Charles L. Sentman

Host MHC class I gene control of NK-cell specificity in the mouse

Summary: The missing self model predicts chat NK cells adapt somatically to the type as well as levels of MHC class I products expressed by their host, Transgenic and gene knock‐out mice have provided conclusive evidence chat MHC class I genes control specificity and tolerance of NK cells. The article describes this control and discusses the POSSIBLE Mechanisms behind it. starting from a genetic model to study how natural resistance to tumors is influenced by MHC class I expression in the host as well as in the target cells. Data on host gene regulation of NK‐cell functional specificity as well as Lγ49 receptor expression are reviewed, leading up to the central question: how does the system develop and maintain “useful” NK cells, while avoiding “harmful” and “useless” ones? The available data can be fitted with in each of two mutually non‐exclusive models: cellular adaptation and clonal selection. Recent studies supporting cellular adaptation bring the focus on different possibilities within this general mechanism, such as energy, receptor calibration and, most importantly, whether the specificity of each NK cell is permanently fixed or subject to continuous regulation.

THE CRYSTAL STRUCTURE OF H-2DD MHC CLASS I COMPLEXED WITH THE HIV-1-DERIVED PEPTIDE P18-I10 AT 2.4 A RESOLUTION : IMPLICATIONS FOR T CELL AND NK CELL RECOGNITION

The structure of H-2Dd complexed with the HIV-derived peptide P18-I10 (RGPGRAFVTI) has been determined by X-ray crystallography at 2.4 A resolution. This MHC class I molecule has an unusual binding motif with four anchor residues in the peptide (G2, P3, R/K/H5, and I/L/F9 or 10). The cleft architecture of H-2Dd includes a deep narrow passage accomodating the N-terminal part of the peptide, explaining the obligatory G2P3 anchor motif. Toward the C-terminal half of the peptide, p5R to p8V form a type I reverse turn; residues p6A to p9T, and in particular p7F, are readily exposed. The structure is discussed in relation to functional data available for T cell and natural killer cell recognition of the H-2Dd molecule.

MHC Class I-Ly49 Interactions Shape the Ly49 Repertoire on Murine NK Cells

This study aims to determine how the interaction of Ly49 receptors with MHC class I molecules shapes the development of the Ly49 repertoire. We have examined the percentage of NK cells that expressed Ly49A, Ly49G2, and Ly49D in single and double Ly49A/C-transgenic mice on four different MHC backgrounds, H-2b, H-2d, H-2b/d, and β2-microglobulin−/−. The results show that the total numbers of NK cells were not different among the strains. The prior expression of a Ly49 receptor capable of binding to self MHC class I altered the percentage of NK cells expressing endogenous Ly49A, Ly49G2, and Ly49D even in mice in which no MHC ligand was present for the latter receptors. The NK cells in the Ly49-transgenic mice expressed the same level of endogenous Ly49 receptors as wild-type mice of a similar MHC background. In contrast, the number of NK T cells was reduced in mice in which the Ly49 transgene could bind to a MHC class I molecule. The onset of Ly49 receptor expression on NK cells during ontogeny was not altered in the presence of transgenic Ly49 receptors. These data support a sequential model and argue against a selection model for Ly49 repertoire development on NK cells.

Expression of Ly49A on T cells alters the threshold for T cell responses

In this study we investigated the balance between activating and inhibitory signals during T cell activation. We have used transgenic mice in which CD8+ T cells expressed an inhibitory receptor, Ly49A, and a specific activating αu2009β TCR. This TCR recognizes an lymphocytic choriomeningitis virus peptide in combination with H‐2Db. We observed a quantitative influence on cellular responses that depended upon the activating signals received through the TCR and the inhibitory signals received through Ly49A. By varying the peptide concentration given to stimulating cells or target cells, we could adjust the amount of ligand available to trigger the TCR. At low doses of peptide, Ly49A‐expressing T cells were unresponsive on target cells that expressed H‐2Dd, but responded against target cells without H‐2Dd. However, this inhibition could be overcome by increasing the peptide concentration or by addition of anti‐Ly49A F(ab)2 fragments. Thus, rather than behaving as simple off switches, our data indicate that Ly49 receptors modulate T cell signaling so that higher amounts of activating signals are required for effector‐cell responses.

Murine class I major histocompatibility complex H–2Dd: expression, refolding and crystallization

A truncated soluble form of the murine class I major histocompatibility antigen complex H-2Dd was cloned using an Escherichia coli based system. It was expressed, refolded in vitro and crystallized in a complex with murine beta2 microglobulin and the peptide RGPGRAFVTI from the V3-loop of the gp160 HIV-1 protein. Crystals belonging to the space group P212121 with cell dimensions a = 51.3, b = 92.5, c = 108.8 A were obtained using two different crystallization conditions. The crystals contain one complex per asymmetric unit and diffract to at least 2.4 A resolution.

