Synergistic polymorphism at two positions distal to the ligand-binding site makes KIR2DL2 a stronger receptor for HLA-C than KIR2DL3

Interactions between HLA-C ligands and inhibitory killer cell Ig-like receptors (KIR) control the development and response of human NK cells. This regulatory mechanism is usually described by mutually exclusive interactions of KIR2DL1 with C2 having lysine 80, and KIR2DL2/3 with C1 having asparagine 80. Consistent with this simple rule, we found from functional analysis and binding assays to 93 HLA-A, HLA-B, and HLA-C isoforms that KIR2DL1*003 bound all C2, and only C2, allotypes. The allotypically related KIR2DL2*001 and KIR2DL3*001 interacted with all C1, but they violated the simple rule through interactions with several C2 allotypes, notably Cw*0501 and Cw*0202, and two HLA-B allotypes (B*4601 and B*7301) that share polymorphisms with HLA-C. Although the specificities of the "cross-reactions" were similar for KIR2DL2*001 and KIR2DL3*001, they were stronger for KIR2DL2*001, as were the reactions with C1. Mutagenesis explored the avidity difference between KIR2DL2*001 and KIR2DL3*001. Recombinant mutants mapped the difference to the Ig-like domains, where site-directed mutagenesis showed that the combination, but not the individual substitutions, of arginine for proline 16 in D1 and cysteine for arginine 148 in D2 made KIR2DL2*001 a stronger receptor than KIR2DL3*001. Neither residue 16 or 148 is part of, or near to, the ligand-binding site. Instead, their juxtaposition near the flexible hinge between D1 and D2 suggests that their polymorphisms affect the ligand-binding site by changing the hinge angle and the relative orientation of the two domains. This study demonstrates how allelic polymorphism at sites distal to the ligand-binding site of KIR2DL2/3 has diversified this receptor's interactions with HLA-C.     

KIR2DL4 is an IL-2-regulated NK cell receptor that exhibits limited expression in humans but triggers strong IFN-gamma production

Killer cell Ig-like receptor (KIR)2DL4 (2DL4, CD158d) was previously described as the only KIR expressed by every human NK cell. It is also structurally atypical among KIRs because it possesses a basic transmembrane residue, which is characteristic of many activating receptors, but also contains a cytoplasmic immunoreceptor tyrosine-based inhibitory motif (ITIM). We expressed epitope-tagged 2DL4 in an NK-like cell line to study receptor function. Three distinct 2DL4 cDNA clones were analyzed: one encoding the "conventional" 2DL4 with the cytoplasmic ITIM (2DL4.1) and two encoding different cytoplasmic truncated forms lacking the ITIM (2DL4.2 and 2DL4(*)). Surprisingly, one truncated receptor (2DL4.2), which is the product of a prevalent human 2DL4 allele, was not expressed on the cell surface, indicating that some individuals may lack functional 2DL4 protein expression. Conversely, both 2DL4.1 and 2DL4(*) were expressed on the cell surface and up-regulated by IL-2. Analysis of primary NK cells with anti-2DL4 mAb confirmed the lack of surface expression in a donor with the 2DL4.2 genotype. Donors with the 2DL4.1 genotype occasionally expressed receptor only on CD56(high) NK cells, although their expression was up-regulated by IL-2. Interestingly, Ab engagement of epitope-tagged 2DL4 triggered rapid and robust IFN-gamma production, but weak redirected cytotoxicity in an NK-like cell line, which was the opposite pattern to that observed upon engagement of another NK cell activating receptor, NKp44. Importantly, both 2DL4.1 and 2DL4(*) exhibited similar activation potential, indicating that the ITIM does not influence 2DL4.1 activating function. The unique activation properties of 2DL4 suggest linkage to a distinct signaling pathway.     

KIR3DL1 polymorphisms that affect NK cell inhibition by HLA-Bw4 ligand

The killer cell Ig-like receptor (KIR) gene family encodes MHC class I receptors expressed by NK cells and several T cell subpopulations. Factors contributing to human KIR haplotype diversity are differences in gene number, gene content, and allelic polymorphism. Whereas functional and clinical consequences of the first two factors are established, knowledge of the effects of KIR gene polymorphism is limited to special cases in which signaling function is reversed or cell surface expression lost. In this study we use retrovirally transduced human cell lines to show that 3DL1*002 is a stronger inhibitory receptor for HLA-Bw4 ligands than 3DL1*007. Analysis of mutant 3DL1*002 and 3DL1*007 molecules demonstrates that residue 238 in the D2 domain and 320 in the transmembrane region contribute to the difference in receptor strength. Neither position 238 nor 320 is predicted to interact directly with HLA-Bw4 ligand. This study also revealed that KIR3DL1 and LILRB1 both contribute to developing an inhibitory response to HLA-Bw4 ligands.     

