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 This information is current as of October 5, 2021. Isabel Wong-Baeza, Anna Ridley, Jackie Shaw, Hiroko Hatano, Oliwia Rysnik, Kirsty McHugh, Christopher Piper, Simon Brackenbridge, Ricardo Fernandes, Anthoni Chan, Paul Bowness and Simon Kollnberger

J Immunol 2013; 190:3216-3224; Prepublished online 25 Downloaded from February 2013; doi: 10.4049/jimmunol.1202926 http://www.jimmunol.org/content/190/7/3216 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2013/02/25/jimmunol.120292 Material 6.DC1 References This article cites 35 articles, 14 of which you can access for free at: http://www.jimmunol.org/content/190/7/3216.full#ref-list-1

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

Isabel Wong-Baeza,*,1 Anna Ridley,*,1 Jackie Shaw,*,1 Hiroko Hatano,* Oliwia Rysnik,* Kirsty McHugh,* Christopher Piper,* Simon Brackenbridge,† Ricardo Fernandes,† Anthoni Chan,‡ Paul Bowness,x and Simon Kollnberger*

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 b2-microglobulin–associated B27 and B27 free H chain forms (FHC), including disulfide-bonded H chain homodimers (termed B272). 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 and B27 FHC bind more strongly to

2 Downloaded from KIR3DL2 than other HLA-class I, including HLA-A3. B272 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 B272 and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B272 and B27 FHC stimulated KIR3DL2CD3«-trans- duced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFN-g secretion and promoted greater survival of KIR3DL2+ CD4 T and NK cells than binding to other HLA-class I. KIR3DL2+ T cells from B27+ http://www.jimmunol.org/ 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. The Journal of Immunology, 2013, 190: 3216–3224.

he human leukocyte Ag HLA-B27 (B27) is strongly as- forms (FHC), including cysteine-67–dependent disulfide-bonded sociated with a group of inflammatory arthritic disorders H chain homodimers (termed B272) (5–7). HLA-class I mole- T collectively known as the spondyloarthritides (SpA) (1). cules bind members of the killer cell Ig-like family (KIR) Several hypotheses have been proposed to explain B27 involvement. (8). B272 binds to different but overlapping groups of immune by guest on October 5, 2021 These include activation of cross-reactive autoimmune T cells by receptors compared with classical b2-microglobulin (b2m)–asso- arthritogenic peptides and stimulation of proinflammatory cytokine ciated B27 (5, 9). We have proposed that differences in the production by induction of endoplasmic reticulum stress resulting strength of binding and specificity of immune receptors binding to from B27 misfolding during assembly (2–4). B27 FHC forms and classical HLA-class I could lead to altered We have shown that B27 can be expressed on the surface of immune regulation and promote inflammation in SpA (10). patient and B27 transgenic rodent leukocytes as B27 free H chain KIRs are expressed by NK, NKT cells, and minor subsets of CD4 and CD8 T cells. KIRs are highly polymorphic and bind to HLA- *Nuffield Department of Rheumatological and Musculoskeletal Sciences, Botnar class I in an allele-specific fashion (11). For example, the cognate Research Centre, Oxford OX3 7LD, United Kingdom; †Medical Research Council KIR for classical HLA-B27 is KIR3DL1, which also binds to Human Immunology Unit, Weatherall Institute of Molecular Medicine, Headington, B27 (5). KIR can be distinguished by the presence or lack of Oxford OX3 9DS, United Kingdom; ‡Royal Berkshire Hospital, Reading RG1 5AN, 2 United Kingdom; and xBiomedical Research Centre, Oxford National Institute for a long cytoplasmic tail incorporating regulatory ITIM motifs. Health Research, Oxford OX3 9DU, United Kingdom These regulatory motifs are phosphorylated upon ligation by class 1I.W.-B., A.R., and J.S. contributed equally to this work. I at immunological synapses. Subsequently, KIR ligation modu- Received for publication October 22, 2012. Accepted for publication January 16, lates cytokine production and promotes immune cell survival 2013. by upregulating the expression of antiapoptotic and down- This work was supported by grants from Arthritis Research U.K. (to S.K., J.S., and regulating expression of proapoptotic genes such as FasL (12). O.R.), the Biomedical Research Centre, Oxford National Institute for Health Re- B27 but not b m-associated B27 binds to KIR3DL2, which has search (to A.R. and P.B.), the Belmont Trust (to P.B.), Consejo Nacional de Ciencia y 2 2 Tecnologı´a Mexico (to I.W.-B.), and the Japan Society for the Promotion of Science also been shown to bind to b2m-associated HLA-A3 and A11 (13, (to H.H.). 14). KIR3DL1 and 2 binding to classical b2m-associated HLA- Address correspondence and reprint requests to Dr. Simon Kollnberger, Nuffield class I is dependent on the sequence of peptide bound to the class I Department of Rheumatological and Musculoskeletal Sciences, Botnar Research molecule (14, 15). By contrast, B27 dimers bind to KIR3DL2 in Centre, Windmill Road, University of Oxford, Oxford OX3 7LD, U.K. E-mail ad- dress: [email protected] a peptide-independent fashion (16). The online version of this article contains supplemental material. Increased proportions of KIR3DL2-expressing NK and CD4 Abbreviations used in this article: AS, ankylosing spondylitis; B27, human leukocyte T cells are present in the blood and peripheral joint synovial fluid of + Ag HLA-B27; FHC, free H chain form; KIR, killer cell Ig-like receptor family; b2m, patients with spondyloarthritis (17, 18). Moreover, KIR3DL2 b2-microglobulin; MFI, mean fluorescence intensity; RA, rheumatoid arthritis; SEB, Th17 account for the majority of IL-17–producing CD4 T cells in staphylococcal enterotoxin B; SpA, spondyloarthritide. SpA patients compared with controls (18). Because KIR3DL2 Copyright Ó 2013 by The American Association of Immunologists, Inc. 0022-1767/13/$16.00 ligation by B272 enhances the survival of NK and CD4 T cells, we www.jimmunol.org/cgi/doi/10.4049/jimmunol.1202926 The Journal of Immunology 3217

