Little is known regarding the ligand specificity of Ly-49 activating receptor subfamily members expressed by NK cells. A new Ly-49 activating receptor related to Ly-49A in its extracellular domain, designated Ly-49P, was recently cloned from 129 strain mice. We independently cloned an apparent allele of Ly-49P expressed by nonobese diabetic and nonobese diabetes-resistant mouse strain NK cells. We found it to be reactive with the A1 Ab thought to recognize a polymorphic epitope expressed only by the Ly-49A inhibitory receptor of the C57BL/6 strain. Rat RNK-16 cells transfected with Ly-49P mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49P can activate NK-mediated lysis. We determined that RNK-16 lysis of Con A blasts induced by Ly-49P was MHC dependent, resulting in efficient lysis of H-2Dd-bearing targets. We found that the Dd α1/α2 domain is required for Ly-49P-mediated RNK-16 activation, as determined by exon shuffling and transfection. Thus, Ly-49P is the second activating Ly-49 receptor demonstrated to induce NK cytotoxicity by recognizing a class I MHC molecule.
The diversity and ligand specificity of activating Ly-49 receptors expressed by murine NK cells are largely unknown. We cloned a new Ly-49-activating receptor, expressed by NK cells of the nonobese diabetic mouse strain, which we have designated Ly-49W. Ly-49W is highly related to the known inhibitory receptor Ly-49G in its carbohydrate recognition domain, exhibiting 97.6% amino acid identity in this region. We demonstrate that the 4D11 and Cwy-3 Abs, thought to be Ly-49G specific, also recognize Ly-49W. Rat RNK-16 cells transfected with Ly-49W mediated reverse Ab-dependent cellular cytotoxicity of FcR-positive target cells, indicating that Ly-49W can activate NK-mediated lysis. We further show that Ly-49W is allo-MHC specific: Ly-49W transfectants of RNK-16 only lysed Con A blasts expressing H-2k or H-2d haplotypes, and Ab-blocking experiments indicated that H-2Dk and Dd are ligands for Ly-49W. Ly-49W is the first activating Ly-49 receptor demonstrated to recognize an H-2k class I product. Ly-49G and Ly-49W represent a new pair of NK receptors with very similar ligand-binding domains, but opposite signaling functions.
The Ly-49 multigene receptor family regulates mouse NK cell functions. A number of Ly-49 genes exhibit allelic variation, but the functional significance of allelic differences in extracellular domains of Ly-49 receptors regarding ligand specificity is largely unknown. Amino acid differences exist in the extracellular domains of the B6 and BALB/c allele products of the inhibitory Ly-49G receptor. We constructed chimeric Ly-49 receptors consisting of common cytoplasmic and transmembrane regions of the activating Ly-49W receptor fused with the ectodomains of the B6 and BALB/c alleles of Ly-49G. Expression of these chimeras in the RNK-16 rat NK cell line allowed us to study the specificity of inhibitory receptor ectodomains as they stimulated NK lytic activity. We found that the ectodomain of the BALB/c allele of Ly-49G recognizes both H-2Dd and Dk class I MHC alleles, whereas the ectodomain of the B6 allele of Ly-49G recognizes Dd, and not Dk. The specificity for Dk as well as Dd of the wild-type Ly-49GBALB/c allele product was confirmed with RNK-16 transfectants of this inhibitory receptor. Furthermore, the ectodomain of the Ly-49GBALB/c allele recognizes a distinct repertoire of xenogeneic ligands that only partially overlaps with that recognized by Ly-49GB6. Our results indicate that allelic variation in Ly-49 extracellular domains can have functional significance by altering Ly-49 receptor specificity for mouse class I MHC and xenogeneic ligands.
CTL lyse target cells through the release of cytolytic granule mediators and expression of the death receptor ligand Fas ligand (FasL). We previously demonstrated that FasL is stored in vesicles distinct from cytolytic granules and is translocated to the cell surface within 15 min of TCR stimulation, followed by a later wave of newly synthesized FasL cell surface expression at 2 h poststimulation. Initial studies suggested that the two FasL responses had different signaling thresholds. To test this possibility directly, we titrated Ag presented to murine CTL to measure FasL and degranulation response thresholds. Stored FasL translocation to the cell surface required substantially lower concentrations of peptide than was required for de novo expression of FasL and degranulation. Furthermore, a low-affinity agonist peptide stimulated strong stored FasL translocation but only limited de novo FasL expression and degranulation. These data imply that the two FasL populations may have distinct functions. We examined bystander killing and found that the rapidly expressed FasL triggered highly specific lysis of target cells, as did degranulation. In contrast, the newly synthesized later wave of FasL mediated extensive Fas-dependent bystander killing. Our data indicate that stored FasL is mobilized in response to low concentrations of Ag to mediate rapid, highly specific lysis of target cells, whereas the later, newly synthesized FasL requires higher concentrations of Ag and mediates indiscriminate lysis. These findings suggest that early and late FasL and degranulation represent nonredundant lytic mechanisms that have been selected for distinct situations, possibly for optimal pathogen clearance.
Ly-49A molecules negatively regulate a subset of mouse natural killer (NK) cells, preventing lysis of H-2Dd-expressing target cells. In the present report, we immunoaffinity-purified Ly-49A from the EL4 lymphoma using the A1 monoclonal antibody (mAb) and examined cell adhesion to immobilized Ly-49A. Adhesion was observed by cells expressing relatively high levels of H-2Dd, but not cells expressing very low or no cell surface Dd, while antibodies specific for Dd or Ly-49A inhibited the cell binding, indicating that Dd and Ly-49A mediate the observed adhesion. The density of immobilized Ly-49A was varied and confirmed by ELISA. Cell binding exhibited a threshold Ly-49A density requirement, and above this threshold, increases in Ly-49A density resulted in substantial increases in cell adhesion to a high maximum cell binding. The density of Ly-49A homodimers required to mediate cell adhesion was found to be quite low: 140-250 molecules/microm2. These results suggest that the avidity of Ly-49A for Dd is relatively high and indicate that small changes in Ly-49A density near the threshold result in large changes in stable Ly-49A receptor engagement. The relatively sharp threshold and marked density dependence presented here for Ly-49A receptor engagement may explain the observation that relatively small differences in Ly-49A expression level on NK cells result in significant differences in functional outcome, i.e. whether a target cell expressing a low level of Dd is spared from lysis or not.
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