derived from murine epidermis and correlated with the expression of the C gamma 4, V delta 6 T cell receptor (TCR). After adaptation of one of the lines (T195/BW) to serum-free culture conditions, cessation of the "spontaneous" production of interleukin 4 (IL-4) was observed and IL-4 production could then by induced by the addition of RGD-containing extracellular matrix (ECM) proteins to the culture. The response to the ECM proteins could be completely inhibited by a mAb to the murine vitronectin receptor (VNR). However, the induction of IL-4 production could also be inhibited by anti-CD3 and by an anti-clonotypic mAb to the TCR-gamma/delta of T195/BW. As TCR-gamma/delta loss mutants of T195/BW also failed to respond to ECM proteins, these data demonstrate that engagement of the VNR by its ligand is necessary, but not sufficient, for the induction of IL-4 production. Furthermore, the VNR is expressed by many other T cell clones (both gamma/delta and alpha/beta), none of which produce lymphokines constitutively. Taken together, these observations strongly favor the view that not only is coexpression of the VNR and TCR required for the induction of IL-4 production, but that the TCR must also be engaged by its ligand, most likely a cell surface antigen expressed by the hybridoma itself.

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.

Ly-49-independent natural killer (NK) cell specificity revealed by NK cell clones derived from p53-deficient mice.

Natural killer (NK) cells are heterogeneous in their specificity and expression of cell surface molecules. In the mouse, the Ly-49A molecule is a primary determinant of NK cell specificity because of its ability to downregulate NK cell activation after physical interaction with target cell MHC class I molecules. Ly-49A is expressed on an NK cell subset, and it belongs to a family of highly related molecules that may similarly dictate major histocompatibility complex (MHC) class I-associated specificity of Ly-49A- NK cells. It is not known, however, whether murine NK cell specificity may occur independently of the Ly-49 family and target cell MHC class I molecules. Similar to the impact of cloned murine T cell lines on molecular description of T cell recognition, derivation of cloned murine NK cells should permit dissection of NK cell specificity but, to date, it has not been possible to produce such effector cells. In this study, we derived NK cell clones from mice that were homozygous for a mutation in the p53 tumor suppressor gene. The cloned cells displayed the molecular, cell surface, and functional phenotype of NK cells. Significantly, the NK cell clones displayed clonal differences in ability to kill a panel of murine tumor targets and did not lyse normal cells. Target lysis was unaffected by target cell MHC class I expression, and none of the clones expressed Ly-49A on the cell surface or transcripts for Ly-49 isoforms. Although consistent with the possibility that NK cell specificity for MHC class I molecules is mediated by the Ly-49 family of molecules, the results indicate that NK cell specificity also is regulated by a mechanism independent of target cell MHC class I and the Ly-49 family.

A natural killer cell receptor specific for a major histocompatibility complex class I molecule.

Target cell expression of major histocompatibility complex (MHC) class I molecules correlates with resistance to lysis by natural killer (NK) cells. Prior functional studies of the murine NK cell surface molecule, Ly-49, suggested its role in downregulating NK cell cytotoxicity by specifically interacting with target cell H-2Dd molecules. In support of this hypothesis, we now demonstrate a physical interaction between H-2Dd and Ly-49 in both qualitative and quantitative cell-cell binding assays employing a stable transfected Chinese hamster ovary (CHO) cell line expressing Ly-49 and MHC class I transfected target cells. Binding occurred only when CHO cells expressed Ly-49 at high levels and targets expressed H-2Dd by transfection. Monoclonal antibody blocking experiments confirmed this interaction. These studies indicate that the specificity of natural killing is influenced by NK cell receptors that engage target cell MHC class I molecules.

Genetic control of natural killing and in vivo tumor elimination by the Chok locus.

The molecular mechanisms underlying target recognition during natural killing are not well understood. One approach to dissect the complexities of natural killer (NK) cell recognition is through exploitation of genetic differences among inbred mouse strains. In this study, we determined that interleukin 2-activated BALB/c-derived NK cells could not lyse Chinese hamster ovary (CHO) cells as efficiently as C57BL/6-derived NK cells, despite equivalent capacity to kill other targets. This strain-determined difference was also exhibited by freshly isolated NK cells, and was determined to be independent of host major histocompatibility haplotype. Furthermore, CHO killing did not correlate with expression of NK1.1 or 2B4 activation molecules. Genetic mapping studies revealed linkage between the locus influencing CHO killing, termed Chok, and loci encoded within the NK gene complex (NKC), suggesting that Chok encodes an NK cell receptor specific for CHO cells. In vivo assays recapitulated the in vitro data, and both studies determined that Chok regulates an NK perforin-dependent cytotoxic process. These results may have implications for the role of NK cells in xenograft rejection. Our genetic analysis suggests Chok is a single locus that affects NK cell-mediated cytotoxicity similar to other NKC loci that also regulate the complex activity of NK cells.

The Ly-49D receptor activates murine natural killer cells.