Human NK cells differ more in their KIR2DL1-dependent thresholds for HLA-Cw6-mediated inhibition than in their maximal killing capacity

In this study we have addressed the question of how activation and inhibition of human NK cells is regulated by the expression level of MHC class I protein on target cells. Using target cell transfectants sorted to stably express different levels of the MHC class I protein HLA-Cw6, we show that induction of degranulation and that of IFN-γ secretion are not correlated. In contrast, the inhibition of these two processes by MHC class-I occurs at the same level of class I MHC protein. Primary human NK cell clones were found to differ in the amount of target MHC class I protein required for their inhibition, rather than in their maximum killing capacity. Importantly, we show that KIR2DL1 expression determines the thresholds (in terms of MHC I protein levels) required for NK cell inhibition, while the expression of other receptors such as LIR1 is less important. Furthermore, using mathematical models to explore the dynamics of target cell killing, we found that the observed delay in target cell killing is exhibited by a model in which NK cells require some activation or priming, such that each cell can lyse a target cell only after being activated by a first encounter with the same or a different target cell, but not by models which lack this feature.     

The protein made from a common allele of KIR3DL1 (3DL1*004) is poorly expressed at cell surfaces due to substitution at positions 86 in Ig domain 0 and 182 in Ig domain 1

KIR3DL1 is an inhibitory HLA-B receptor of human NK and T cells that exhibits genetic and phenotypic polymorphism. KIR3DL1*004, a common allotype, cannot be detected on the surface of PBLs using the KIR3DL1-specific Ab DX9. The nature of this phenotype was investigated through comparison of 3DL1*004 with 3DL1*002, an allele giving high DX9 binding to cell surfaces. Analysis of Jurkat T cell transfectants with 3DL1*004 cDNA showed that 3DL1*004 is poorly expressed at the cell surface, but detectable intracellularly. Analysis of recombinant mutants made between 3DL1*004 and 3DL1*002 showed that polymorphism in Ig domains 0 and 1 (D0 and D1) causes the intracellular retention of 3DL1*004. Reciprocal point mutations were introduced into 3DL1*004 and 3DL1*002 at positions 44 and 86 of the D0 domain, where 3DL1*004 has unique residues, and at position 182 of the D1 domain, where 3DL1*004 resembles 3DL1*005, an allotype giving low DX9-binding phenotype. Leucine 86 in 3DL1*004 is the principal cause of its intracellular retention, with a secondary and additive contribution from serine 182. By contrast, glycine 44, which is naturally present in 3DL1*004, slightly increased cell surface expression when introduced into 3DL1*002. In 3DL1*004, the presence of leucine at position 86 corrupts the WSXPS motif implicated in proper folding of the KIR D0 Ig-like domain. This study demonstrates how a difference between KIR3DL1 allotypes in the D0 domain profoundly affects cell surface expression and function.     

Human immunodeficiency virus type 1 infection is associated with increased NK cell polyfunctionality and higher levels of KIR3DL1+ NK cells in ugandans carrying the HLA-B Bw4 motif

Natural killer (NK) cells are important innate effector cells controlled by an array of activating and inhibitory receptors. Some alleles of the inhibitory killer-cell immunoglobulin-like receptor KIR3DL1 in combination with its HLA class I ligand Bw4 have been genetically associated with slower HIV-1 disease progression. Here, we observed that the presence of HLA-B Bw4 was associated with elevated frequencies of KIR3DL1(+) CD56(dim) NK cells in chronically HIV-1-infected individuals from the rural district of Kayunga, Uganda. In contrast, levels of KIR2DL1(+) CD56(dim) NK cells were decreased, and levels of KIR2DL3(+) CD56(dim) NK cells were unchanged in infected subjects carrying their respective HLA-C ligands. Furthermore, the size of the KIR3DL1(+) NK cell subset correlated directly with viral load, and this effect occurred only in HLA-B Bw4(+) patients, suggesting that these cells expand in response to viral replication but may have relatively poor antiviral capacity. In contrast, no association with viral load was present for KIR2DL1(+) and KIR2DL3(+) NK cells. Interestingly, chronic HIV-1 infection was associated with an increased polyfunctional response in the NK cell compartment, and, upon further investigation, KIR3DL1(+) CD56(dim) NK cells exhibited a significantly increased functional response in the patients carrying HLA-B Bw4. These results indicate that chronic HIV-1 infection is associated with increased NK cell polyfunctionality and elevated levels of KIR3DL1(+) NK cells in Ugandans carrying the HLA-B Bw4 motif.     

