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.

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-D d . To compare Ly-49D and Ly-49A interactions with H2-D d , we created mutations in H2-D d 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-D d α 1 helix or the α 2 helix abrogated Ly-49D–mediated cytotoxicity, but these changes had no significant effect on Ly-49A–dependent inhibition. Each of three α 2 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-D d interactions require distinct determinants compared with Ly-49A/H2-D d interactions. These differences have important implications for the integration of activating and inhibitory signals in NK cells.

Inhibitory receptors expressed on natural killer (NK) cells abrogate positive signals upon binding corresponding major histocompatibility complex (MHC) class I molecules on various target cells. By directly micromanipulating the effector–target cell encounter using an optical tweezers system which allowed temporal and spatial control, we demonstrate that Ly49–MHC class I interactions prevent characteristic cellular responses in NK cells upon binding to target cells. Furthermore, using this system, we directly demonstrate that an NK cell already bound to a resistant target cell may simultaneously bind and kill a susceptible target cell. Thus, although Ly49-mediated inhibitory signals can prevent many types of effector responses, they do not globally inhibit cellular function, but rather the inhibitory signal is spatially restricted towards resistant targets.

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-2D d , D b , K k , or K b . RNK.mLy-49D cells lysed YB2/0.D d targets more efficiently than untransfected YB2/0 or YB2/0 transfected with D b , K k , or K b . This augmented lysis of H-2D d targets was specifically inhibited by F(ab′) 2 anti–Ly-49D (12A8) and F(ab′) 2 anti–H-2D d (34-5-8S). RNK.mLy-49D effectors were also able to specifically lyse Concanavalin A blasts isolated from H-2 d mice (BALB/c, B10.D2, and DBA/2) but not from H-2 b or H-2 k mice. These experiments show that the activating receptor Ly-49D specifically interacts with the MHC I antigen, H-2D d , demonstrating the existence of alloactivating receptors on murine NK cells.

The lytic activity of natural killer (NK) cells is inhibited by the expression of class I major histocompatibility complex (MHC) antigens on target cells. In murine NK cells, Ly-49A mediates inhibition of cytotoxicity in response to the class I MHC antigen H-2D d . In this report, we studied the function of mouse Ly-49A in both the rat NK cell tumor line, RNK-16, transfected with Ly-49A cDNA, and in primary NK cells. We show that ligation of Ly-49A by H-2D d inhibits early signaling events during target cell stimulation, including polyphosphoinositide turnover and tyrosine phosphorylation. We also show that Ly-49A directly associates with the cytoplasmic tyrosine phosphatase SHP-1, and that Ly-49A function is impaired in NK cells from SHP-1 mutant viable motheaten mice and from SHP-1–deficient motheaten mice. Finally, we demonstrate that mutational substitution of the tyrosine within the proposed SHP-1 binding motif in Ly-49A completely abrogates inhibition of NK cell cytotoxicity through this receptor. These results demonstrate that Ly-49A interrupts early activating signals in NK cells, and that SHP-1 is an important mediator of Ly-49A function.