Miguel López-Botet

Implications for immunosurveillance of altered HLA class I phenotypes in human tumours

HLA class I downregulation is a frequent event associated with tumour invasion and development. Altered HLA class I tumour phenotypes can have profound effects on T-cell and natural killer (NK)-cell antitumour responses. Here, Federico Garrido and colleagues analyse these altered tumour phenotypes in detail, indicating their potential relevance for implementation of immunotherapeutic protocols and strategies to overcome tumour escape mechanisms.

The ILT2(LIR1) and CD94/NKG2A NK cell receptors respectively recognize HLA-G1 and HLA-E molecules co-expressed on target cells.

Previous studies on NK recognition of HLA‐G1 employed as targets 721.221 transfectants (.221‐G1) that unknowingly co‐expressed the HLA‐E molecule, subsequently found to be a major ligand for the CD94/NKG2 receptors. In the present study we re‐evaluated the relative role played by CD94/NKG2 and ILT2(LIR1) molecules in recognition of HLA‐G1 by NK clones. We employed as targets .221‐G1 cells and a surface HLA‐E‐negative transfectant, .221‐G1(Eneg), generated by site‐directed mutagenesis of the HLA‐G1 leader sequence. The antagonistic effects of receptor‐ (i.e. CD94/NKG2A, ILT2) and ligand‐specific mAb (i.e. HLA‐G, HLA‐E) were assessed. In addition, binding of an ILT2‐Ig fusion protein to the .221‐AEH, expressing only HLA‐E, and the .221‐G1(Eneg) transfectants was analyzed. Our data demonstrate that NK recognition of cells expressing HLA‐G1 involves at least two non‐overlapping receptor‐ligand systems: the CD94/NKG2 interaction with HLA‐E, and the engagement of the ILT2(LIR1) receptor by HLA‐G1 molecules.

HLA-E-BOUND PEPTIDES INFLUENCE RECOGNITION BY INHIBITORY AND TRIGGERING CD94/NKG2 RECEPTORS : PREFERENTIAL RESPONSE TO AN HLA-G-DERIVED NONAMER

The HLA‐E class Ib molecule constitutes a major ligand for the lectin‐like CD94/NKG2 natural killer (NK) cell receptors. Specific HLA class I leader sequence‐derived nonapeptides bind to endogenous HLA‐E molecules in the HLA‐defective cell line 721.221, inducing HLA‐E surface expression, and promote CD94/NKG2A‐mediated recognition. We compared the ability of NK clones which expressed either inhibitory or activating CD94/NKG2 receptors to recognize HLA‐E molecules on the surface of 721.221 cells loaded with a panel of synthetic nonamers derived from the leader sequences of most HLA class I molecules. Our results support the notion that the primary structure of the HLA‐E‐bound peptides influences CD94/NKG2‐mediated recognition, beyond their ability to stabilize surface HLA‐E. Further, CD94/NKG2A+ NK clones appeared more sensitive to the interaction with most HLA‐E‐peptide complexes than did effector cells expressing the activating CD94/NKG2C receptor. However, a significant exception to this pattern was HLA‐E loaded with the HLA‐G‐derived nonamer. This complex triggered cytotoxicity very efficiently over a wide range of peptide concentrations, suggesting that the HLA‐E/G‐nonamer complex interacts with the CD94/NKG2 triggering receptor with a significantly higher affinity. These results raise the possibility that CD94/NKG2‐mediated recognition of HLA‐E expressed on extravillous cytotrophoblasts plays an important role in maternal‐fetal cellular interactions.

Kinetics and peptide dependency of the binding of the inhibitory NK receptor CD94/NKG2-A and the activating receptor CD94/NKG2-C to HLA-E

