Regulation of the natural killer cell receptor repertoire.

Natural killer cells express inhibitory receptors specific for MHC class I proteins and stimulatory receptors with diverse specificities. The MHC-specific receptors discriminate among different MHC class I alleles and are expressed in a variegated, overlapping fashion, such that each NK cell expresses several inhibitory and stimulatory receptors. Evidence suggests that individual developing NK cells initiate expression of inhibitory receptor genes in a sequential, cumulative, and stochastic fashion. Superimposed on the receptor acquisition process are multiple education mechanisms, which act to coordinate the stimulatory and inhibitory specificities of developing NK cells. One process influences the complement of receptors expressed by individual NK cells. Other mechanisms may prevent NK cell autoaggression even when the developing NK cell fails to express self-MHC-specific inhibitory receptors. Together, these mechanisms ensure a self-tolerant and maximally discriminating NK cell population. Like NK cells, a fraction of memory phenotype CD8(+) T cells, as well as other T cell subsets, express inhibitory class I--specific receptors in a variegated, overlapping fashion. The characteristics of these cells suggest that inhibitory receptor expression may be a response to prior antigenic stimulation as well as to poorly defined additional signals. A unifying hypothesis is that both NK cells and certain T cell subsets initiate expression of inhibitory receptors in response to stimulation.

Analysis of Qa-1(b) peptide binding specificity and the capacity of CD94/NKG2A to discriminate between Qa-1-peptide complexes.

The major histocompatibility complex class Ib protein, Qa-1(b), serves as a ligand for murine CD94/NKG2A natural killer (NK) cell inhibitory receptors. The Qa-1(b) peptide-binding site is predominantly occupied by a single nonameric peptide, Qa-1 determinant modifier (Qdm), derived from the leader sequence of H-2D and L molecules. Five anchor residues were identified in this study by measuring the peptide-binding affinities of substituted Qdm peptides in experiments with purified recombinant Qa-1(b). A candidate peptide-binding motif was determined by sequence analysis of peptides eluted from Qa-1 that had been folded in the presence of random peptide libraries or pools of Qdm derivatives randomized at specific anchor positions. The results indicate that Qa-1(b) can bind a diverse repertoire of peptides but that Qdm has an optimal primary structure for binding Qa-1(b). Flow cytometry experiments with Qa-1(b) tetramers and NK target cell lysis assays demonstrated that CD94/NKG2A discriminates between Qa-1(b) complexes containing peptides with substitutions at nonanchor positions P4, P5, or P8. Our findings suggest that it may be difficult for viruses to generate decoy peptides that mimic Qdm and raise the possibility that competitive replacement of Qdm with other peptides may provide a novel mechanism for activation of NK cells.

Cutting edge: cutting edge commentary: a Copernican revolution? Doubts about the danger theory.

The immune system is often said to function by "self-nonself" discrimination. Recently, some have argued that it actually detects "danger" or "strangers". There are problems with all of these points of view. Given that the immune system has been cobbled together throughout evolution and uses a diverse array of innate and adaptive defense mechanisms, it may not be possible to account for immunity within one "paradigm" or another.

NK cell expression of the killer cell lectin-like receptor G1 (KLRG1), the mouse homolog of MAFA, is modulated by MHC class I molecules.

Using a new mAb, 2F1, we characterize a mouse natural killer (NK) cell antigen termed 'killer cell lectin-like receptor G1' (KLRG1; formerly mouse MAFA or 2F1-Ag). KLRG1 is expressed on 30-60% of murine NK cells, and a small fraction of T cells, and is composed of a homodimer of glycosylated 30-38-kDa subunits. Strikingly, cell surface expression of KLRG1 by NK cells was substantially down-regulated in mice deficient for expression of class I molecules, in contrast to the Ly49 lectin-like NK receptors, which are up-regulated in class I-deficient mice. We could not demonstrate binding of KLRG1 to class I molecules in a cell-cell adhesion assay. Transgenic expression of KLRG1 under heterologous transcription elements was unaffected by class I deficiency, indicating that class I molecules do not affect the KLRG1 protein directly, and suggesting that regulation is at the level of expression of the endogenous KLRG1 gene. Evidence is presented that class I molecules regulate KLRG1 via interactions with class I-specific inhibitory Ly49 molecules and SHP-1 signaling. Thus, although KLRG1 and Ly49 molecules are both lectin-like inhibitory receptors that are regulated by class I expression, the effects of class I on the cell surface expression of the molecules are opposing, and the underlying regulatory mechanisms are distinct.

Recognition of the class Ib molecule Qa-1(b) by putative activating receptors CD94/NKG2C and CD94/NKG2E on mouse natural killer cells.

