The protein HLA-E is a non-classical major histocompatibility complex (MHC) molecule of limited sequence variability. Its expression on the cell surface is regulated by the binding of peptides derived from the signal sequence of some other MHC class I molecules. Here we report the identification of ligands for HLA-E. We constructed tetramers in which recombinant HLA-E and beta2-microglobulin were refolded with an MHC leader-sequence peptide, biotinylated, and conjugated to phycoerythrin-labelled Extravidin. This HLA-E tetramer bound to natural killer (NK) cells and a small subset of T cells from peripheral blood. On transfectants, the tetramer bound to the CD94/NKG2A, CD94/NKGK2B and CD94/NKG2C NK cell receptors, but did not bind to the immunoglobulin family of NK cell receptors (KIR). Surface expression of HLA-E was enough to protect target cells from lysis by CD94/NKG2A+ NK-cell clones. A subset of HLA class I alleles has been shown to inhibit killing by CD94/NKG2A+ NK-cell clones. Only the HLA alleles that possess a leader peptide capable of upregulating HLA-E surface expression confer resistance to NK-cell-mediated lysis, implying that their action is mediated by HLA-E, the predominant ligand for the NK cell inhibitory receptor CD94/NKG2A.

The nonclassical major histocompatibility complex (MHC) class I molecule HLA-E inhibits natural killer (NK) cell-mediated lysis by interacting with CD94/NKG2A receptors. Surface expression of HLA-E depends on binding of conserved peptides derived from MHC class I molecules. The same peptide is present in the leader sequence of the human cytomegalovirus (HCMV) glycoprotein UL40 (gpUL40). It is shown that, independently of the transporter associated with antigen processing, gpUL40 can up-regulate expression of HLA-E, which protects targets from NK cell lysis. While classical MHC class I molecules are down-regulated, HLA-E is up-regulated by HCMV. Induction of HLA-E surface expression by gpUL40 may represent an escape route for HCMV.

Ig-like transcript 4 (ILT4) (also known as leukocyte Ig-like receptor 2, CD85d, and LILRB2) is a cell surface receptor expressed mainly on myelomonocytic cells, whereas ILT2 (also known as leukocyte Ig-like receptor 1, CD85j, and LILRB1) is expressed on a wider range of immune cells including subsets of natural killer and T cells. Both ILTs contain immunoreceptor tyrosine-based inhibitory receptor motifs in their cytoplasmic tails that inhibit cellular responses by recruiting phosphatases such as SHP-1 (Src homology 2 domain containing tyrosine phosphatase 1). Although these ILTs have been shown to recognize a broad range of classical and nonclassical human MHC class I molecules (MHCIs), their precise binding properties remain controversial. We have used surface plasmon resonance to analyze the interaction of soluble forms of ILT4 and ILT2 with several MHCIs. Although the range of affinities measured was quite broad (K d ‫؍‬ 2-45 M), some interesting differences were observed. ILT2 generally bound with a 2-to 3-fold higher affinity than ILT4 to the same MHCI. Furthermore, ILT2 and ILT4 bound to HLA-G with a 3-to 4-fold higher affinity than to classical MHCIs, suggesting that ILT͞HLA-G recognition may play a dominant role in the regulation of natural killer, T, and myelomonocytic cell activation. Finally, we show that ILT2 and ILT4 effectively compete with CD8 for MHCI binding, raising the possibility that ILT2 modulates CD8 ؉ T cell activation by blocking the CD8 binding as well as by recruiting inhibitory molecules through its immunoreceptor tyrosine-based inhibitory receptor motif.leukocyte Ig-like receptors ͉ major histocompatibility complex ͉ surface plasmon resonance ͉ natural killer cell ͉ coreceptor I g-like transcripts (ILTs) (also called leukocyte Ig-like receptors, CD85, or LILRB) are encoded by a family of immunoreceptor genes located at human chromosome 19q13.4. This locus is called the leukocyte receptor complex and includes, in addition to ILT genes, the genes encoding killer cell Ig-like receptors (KIRs), leukocyte-associated Ig-like receptors, NKp46, and the Fc␣ receptor (1). Although ILT2 is broadly expressed on monocytes, B cells, dendritic cells, and subsets of natural killer (NK) and T cells, ILT4 expression is largely confined to the myelomonocytic lineage (2-8). Both ILT2 and ILT4 have four tandem Ig-like extracellular domains and four and three immunoreceptor tyrosine-based inhibitory receptor motifs, respectively, in their cytoplasmic tails. Immunoreceptor tyrosine-based inhibitory receptor motifs recruit the protein tyrosine phosphatase SHP-1 (Src homology 2 domain containing phosphatase 1), which is thought to inhibit early signaling events triggered by stimulatory receptors. Indeed engagement of ILT2 on T cells has been shown to inhibit T cell antigen receptor (TCR) signaling and downstream events such as actin reorganization (9). Studies on CD8 ϩ cells suggest that ILT2 is expressed early on in contrast to KIRs, which are expressed primarily on the subset of stimulated CD8 ϩ cells tha...