have proposed that KIR3DL2-B272 interactions promote the sur- inhibitors (Thermo Scientific). KIR3DL2 was subsequently immunopre- vival of proinflammatory leukocytes in SpA (17, 18). cipitated by tumbling with anti-mouse Ig Dynal beads (Dynal). Immuno- By contrast with HLA-B27, HLA-A3 is not strongly associated precipitates were washed and resolved by reducing SDS-PAGE. Resolved lysates were Western blotted with a mix of anti-phosphotyrosine mAbs with spondyloarthritis. We hypothesized that differences between (PY20, Invitrogen; P-Tyr 100, Cell Signaling) and were subsequently the strength of binding of B272 and B27 FHC and HLA-A3 to reprobed with anti-hemagglutinin mAb (HA-7; Sigma-Aldrich). KIR3DL2 could explain the differential disease association of KIR3DL2CD3« reporter cell assay these different class I molecules. We predicted that stronger interactions of B27 FHC with A KIR3DL2CD3ε PHR-SIN lentiviral expression cassette was generated KIR3DL2 compared with HLA-A3 and other ligands would result in by overlapping PCR with primers designed to amplify the extracellular and transmembrane portions of KIR3DL2*0101 and the cytoplasmic region of stronger effects on downstream functions modulated by KIR ligation. CD3ε. KIR2DL3CD3ε PHR-SIN has been previously described (19). In this study, we compare the strength of interaction of B272 and Subsequently, KIR3DL2CD3ε and KIR2DL3CD3ε lentiviruses were made B27 FHC and HLA-A3 and other HLA-class I with KIR3DL2. We as described previously for LILRB2 (20). compare KIR3DL2 binding to HLA-B27 and other HLA-class I All experiments for cytokine assay were set up in RPMI 1640 medium using KIR3DL2 reporter cells and class I tetramer and KIR3DL2Fc (Sigma-Aldrich) supplemented with 10% FCS and antibiotics (R10) (16). A total of 200,000 transduced T cells was incubated with 200,000 221B27 staining of transfected cells. We also study the effect of KIR3DL2 or parental 221 cells, EBV-immortalized B cell lines, or MACS-sorted ligation by HLA-B27 and other ligands on receptor phosphoryla- (Miltenyi Biotec) CD14+ monocytes for 24 h with/without HC10, W632, tion, cell proliferation and survival, and cytokine production. We ME1, or isotype control IgG1/G2a mAbs (MOPC 123 and MG1-45; Bio- show that cell surface B27 FHC (which include B27 ) are ligands Legend) at 50 mg/ml. Subsequently, supernatants were harvested for IL-2 2 ELISA, according to the manufacturer’s instructions (eBioscience). for KIR3DL2. KIR3DL2 bind more strongly to B27 FHC than other

The tetramer and KIR3DL2 constructs used in this study are summarized Downloaded from characterized ligands. KIR3DL2 binding to B27 FHC inhibits IFN- in a schematic in Supplemental Fig. 1. g production and promotes the survival of leukocytes to a greater Generation of T and NK cell lines and clones: coculture of T or extent than binding to other HLA-class I, including HLA-A3. NK cell lines with HLA-B27–expressing APC T and NK cell lines and clones were generated from FACS-sorted CD4 Materials and Methods + Recombinant expression T cells from B27 SpA patients and maintained, as described previously

