HLA-B*27 Heavy Chain Homo-Oligomers Promote the Cytotoxicity of NK Cells via Activation of PI3K/AKT Signaling.

Background and Objectives: Ankylosing spondylitis (AS) is a chronic inflammatory disease and is highly linked with the expression of the human leukocytic antigen-B*27 (HLA-B*27) genotype. HLA-B*27 heavy chain (B*27-HC) has an innate characteristic to slowly fold, resulting in the accumulation of the misfolded B*27-HC and the formation of homo-oligomeric B*27-HC molecules. The homo-oligomeric B*27-HC can act as a ligand of KIR3DL2. Interaction of the homo-oligomeric B*27-HC molecules with KIR3DL2 will trigger the survival and activation of KIR3DL2-positive NK cells. However, the effects of homo-oligomeric B*27-HC molecules associated with KIR3DL2 on the cytotoxic activity of NK cells and their cytokine expressions remain unknown. Materials and Methods: HLA-B*-2704-HC was overexpressed in the HMy2.C1R (C1R) cell line. Western blotting and quantitative RT-PCR were used to analyze the protein expression and cytokine expression, respectively, when C1R-B*-2704 cells that overexpress B*2704-HC were co-cultured with NK-92MI cells. Flow cytometry was used to analyze the cytotoxicity mediated by NK-92MI cells. Results: Our results revealed that NK-92MI cells up-regulated the expression of perforin and enhanced the cytotoxic activity via augmentation of PI3K/AKT signaling after co-culturing with C1R-B*2704 cells. Suppression of the dimerized B*27-HC formation or treatment with an inhibitor of PI3K, LY294002, or with an anti-B*27-HC monoclonal antibody can reduce the perforin expression of NK-92MI after co-culturing with C1R-B*-2704. Co-culturing with C1R-B*-2704 cells suppressed the TNF-α and IL6 expressions of NK-92MI cells. Conclusion: Stimulation of NK cell-mediated cytotoxicity by homo-oligomeric B*27-HC molecules may contribute to the pathogenesis of AS.

An immune-molecular hypothesis supporting infectious aetiopathogenesis of Kawasaki disease in children.

The competitive binding between CpG-ODN (single-stranded DNA from pathogens) and HLA-B and HLA-A ligands for the inhibitory Killer Immunoglobulin-like Receptors (KIR)3DL1/2 may lead to possible hypo-sensing of pathogens and ineffective clearance. We observed an overabundance of HLA ligands for inhibitory KIR with three domains in KD subjects.

Non-conventional forms of HLA-B27 are expressed in spondyloarthritis joints and gut tissue.

OBJECTIVES: Human leukocyte antigen (HLA)-B27 (B27) is the strongest genetic factor associated with development of Ankylosing Spondylitis and other spondyloarthropathies (SpA), yet the role it plays in disease pathogenesis remains unclear. We investigated the expression of potentially pathogenic non-conventional heavy chain forms (NC) of B27 in synovial and intestinal tissues obtained from SpA patients. We also determined the presence of NC-B27 in joints, lymphoid and gastrointestinal tissue from B27 transgenic (TG(1)) rats with M.tuberculosis-induced SpA. METHODS: Expression of NC-B27 in human SpA joints and gut and in (21-3 × 283-2)F1 HLA-B27/Huß2m rat tissue was determined by immunohistochemistry, flow cytometry and confocal microscopy analysis using HC10 and HD6 antibodies. RESULTS: Both HC10- and HD6-reactive HLA molecules were present in synovial tissue from SpA patients. Both NC-B27 and KIR3DL2, a ligand for NC-B27, were expressed in inflamed terminal ileal tissues in patients with early SpA. Infiltrating cells in inflamed joint tissues isolated from B27 TG(1) rats expressed high levels of NC-B27. NC-B27 were also expressed in joint-resident cells from ankle and tail joints of B27 TG(1) rats prior to clinical arthritis. The expression of NC-B27 on B27 TG(1) rat CD11b/c(+), CD8α(+), cells from spleens and LNs increased with animal age and disease progression. CONCLUSIONS: Non-conventional HLA class 1 molecules are expressed on resident and infiltrating cells in both synovial and GI tissues in human SpA. NC-B27 expression in joints and lymphoid tissues from B27 TG(1) rats prior to the onset of arthritis is consistent with the hypothesis that they play a pathogenic role in SpA.

