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Killer cell immunoglobulin-like receptors (KIRs) are expressed predominantly on natural killer cells, where they play a key role in the regulation of innate immune responses. Recent studies show that inhibitory KIRs can also affect adaptive T cell–mediated immunity. In mice and in human T cells in vitro, inhibitory KIR ligation enhanced CD8+ T cell survival. To investigate the clinical relevance of these observations, we conducted an extensive immunogenetic analysis of multiple independent cohorts of HIV-1–, hepatitis C virus (HCV)–, and human T cell leukemia virus type 1 (HTLV-1)–infected individuals in conjunction with in vitro assays of T cell survival, analysis of ex vivo KIR expression, and mathematical modeling of host-virus dynamics. Our data suggest that functional engagement of inhibitory KIRs enhances the CD8+ T cell response against HIV-1, HCV, and HTLV-1 and is a significant determinant of clinical outcome in all three viral infections.

Objective. HLA-B27 and ERAP1 are the two strongest predisposing genetic factors to Ankylosing Spondylitis (AS). A key aminopeptidase in MHC class I presentation, ERAP1 potentially contributes to AS pathogenesis through altering HLA-B27 peptide presentation. We studied the effects of ERAP1 on the HLA-B27 peptide repertoire and peptide presentation to Cytotoxic T lymphocytes (CTLs). Methods. ERAP1-silenced and -competent HeLa.B27/C1R.B27 cells were isotope labeled, mixed, lysed and then immuno-precipitated using W6/32 or ME1. Peptides bound to HLA-B27 were eluted and analyzed by tandem Mass Spectrometry. Selected peptides were synthesized and tested for HLA-B27 binding ability. The effect of ERAP1 silencing/mutation on presentation of an immunodominant viral HLA-B27 epitope, KK10, to CTL was also studied. Results. In both HeLa.B27 and C1R.B27 cells, the proportion of 9mer HLA-B27-bound peptides was decreased by ERAP1 silencing, whereas the percentage of longer peptides (11-13mers) increased. Surprisingly, following ERAP1 silencing, C-terminally extended peptides were readily identified. These were better able to bind to HLA-B27 than N-terminally extended peptides lacking a P2 Arginine. In both HeLa.B27 and mouse fibroblasts expressing HLA-B27, the absence of ERAP1 reduced recognition by HLA-B27-restricted KK10-specific CTLs following recombinant vaccinia viral infection or transfection with minigenes expressing KK10 precursors. Lastly, an AS protective variant, K528R-ERAP1, reduced KK10 CTL recognition following extended-KK10 minigene transfection compared to WT-ERAP1. Conclusion. Our study shows that ERAP1 directly alters peptide binding and presentation by HLA-B27, supporting a pathogenic mechanism in AS. ERAP1 inhibition could potentially be used for treatment of AS and other ERAP1-associated diseases. © 2013 American College of Rheumatology.

OBJECTIVE: HLA-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are the two strongest genetic factors predisposing to ankylosing spondylitis (AS). A key aminopeptidase in class I major histocompatibility complex presentation, ERAP1 potentially contributes to the pathogenesis of AS by altering HLA-B27 peptide presentation. The aim of this study was to analyze the effects of ERAP1 on the HLA-B27 peptide repertoire and peptide presentation to cytotoxic T lymphocytes (CTLs). METHODS: ERAP1-silenced and -competent HeLa.B27 and C1R.B27 cells were isotope-labeled, mixed, lysed, and then immunoprecipitated using W6/32 or ME1 antibodies. Peptides bound to HLA-B27 were eluted and analyzed by tandem mass spectrometry. Selected peptides were synthesized and tested for HLA-B27 binding ability. The effect of ERAP1 silencing/mutation on presentation of an immunodominant viral HLA-B27 epitope, KK10, to CTLs was also studied. RESULTS: In both HeLa.B27 and C1R.B27 cells, the proportion of 9-mer HLA-B27-bound peptides was decreased by ERAP1 silencing, whereas the percentages of longer peptides (11-13 mer) were increased. Surprisingly, following ERAP1 silencing, C-terminally extended peptides were readily identified. These were better able to bind to HLA-B27 than were N-terminally extended peptides lacking an arginine at position 2. In both HeLa.B27 cells and mouse fibroblasts expressing HLA-B27, the absence of ERAP1 reduced peptide recognition by HLA-B27-restricted KK10-specific CTLs following infection with recombinant vaccinia virus or transfection with minigenes expressing KK10 precursors. Presence of an AS-protective variant of ERAP1, K528R, as compared to wild-type ERAP1, reduced the peptide recognition by KK10 CTLs following transfection with extended KK10 minigenes. CONCLUSION: These results show that ERAP1 directly alters peptide binding and presentation by HLA-B27, thus demonstrating a potential pathogenic mechanism in AS. Inhibition of ERAP1 could potentially be used for treatment of AS and other ERAP1-associated diseases.

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.