(17, 18). LBL.721.220 and LBL.721.221 parental B lymphocyte–derived http://www.jimmunol.org/ Recombinant class I were expressed as inclusion bodies, refolded cell lines (abbreviated to 220 or 221) transfected with HLA-A3, HLA-B7, as either heterotrimeric or homodimer complexes and purified by gel ex- HLA-B8, HLA-B27, HLA-B27C67S, HLA-B35, HLA-B44, and HLA- clusion chromatography, as previously described (5). Heterotrimers of B27 together with human tapasin have been described previously (21, HLA-class I comprise b2m, peptide class I H chains. HLA-A3 and HLA- 22). CD4 T or NK cell lines were labeled with CFSE, according to the B27 heterotrimer and homodimer tetramers were made as previously de- manufacturer’s instructions (Invitrogen). T and NK cell cocultures with scribed (16). 221 APC were set up, as previously described for 220 cells (17, 23). A total of 1 3 106 T or NK cells was cocultured with 0.5 3 106 221 cells. On FACS staining with tetramers and tetramer competition day 5, cells were stained for KIR3DL2 expression with the DX31 mAb, experiments annexin V allophycocyanin (BD Biosciences) and pacific blue Live Dead stain (InVitrogen) in annexin V buffer, according to the manufacturer’s Tetrameric complexes were used to stain Baf3 cells transduced with instructions (BD Biosciences). Alternatively, in CFSE proliferation by guest on October 5, 2021 KIR3DL1 and KIR3DL2, as previously described (16). For tetramer experiments, dead cells stained with Live Dead were excluded before competition experiments, cells were first stained with a saturating con- FACS analysis. Total viable numbers of KIR3DL2+ CD4 T cells were centration of PE-labeled tetramer before competition at room temperature enumerated with fluocount beads (Beckman Coulter). with a 2-fold excess of tetramer without fluorescent label. Subsequently, Supernatants for IFN-g ELISA (eBioscience) were harvested from NK FACS staining with fluorescent-conjugated tetramer was assessed at dif- cells after 0.24-h stimulation in R10 medium. ferent times after adding unlabeled tetramer. KIR3DL2Fc expression and purification, FACS staining, and Results pull-downs B27 dimer tetramers bind more strongly to KIR3DL2 than HLA-A3 A KIR3DL2Fc lentiviral expression cassette was constructed by overlapping PCR to generate cDNA encoding the D0, D1, and D2 domains of HLA-class I molecules are expressed as heterotrimeric complexes KIR3DL2*0101 fused to the Fc portion of human IgG1 cloned in PHR-SIN. with H chain, b m,andpeptide.Inadditiontob m-associated het- Lentiviruses were used to transduce 293T cells and soluble KIR3DL2Fc 2 2 purified from supernatants on protein A Sepharose. erotrimer, HLA-B27 isalsoexpressedasb2m-FHC dimers (termed A total of 200,000 LBL.721.221 (221) cells transfected with HLA-B27 or B272). We first compared staining of KIR3DL2-transfected Baf3 control HLA was stained for FACS analysis with 5 mg KIR3DL2 or control cells with HLA-A3 heterotrimers and B272 tetramers. B272 tetramer DR5 Fc protein, washed, and stained with PE-conjugated consistently stained KIR3DL2 transfectants more strongly than goat anti-human Igs (BioLegend). HLA-A3 heterotrimer tetramers (Fig. 1A, 1B). B27 tetramers did A total of 2 3 107 parental 221 cells or transfected cells was stained 2 with 50 mg KIR3DL2Fc or control DR5Fc. Cells were washed twice in ice- not stain parental Baf3 cells (Fig. 1A). B272 tetramers may have an cold PBS and lysed in lysis buffer (1% Nonidet P-40, 20 mM Tris, 150 mM enhanced avidity for KIR3DL2 because each dimer tetramer incor- NaCl, 0.5 mM iodoacetamide, 1 mM EDTA with peptidase inhibitors; porates eight molecules of HLA-B27 compared with HLA-A3, which Roche), and bound proteins were precipitated with protein G Dynal beads incorporates four molecules. Because of this, we stained KIR3DL2- (Dynal) washed six times with lysis buffer and resolved by nonreducing/ reducing SDS-PAGE, as previously described. Resolved lysates were expressing cells with nontetramerized recombinant B27 dimer, HLA- Western blotted with the anti–HLA-class I H chain mAb HC10. A3, or control HLA-class I protein. Subsequently, bound protein was detected by staining with extravidin PE. Recombinant B27 dimer Analysis of KIR3DL2 phosphorylation protein stained KIR3DL2 transfectants more strongly than HLA- Transduced T cells were starved overnight in DMEM without supplements A3 and other HLA-class I proteins (Fig. 1C). (D0). A total of 5 3 106 Jurkat cells transduced with C-terminal Next, we studied the ability of B27 dimer and HLA-A3 heter- hemagglutinin-tagged KIR3DL2 was stimulated with 10 ng/ml staphylo- otrimer to compete for binding to KIR3DL2. We stained KIR3DL2- coccal enterotoxin E and 5 3 106 parental or transfected 221 cells in DMEM without supplements for 20 min prior to staining with anti-KIR3DL2 expressing cell lines with fluorescent-conjugated HLA-A3 heter- mAb for 10 min on ice (clone 1; Innate Pharma, Marseille, France). After otrimer or B27 dimer tetramers and then measured remaining washing in ice-cold TBS, cells were lysed in lysis buffer with phosphatase bound tetramer at 30 and 60 min after adding unlabeled tetramer. 3218 STRONGER B27 FREE H CHAIN INTERACTIONS WITH KIR3DL2 Downloaded from http://www.jimmunol.org/ by guest on October 5, 2021

FIGURE 1. B272 protein and tetramers bind to KIR3DL2-transfected cells more strongly than HLA-A3. (A) Representative FACS stain of KIR3DL2- transfected Baf3 cells with saturating concentrations of B27 dimer and HLA-A3 tetramers. Geometric MFIs for staining were 206 and 52 for B27 dimer and

HLA-A3 tetramer staining, respectively. Representative of one of four independent experiments. (B) Titration of PE-labeled B272 and HLA-A3 tetramer in FACS staining of KIR3DL2-transfected Baf3 cells. Representative stain from one of four independent experiments. (C) FACS staining of KIR3DL2 Baf3 cells with B27 dimer (B272) HLA-A3, HLA-B8, and HLA-B27 protein. Representative staining of one of three independent experiments. (D) B272 tet- ramers compete for binding to KIR3DL2 more strongly than HLA-A3. Left-hand top panel, B272 tetramer competition with PE-labeled HLA-A3 tetramer bound to KIR3DL2 Baf3 cells. Staining with HLA-A3 tetramer at 0, 30, and 60 min after addition of HLA-A3 or B272 tetramer. Left-hand bottom panel, HLA-A3 tetramer competition with PE-labeled B272 tetramer bound to KIR3DL2 Baf3 cells. Staining with B272 tetramer at 0, 30, and 60 min after addition of HLA-A3 or B272 tetramer. Results are presented as the mean reduction in the geometric MFI of tetramer staining from three independent experiments 6 1 SD.