Activation-Induced Killer Cell Immunoglobulin-like Receptor 3DL2 Binding to HLA-B27 Licenses Pathogenic T Cell Differentiation in Spondyloarthritis.

OBJECTIVE: In the spondyloarthritides (SpA), increased numbers of CD4+ T cells express killer cell immunoglobulin-like receptor 3DL2 (KIR-3DL2). The aim of this study was to determine the factors that induce KIR-3DL2 expression, and to characterize the relationship between HLA-B27 and the phenotype and function of KIR-3DL2-expressing CD4+ T cells in SpA. METHODS: In total, 34 B27+ patients with SpA, 28 age- and sex-matched healthy controls (20 B27- and 8 B27+), and 9 patients with rheumatoid arthritis were studied. KIR-3DL2 expression and other phenotypic characteristics of peripheral blood and synovial fluid CD4+ T cells were studied by flow cytometry, quantitative polymerase chain reaction, and Western blotting. T cell receptor clonality was determined by template-switch anchored reverse transcription-polymerase chain reaction and sequencing analysis. Cytokines were measured by enzyme-linked immunosorbent assay. RESULTS: Cellular activation induced KIR-3DL2 expression on both naive and effector CD4+ T cells. KIR-3DL2 binding to B27+ cells promoted expression of KIR-3DL2, the Th17-specific transcription factor retinoic acid receptor-related orphan nuclear receptor γt, and the antiapoptotic factor B cell lymphoma 2. KIR-3DL2+CD4+ T cells in patients with ankylosing spondylitis were oligoclonal and enriched for markers of T cell activation and for the gut homing receptor CCR9. In the presence of B27+ antigen-presenting cells, KIR-3DL2+CD4+ T cells produced less interleukin-2 (IL-2) but more IL-17. This effect was blocked by HC10, an antibody that inhibits the binding of KIR-3DL2 to B27 heavy chains. CONCLUSION: KIR-3DL2 binding to HLA-B27 licenses Th17 cell differentiation in SpA. These findings raise the therapeutic potential of targeting HLA-B27-KIR-3DL2 interactions for the treatment of B27+ patients with SpA.

The D0 Ig-like domain plays a central role in the stronger binding of KIR3DL2 to B27 free H chain dimers.

We proposed that the killer cell Ig-like receptor KIR3DL2 binding more strongly to HLA-B27 (B27) ß2-microglobulin free H chain (FHC) dimers than other HLA-class I molecules regulates lymphocyte function in arthritis and infection. We compared the function of B27 FHC dimers with other class I H chains and identified contact residues in KIR3DL2. B27 FHC dimers interacted functionally with KIR3DL2 on NK and reporter cells more strongly than did other class I FHCs. Mutagenesis identified key residues in the D0 and other Ig-like domains that were shared and distinct from KIR3DL1 for KIR3DL2 binding to B27 and other class I FHCs. We modeled B27 dimer binding to KIR3DL2 and compared experimental mutagenesis data with computational "hot spot" predictions. Modeling predicts that the stronger binding of B27 dimers to KIR3DL2 is mediated by nonsymmetrical complementary contacts of the D0 and D1 domains with the α1, α2, and α3 domains of both B27 H chains. In contrast, the D2 domain primarily contacts residues in the α2 domain of one B27 H chain. These findings provide novel insights about the molecular basis of KIR3DL2 binding to B27 and other ligands and suggest an important role for KIR3DL2-B27 interactions in controlling the function of NK cells in B27(+) individuals.

KIR3DL2/CpG ODN interaction mediates Sézary syndrome malignant T cell apoptosis.

We previously identified the NK cell receptor KIR3DL2 as a valuable diagnostic and prognostic marker for the detection of the tumoral T cell burden of Sézary syndrome (SS) patients. However, the function of this receptor on the malignant T lymphocyte population remained unexplored. We here demonstrate that engagement of KIR3DL2 by its recently identified ligand CpG oligodeoxynucleotide (ODN) induces the internalization of the receptor and leads to a caspase-dependent apoptosis of malignant T cells. This process of cellular death is correlated to a dephosphorylation of the transcription factor STAT3 (signal transducer and activator of transcription 3), which is found constitutively phosphorylated and activated in Sézary cells. Our results indicate that KIR3DL2 can directly promote SS malignant cell death through the use of CpG ODN.

