Crystal structure of the HLA-Cw3 allotype-specific killer cell inhibitory receptor KIR2DL2.
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Killer cell inhibitory receptors (KIR) protect class I HLAs expressing target cells from natural killer (NK) cell-mediated lysis. To understand the molecular basis of this receptor-ligand recognition, we have crystallized the extracellular ligand-binding domains of KIR2DL2, a member of the Ig superfamily receptors that recognize HLA-Cw1, 3, 7, and 8 allotypes. The structure was determined in two different crystal forms, an orthorhombic P212121 and a trigonal P3221 space group, to resolutions of 3.0 and 2.9 A, respectively. The overall fold of this structure, like KIR2DL1, exhibits K-type Ig topology with cis-proline residues in both domains that define beta-strand switching, which sets KIR apart from the C2-type hematopoietic growth hormone receptor fold. The hinge angle of KIR2DL2 is approximately 80 degrees, 14 degrees larger than that observed in KIR2DL1 despite the existence of conserved hydrophobic residues near the hinge region. There is also a 5 degrees difference in the observed hinge angles in two crystal forms of 2DL2, suggesting that the interdomain hinge angle is not fixed. The putative ligand-binding site is formed by residues from several variable loops with charge distribution apparently complementary to that of HLA-C. The packing of the receptors in the orthorhombic crystal form offers an intriguing model for receptor aggregation on the cell surface. (+info)
Trophoblast cell line resistance to NK lysis mainly involves an HLA class I-independent mechanism.
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The lack of classical HLA molecules on trophoblast prevents allorecognition by maternal T lymphocytes, but poses the problem of susceptibility to NK lysis. Expression of the nonclassical class I molecule, HLA-G, on cytotrophoblast may provide the protective effect. However, the class I-negative syncytiotrophoblast escapes NK lysis by maternal PBL. In addition, while HLA-G-expressing transfectants of LCL.721.221 cells are protected from lymphokine-activated killer lysis, extravillous cytotrophoblast cells and HLA-G-expressing choriocarcinoma cells (CC) are not. The aim of this work was therefore to clarify the role of HLA class I expression on trophoblast cell resistance to NK lysis and on their susceptibility to lymphokine-activated killer lysis. Our results showed that both JAR (HLA class I-negative) and JEG-3 (HLA-G- and HLA-Cw4-positive) cells were resistant to NK lysis by PBL and were equally lysed by IL-2-stimulated PBL isolated from a given donor. In agreement, down-regulating HLA class I expression on JEG-3 cells by acid treatment, masking these molecules or the putative HLA-G (or HLA-E) receptor CD94/NKG2 and the CD158a/p58.1 NKR with mAbs, and inducing self class I molecule expression on JAR cells did not affect NK or LAK lysis of CC. These results demonstrate that the resistance of CC to NK lysis mainly involves an HLA class I-independent mechanism(s). In addition, we show that the expression of a classical class I target molecule (HLA-B7) on JAR cells is insufficient to induce lysis by allospecific polyclonal CTL. (+info)
Killer cell immunoglobulin receptors and T cell receptors bind peptide-major histocompatibility complex class I with distinct thermodynamic and kinetic properties.