B272 tetramers effectively competed HLA-A3 heterotrimer tetra- tetramer weakly. HLA-A3 tetramers also competed for bound mer binding to KIR3DL2 transfectants, whereas HLA-A3 tet- HLA-A3 tetramer weakly (Fig. 1D). B272 tetramer staining of ramers had little effect on B272 binding (Fig. 1D, upper and lower KIR3DL2-expressing cell lines was strongly inhibited by H chain panels). B27 dimer tetramers competed for bound B27 dimer mAb, which recognizes B27 dimers compared with HLA-A3 The Journal of Immunology 3219 tetramer staining, which was only weakly inhibited (HC10 or HCA2; Supplemental Fig. 1). KIR3DL2 staining by both tetramers was inhibited by anti-KIR3DL2 mAb (DX31) (Supplemental Fig. 1). KIR3DL2 binds more strongly to cell surface B27 FHC than other HLA-class I We next compared binding of KIR3DL2Fc fusion protein to HLA- B27 and other HLA-class I molecules. KIR3DL2 Fc consistently stained HLA-B27–transfected LBL.721.221 cells (hereafter referred to as 221B27 cells) more strongly than parental 221 cells or 221A3 and 221B35 cells (Fig. 2A, 2B). Fig. 2B shows KIR3DL2Fc staining of transfected cells expressed as a ratio with the mean fluorescence intensity (MFI) for W632 staining after subtracting the MFIs for background KIR3DL2 staining of parental 221 cells. The 221B35 cells did not stain with KIR3DL2Fc significantly above background staining of parental 221 cells. We hypothesized that the increased staining of B27-transfected cells with KIR3DL2Fc could result from KIR3DL2 binding to B27 FHC expressed by these cells (22). Thus, we stained 221B27 with KIR3DL2Fc and ana- lyzed bound protein precipitated with protein G from cell lysates Downloaded from by SDS-PAGE and Western blot with HC10 mAb. Bands corre- sponding to B27 dimers, monomers, and multimers were detec- ted by Western blot (Fig. 2B). No bands were detected when Western blots were reprobed with the anti-b2m Ab BBM-1 (results not shown). We reasoned that stronger binding of B27 FHC would stimulate http://www.jimmunol.org/ greater KIR3DL2 ITIM phosphorylation compared with other class I ligands. Thus, we compared KIR3DL2 tyrosine phosphorylation in superantigen-activated KIR3DL2-expressing Jurkat cells with transfected 221 cells. HLA-B27 ligation consistently stimulated greater phosphorylation of KIR3DL2 compared with HLA-A3, HLA-B35, or parental 221 cells (Fig. 2C). We then determined whether the stronger binding of B27 dimers to KIR3DL2 in vitro could be translated into differences in fun- by guest on October 5, 2021 ctional interactions with this receptor. We transduced Jurkat T cells with KIR3DL2CD3ε and measured functional interactions with plate-immobilized recombinant B27 dimers and HLA-class I het- erotrimers by measuring IL-2 production by ELISA. B27 dimers, but not control HLA-B27 heterotrimers, consistently stimulated IL- 2 production above background levels in the presence of PMA and FIGURE 2. KIR3DL2Fc binds B272 more strongly than other HLA- class I. (A) FACS staining of nontransfected LBL.721.221 (221) cells and anti-CD28 (Fig. 3A). Equivalent quantities of immobilized B27 221 cells transfected with HLA-B27, HLA-A3, and HLA-B35 with dimer consistently stimulated greater production of IL-2 than HLA- KIR3DL2Fc. The 221B27 cells stained with control DR5Fc fusion proteins A3 heterotrimers. are also shown. Geometric MFIs for staining of 221, 221B27, 221A3, and Subsequently, we studied whether cell surface HLA-B27 bound 221B35 were 18, 46, 22, and 17, respectively. Representative FACS stain to KIR3DL2CD3ε-Jurkat T cells. The 221B27 cells stimulated greater from one of three independent experiments. (B) Ratio of KIR3DL2Fc MFI: IL-2 secretion than 221A2, 221A3, 221B7, 221B35, 221B27C67S, W632 MFI for staining of 221 transfectants. Ratios were calculated after and parental 221 cells (Fig. 3B). HLA-class I transfectants ex- subtracting the MFI for KIR3DL2Fc staining of parental 221 cells. Ratios pressed similar quantities of class I, as assessed by FACS staining were calculated from the mean MFIs from three independent experiments. C with W632 mAb (Supplemental Fig. 2). Parental Jurkat T cells ( ) Nonreducing SDS-PAGE gel and Western blot with HC10 of precip- and KIR2DL3CD3ε-transduced Jurkat T cells produced negligible itated proteins from 221B27 cells with control DR5Fc (lane I), and with KIR3DL2Fc from 221B27 (lane II), and 221B35-transfected cells (lane quantities of IL-2 in response to these stimuli (Supplemental ε III)(left-hand panel). Positions of multimeric, M, B27 dimer, B272, and Fig. 3). IL-2 production by KIR3DL2CD3 -transduced Jurkat T monomeric B27 H chains, B27, are indicated. The right-hand panel shows stimulated with 221B27 cells was inhibited by KIR3DL2-specific the same blot reprobed with HRP-conjugated anti-human Igs. The position mAb (DX31), H chain (HC10), and W632 Abs (Fig. 3B). By con- of KIR3DL2Fc is indicated. Representative Western blots from one of trast, the B27-specific Ab ME1 had no significant effect (Fig. 3B). three independent experiments. (D) Upper panel, Phosphotyrosine Western LBL.721.220 cells transfected with HLA-B27 (hereafter referred blots of immunoprecipitates of KIR3DL2-transduced Jurkat T cells (lane to as 220B27) also express cell surface B27 H chain dimers and I), or T cells stimulated with superantigen and parental 221 cells (lane II), multimers (22). These cells lack functional human tapasin and as or HLA-B35 (lane III), HLA-A3 (lane IV), or HLA-B27 transfectants a consequence form more unstable B27 heterotrimers on their cell (lane V). Lane VI, 221B27 cells alone. Lower panel, Western blot reprobed surface. B27 dimers and multimers form from unstable cell sur- with anti-hemagglutinin. Representative blots from one of three indepen- dent experiments. face B27 heterotrimers (22). Supertransfection of 220B27 cells with human tapasin (220B27 huTPN), which optimizes peptide cargo and stabilizes B27 heterotrimers, reduces the level of cell surface B27 dimer expression (16). As a consequence, 220B27huTPN cells 3220 STRONGER B27 FREE H CHAIN INTERACTIONS WITH KIR3DL2