Proteins encoded by members of the Ly-49 gene family are predominantly expressed on murine natural killer (NK) cells. Several members of this gene family have been demonstrated to inhibit NK cell lysis upon recognizing their class I ligands on target cells. In this report, we present data supporting that not all Ly-49 proteins inhibit NK cell function. Our laboratory has generated and characterized a monoclonal antibody (mAb) (12A8) that can be used to recognize the Ly-49D subset of murine NK cells. Transfection of Cos-7 cells with known members of the Ly-49 gene family revealed that 12A8 recognizes Ly-49D, but also cross-reacts with the Ly-49A protein on B6 NK cells. In addition, 12A8 demonstrates reactivity by both immuno-precipitation and two-color flow cytometry analysis with an NK cell subset that is distinct from those expressing Ly-49A, C, or G2. An Ly-49D+ subset of NK cells that did not express Ly-49A, C, and G2 was isolated and examined for their functional capabilities. Tumor targets and concanovalin A (ConA) lymphoblasts from a variety of H2 haplotypes were examined for their susceptibility to lysis by Ly-49D+ NK cells. None of the major histocompatibility complex class I-bearing targets inhibited lysis of Ly-49D+ NK cells. More importantly, we demonstrate that the addition of mAb 12A8 to Ly-49D+ NK cells can augment lysis of Fc gamma R+ target cells in a reverse antibody-dependent cellular cytotoxicity-type assay and induces apoptosis in Ly-49D+ NK cells. Furthermore, the cytoplasmic domain of Ly-49D does not contain the V/Ix-YxxL immunoreceptor tyrosine-based inhibitory motif found in Ly-49A, C, or G2 that has been characterized in the human p58 killer inhibitory receptors. Therefore, Ly-49D is the first member of the Ly-49 family characterized as transmitting positive signals to NK cells, rather than inhibiting NK cell function.

Nonstochastic coexpression of activation receptors on murine natural killer cells.

Murine natural killer cells (NK) express lectin-like activation and inhibitory receptors, including the CD94/NKG2 family of receptors that bind Qa-1, and the Ly-49 family that recognizes major histocompatibility complex class I molecules. Here, we demonstrate that cross-linking of NK cells with a new specific anti-Ly-49H mAb induced NK cell cytotoxicity and cytokine production. Ly-49H is expressed on a subset of NK cells and can be coexpressed with Ly-49 inhibitory receptors. However, unlike Ly-49 inhibitory receptors, Ly-49H is not detectable on naive splenic CD3(+) T cells, indicating that Ly-49H may be an NK cell-specific activation receptor. In further contrast to the stochastically expressed Ly-49 inhibitory receptors, Ly-49H is preferentially expressed with the Ly-49D activation receptor, and expression of both Ly-49H and Ly-49D is augmented on NK cells that lack receptors for Qa-1 tetramers. On developing splenic NK1.1(+) cells, Ly-49D and Ly-49H are expressed later than the inhibitory receptors. These results directly demonstrate that Ly-49H activates primary NK cells, and suggest that expression of Ly-49 activation receptors by NK cells may be specifically regulated on NK cell subsets. The simultaneous expression of multiple activation receptors by individual NK cells contrasts with that of T cell antigen receptors and is relevant to the role of NK cells in innate immunity.

Murine T cells express a cell surface receptor for multiple extracellular matrix proteins. Identification and characterization with monoclonal antibodies.

Cell-cell and cell-extracellular (ECM) protein interactions are mediated through heterodimers termed integrins. We have demonstrated that dendritic epidermal T cell (DETC) lines adhere to the ECM proteins, fibronectin, fibrinogen, and vitronectin but not to collagen, laminin, or control proteins. This adhesion was blocked by the tetrapeptide RGDS, but not the control peptide, RGES. We have derived a hamster mAb H9.2B8, and a rat mAb, 8.18E12, from immunizations with DETC lines. The mAbs in combination, but not individually, specifically inhibited the adhesion of DETC lines to fibronectin, fibrinogen, and vitronectin. Immunoprecipitation analysis revealed that both mAbs reacted with a heterodimer composed of noncovalently linked 140- and 95-kD subunits. The 140-kD subunit can be reduced to 120- and 23-kD fragments. Although the two mAbs did not cross-compete for binding to DETC, sequential immunoprecipitation studies indicated that they react with the same 120-kD fragment. While all DETC cell lines and several T cell clones were reactive with the mAbs, the mAbs were not reactive with normal spleen, lymph node, thymus, or skin. Stimulation of splenic T cells with Con A or allogeneic cells induced mAb reactivity after 1 wk in vitro. These data demonstrate that a single lymphocyte receptor, with biochemical features characteristic of integrins, mediates RGD-dependent binding to the ECM proteins, fibronectin, fibrinogen, and vitronectin. Furthermore, since this integrin is expressed by long-term activated T cells, this receptor may play a physiological role in T cell function.

Production of a T cell hybridoma that expresses the T cell receptor gamma/delta heterodimer.

We have produced a T cell hybridoma line by fusion of an IL-2-dependent, long-term T cell receptor (TCR) gamma/delta+ Thy-1+, bone marrow-derived, dendritic epidermal cell line to the BW5147 tumor line. The resultant hybridoma was rapidly growing, lymphokine independent, and expressed T3 in association with the TCR gamma/delta heterodimer. Several subclones of the hybridoma line produced easily detectable levels of IL-2 after stimulation by anti-T3 or Con A. The availability of these cloned cell lines should greatly facilitate further functional, biochemical, and molecular studies of the TCR delta chain.