Ly49A inhibitory receptors redistribute on natural killer cells during target cell interaction.

When T effector cells meet antigen‐bearing target cells, there is a specific accumulation of T‐cell receptors, co‐receptors and structural proteins at the point of cell–cell contact. Ly49 inhibitory receptors bind to murine major histocompatibility complex (MHC) class I molecules and prevent natural killer‐(NK) cell cytotoxicity. In this study we have tested whether inhibitory receptors accumulate at the point of cell–cell contact when NK cells encounter target cells bearing MHC class I ligands for those inhibitory receptors. We have used RNK‐16 effector cells that express Ly49A receptors and have found that there was a specific accumulation of Ly49A receptors at the point of NK cell–target cell contact when the target cells expressed H‐2Dd. We also observed that engagement of Ly49A on NK cells resulted in an altered redistribution of potential triggering receptors CD2 and NKR‐P1. These data indicate that inhibitory receptors, like activating receptors, may specifically aggregate at the point of cell–cell contact which may be necessary for them to mediate their full inhibitory effect.

Purified MHC class I molecules inhibit activated NK cells in a cell-free system in vitro.

Natural killer cells have been shown to interact with MHC class I molecules via inhibitory receptors. However, it is not known whether the inhibition induced by MHC class I molecules requires other NK cell‐target cell interactions. Thus, we examined whether purified MHC class I molecules alone were able to inhibit NK cell function. Purified H‐2Kb and H‐2Db molecules inhibited the release of IFN‐γ from spleen (H‐2b)‐derived lymphokine‐activated killer (LAK) cell cultures stimulated by anti‐NK1.1 antibody in a concentration‐dependent manner. LAK cells generated from newborn mice that express low levels of MHC class I binding Ly49 inhibitory receptors were significantly less sensitive to inhibition by H‐2Kb compared to LAK cells from adult mice. Furthermore, LAK cells generated from spleen cells of Ly49C‐transgenic mice were significantly more sensitive to inhibition by H‐2Kb compared to non‐transgenic littermates. Taken together, the data indicate that MHC class I induced inhibition of NK cell mediated effector functions, as assessed by IFN‐γ release after NK1.1 triggering, does not require additional cell surface molecules other than MHC class I.

H-2Dd engagement of Ly49A leads directly to Ly49A phosphorylation and recruitment of SHP1

We have used a number of in vitro and in vivo techniques to identify the molecules that can bind to the cytoplasmic tail of the Ly49A receptor. Affinity chromatography using peptides corresponding to the N‐terminal 18 amino acids of Ly49A allowed the recovery of a number of proteins that bound preferentially to the tyrosine‐phosphorylated peptide, including SH2‐containing phosphatase‐1 (SHP1) and the SH2‐containing inositol 5′ phosphatase (SHIP). In another approach, using the entire cytoplasmic domain of the Ly49A receptor, we found that SHP2 also interacted with the tyrosine‐phosphorylated form of the Ly49A cytoplasmic tail. Using BIACORE®2000 analysis, we determined that both SHP1 and SHP2 bound to the tyrosine‐phosphorylated cytoplasmic tail of Ly49A with affinities in the nanomolar range, whilst SHIP showed no binding. Mutation of tyrosine‐36 to phenylalanine did not significantly affect the affinities of these proteins for the tyrosine‐phosphorylated cytoplasmic tail of Ly49A. In addition, using a whole‐cell system with T‐cell lymphoma cell lines that expressed the Ly49A receptor or its H‐2Dd ligand, we determined that engagement of Ly49A by its major histocompatibility complex (MHC) ligand leads to tyrosine‐phosphorylation events and recruitment of SHP1. Recruitment of SHP1 was rapid and transient, reaching a maximum after 5u2003min. These data suggest that mechanisms for the inhibitory signal are generated following receptor engagement. They also provide direct evidence that ligand engagement of the Ly49A receptor is responsible for recruitment of downstream signalling molecules.

Modulation of Ly49A receptors on mature cells to changes in major histocompatibility complex class I molecules.

The expression of murine Ly49 receptors on natural killer (NK) cells and NK1.1+ Tu2003cells is believed to prevent these cells from responding against normal self‐tissues. In this report we investigated whether the expression level of Ly49A was fixed on mature cells or if it could be adapted as the major histocompatibility complex (MHC) class I environment changed in vivo. By transferring peripheral Tu2003cells from Ly49A transgenic mice into BALB/c nude/nude and B6 nude/nude mice, we demonstrated that mature cells modulate their Ly49A receptor expression relative to the in vivo MHC class I environment. These results indicated that the expression of the inhibitory Ly49A receptor is not permanently fixed during a maturation and/or education process but rather is adapted to MHC class I changes on the surrounding cells.