FIGURE 3. B27 dimers and FHC stimulate KIR3DL2CD3ε reporter Downloaded from T cells more strongly than control HLA class I. (A) IL-2 production by KIR3DL2CD3ε-transduced T cells stimulated with recombinant HLA-B27 dimer and HLA-B27 or HLA-A3 heterotrimers. Results, presented as mean values from triplicate samples (pg/ml) 6 1 SD. (B) IL-2 secretion by KIR3DL2CD3ε-transduced Jurkat T cells, stimulated with parental LBL.721.221 (221) cells and cells transfected with HLA-B27, HLA- B27C67S, HLA-B7, HLA-B35, HLA-A2, and HLA-A3. Results presented http://www.jimmunol.org/ as mean values from five independent experiments (pg/ml) 6 1 SEM. (C) IL-2 production by KIR3DL2CD3ε-transduced T cells stimulated with 221B27 cells with HC10, W632, ME1, and IgG2a and IgG1 isotype control mAbs or the anti-KIR3DL2 mAb (DX31). Results presented as mean values from triplicate samples (pg/ml) 6 1 SD are representative of four independent experiments. (D) IL-2 production by KIR3DL2CD3ε- transduced T cells stimulated with parental LBL.221.220 cells, 220B8, 220B27 cells with HC10, W632, ME1, and IgG2a and IgG1 isotype control mAbs and 220B27 cells transfected with human tapasin (HuTPN). by guest on October 5, 2021 Results presented as mean values from triplicate samples (pg/ml) 6 1SD are representative of four independent experiments. express higher levels of ME1 and W632-reactive B27 and lower levels of HC10-reactive B27 FHC. As expected, 220B27 cells stimulated production of IL-2 by KIR3DL2CD3ε-transduced T cells to a greater extent than parental 220 cells or 220 cells transfected with control HLA-A1, HLA-B8, or HLA-B44 (Fig. 3D). The 220B27 cells transfected with human tapasin stimulated less IL-2 production by KIR3DL2CD3ε reporter cells compared with 220B27 transfectants (Fig. 3D). As seen for the 221B27 cells, production of IL-2 by reporter cells was reduced by Abs, which inhibit KIR3DL2 interactions with B27 FHC and dimers (HC10 and W632, respectively; Fig. 3D). FIGURE 4. B cell lines and monocytes from B27+ SpA patients and We have shown that B27 dimers and H chains are expressed by B27+ controls express HLA-class I ligands for KIR3DL2. (A) Represen- + APC in B27 SpA patient peripheral blood and synovial fluid tative FACS stain with W632, HC10, and ME1 Abs of an EBV-trans- (5, 7). We sought to determine whether HLA-class I on the surface formed B cell line from a B27+ SpA patient. Representative stain from one of APC from B27+ SpA patients and B27+ healthy controls could of four different B cell lines. Geometric MFIs for staining with HC10, interact with KIR3DL2 on KIR3DL2CD3ε Jurkat T cells. The ME1, and W632 were 406, 1443, and 3464, respectively. (B) Represen- + representative FACS stains in Fig. 4A and 4B show the relative tative FACS stain with HC10 and ME1 Abs of CD14 monocytes from + levels of expression of HC10 and W632-reactive forms of HLA- a B27 SpA patient. Representative stain from one of four monocyte class I by EBV-transformed B cells and purified monocytes from samples. Geometric MFIs for staining with HC10, ME1, and W632 were + + 82, 1315, and 2724, respectively. (C) IL-2 production by KIR3DL2CD3ε B27 individuals. B cell lines and monocyte lines from B27 D + reporter cells stimulated with primary B cell lines and ( ) primary healthy controls and B27 SpA patients both stimulated IL-2 monocyte lines from a B27+ healthy control and a B27+ AS patient. IL-2 ε production by KIR3DL2CD3 -transduced T cells (Fig. 4C, 4D). production by reporter cells is inhibited by anti-H chain (HC10), anti- Reporter cell IL-2 production was inhibited by HC10 and W632 KIR3DL2, and W632 monoclonals. Representative data from experiments mAbs, but not isotype control mAb (Fig. 4A, 4B). B cell lines with four different B cell lines and three monocyte lines. Results with B from healthy controls also weakly stimulated IL-2 production by cell lines were repeated on three independent occasions. Results are KIR3DL2CD3ε-transduced T cells (data not shown). expressed as mean values of IL-2 pg/ml 6 1 SD. *p , 0.05 by ANOVA. The Journal of Immunology 3221

Ligation of KIR3DL2 by HLA-B27 inhibits NK IFN-g cells were inhibited with anti-KIR3DL2 (DX31), anti-class I H production and promotes NK cell survival to a greater extent chain (HC10), and W632 mAbs, but not isotype control IgG2a than binding to HLA-A3, HLA-B7, and HLA-B35 mAb (Fig. 6B). We have previously shown increased proportions of CD4 T cells We next investigated the effect of KIR3DL2 binding to HLA-B27 + on cytokine production and survival of primary NK cells. The expressing KIR3DL2 in B27 SpA patients (17, 23). We hypothe- 221B27 cells inhibited the production of IFN-g by KIR3DL2- sized HLA-B27 on APC could promote the survival of Ag-stimulated KIR3DL2+-expressing leukocytes in ankylosing spondylitis (AS). expressing NK cells to a greater extent than cells expressing + control HLA-class I (Fig. 5A). Inhibition of NK cytokine pro- Thus, we compared the survival of CFSE-labeled KIR3DL2 duction by 221B27 cells was reduced by anti-KIR3DL2 and HC10 CD4 T cells stimulated with superantigen presented by APCs in PBMC samples from B27+ SpA patients and healthy B27+ and mAbs, but not isotype control mAb (Fig. 5B). Stimulation with 2 221B27 cells promoted the survival of KIR3DL2-expressing NK B27 controls and patients with rheumatoid arthritis (RA). PBMC cells to a greater degree than parental 221 cells and cells ex- were labeled with CFSE and stimulated with SEB for 5 d. Viable pressing control HLA-class I (Fig. 5C). The effect of 221B27 cells proliferating CD4 T cells expressing KIR3DL2 or other KIR were in promoting NK cell survival could be inhibited by anti-class I determined by FACS staining. (HC10 and W632) and anti-KIR3DL2 (DX31) mAbs (Fig. 5D). Stimulation with HLA-B27 enhances the survival of KIR3DL2+ CD4 T cells compared with stimulation with HLA-A3 and other HLA-class I: increased expansion of Ag-stimulated KIR3DL2+ T cells in B27+ SpA patients Downloaded from We next studied proliferation and survival of primary KIR3DL2+ CD4 T cells, stimulated with 221B27 cells by CFSE dilution. The 221B27 cells promoted the survival of staphylococcal enterotoxin B (SEB)-activated KIR3DL2+ CD4 T cells to a greater degree than parental 221 and 221A3 (Fig. 6A). Superantigen-driven proliferation and survival of KIR3DL2+ CD4 T cells with 221B27-transfected http://www.jimmunol.org/ by guest on October 5, 2021