Objective: Human leucocyte antigen (HLA)-B27 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly associated with ankylosing spondylitis (AS). ERAP1 is a key aminopeptidase in HLA class I presentation and can potentially alter surface expression of HLA-B27 free heavy chains (FHCs). We studied the effects of ERAP1 silencing/inhibition/variations on HLA-B27 FHC expression and Th17 responses in AS. Methods: Flow cytometry was used to measure surface expression of HLA class I in peripheral blood mononuclear cells (PBMCs) from patients with AS carrying different ERAP1 genotypes (rs2287987, rs30187 and rs27044) and in ERAP1-silenced/inhibited/mutated HLA-B27-expressing antigen presenting cells (APCs). ERAP1-silenced/inhibited APCs were cocultured with KIR3DL2CD3ϵ-reporter cells or AS CD4+ T cells. Th17 responses of AS CD4+ T cells were measured by interleukin (IL)-17A ELISA and Th17 intracellular cytokine staining. FHC cell surface expression and Th17 responses were also measured in AS PBMCs following ERAP1 inhibition. Results: The AS-protective ERAP1 variants, K528R and Q730E, were associated with reduced surface FHC expression by monocytes from patients with AS and HLA-B27-expressing APCs. ERAP1 silencing or inhibition in APCs downregulated HLA-B27 FHC surface expression, reduced IL-2 production by KIR3DL2CD3ϵ-reporter cells and suppressed the Th17 expansion and IL-17A secretion by AS CD4+ T cells. ERAP1 inhibition of AS PBMCs reduced HLA class I FHC surface expression by monocytes and B cells, and suppressed Th17 expansion. Conclusions: ERAP1 activity determines surface expression of HLA-B27 FHCs and potentially promotes Th17 responses in AS through binding of HLA-B27 FHCs to KIR3DL2. Our data suggest that ERAP1 inhibition has potential for AS treatment.

OBJECTIVE: Spondylarthritides (SpA) including ankylosing spondylitis (AS) are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLA B27 normally forms complexes with ß(2) microglobulin (ß(2) m) and peptide to form heterotrimers. However an unusual characteristic of HLA B27 is its ability to form ß(2) m free heavy chain homodimers (HLA B27(2) ) which unlike classic HLA B27 bind to killer cell immunoglobulin like receptor 3DL2 (KIR 3DL2). Binding of HLA B27(2) to KIR 3DL2 positive CD4+ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLA B27(2) in order to confirm its expression in SpA and to inhibit its proinflammatory properties. METHODS: We generated monoclonal antibodies by screening a human phage display library positively against B27(2) and negatively against B27 heterotrimers. Specificity was tested by enzyme linked immunosorbent assay (ELISA) surface plasmon resonance (SPR) assay and fluorescence activated cell sorting (FACS) analysis of B27(2) expressing cell lines and peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with SpA. Functional inhibition of KIR 3DL2 B27(2) interactions was tested using cell lines and PBMCs from patients with SpA. RESULTS: Monoclonal antibody HD6 specifically recognized recombinant HLA B27(2) by ELISA and by SPR assay. HD6 bound to cell lines expressing B27(2) . FACS revealed binding of HD6 to PBMCs and SFMCs from patients with AS but not from controls. HD6 inhibited both the binding of HLA B27(2) to KIR 3DL2 and the survival and proliferation of KIR 3DL2 positive NK cells. Finally HD6 inhibited production of the proinflammatory disease associated cytokine interleukin 17 by PBMCs from patients with AS. CONCLUSION: These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27 associated spondylarthritides.

Reports of the use of HLA-B27/peptide tetrameric complexes to study peptide-specific CD8+ T cells in HLA-B27+-related diseases are rare. To establish HLA-B27 tetramers we first compared the function of HLA-B27 tetramers with HLA-A2 tetramers by using viral epitopes. HLA-B27 and HLA-A2 tetramers loaded with immunodominant peptides from Epstein-Barr virus were generated with comparable yields and both molecules detected antigen-specific CD8+ T cells. The application of HLA-B27 tetramers in HLA-B27-related diseases was performed with nine recently described Chlamydia-derived peptides in synovial fluid and peripheral blood, to examine the CD8+ T cell response against Chlamydia trachomatis antigens in nine patients with Chlamydia-triggered reactive arthritis (Ct-ReA). Four of six HLA-B27+ Ct-ReA patients had specific synovial T cell binding to at least one HLA-B27/Chlamydia peptide tetramer. The HLA-B27/Chlamydia peptide 195 tetramer bound to synovial T cells from three of six patients and HLA-B27/Chlamydia peptide 133 tetramer to synovial T cells from two patients. However, the frequency of these cells was low (0.02-0.09%). Moreover, we demonstrate two methods to generate HLA-B27-restricted T cell lines. First, HLA-B27 tetramers and magnetic beads were used to sort antigen-specific CD8+ T cells. Second, Chlamydia-infected dendritic cells were used to stimulate CD8+ T cells ex vivo. Highly pure CD8 T cell lines could be generated ex vivo by magnetic sorting by using HLA-B27 tetramers loaded with an EBV peptide. The frequency of Chlamydia-specific, HLA-B27 tetramer-binding CD8+ T cells could be increased by stimulating CD8+ T cells ex vivo with Chlamydia-infected dendritic cells. We conclude that HLA-B27 tetramers are a useful tool for the detection and expansion of HLA-B27-restricted CD8+ T cells. T cells specific for one or more of three Chlamydia-derived peptides were found at low frequency in synovial fluid from HLA-B27+ patients with Ct-ReA. These cells can be expanded ex vivo, suggesting that they are immunologically functional.