Cutting edge: induction of IFN-gamma production but not cytotoxicity by the killer cell Ig-like receptor KIR2DL4 (CD158d) in resting NK cells

Activated NK cells lyse tumor cells and virus-infected cells and produce IFN-gamma upon contact with sensitive target cells. The regulation of these effector responses in resting NK cells is not well understood. We now describe a receptor, KIR2DL4, that has the unique property of inducing IFN-gamma production, but not cytotoxicity, by resting NK cells in the absence of cytokines. In contrast, the NK cell-activation receptors CD16 and 2B4 induced cytotoxicity but not IFN-gamma production. The induction by KIR2DL4 of IFN-gamma production by resting NK cells was blocked by an inhibitor of the p38 mitogen-activated protein kinase signaling pathway, in contrast to the IL-2-induced IFN-gamma secretion that was sensitive to inhibition of the extracellular signal-regulated kinase mitogen-activated protein kinase pathway. These results reveal a functional dichotomy (cytokine production vs cytotoxicity) in the response of resting NK cells, as dictated by the signals of individual receptors.     

Interactions of NK cell receptor KIR3DL1*004 with chaperones and conformation-specific antibody reveal a functional folded state as well as predominant intracellular retention

Variable interaction between the Bw4 epitope of HLA-B and the polymorphic KIR3DL1/S1 system of inhibitory and activating NK cell receptors diversifies the development, repertoire formation, and response of human NK cells. KIR3DL1*004, a common KIR3DL1 allotype, in combination with Bw4(+) HLA-B, slows progression of HIV infection to AIDS. Analysis in this study of KIR3DL1*004 membrane traffic in NK cells shows this allotype is largely misfolded but stably retained in the endoplasmic reticulum, where it binds to the chaperone calreticulin and does not induce the unfolded protein response. A small fraction of KIR3DL1*004 folds correctly and leaves the endoplasmic reticulum to be expressed on the surface of primary NK and transfected NKL cells, in a form that can be triggered to inhibit NK cell activation and secretion of IFN-γ. Consistent with this small proportion of correctly folded molecules, trace amounts of MHC class I coimmunoprecipitated with KIR3DL1*004. There was no indication of any extensive intracellular interaction between unfolded KIR3DL1*004 and cognate Bw4(+) HLA-B. A similarly limited interaction of Bw4 with KIR3DL1*002, when both were expressed by the same cell, was observed despite the efficient folding of KIR3DL1*002 and its abundance on the NK cell surface. Several positions of polymorphism modulate KIR3DL1 abundance at the cell surface, differences that do not necessarily correlate with the potency of allotype function. In this context, our results suggest the possibility that the effect of Bw4(+) HLA-B and KIR3DL1*004 in slowing progression to AIDS is mediated by interaction of Bw4(+) HLA-B with the small fraction of cell surface KIR3DL1*004.     

Cutting Edge: KIR3DS1, a gene implicated in resistance to progression to AIDS, encodes a DAP12-associated receptor expressed on NK cells that triggers NK cell activation

The killer cell Ig-like receptor (KIR) gene, KIR3DS1, has been implicated in slowing disease progression in HIV infection; however, little is known about its expression, function, or ligand specificity. Using retrovirally transduced NKL cells and peripheral blood NK cells from KIR3DS1-positive donors we assessed expression of this gene by flow cytometry and its function by in vitro assays measuring KIR3DS1-induced cell-mediated cytotoxicity and cytokine production. In the present study, we demonstrate that KIR3DS1 is expressed on peripheral blood NK cells and triggers both cytotoxicity and IFN-gamma production. Using cotransfection and coimmunoprecipitation, we found that KIR3DS1 associates with the ITAM-bearing adaptor, DAP12. Soluble KIR3DS1-Ig fusion proteins did not bind to EBV-transformed B lymphoid cell lines transfected with HLA-Bw4 80I or 80T allotypes, suggesting that if KIR3DS1 does recognize HLA-Bw4 ligands, this may be peptide dependent.     