Two new cases of KIR3DP1, KIR2DL4-negative genotypes, one of which is also lacking KIR3DL2.

The killer immunoglobulin-like receptor (KIR) genes KIR2DL4, KIR3DL2, and KIR3DP1 are present in virtually all humans. KIR2DL4 encodes a receptor present on uterine and decidual natural killer (NK) cells and some peripheral blood NK cells. Its only known ligand is the human leukocyte antigen-G molecule expressed on extravillous trophoblasts, and on tissues in some diseases. KIR3DL2 binds HLA-A*03 and HLA-A*11 as well as HLA-B*27 dimers, and microbial CpG DNA. KIR3DP1 is a pseudogene. During our immunogenetic studies we found two individuals, one from Lower Silesia district in Poland, and another from Western Ukraine, who were reproducibly negative for KIR2DL4 and KIR3DP1 genes, using three different PCR systems. Both individuals displayed very similar genotypes, possessing only KIR3DL3, KIR2DL3, KIR2DP1, KIR2DS1, and probably a rare variant of KIR2DL1. The Pole had also KIR3DL2, which the Ukrainian was apparently lacking. The Lower Silesia has been populated after the Second World War by a remarkable percentage with displaced people from Western Ukraine, which might contribute to genetic similarity of the two individuals described here.

Expression of aberrant HLA-B27 molecules is dependent on B27 dosage and peptide supply.

OBJECTIVES: Cellular expression of non-classical forms of human leukocyte antigen (HLA)-B27 (NC-B27) may be involved in spondyloarthritis (SpA) pathogenesis. We used a novel B27-specific monoclonal antibody, HD6, to ask if B27 transgenic (TG) rat splenocytes express these NC-B27 molecules. We also investigated whether B27-binding peptides could affect the expression and functional immune recognition of HD6-reactive B27 molecules. METHODS: Splenocytes from B27-TG, B7-TG and non-transgenic rats, and HLA-B27+ cell lines were stained with monoclonal antibodies recognising classical (ME-1, HLA-ABC-m1) and non-classical (HD6, HC10) B27. Cells were further cultured in the presence of HLA-B27-binding peptides, or subjected to brief low pH treatment prior to mAb staining and/or immunoprecipitation or co-culture with KIR3DL2-CD3ε-expressing Jurkat reporter cells. RESULTS: HD6-reactive molecules were detected in the majority of adult B27-TG rat splenocyte cell subsets, increasing with age and concomitant increased B27 expression. HD6 staining was inhibited by incubation with B27-binding peptides and induced by low pH treatment. HD6 staining correlated with KIR3DL2-CD3ε-expressing Jurkat reporter cell activity. Thus, IL-2 production was decreased when B27-expressing antigen-presenting cells were preincubated with B27-binding peptides, but increased following pretreatment with low pH buffer. CONCLUSIONS: Surface expression of HD6-reactive B27 molecules on B27-TG rat splenocytes is consistent with a pathogenic role for NC-B27 in SpA. Interaction of NC-B27 with innate immune receptors could be critical in SpA pathogenesis, and we show that this may be influenced by the availability and composition of the B27-binding peptide pool.

The biochemistry and immunology of non-canonical forms of HLA-B27.

HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 is expressed at the cell surface of antigen presenting cells (APC) both as canonical ß2m-associated and non-canonical ß2m-free heavy chain (FHC) forms which include B27 dimers (termed B272). B27 FHC forms arise in an endosomal compartment from recycling ß2m-associated B27. Formation of cell surface FHC dimers is critically dependent on an unpaired reactive cysteine 67 in the α1 helix of the class I heavy chain. HLA-B27 also form redox-inducible ß2m-associated dimers on exosomes and apoptosing cells. By contrast with cell surface expressed cysteine 67-dependent heavy chain dimers these dimers are dependent on a cytoplasmic cysteine 325 for their formation. HLA-B27 binds to immunoregulatory receptors including members of the Killer cell Immunoglobulin-like (KIR) and Leukocyte Immunoglobulin-like receptor family. B27 FHC bind to different but overlapping sets of these immunoreceptors compared to classical ß2m-associated HLA-B27. B27 FHC bind more strongly to KIR3DL2 and LILRB2 immune receptor than other ß2m-associated HLA-class I ligands. Genetic studies have implicated genes which control production of the important proinflammatory cytokine IL-17 in the pathogenesis of spondyloarthritis. Cell surface HLA-B27 FHC binding to these immune receptors or acting through other mechanisms could impact on the pathogenesis of spondyloarthritis by promoting immune cell production of IL-17. Here we review the literature on these non-canonical forms of HLA-B27 and the immune receptors they bind to and discuss the possible relevance of these interactions to the pathogenesis of spondyloarthropathy.

