Activating Ly-49d and Inhibitory Ly-49a Natural Killer Cell Receptors Demonstrate Distinct Requirements for Interaction with H2-Dd

The activating Ly-49D receptor and the inhibitory Ly-49A receptor mediate opposing effects on natural killer (NK) cell cytotoxicity after interaction with the same major histocompatibility complex ligand, H2-Dd. To compare Ly-49D and Ly-49A interactions with H2-Dd, we created mutations in H2-Dd and examined the functional ability of these mutants to activate lysis through Ly-49D or to inhibit lysis through Ly-49A. Specific single amino acid changes in either the H2-Dd α1 helix or the α2 helix abrogated Ly-49D–mediated cytotoxicity, but these changes had no significant effect on Ly-49A–dependent inhibition. Each of three α2 domain mutations in the floor of the peptide binding groove reduced functional recognition by either Ly-49D or Ly-49A, but all three were required to fully abrogate inhibition by Ly-49A. Our studies indicate that Ly-49D/H2-Dd interactions require distinct determinants compared with Ly-49A/H2-Dd interactions. These differences have important implications for the integration of activating and inhibitory signals in NK cells

Mouse Ly-49D Recognizes H-2Dd and Activates Natural Killer Cell Cytotoxicity

Although activation of natural killer (NK) cytotoxicity is generally inhibited by target major histocompatibility complex (MHC) class I expression, subtle features of NK allorecognition suggest that NK cells possess receptors that are activated by target MHC I. The mouse Ly-49D receptor has been shown to activate NK cytotoxicity, although recognition of MHC class I has not been demonstrated previously. To define Ly-49D–ligand interactions, we transfected the mouse Ly-49D receptor into the rat NK line, RNK-16 (RNK.mLy-49D). As expected, anti– Ly-49D monoclonal antibody 12A8 specifically stimulated redirected lysis of the Fc receptor– bearing rat target YB2/0 by RNK.mLy-49D transfectants. RNK.mLy-49D effectors were tested against YB2/0 targets transfected with the mouse MHC I alleles H-2Dd, Db, Kk, or Kb. RNK.mLy-49D cells lysed YB2/0.Dd targets more efficiently than untransfected YB2/0 or YB2/0 transfected with Db, Kk, or Kb. This augmented lysis of H-2Dd targets was specifically inhibited by F(ab′)2 anti–Ly-49D (12A8) and F(ab′)2 anti–H-2Dd (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2d mice (BALB/c, B10.D2, and DBA/2) but not from H-2b or H-2k mice. These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2Dd, demonstrating the existence of alloactivating receptors on murine NK cells

TIM-2 is expressed on B cells and in liver and kidney and is a receptor for H-ferritin endocytosis

T cell immunoglobulin-domain and mucin-domain (TIM) proteins constitute a receptor family that was identified first on kidney and liver cells; recently it was also shown to be expressed on T cells. TIM-1 and -3 receptors denote different subsets of T cells and have distinct regulatory effects on T cell function. Ferritin is a spherical protein complex that is formed by 24 subunits of H- and L-ferritin. Ferritin stores iron atoms intracellularly, but it also circulates. H-ferritin, but not L-ferritin, shows saturable binding to subsets of human T and B cells, and its expression is increased in response to inflammation. We demonstrate that mouse TIM-2 is expressed on all splenic B cells, with increased levels on germinal center B cells. TIM-2 also is expressed in the liver, especially in bile duct epithelial cells, and in renal tubule cells. We further demonstrate that TIM-2 is a receptor for H-ferritin, but not for L-ferritin, and expression of TIM-2 permits the cellular uptake of H-ferritin into endosomes. This is the first identification of a receptor for ferritin and reveals a new role for TIM-2

Binding and uptake of H-ferritin are mediated by human transferrin receptor-1

Ferritin is a spherical molecule composed of 24 subunits of two types, ferritin H chain (FHC) and ferritin L chain (FLC). Ferritin stores iron within cells, but it also circulates and binds specifically and saturably to a variety of cell types. For most cell types, this binding can be mediated by ferritin composed only of FHC (HFt) but not by ferritin composed only of FLC (LFt), indicating that binding of ferritin to cells is mediated by FHC but not FLC. By using expression cloning, we identified human transferrin receptor-1 (TfR1) as an important receptor for HFt with little or no binding to LFt. In vitro, HFt can be precipitated by soluble TfR1, showing that this interaction is not dependent on other proteins. Binding of HFt to TfR1 is partially inhibited by diferric transferrin, but it is hindered little, if at all, by HFE. After binding of HFt to TfR1 on the cell surface, HFt enters both endosomes and lysosomes. TfR1 accounts for most, if not all, of the binding of HFt to mitogen-activated T and B cells, circulating reticulocytes, and all cell lines that we have studied. The demonstration that TfR1 can bind HFt as well as Tf raises the possibility that this dual receptor function may coordinate the processing and use of iron by these iron-binding molecules