The heterodimeric CD94/NKG2A receptor, expressed by mouse natural killer (NK) cells, transduces inhibitory signals upon recognition of its ligand, Qa-1(b), a nonclassical major histocompatibility complex class Ib molecule. Here we clone and express two additional receptors, CD94/NKG2C and CD94/NKG2E, which we show also bind to Qa-1(b). Within their extracellular carbohydrate recognition domains, NKG2C and NKG2E share extensive homology with NKG2A (93-95% amino acid similarity); however, NKG2C/E receptors differ from NKG2A in their cytoplasmic domains (only 33% similarity) and contain features that suggest that CD94/NKG2C and CD94/NKG2E may be activating receptors. We employ a novel blocking anti-NKG2 monoclonal antibody to provide the first direct evidence that CD94/NKG2 molecules are the only Qa-1(b) receptors on NK cells. Molecular analysis reveals that NKG2C and NKG2E messages are extensively alternatively spliced and approximately 20-fold less abundant than NKG2A message in NK cells. The organization of the mouse Cd94/Nkg2 gene cluster, presented here, shows striking similarity with that of the human, arguing that the entire CD94/NKG2 receptor system is relatively primitive in origin. Analysis of synonymous substitution frequencies suggests that within a species, NKG2 genes may maintain similarities with each other by concerted evolution, possibly involving gene conversion-like events. These findings have implications for understanding NK cells and also raise new possibilities for the role of Qa-1 in immune responses.

Impaired anti-viral T cell responses due to expression of the Ly49A inhibitory receptor.

Inhibitory receptors specific for alleles of MHC class I proteins play an important role in determining the reactivity and specificity of NK cells. To determine whether these receptors are also able to regulate T cell functions, we have studied anti-viral immune responses in mice transgenic for a class I-specific inhibitory receptor, Ly49A. Although nontransgenic mice express Ly49A primarily on NK cells and some T cells, the Ly49A transgenic mice express Ly49A on all lymphocytes, including T cells. We have assessed the activation, expansion, cytokine production, and cytotoxic activity of CD8 T cells in both transgenic and nontransgenic mice following infection with lymphocytic choriomeningitis virus. As expected, nontransgenic mice made a potent virus-specific CD8 T cell response following virus infection. However, as measured in cytolysis assays and by cytokine production, virus-specific CD8 T cell activity was reduced in Ly49A transgenic mice. This inhibition was largely, but not always exclusively, dependent upon the presence, either in vivo or in vitro, of the Ly49A ligand, H-2Dd. Strikingly Ly49A transgenic mice have reduced capacity to control infection with the virulent lymphocytic choriomeningitis virus variant clone 13. Overall, these studies demonstrate that expression of killer inhibitory receptors can modulate anti-viral T cell responses in vivo and in vitro.

2F1 antigen, the mouse homolog of the rat "mast cell function-associated antigen", is a lectin-like type II transmembrane receptor expressed by natural killer cells.

Inhibitory lectin-like receptors expressed on the surface of hematopoietic cells are critically involved in regulation of their effector functions. Here we report that a novel mAb specific for mouse NK cells, 2F1, recognizes the mouse homolog of the mast cell function-associated antigen (MAFA), an inhibitory lectin-like transmembrane receptor expressed on rat mast cells. The 2F1 antigen (2F1-Ag) and rat MAFA are structurally highly conserved and contain a cytoplasmic motif similar to the immunoreceptor tyrosine-based inhibitory motif that is presumably utilized for inhibitory signaling. We also identified a human homolog that is closely related to the rodent MAFA/2F1-Ag proteins. Like rat MAFA, 2F1-Ag is probably encoded by a single gene, which exhibits relatively little polymorphism. Strikingly, while rat MAFA is considered a mast cell antigen, we have been unable to detect cell surface expression of 2F1-Ag by mouse mast cell lines, bone marrow-derived mast cells, or peritoneal mast cells. Furthermore, mouse bone marrow-derived mast cells were devoid of 2F1-Ag mRNA. Instead, we find that approximately 40% of mouse NK cells express 2F1-Ag. Thus, MAFA/2F1-Ag may modulate immunological responses on at least two different cell types bridging the specific and innate immune system.

Mouse CD94/NKG2A is a natural killer cell receptor for the nonclassical major histocompatibility complex (MHC) class I molecule Qa-1(b).

Natural killer (NK) cells preferentially lyse targets that express reduced levels of major histocompatibility complex (MHC) class I proteins. To date, the only known mouse NK receptors for MHC class I belong to the Ly49 family of C-type lectin homodimers. Here, we report the cloning of mouse NKG2A, and demonstrate it forms an additional and distinct class I receptor, a CD94/NKG2A heterodimer. Using soluble tetramers of the nonclassical class I molecule Qa-1(b), we provide direct evidence that CD94/NKG2A recognizes Qa-1(b). We further demonstrate that NK recognition of Qa-1(b) results in the inhibition of target cell lysis. Inhibition appears to depend on the presence of Qdm, a Qa-1(b)-binding peptide derived from the signal sequences of some classical class I molecules. Mouse NKG2A maps adjacent to CD94 in the heart of the NK complex on mouse chromosome six, one of a small cluster of NKG2-like genes. Our findings suggest that mouse NK cells, like their human counterparts, use multiple mechanisms to survey class I expression on target cells.