The lytic function of human natural killer (NK) cells is markedly influenced by recognition of class I major histocompatibility complex (MHC) molecules, a process mediated by several types of activating and inhibitory receptors expressed on the NK cell. One of the most important of these mechanisms of regulation is the recognition of the non‐classical class I MHC molecule HLA‐E, in complex with nonamer peptides derived from the signal sequences of certain class I MHC molecules, by heterodimers of the C‐type lectin‐like proteins CD94 and NKG2. Using soluble, recombinant HLA‐E molecules assembled with peptides derived from different leader sequences and soluble CD94/NKG2‐A and CD94/NKG2‐C proteins, the binding of these receptor–ligand pairs has been analysed. We show first that these interactions have very fast association and dissociation rate constants, secondly, that the inhibitory CD94/NKG2‐A receptor has a higher binding affinity for HLA‐E than the activating CD94/NKG2‐C receptor and, finally, that recognition of HLA‐E by both CD94/NKG2‐A and CD94/NKG2‐C is peptide dependent. There appears to be a strong, direct correlation between the binding affinity of the peptide–HLA‐E complexes for the CD94/NKG2 receptors and the triggering of a response by the NK cell. These data may help to understand the balance of signals that control cytotoxicity by NK cells.

Human Cytomegalovirus Infection Is Associated with Increased Proportions of NK Cells That Express the CD94/NKG2C Receptor in Aviremic HIV-1–Positive Patients

In healthy blood donors, serological positivity for human cytomegalovirus (HCMV) is associated with an increased proportion of NK cells bearing the CD94/NKG2C NK cell receptor (NKR). The expression of the activating CD94/NKG2C NKR and of the inhibitory CD94/NKG2A NKR was studied in a cohort of 45 aviremic human immunodeficiency virus type 1 (HIV-1)-positive patients receiving highly active antiretroviral therapy. The proportions of NKG2C+ NK cells were significantly increased in HIV-1-positive patients (mean +/- SD, 25.9% +/- 23.0%), compared with those in 31 healthy individuals (mean +/- SD, 16.1% +/- 20.7%). Yet, the association vanished when HCMV serological status was considered in a multivariate regression model. These results support the conclusion that changes in the NKR repertoire in HIV1-positive patients are related to a concomitant HCMV infection.

A novel family of Ig-like receptors for HLA class I molecules that modulate function of lymphoid and myeloid cells.

We review what is presently known about structure, cellular distribution, biochemical characteristics, and function of a new family of human cell‐surface receptors referred to as immunoglobulin‐like transcripts (ILTs), leukocyte Ig‐like receptors (LIRs), or monocyte/macrophage Ig‐like receptors (MIRs). These receptors are genetically, structurally, and functionally related to a group of natural killer (NK) cell receptors for HLA class I molecules known as killer cell Ig‐like receptors (KIRs). Distinct ILT/LIR/MIR isotypes are differentially expressed on lymphocytes, monocytes, macrophages, dendritic cells, and granulocytes; at least some of them recognize HLA class I molecules. Whereas some isotypes either inhibit or induce cell activation, others may be secreted as soluble receptors. ILT/LIR/MIR receptors may allow all immune cells to monitor class I expression on other cells and to respond in its absence, just as NK cells do. In addition, they may contribute to homeostasis by establishing activation thresholds that can be overcome only by relevant triggering stimuli and not by bystander cells. J. Leukoc. Biol. 66: 375–381; 1999.

NATURAL KILLER CELL ACTIVATION AND INHIBITION BY RECEPTORS FOR MHC CLASS I

Several recent advances have been made in our understanding of the mechanisms which human natural killer cells recognize MHC class I molecules. Three are of special relevance: the identification of a novel molecule (DAP12) with a key role in the activation pathways; the observation that certain immunoglobulin-like receptors for HLA class I molecules are also utilized by other leucocyte lineages; and the definition of MHC class Ib proteins (i.e. HLA-E and Qa-1b) as specific ligands for the phylogenetically conserved CD94-NKG2 lectin-like receptors.

Structure and function of the CD94 C‐type lectin receptor complex involved in recognition of HLA class I molecules