Human histocompatibility leukocyte antigen E (HLA-E) and mouse major histocompatibility complex (MHC) class Ib antigen, Qa-1, share the same substitutions at two normally conserved positions 143 and 147, which are likely to affect binding of the C terminus of peptides. Qa-1 is able to bind a peptide derived from the leader sequence of H-2 D and H-2 L molecules. We developed a peptide binding assay in vitro to compare the binding specificity of HLA-E with the mouse MHC class Ib molecule Qa-1. We demonstrate that HLA-E binds, although poorly, the peptide which binds to Qa-1 and that it also binds nonamer signal sequence-derived peptides from human MHC class I molecules. Using alanine and glycine substitutions, we could define primary anchor residues at positions 2 and 9 and secondary anchor residues at position 7 and possibly 3.

Celiac disease is an intestinal inflammatory disorder induced by dietary gluten in genetically susceptible individuals. The mechanisms underlying the massive expansion of interferon γ–producing intraepithelial cytotoxic T lymphocytes (CTLs) and the destruction of the epithelial cells lining the small intestine of celiac patients have remained elusive. We report massive oligoclonal expansions of intraepithelial CTLs that exhibit a profound genetic reprogramming of natural killer (NK) functions. These CTLs aberrantly expressed cytolytic NK lineage receptors, such as NKG2C, NKp44, and NKp46, which associate with adaptor molecules bearing immunoreceptor tyrosine-based activation motifs and induce ZAP-70 phosphorylation, cytokine secretion, and proliferation independently of T cell receptor signaling. This NK transformation of CTLs may underlie both the self-perpetuating, gluten-independent tissue damage and the uncontrolled CTL expansion leading to malignant lymphomas in severe forms of celiac disease. Because similar changes were detected in a subset of CTLs from cytomegalovirus-seropositive patients, we suggest that a stepwise transformation of CTLs into NK-like cells may underlie immunopathology in various chronic infectious and inflammatory diseases.

Human NK cells and subsets of T cells or NKT cells express the orphan C-type lectin receptor CD161 (NKR-P1A) of unknown function. In contrast to rodents that possess several NKR-P1 genes coding for either activating or inhibitory receptors, the nature of signals delivered by the single human NKR-P1A receptor is still to be clarified. In this article, we show that the lectin-like transcript 1 (LLT1) molecule is a ligand for the CD161 receptor. Engagement of CD161 on NK cells with LLT1 expressed on target cells inhibited NK cell-mediated cytotoxicity and IFN-γ secretion. Conversely, LLT1/CD161 interaction in the presence of a TCR signal enhanced IFN-γ production by T cells. These findings identify a novel ligand/receptor pair that differentially regulate NK and T cell functions.

The recognition of MHC class I molecules by killer cell immunoglobulin-like receptors (KIR) is central to the control of NK cell function and can also modulate the CTL activation threshold. Among KIR receptors, KIR3DL2 is thought to interact with HLA-A3 and -A11, although direct evidence has been lacking. In this study, we show that HLA-A3 and -A11 tetramers specifically bind to KIR3DL2*001 transfectants and that this recognition is peptide-specific. Single amino acid substitutions in the nonamer peptide underline a critical role for residue 8 in recognition of KIR3DL2. However, the role of this interaction in vivo still remains to be established.

These experiments establish that surface expression of HLA-E is regulated by the binding of a restricted pool of peptides from the leader sequence of MHC class I molecules. The correlation between HLA-E and MHC class I surface expression might be relevant to the function of HLA-E. Our results also show that, although these HLA-E binding peptides are derived from signal sequences, they may be released back into the cytosol and subsequently translocated by the TAP complex and loaded onto HLA-E molecules.