The acidic cluster of the CK2 site of the cation-dependent mannose 6-phosphate receptor (CD-MPR) but not its phosphorylation is required for GGA1 and AP-1 binding

Lysosomal biogenesis depends on proper transport of lysosomal enzymes by the cation-dependent mannose 6-phosphate receptor (CD-MPR) from the trans-Golgi network (TGN) to endosomes. Trafficking of the CDMPR is mediated by sorting signals in its cytoplasmic tail. GGA1 (Golgi-localizing, gamma-ear-containing, ARF-binding protein-1) binds to CD-MPR in the TGN and targets the receptor to clathrin-coated pits for transport from the TGN to endosomes. The motif of the CD-MPR that interacts with GGA1 was shown to be 61DXXLL65. Reports on increased affinity of cargo, when phosphorylated by casein kinase 2 (CK2), to GGAs focused our interest on the effect of the CD-MPR CK2 site on binding to GGA1. Here we demonstrate that Glu58 and Glu59 of the CK2 site are essential for high affinity GGA1 binding in vitro, whereas the phosphorylation of Ser57 of the CD-MPR has no influence on receptor binding to GGA1. Furthermore, the in vivo interaction between GGA1 and CD-MPR was abolished only when all residues involved in GGA1 binding were mutated, namely, Glu58, Glu59, Asp61, Leu64, and Leu65. In contrast, the binding of adaptor protein-1 (AP-1) to CD-MPR required all the glutamates surrounding the phosphorylation site, namely, Glu55, Glu56, Glu58, and Glu59, but like GGA1 binding, was independent of the phosphorylation of Ser57. The binding affinity of GGA1 to the CD-MPR was found to be 2.4-fold higher than that of AP-1. This could regulate the binding of the two proteins to the partly overlapping sorting signals, allowing AP-1 binding to the CD-MPR only when GGA1 is released upon autoinhibition by phosphorylation.     

Mutation of Tyr697, a GRB2-binding site, and Tyr721, a PI 3-kinase binding site, abrogates signal transduction by the murine CSF-1 receptor expressed in Rat-2 fibroblasts.

The receptor for the myeloid cell growth factor colony stimulating factor 1 (CSF-1) is a protein tyrosine kinase that is closely related to the PDGF receptor. Ligand binding results in kinase activation and autophosphorylation. Three autophosphorylation sites, Tyr697, Tyr706 and Tyr721, have been mapped to the kinase insert domain. Deletion of the entire kinase insert domain completely abrogates signal transduction by the CSF-1 receptor expressed in Rat-2 fibroblasts. To investigate the function of individual phosphorylation sites present in the CSF-1 receptor kinase insert domain, a number of phosphorylation site mutants were expressed in Rat-2 fibroblasts. Mutation of either Tyr697 or Tyr721 compromised signal transduction by the CSF-1 receptor. A mutant receptor, in which both Tyr697 and Tyr721 were replaced by phenylalanine, has lost all ability to induce changes in morphology or to increase cell growth rate in response to CSF-1. Tyr721 has been identified recently as the binding site for PI 3-kinase. Here we report that GRB2 associates with the CSF-1 receptor upon ligand binding. The phosphorylation on tyrosine of SHC and several other GRB2-associated proteins increased upon stimulation with CSF-1. Tyr697 was identified as a binding site for GRB2. We suggest that PI 3-kinase, GRB2 and some of the GRB2-associated proteins could play an important role in signal transduction by the CSF-1 receptor.     

MPTP lesions of the nigrostriatal dopaminergic projection decrease [3H]1-[1-(2-thienyl)cyclohexyl]-piperidine binding to PCP site 2: Further evidence that PCP site 2 is associated with the biogenic amine reuptake complex

Our previous studies have demonstrated that, using membranes of guinea pig brain, [³H]1-[1-(2-thienyl)cyclohexyl]piperidine ([³H]TCP) labels not only the phencyclidine binding site associated with the NMDA receptor (PCP site 1), but also a second high affinity binding site which is associated with the biogenic amine reuptake carrier (termed PCP site 2). To test this hypothesis, the binding of [³H]GBR12935 to the dopamine transporter, and [³H]TCP binding to PCP sites 1 and 2 were measured in caudates harvested from control MPTP-treated and reserpine-treated dogs. MPTP treatment decreased dopamine levels by over 99%, decreased [³H]GBR12935 binding by over 90%, decreased [³H]TCP binding to PCP site 2 by about 50%, and had no significant effect on [³H]TCP binding to PCP site 1. These data are consistent with hypothesis that a portion of PCP site 2 is associated with dopaminergic nerve, terminals in dog caudate.     