FIGURE 6. Enhanced survival of KIR3DL2-expressing T cells stimu- lated with 221B27 cells. Increased expansion of Ag-stimulated KIR3DL2+ CD4 T cells in B27+ SpA patients. (A) Viable CFSE-labeled primary KIR3DL2+ CD4 T cells after stimulation with parental 221 cells and 221B27, -B7, -B35, and -A3 and superantigen (SEB) or with 221B27 cells without superantigen. Geometric MFIs are 439 (221B27 + SEB), 998 (221 + SEB), 851 (221A3 + SEB), 1214 (221B7 + SEB), 1935 (221B35 + SEB), and 2876 (221B27). Representative FACS stain from one of five independent experiments. (B) Numbers of viable CFSE-labeled primary KIR3DL2+ CD4 T cells after 5-d stimulation with 221B27 cells without stimulus (2) or parental 221 cells, 221-A3, -B7, -B35, -B27C67S, and -B27 cells with SEB. Numbers of viable CD4 T cells after 5-d stimulation with SEB and FIGURE 5. KIR3DL2 ligation by HLA-B27 inhibits NK cell IFN-g and 221B27 cells with control (IgG2a), anti–HLA-class I (HC10 or W632), or promotes NK cell survival. (A) IFN-g production by KIR3DL2-expressing anti-KIR3DL2 (DX31) mAbs are also shown. Mean data 6 1 SEM from NK cells stimulated with parental 221 cells and 221 cells transfected with three independent experiments. *p , 0.05 by ANOVA for T cells stim- HLA-B27, HLA-A3, HLA-B7, HLA-B35, or HLA-B27C67S. Represen- ulated with SEB and 221B27 or SEB 221B27 and isotype mAb (IgG2a) tative data from triplicate samples from one of three independent experi- compared with other stimuli. (C) Representative FACS stains of CFSE- ments. (B) IFN-g production by KIR3DL2-expressing NK cells stimulated labeled PBMC from an AS and RA patient and a healthy B272 control with parental 221B27 cells and anti-KIR3DL2 (DX31), anti–HLA-class I and healthy B27+ control showing proliferation of KIR3DL2+ and H chain (HC10), or isotype control mAbs (IgG2a). Representative data KIR3DL22 CD4 T cells following 5-d stimulation with superantigen from triplicate samples from one of three independent experiments. (C) (SEB). Representative FACS stains from 10 independent experiments. Annexin V and Live Dead (Dead) staining of KIR3DL2+ NK cells stim- (D) Fold increase in peripheral blood KIR3DL2 CD4 T cells from AS ulated for 7 d with parental 221 cells or 221A3, HLA-B27 or B7 cells. patients, B272 and B27+ healthy controls, and RA patients following 5-d Representative data from one of three independent experiments. Lower stimulation with SEB. Right-hand panel, Mean fold increases in absolute left-hand quadrants, Indicate percentages of viable NK cells remaining at KIR3DL2+ CD4 T cell numbers with interquartile ranges for each of the the end of the assay. (D) Annexin V and Live Dead staining of KIR3DL2+ group studies are 8.6 and 9.3 (n = 16) for B27+ SpA, 3.2 and 3.4 for B272 NK cells stimulated for 7 d with 221B27 cells and anti-KIR3DL2 (DX31), healthy controls (n = 12), 7.2 and 9 for B27+ healthy controls (n =6),and anti–HLA-class I mAbs (HC10 and W632), or isotype control mAb. 3.8 and 4.2 for RA patients (n =13). 3222 STRONGER B27 FREE H CHAIN INTERACTIONS WITH KIR3DL2