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Human natural killer cells and a subset of T cells express a repertoire of killer cell immunoglobulin receptors (KIRs) that recognize major histocompatibility complex (MHC) class I molecules. KIRs and T cell receptors (TCRs) bind in a peptide-dependent manner to overlapping regions of peptide-MHC class I complexes. KIRs with two immunoglobulin domains (KIR2Ds) recognize distinct subsets of HLA-C alleles. Here we use surface plasmon resonance to study the binding of soluble forms of KIR2DL1 and KIR2DL3 to several peptide-HLA-Cw7 complexes. KIR2DL3 bound to the HLA-Cw7 allele presenting the peptide RYRPGTVAL with a 1:1 stoichiometry and an affinity (K(d) approximately 7 microM at 25 degrees C) within the range of values measured for other cell-cell recognition molecules, including the TCR. Although KIR2DL1 is reported not to recognize the HLA-Cw7 allele in functional assays, it bound RYRPGTVAL/HLA-Cw7, albeit with a 10-20-fold lower affinity. TCR/peptide-MHC interactions are characterized by comparatively slow kinetics and unfavorable entropic changes (Willcox, B. E., Gao, G. F., Wyer, J. R. , Ladbury, J. E., Bell, J. I., Jakobsen, B. K., and van der Merwe, P. A. (1999) Immunity 10, 357-365), suggesting that binding is accompanied by conformational adjustments. In contrast, we show that KIR2DL3 binds RYRPGTVAL/HLA-Cw7 with fast kinetics and a favorable binding entropy, consistent with rigid body association. These results indicate that KIR/peptide-MHC class I interactions have properties typical of other cell-cell recognition molecules, and they highlight the unusual nature of TCR/peptide-MHC recognition. (+info)
Killer-cell inhibitory receptors, CD158a/b, are upregulated by interleukin-2, but not interferon-gamma or interleukin-4.
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Although it is now accepted that killer-cell inhibitory receptors (KIRs), which were molecularly cloned in 1995, deliver negative signals to natural killer (NK) cells regarding the recognition of target cells, it is still unclear how the expression of these receptors on lymphocytes is regulated. Therefore, we investigated the regulation of expression of representative KIRs, CD158a and CD158b, by cytokines such as interleukin-2 (IL-2), IL-4 and interferon-gamma (IFN-gamma). Neither IL-4 nor IFN-gamma affected the expression of CD158a/b, but incubation for 48 h with IL-2, which enhances the killer activity of NK cells, upregulated the expression of the KIRs. This upregulation by IL-2 was also observed in CD16-positive cells sorted from total lymphocytes. In contrast, IL-4, which is a down-regulator of IL-2-induced killer responses, did not change the level of CD158a/b expression when added after the IL-2 treatment. These findings suggest that IL-2 plays an important role in the regulation of CD158a/b expression, and might be involved in controlling NK activity via regulating expression of these molecules. (+info)
Cobalt-mediated dimerization of the human natural killer cell inhibitory receptor.
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Upon engagement of specific class I major histocompatibility complex (MHC) molecules on target cells, inhibitory receptors on natural killer (NK) cells deliver a negative signal that prevents the target cell lysis by NK cells. In humans, killer cell immunoglobulin-related receptors (KIR) with two immunoglobulin-like domains (KIR2D) modulate the lysis of target cells bearing specific HLA-C alleles (Moretta, A., Vitale, M., Bottino, C., Orengo, A. M., Morelli, L., Augugliaro, R., Barbaresi, M., Ciccone, E., and Moretta, L. (1993) J. Exp. Med. 178, 597-604). The transduction of inhibitory signals by KIR2D molecules is impaired by the zinc chelator, 1,10-phenanthroline, and mutation of a putative zinc-binding site (Rajagopalan, S., and Long, E. O. (1998) J. Immunol. 161, 1299-1305), but the mechanism by which zinc may affect the function of KIR remains unknown. In this study, the inhibitory NK receptor KIR2DL1 was discovered to dimerize in the presence of Co(2+) as observed on native gel electrophoresis and by gel filtration column chromatography. Furthermore, Co(2+)-mediated KIR2DL1 dimer binds to HLA-Cw4 with higher affinity than the wild type KIR2DL1 monomer. Replacement of the amino-terminal His residue by Ala abolishes the ability of KIR2DL1 to bind Co(2+), indicating that Co(2+)-mediated KIR2DL1 dimerization involves pairing of the D1 domain. Although not observed on native gels, the inhibitory receptor KIR2DL1 can be chemically cross-linked into dimers in the presence of Zn(2+) and its related divalent metal ions, suggesting that Co(2+)-mediated dimerization of KIR2DL1 may mimic a weaker interaction between KIR2DL1 and zinc in vivo. (+info)
NK cell inhibitory receptors prevent tyrosine phosphorylation of the activation receptor 2B4 (CD244).