3D computation modelling of the influence of cytokine secretion on Th-cell development suggests that negative selection (inhibition of Th1 cells) is more effective than positive selection by IL-4 for Th2 cell dominance

Th-cell development has been suggested to include selective mechanisms in which certain cytokines select either Th1 or Th2 cells to proliferate and grow. The selective theory is based on the observation that Th2 cells secrete IL-4, a cytokine that promotes Th2 development, whereas Th1 cells secrete interferon-gamma (IFN-gamma) that favours Th1 development, and both positive and negative selective influences have been suggested to operate. In this study, we investigate the role of autocrine secretion and utilization of IL-4 by Th2 cells and address the question of whether an activated Th2 cell can be positively selected by IL-4 secreted from other Th2 cells. We present a spatial three dimensional (3D) modelling approach to simulate the interaction between the IL-4 ligand and its IL-4 receptors expressed on discrete IL-4 secreting cells. The simulations, based on existing experimental data on the IL-4 receptor-ligand system, illustrate how Th-cell development is highly dependent on the distance between cells that are communicating. The model suggests that a single Th2 cell is likely to communicate with possible target cells within a range of approximately 100 microm and that an activated Th2 cell manages to fill most of its own IL-4 receptors, even at a low secretion rate. The predictions made by the model suggest that negative selection against Th1 cells is more effective than positive selection by IL-4 for promoting Th2 dominance.     

The epicenter of chromosomal fragility of Fra14A2, the mouse ortholog of human FRA3B common fragile site,is largely attached to the nuclear matrix in lymphocytes but not in other cell types that do not express such a fragility

Common fragile sites (CFSs) correspond to chromosomal regions susceptible to present breaks, discontinuities or constrictions in metaphase chromosomes from cells subjected to replication stress. They are considered as genomic regions intrinsically difficult to replicate and they are evolutionary conserved at least in mammals. However, the recent discovery that CFSs are cell-type specific indicates that DNA sequence by itself cannot account for CFS instability. Nevertheless, the large gene FHIT that includes FRA3B, the most highly expressed CFS in human lymphocytes, is commonly deleted in a variety of tumors suggesting a tumor suppressor role for its product. Here, we report that the epicenter of fragility of Fra14A2/Fhit, the mouse ortholog of human FRA3B/FHIT that like its human counterpart is the most highly expressed CFS in mouse lymphocytes, is largely attached to the nuclear matrix compartment in naive B lymphocytes but not in primary hepatocytes or cortical neurons that do not express such a CFS. Our results suggest a structural explanation for the difficult-to-replicate nature of such a region and so for its common fragility in lymphocytes, that is independent of the possible tumor suppressor role of the gene harboring such CFS.     

CD1d-independent developmental acquisition of prompt IL-4 gene inducibility in thymus CD161(NK1)-CD44lowCD4+CD8- T cells is associated with complementarity determining region 3-diverse and biased Vbeta2/Vbeta7/Vbeta8/Valpha3.2 T cell receptor usage

Among Ag-inexperienced naive T cells, the CD1d-restricted NKT cell that uses invariant TCR-alpha-chain is the most widely studied cell capable of prompt IL-4 inducibility. We show in this study that thymus CD161-CD44lowCD4+CD8- T cells promptly produce IL-4 upon TCR stimulation, a response that displays biased Vbeta(2/7/8) and Valpha3.2 TCR usage. The association of Vbeta family bias and IL-4 inducibility in thymus CD161-CD44lowCD4+CD8- T cells is found for B6, B10, BALB/c, CBA, B10.A(4R), and ICR mouse strains. Despite reduced IL-4 inducibility, there is a similarly biased Vbeta(2/7/8) TCR usage by IL-4 inducibility+ spleen CD161-CD44lowCD4+CD8- T cells. Removal of alpha-galacotosylceramide/CD1d-binding cells from CD161-CD44lowCD4+CD8- thymocytes does not significantly affect their IL-4 inducibility. The development of thymus CD161-CD44lowCD4+CD8- T cells endowed with IL-4 inducibility and their associated use of Vbeta(2/7/8) are beta2-microglobulin-, CD1d-, and p59fyn-independent. Thymus CD161-CD44lowCD4+CD8- T cells produce low and no IFN-gamma inducibility in response to TCR stimulation and to IL-12 + IL-18, respectively, and they express diverse complementarity determining region 3 sequences for both TCR-alpha- and -beta-chains. Taken together, these results demonstrate the existence of a NKT cell distinct, TCR-repertoire diverse naive CD4+ T cell subset capable of prompt IL-4 inducibility. This subset has the potential to participate in immune response to a relatively large number of Ags. The more prevalent nature of this unique T cell subset in the thymus than the periphery implies roles it might play in intrathymic T cell development and may provide a framework upon which mechanisms of developmentally regulated IL-4 gene inducibility can be studied.