HLA-F and MHC class I open conformers are ligands for NK cell Ig-like receptors.

Killer Ig-like receptors (KIRs) are innate immune receptors expressed by NK and T cells classically associated with the detection of missing self through loss of their respective MHC ligand. Some KIR specificities for allelic classical class I MHC (MHC-I) have been described, whereas other KIR receptor-ligand relationships, including those associated with nonclassical MHC-I, have yet to be clearly defined. We report in this article that KIR3DL2 and KIR2DS4 and the nonclassical Ag HLA-F, expressed as a free form devoid of peptide, physically and functionally interact. These interactions extend to include classical MHC-I open conformers as ligands, defining new relationships between KIR receptors and MHC-I. The data collectively suggest a broader, previously unrecognized interaction between MHC-I open conformers--including prototypical HLA-F--and KIR receptors, acting in an immunoregulatory capacity centered on the inflammatory response.

The arthritis-associated HLA-B*27:05 allele forms more cell surface B27 dimer and free heavy chain ligands for KIR3DL2 than HLA-B*27:09.

OBJECTIVES: HLA-B*27:05 is associated with AS whereas HLA-B*27:09 is not associated. We hypothesized that different interactions with KIR immune receptors could contribute to the difference in disease association between HLA-B*27:05 and HLAB*27:09. Thus, the objective of this study was to compare the formation of ß2m-free heavy chain (FHC) including B27 dimers (B272) by HLA-B*27:05 and HLA-B*27:09 and their binding to KIR immunoreceptors. METHODS: We studied the formation of HLA-B*27:05 and HLA-B*27:09 heterotrimers and FHC forms including dimers in vitro and in transfected cells. We investigated HLA-B*27:05 and HLA-B*27:09 binding to KIR3DL1, KIR3DL2 and LILRB2 by FACS staining with class I tetramers and by quantifying interactions with KIR3DL2CD3ε-reporter cells and KIR3DL2-expressing NK cells. We also measured KIR expression on peripheral blood NK and CD4 T cells from 18 HLA-B*27:05 AS patients, 8 HLA-B27 negative and 12 HLA-B*27:05+ and HLA-B*27:09+ healthy controls by FACS staining. RESULTS: HLA-B*27:09 formed less B272 and FHC than HLA-B*27:05. HLA-B*27:05-expressing cells stimulated KIR3DL2CD3ε-reporter T cells more effectively. Cells expressing HLA-B*27:05 promoted KIR3DL2+ NK cell survival more strongly than HLA-B*27:09. HLA-B*27:05 and HLA-B*27:09 dimer tetramers stained KIR3DL1, KIR3DL2 and LILRB2 equivalently. Increased proportions of NK and CD4 T cells expressed KIR3DL2 in HLA-B*27:05+ AS patients compared with HLA-B*27:05+, HLA-B*27:09+ and HLA-B27- healthy controls. CONCLUSION: Differences in the formation of FHC ligands for KIR3DL2 by HLA-B*27:05 and HLA-B*27:09 could contribute to the differential association of these alleles with AS.

KIR3DL2 binds to HLA-B27 dimers and free H chains more strongly than other HLA class I and promotes the expansion of T cells in ankylosing spondylitis.