Summary: A multigene family of immunoglobulin superfamily (Ig‐SF) killer cell inhibitory receptors (KIRs) specifically recognize HLA class I molecules, while the interaction with H‐2 products is mediated by members of the murine Ly49 C‐type lectin family. A common structural feature of these receptors with inhibitory function is the presence of cytoplasmic immunoreceptor tyrosine‐based inhibitory motifs (ITIMs) that couple them to SHP phosphatases. Strong support for the involvement of the CD94 C‐type lectin receptor complex in NK cell‐mediated recognition of Bw6+ HLA‐B, HLA A and HLA‐C alleles has been obtained. The cloned CD94 molecule covalently assembles with at least two different glyco‐proteins (43 kDa and 39 kDa) to form functional receptors. NK cells inhibited upon HLA recognition express the CD94/p43 dimer, whose specificity for HLA molecules partially overlaps the Ig‐SF receptor system. By contrast. NK clones bearing the homologous CD94/p39 receptor are triggered upon its ligation by CD94‐specific mAbs. Remarkably, a set of Ig‐SF receptors (p50) homologous to p58 KIRs also display an activating function. CD94‐associated molecules belong to the NKG2 family of C‐type lectins; the NKG2‐A gene encodes for the p43 subunit. which contains cytoplasmic ITIMS. Expression of the different CD94 heterodimeric receptors will enable precise analysis of their putative interaction with HLA class I molecules.

The Human Cytomegalovirus MHC Class I Homolog UL18 Inhibits LIR-1+ but Activates LIR-1− NK Cells

The inhibitory leukocyte Ig-like receptor 1 (LIR-1, also known as ILT2, CD85j, or LILRB1) was identified by its high affinity for the human CMV (HCMV) MHC class I homolog gpUL18. The role of this LIR-1-gpUL18 interaction in modulating NK recognition during HCMV infection has previously not been clearly defined. In this study, LIR-1+ NKL cell-mediated cytotoxicity was shown to be inhibited by transduction of targets with a replication-deficient adenovirus vector encoding UL18 (RAd-UL18). Fibroblasts infected with an HCMV UL18 mutant (ΔUL18) also exhibited enhanced susceptibility to NKL killing relative to cells infected with the parental virus. In additional cytolysis assays, UL18-mediated protection was also evident in the context of adenovirus vector transduction and HCMV infection of autologous fibroblast targets using IFN-α-activated NK bulk cultures derived from a donor with a high frequency of LIR-1+ NK cells. A single LIR-1high NK clone derived from this donor was inhibited by UL18, while 3 of 24 clones were activated. CD107 mobilization assays revealed that LIR-1+ NK cells were consistently inhibited by UL18 in all tested donors, but this effect was often masked in the global response by UL18-mediated activation of a subset of LIR-1− NK cells. Although Ab-blocking experiments support UL18 inhibition being induced by a direct interaction with LIR-1, the UL18-mediated activation is LIR-1 independent.

Specific engagement of the CD94/NKG2-A killer inhibitory receptor by the HLA-E class Ib molecule induces SHP-1 phosphatase recruitment to tyrosine-phosphorylated NKG2-A: Evidence for receptor function in heterologous transfectants

It has been recently demonstrated that the CD94/NKG2‐A killer inhibitory receptor (KIR) specifically recognizes the HLA‐E class Ib molecule. Moreover, the apparent CD94‐mediated specific recognition of different HLA class Ia allotypes, transfected into the HLA‐defective cell line 721.221, indeed depends on their selective ability to concomitantly stabilize the surface expression of endogenous HLA‐E molecules, which confer protection against CD94/NKG2‐A+ effector cells. In the present study, we show that a selective engagement of the CD94/NKG2‐A inhibitory receptor with a specific monoclonal antibody (mAb) (Z199) was sufficient to induce tyrosine phosphorylation of the NKG2‐A subunit and SHP‐1 recruitment. These early biochemical events, commonly related to negative signaling pathways, were also detected upon the specific interaction of NK cells with an HLA‐E+ 721.221 transfectant (.221‐AEH), and were prevented by pre‐incubation of .221‐AEH with an anti‐HLA class I mAb. Furthermore, mAb cross‐linking of the CD94/NKG2‐A receptor, segregated from other NK‐associated molecules by transfection into a rat basophilic leukemia cell line (RBL‐2H3), promoted tyrosine phosphorylation of NKG2‐A and co‐precipitation of SHP‐1, together with an inhibition of secretory events triggered via FcϵRI. Remarkably, interaction of CD94/NKG2‐A+ RBL cells with the HLA‐E+ .221‐AEH transfectant specifically induced a detectable association of SHP‐1 with NKG2‐A, constituting a more formal evidence for the receptor‐HLA class I interaction.