T cell receptor gamma/delta chain diversity.

The TCR-gamma and -delta chains of six murine hybridomas were compared by one-dimensional SDS-PAGE and two-dimensional NEPHGE/SDS-PAGE analysis. This allowed the identification of three distinct gamma chains (gamma a, gamma b, and gamma c) and three distinct delta chains (delta a, delta b, and delta c). Four gamma/delta chain combinations (gamma a delta a, gamma b delta b, gamma b delta c, and gamma c delta a) were observed. These results indicate that multiple forms of the delta chain are expressed and suggest that the delta chains are encoded for by an Ig-like rearranging gene. This delta chain polymorphism significantly enhances the potential diversity of TCR-gamma/delta, which may be of importance for a better understanding of the putative ligand(s) recognized by this receptor.

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.

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.

Requirement for type 2 NO synthase for IL-12 signaling in innate immunity.

Interleukin-12 (IL-12) and type 2 NO synthase (NOS2) are crucial for defense against bacterial and parasitic pathogens, but their relationship in innate immunity is unknown. In the absence of NOS2 activity, IL-12 was unable to prevent spreading of Leishmania parasites, did not stimulate natural killer (NK) cells for cytotoxicity or interferon-gamma (IFN-gamma) release, and failed to activate Tyk2 kinase and to tyrosine phosphorylate Stat4 (the central signal transducer of IL-12) in NK cells. Activation of Tyk2 in NK cells by IFN-alpha/beta also required NOS2. Thus, NOS2-derived NO is a prerequisite for cytokine signaling and function in innate immunity.

Identification of a T3-associated gamma delta T cell receptor on Thy-1+ dendritic epidermal Cell lines.

The murine epidermis contains a subpopulation of bone marrow-derived lymphocytes that have a dendritic morphology and that express Thy-1 and T3 cell-surface antigens but not other markers (L3T4 or Lyt-2) characteristic of mature peripheral T lymphocytes. An alternative type of T cell receptor was earlier identified on a subpopulation of murine thymocytes with a similar phenotype (T3+, L3T4-, Lyt-2-), but not on peripheral murine T lymphocytes. Two independently derived Thy-1+, L3T4-, and Lyt-2- dendritic cell lines of epidermal origin that express a T3-associated disulfide-linked heterodimer composed of a 34-kilodalton gamma-chain and 46-kilodalton partner (the delta chain) have now been identified. Analysis of N-linked glycosylation revealed that this receptor is similar to that detected on thymocytes. These results demonstrate that Thy-1+ dendritic epidermal cell lines can express gamma delta T cell receptors in vitro and suggest that Thy-1+ dendritic epidermal cells express such receptors in vivo. The localization of these gamma delta T cell receptor-expressing cells in the epidermis may be of importance for understanding the function of these receptors.

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.

Natural killer cells. Right-side-up and up-side-down NK-cell receptors.

Recent cDNA cloning of the receptors on human natural killer cells for MHC class I molecules reveals that human and mouse receptors have inverted structural orientations. Are there two distinct receptor systems?

Natural killer cell receptors.

In the past year, significant strides have been made in understanding natural killing, the process whereby natural killer cells lyse target cells. Recognition of susceptible targets and activation now appear to be mediated by a natural killer cell receptor that binds carbohydrate determinants on target cells and initiates target lysis. Conversely, receptors have been identified that bind MHC class I molecules on targets that inhibit natural killer cell activation. These findings provide the basis for understanding the molecular processes in the initial steps of natural killing.

Natural killer cell receptors.

In killing of cellular targets, natural killer cells employ receptors that activate them and receptors specific for MHC class I that inhibit their activation. Progress in understanding the inhibitory receptors has been rapid, and indications are that they fall into two distinct structural types that appear to utilize the same inhibitory signaling cascade; meanwhile, components of the activation cascade are being elucidated, permitting us to integrate the pathways involved.

Recognition structures on natural killer cells.

Recent studies have identified cell surface molecules that appear to play important roles in natural killer cell specificity for their targets. Natural killer cells display activation 'receptors', such as NKR-P1 molecules in rodents, that may activate cytotoxicity by transducing biochemical signals. These molecules presumably interact with target cell surface ligands but these structures have not been elucidated. Natural killer cells display other molecules, such as Ly-49 in mice, that appear to be 'inhibitory' receptors that engage target cell MHC class I molecules and deliver signals, negatively regulating natural killer cell cytotoxic activity. The murine NKR-P1 and Ly-49 molecules are structurally similar and encoded by members of polymorphic gene families that reside in the natural killer gene complex on the distal region of mouse chromosome 6. Additional molecules have been serologically defined and studied functionally in murine and human systems. Thus, the specificity of an individual natural killer cell may be determined by its expressed repertoire of these molecules. The complexities of this recognition system are beginning to be appreciated at the molecular level.