We observed increased numbers of viable proliferating KIR3DL2 that B27 FHC form from b2m-associated HLA-B27 with subop- CD4 T cells from SpA patients after stimulation with superantigen timal peptides (22). Thus, 220B27 cells express increased levels of compared with KIR3DL2 CD4 T cells from healthy and RA disease B27 dimers and other FHC species because of the presence of controls (Fig. 6C). By contrast, we did not see similar expansions of more B27 peptide MHC complexes suboptimally loaded with CD4 T cells expressing other KIR (Supplemental Fig. 4A). Because peptide. Consistent with this hypothesis, 220B27 cells transfected of variability in the percentages of T cells expressing KIR and with human tapasin stimulated less IL-2 production by reporter responding to superantigen between individuals, we counted num- cells compared with 220B27 cells. bers of KIR3DL2+ CD4 T cells before and after stimulation using The 221B27 cells inhibited IFN-g production by KIR3DL2- fluocount beads. We then compared the fold increase in KIR3DL2+ expressing NK cells to a greater extent than cells expressing CD4 T cells between AS patients and controls when cells were other HLA-class I. The 221B27 inhibition of IFN-g production was stimulated with superantigen. Fig. 6D shows that there was reduced by H chain and anti-KIR3DL2 Abs, suggesting that at least a greater increase in numbers of KIR3DL2 CD4 T cells in patients part of this effect was due to KIR3DL2 binding to B27 FHC spe- compared with B272 healthy controls and RA disease controls. cies. The 221B27 cells also promoted the survival of KIR3DL2- Expansion of KIR3DL2+ CD4 T cells in AS patients was not due expressing NK and T cells more strongly than HLA-A3 and other to Vb bias toward SEB-binding Vb in this population in patients HLA-class I–transfected cells. We have shown that KIR3DL2- as FACS staining showed no enrichment for SEB-reactive Vb in expressing leukocytes are expanded in B27+ spondyloarthritis patients (Supplemental Fig. 4B). We also observed a greater fold patients (17, 18). KIR ligation inhibits activation-induced cell death increase in the number of CD4-negative T cells between patients of leukocytes (17, 26). The increased proportions of KIR3DL2+-ex- and controls (Supplemental Fig. 4C). SEB-driven expansion of pressing leukocytes in SpA patients could thus be due to stronger KIR3DL2+ CD4 T cells in AS patients was inhibited by anti- KIR3DL2 binding to B27 promoting immune cell survival. Downloaded from KIR3DL2 mAb (DX31) and B27 FHC-specific mAb (HD6) Whereas HLA-A3 binding to KIR3DL2 is dependent on the (Supplemental Fig. 4D). By contrast, B27 FHC-specific mAb had sequence of complexed peptide, B27 dimers bind to KIR3DL2 in little effect on expansion of KIR3DL2+ CD4 T cells in B272 a peptide-independent fashion (14, 16). This could also result in healthy controls (Supplemental Fig. 4D). higher densities of B27 ligand at the surface of APC available for binding to KIR3DL2 compared with HLA-A3. Higher densities of Discussion ligand could also result in stronger interactions with KIR3DL2. http://www.jimmunol.org/ In this study, we show that KIR3DL2 binds more strongly to HLA- HLA-A3–expressing individuals have NK cells with poor effector B27 FHC species (which include B27 dimers) than to HLA-A3 and function, suggesting that HLA-A3 may only interact weakly with other HLA-class I. B27 dimer tetramers bound more strongly to KIR3DL2 in vivo in “licensing” NK cells (27). Our findings suggest KIR3DL2 and competed more effectively for binding to KIR3DL2 that studying how HLA-B27 expression could “license” better than HLA-A3 tetramers, suggesting that B27 dimers are stronger KIR3DL2+ NK cell effector function merits further investigation. ligands. The fact that nontetramerized B27 dimer protein also We hypothesize that KIR3DL2-B27 interactions could promote bound more strongly to KIR3DL2-expressing cell lines than HLA- the expansion of T cells and other leukocytes in vivo. In support of A3 suggests that this was not simply due to differences in tetramer this, superantigen-activated KIR3DL2+-expressing T cells in by guest on October 5, 2021 stoichiometry. Moreover, B27 dimer tetramer binding to KIR3DL2 PBMC samples from SpA patients proliferated more than the same was strongly inhibited by the H chain–specific monoclonal HC10. immune subset in control samples. We observed greater prolifera- The HLA-A H chain mAbs HCA2 and HC10 mAb only slightly tion of both CD4+ and CD42 T cells expressing KIR3DL2 in SpA reduced HLA-A3 binding to KIR3DL2, suggesting that binding of patients. Although we have previously shown enrichment for IL- B27 FHC and HLA-A3 to this receptor is distinct. HCA2 binds 23R expression on KIR3DL2-expressing CD4 T cells, we did not within and HC10 binds close to a region of HLA-class 1, which observe similar enrichment on CD42 T (18). This suggests that the binds to the D0 domain of KIR3DL1 (23–25). KIR3DL1 has increased expansion of KIR3DL2 T cells in these assays is not a related structure to KIR3DL2. KIR3DL2Fc stained 221B27 cells simply because of increased levels of IL-23 in SpA patients pro- more strongly than HLA-A3 and control HLA-class I–transfected moting the survival of these cells. We did not observe significantly cells. increased proliferation of KIR3DL2+-expressing T cells from We also observed greater phosphorylation of KIR3DL2 when B27+ healthy controls compared with other control samples. We T cells were stimulated with 221B27 cells compared with stim- have previously reported intermediately increased proportions of ulation with parental 221, 221A3, and 221B35 cells. This increased KIR3DL2+ CD4 T cells in healthy B27+ controls compared with phosphorylation of KIR3DL2 ITIMs by B27 also suggests stronger B272 controls (18). The difference in proliferation of KIR3DL2 binding compared with other HLA-class I. CD4 T cells between B27+ SpA patients and B27+ controls might Recombinant B27 