Cloning of a mouse homolog of CD94 extends the family of C-type lectins on murine natural killer cells.

Two families of major histocompatibility complex (MHC) class I-specific receptors are found on natural killer (NK) cells: immunoglobulin-like receptors and C-type lectin receptors. In mice, the latter category is represented by the Ly49 family of receptors, whereas in humans, NK cells express the distantly related CD94, which forms MHC class I-specific heterodimers with NKG2 family members. Humans also express the MHC class I-specific p50/p58/p70 family of immunoglobulin-like receptors, but these have not been identified in mice. Hence, there is no known instance of an MHC class I-specific receptor that is expressed by both human and murine NK cells. Here we report the cloning of CD94 from the CB.17 and C57BL/6 strains of mice. Mouse CD94 is 54% identical and 66% similar to human CD94, and is also a member of the C-type lectin superfamily. Mouse CD94 is expressed efficiently on the cell surface of cells transiently transfected with the corresponding cDNA, but surface CD94 was unable to mediate detectable binding to MHC class I-expressing ConA blasts. Notably, mouse CD94, like human CD94, has a very short cytoplasmic tail, suggesting the existence of partner chains that may play a role in ligand binding and signaling. Like many other C-type lectins expressed by NK cells, mouse CD94 maps to the NK complex on distal chromosome 6, synteneic to human CD94. We also demonstrate that mouse CD94 is highly expressed specifically by mouse NK cells, raising the possibility that mice, like humans, express multiple families of MHC class I-specific receptors on their NK cells. Murine homologs of human NKG2 family members have not yet been identified, but we report here the existence of a murine NKG2D-like sequence that also maps to the murine NK complex near CD94 and Ly49 family members.

Inhibition of liver microsome-mediated mutagenesis, metabolism and DNA-binding of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in the rat following glucose administration.

Aroclor 1254-induced rat liver microsomes prepared from control and glucose-treated rats (30% glucose in drinking water 48 h prior to sacrifice) were used in studies of benzo[a]pyrene (BaP) and BaP 7,8-dihydrodiol (BaP 7,8-DHD)-induced mutagenesis in Salmonella typhimurium TA100. Microsome-dependent metabolism and metabolite binding of BaP and BaP 7,8-DHD to calf thymus DNA was also investigated. BaP-induced mutagenesis in TA100 was inhibited 27% and BaP 7,8-DHD-induced mutagenesis was inhibited 55% by microsomes from glucose-treated rats. [3H]BaP and [3H]BaP 7,8-DHD metabolite binding to DNA was inhibited 17% and 20%, respectively. High performance liquid chromatographic (hplc) analysis of enzyme-hydrolyzed DNA yielded 7R and 7S-diol epoxide-1 deoxyguanosine (BPDE-1:dG) adducts and BPDE-2:dG adducts of [3H]BaP and [3H]BaP 7,8-DHD. These adducts were inhibited 38% and 50%, respectively, by microsomes from glucose-treated rats. Hplc analysis of organosoluble metabolites of [3H]BaP and [3H]BaP 7,8-DHD showed an inhibition of metabolism of 28% and 50%, respectively, by microsomes from glucose-treated rats. The inhibition of metabolism correlated with the effect of glucose treatment on inhibition of BaP and BaP 7,8-DHD-induced mutagenesis and adduct formation. These results suggest that the mechanism by which glucose produces its effects on mutagenesis, DNA-binding and adduct formation is by an inhibition of microsome-mediated metabolism of BaP and BaP 7,8-DHD.

Effect of tannic acid on rat liver S9 mediated mutagenesis, metabolism and DNA binding of benzo[a]pyrene.

Tannic acid, a naturally occurring plant phenol, inhibited rat liver S9 mediated mutagenesis of benzo[a]pyrene in Salmonella typhimurium by 32-77% at concentrations of 5-50 micrograms/mutagenesis plate. Tannic acid (10-40 microM) had no affect on the formation of organosoluble metabolites of benzo[a]pyrene or of its water-soluble conjugates. It did, however, inhibit benzo[a]pyrene (B[a]P) metabolite binding to calf thymus DNA by 40% at a concentration of 40 microM and inhibited benzo[a]pyrene 7,8-dihydrodiol-9,10-epoxide (BPDE): deoxyguanosine adduct formation in calf thymus DNA by 12-54% at concentrations of 10-40 microM. These results suggest that the antimutagenic effect of tannic acid and inhibition of B[a]P metabolite binding to DNA is by a previously described scavenging mechanism and/or by a DNA-affinity binding mechanism that prevents BPDE interaction with DNA as previously described for ellagic acid.