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2B4 is an NK cell activation receptor that can provide a co-stimulatory signal to other activation receptors and whose mode of signal transduction is still unknown. We show that cross-linking of 2B4 on NK cells results in its rapid tyrosine phosphorylation, implying that this initial step in 2B4 signaling does not require coligation of other receptors. Ligation of 2B4 in the context of an NK cell-target cell interaction leads to 2B4 tyrosine phosphorylation, target cell lysis, and IFN-gamma release. Coligation of 2B4 with the inhibitory receptors killer cell Ig-like receptor (KIR)2DL1 or CD94/NKG2 completely blocks NK cell activation. The rapid tyrosine phosphorylation of 2B4 observed upon contact of NK cells with sensitive target cells is abrogated when KIR2DL1 or CD94/NKG2 are engaged by their cognate MHC class I ligand on resistant target cells. These results demonstrate that NK inhibitory receptors can interfere with a step as proximal as phosphorylation of an activation receptor. (+info)
Functional inhibitory receptors expressed by a cutaneous T cell lymphoma-specific cytolytic clonal T cell population.
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Inhibitory receptors on natural killer cells and on a minority of T lymphocytes are major histocompatibility complex class Ia or Ib specific. We have previously reported several tumor-specific cytotoxic T cell clones infiltrating a CD4(+) V beta 13(+) cutaneous T cell lymphoma. These clones mediated a specific major histocompatibility complex class I-restricted cytotoxic activity toward the uncultured tumor cells and autologous long-term tumor T cell lines. In this study, we cultured with interleukin-2 the peripheral blood lymphocytes of the same patient a few weeks before invasion of the blood by tumor cells. We report the rapid and selective expansion of a CD8(+) V beta 13(+) lymphoid population. This population was clonal, as it expressed a unique T cell receptor-V beta junctional region. V beta 13(+) tumor cells and V beta 13(+) reactive T cells were shown to have different junctional sequences. The CD8(+) reactive clone was functional, as it had a specific autologous tumor-specific, human leukocyte antigen-A2 restricted, cytotoxic activity. This clone coexpressed high levels of CD158a, CD158b, p70, and CD94/NKG2A inhibitory receptors. Interestingly, we found that anti-CD158a and anti-CD158b monoclonal antibodies could inhibit anti-CD3 redirected cytotoxicity mediated by the reactive clonal population. Further, an anti-human leukocyte antigen-B/C monoclonal antibody enhanced the specific cytotoxic activity of the clone against autologous tumor cells. These results are the first evidence that inhibitory receptor expression can lead to the inhibition of cutaneous T cell lymphoma-specific T cell responses. (+info)
Effect of interleukin 2 on killer cell inhibitory receptors in patients with rheumatoid arthritis.
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OBJECTIVE: The genes for killer cell inhibitory receptors (KIRs) have been cloned and their functions and responses to other molecules, including cytokines, have been partially clarified. However, the expression of KIRs has not been analysed in patients with autoimmune diseases, such as rheumatoid arthritis (RA), who are highly susceptible to microbial infection. Therefore, KIR expression on lymphocytes in patients with RA, and the regulation of KIR expression by interleukin 2 (IL2) in RA was investigated. METHODS: CD158a/b expression on peripheral blood mononuclear cells (PBMC) obtained from 25 patients with RA and 14 healthy subjects was analysed by flow cytometry. Additionally, PBMC from the two groups of subjects were cultured in RPMI 1640 medium with or without IL2 for 48 hours, and then their CD158a/b expression was analysed. RESULTS: The rate of CD158a expression on the CD8+ cells was lower in patients with RA than in healthy subjects, though there was no significant difference in the CD158a/b expression on the CD16+ cells between the two groups. The upregulation of CD16+CD158a/b+ cells in response to IL2 was significantly reduced in patients with RA compared with healthy subjects. CONCLUSION: The reduced induction of KIR expression in response to IL2 may provide insight into the reason for the high susceptibility of patients with RA to microbial infection. (+info)