The human leukocyte Ag HLA-B27 (B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of APC as both classical ß2-microglobulin-associated B27 and B27 free H chain forms (FHC), including disulfide-bonded H chain homodimers (termed B27(2)). B27 FHC forms, but not classical B27, bind to KIR3DL2. HLA-A3, which is not associated with spondyloarthritis (SpA), is also a ligand for KIR3DL2. In this study, we show that B27(2) and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B27(2) tetramers bound KIR3DL2-transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric, and monomeric FHC from HLA-B27-expressing cell lines. Binding to B27(2) and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B27(2) and B27 FHC stimulated KIR3DL2CD3ε-transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFN-γ secretion and promoted greater survival of KIR3DL2(+) CD4 T and NK cells than binding to other HLA-class I. KIR3DL2(+) T cells from B27(+) SpA patients proliferated more in response to Ag presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27(+) SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC.

CD158k/KIR3DL2 and NKp46 are frequently expressed in transformed mycosis fungoides.

Malignant Sezary cells express the natural killer (NK) receptors KIR3DL2 (CD158k) and Nkp46 and may co-express activating killer immunoglobulin-like receptors (KIR) that may participate to neoplastic T-cell activation through the JNK pathway. Little is known regarding NK receptor expression in other cutaneous T-cell lymphomas. We studied the expression of KIR and natural cytotoxicity receptor (NCR) transcripts, and KIR3DL1/2 at the protein level, in 16 skin biopsies from 10 patients with transformed mycosis fungoides (tMF). Some KIR and NCR transcripts were found in all cases, with various repertoires. Two to nine different KIR receptors were expressed in a single biopsy. Among them, KIR3DL2 was the most frequent, with the highest level of expression in quantitative analyses and correlated with in situ protein expression, while phosphorylated JNK was never detected. Among NCR, NKp46 was expressed in all investigated cases. The role of KIR3DL2 and NKp46 in tMF oncogenesis remains to be studied.

A novel KIR-associated function: evidence that CpG DNA uptake and shuttling to early endosomes is mediated by KIR3DL2.

Human natural killer (NK) cells express Toll-like receptor 9 (TLR9) transcript and, upon exposure to microbial CpG oligodeoxynucleotide (ODN), release cytokines and kill target cells. Here we show that NK cell treatment with CpG ODN results in down-modulation of KIR3DL2 inhibitory receptor from the cell surface and in its cointernalization with CpG ODN. CpG ODN-induced interferon-γ (IFN-γ) release is mostly confined to KIR3DL2(+) NK cells, thus suggesting a crucial role of KIR3DL2 in CpG ODN-mediated NK responses. Using soluble receptor molecules, we demonstrate the direct binding of KIR3DL2 to ODNs and we show that the D0 domain is involved primarily in this interaction. KIR3DL2 modulation is also induced in malignant cells of Sézary cutaneous T-cell lymphoma, a disease in which KIR3DL2 represents a typical marker of malignant T cells. Confocal microscopy analysis suggests that, in human NK cells, CpG ODN can encounter TLR9 in early endosomes after being shuttled to these sites by KIR3DL2, which functions as a CpG ODN receptor at the cell surface. This novel KIR-associated function emphasizes the antimicrobial role of NK cells in the course of infection.

[Effect of demethylating treatment on cytotoxicity of NK-92MI cells].

In order to investigate the effect of demethylating treatment on the expression of inhibitory KIR and the cytolytic activity of NK-92MI cells, and to study the possible relationship between the demethylation of inhibitory kir gene and the function of NK cells. NK-92MI cells were treated with 5-azacytidine to induce DNA demethylation. The expression of KIR3DL1, KIR2DL2/KIR2DL3, KIR2DL1 and the viability of NK-92MI cells were detected by flow cytometry. The KIR3DL1 positive and the KIR3DL1 negative NK-92MI cells were also sorted by flow cytometry. The cytotoxicity of NK-92MI against K562 cells was detected by the LDH release assay. The results demonstrated that the expressions of KIR3DL1, KIR2DL2/KIR2DL3 and KIR2DL1 in NK-92MI cells all increased after treating with 1.0, 2.5 and 5 micromol/L of 5-azacytidine for 72 hours. And the cytotoxicity of NK-92MI against K562 cells decreased. In these dose range, 5-azacytidine did not influence the viability of NK-92MI cells. Additionally, the cytotoxicity of KIR3DL1 positive NK-92MI cells was lower than that of the KIR3DL1 negative cells. It is concluded that the demethylating treatment suppresses the cytotoxicity of NK-92MI cells through increasing the expression of inhibitory KIR.