dimers stimulated greater production of IL-2 be explained by higher levels of expression of B27 FHC ligands by KIR3DL2 reporter cells compared with HLA-A3 and HLA-B27 for KIR3DL2 expressed by APC from B27+ SpA. Indeed, in heterotrimers. Moreover, 221B27 cells stimulated greater pro- support of this, we and others have reported higher levels of ex- duction of IL-2 by KIR3DL2CDε reporter cells compared with pression of B27 and other FHC on the surface of peripheral blood 221A3, parental 221 cells, and control HLA-class I–transfected monocytes from B27+ SpA patients (5, 7, 28, 29). cells. The 221A3 cells stimulated production of more IL-2 by KIR3DL2CD3ε Τ cell interactions with B27 were inhibited by KIR3DL2CDε-reporter cells compared with stimulation with 221 HC10, W632, and HD6, but not by ME1 Abs (this study) (7). cells expressing control HLA-class I, but not HLA-B27. These W632 and HD6 Abs inhibited KIR3DL2 binding more weakly results suggest that peptide MHC complexes and possibly other than HC10 in these assays. HC10 was most effective at inhibiting forms of HLA-A3 are weaker ligands for KIR3DL2 than HLA- KIR3DL2CD3ε Τ cell interactions, suggesting that the different B27 H chain forms. inhibitory Abs recognize different B27 species that share the The 220B27 cells also stimulated greater production of IL-2 by HC10 epitope. By contrast, ME1 recognizes a population of b2m- KIR3DL2CD3ε-transduced T cells compared with cells express- associated HLA-B27 molecules and did not inhibit KIR3DL2 inter- ing control HLA-class I. The 220B27 cells lack functional tapasin, actions. W632 and ME1 recognize distinct epitopes of b2m-associated which optimizes peptides bound to HLA-class I. We have shown HLA-class I. W632 recognizes a conformational-dependent epi- The Journal of Immunology 3223 tope in the a2 and 3 domains of HLA-class I close to residues 86 spondylitis in the white population in the United Kingdom. Ann. Rheum. Dis. 55: 268–270. and 121 of class I H chains (30). By contrast, ME1 binds to a 2. Colbert, R. A., M. L. DeLay, G. Layh-Schmitt, and D. P. Sowders. 2009. HLA- conformational b2m-dependent epitope in the a1 domain of HLA- B27 misfolding and spondyloarthropathies. Adv. Exp. Med. Biol. 649: 217–234. B27 that is critically dependent on interactions between residues 3. Fiorillo, M. T., and R. Sorrentino. 2009. T-cell responses against viral and self- epitopes and HLA-B27 subtypes differentially associated with ankylosing 67 and 71 (31). W632 has also been reported to recognize a pop- spondylitis. Adv. Exp. Med. Biol. 649: 255–262. ulation of B27 dimers on transfected cells and b2m-free HLA 4. Lo´pez de Castro, J. A. 2007. HLA-B27 and the pathogenesis of spondyloar- class 1 on transfected and Daudi cells, but does not inhibit B27 thropathies. Immunol. Lett. 108: 27–33. 5. Kollnberger, S., L. Bird, M. Y. Sun, C. Retiere, V. M. Braud, A. McMichael, and dimer tetramer binding to KIR3DL2 (32, 33). P. Bowness. 2002. Cell-surface expression and recognition of Our results suggest that B27 FHC ligands for KIR3DL2 are HLA-B27 homodimers. Arthritis Rheum. 46: 2972–2982. heterogeneous. KIR3DL2Fc pulled down monomeric, dimeric, and 6. Kollnberger, S., L. A. Bird, M. Roddis, C. Hacquard-Bouder, H. Kubagawa, H. C. Bodmer, M. Breban, A. J. McMichael, and P. Bowness. 2004. HLA-B27 multimeric H chain forms from B27-expressing cells. Dimerization heavy chain homodimers are expressed in HLA-B27 transgenic rodent models of and multimerization of B27 H chains could increase the binding spondyloarthritis and are ligands for paired Ig-like receptors. J. Immunol. 173: 1699–1710. avidity for KIR3DL2. Alternatively, monomeric B27 H chains could 7. Payeli, S. K., S. Kollnberger, O. Marroquin Belaunzaran, M. Thiel, K. McHugh, bind more strongly to this receptor than other HLA-class I H chains. J. Giles, J. Shaw, S. Kleber, A. Ridley, I. Wong-Baeza, et al. 2012. Inhibiting Monocytes and B cells from B27+ SpA patients and B27+ healthy HLA-B27 homodimer-driven immune cell inflammation in spondylarthritis. ε Arthritis Rheum. 64: 3139–3149. controls stimulated production of IL-2 by KIR3DL2CD3 -trans- 8. Lanier, L. L. 1998. Follow the leader: NK cell receptors for classical and non- duced T cells, which was inhibited by HC10 and W632 mAbs. classical MHC class I. Cell 92: 705–707. 9. Allen, R. L., T. Raine, A. Haude, J. Trowsdale, and M. J. Wilson. 2001. Leu- Thus, b2m-free class I H chains and other class I species on cells kocyte receptor complex-encoded immunomodulatory receptors show differing that express physiological levels of HLA-class I can interact with specificity for alternative HLA-B27 structures. J. Immunol. 167: 5543–5547. Downloaded from KIR3DL2. Our results with monocytes and immortalized B cell 10. Kollnberger, S., and P. Bowness. 2009. The role of B27 heavy chain dimer lines do not distinguish between B27 and other HLA-class I H immune receptor interactions in spondyloarthritis. Adv. Exp. Med. Biol. 649: 277–285. chains binding to KIR3DL2. Indeed, interestingly, our observations 11. Lanier, L. L. 2005. NK cell recognition. Annu. Rev. Immunol. 23: 225–274. suggest that KIR3DL2 can also weakly interact with other HLA- 12. Chwae, Y. J., M. J. Chang, S. M. Park, H. Yoon, H. J. Park, S. J. Kim, and J. Kim. 2002. Molecular mechanism of the activation-induced cell death inhibition class I H chains in addition to HLA-B27 on transfected cells and mediated by a p70 inhibitory killer cell Ig-like receptor in Jurkat T cells. J. EBV-transformed B cell lines (our unpublished observations). Immunol. 169: 3726–3735. We propose that HLA-B27 forms higher levels of high-avidity 13. Do¨hring, C., D. Scheidegger, J. Samaridis, M. Cella, and M. Colonna. 1996. A http://www.jimmunol.org/ human killer inhibitory receptor specific for HLA-A1,2. J. Immunol. 156: 3098– FHC ligands for KIR3DL2 under inflammatory conditions com- 3101. pared with other HLA-class I. In support of this, both we and other 14. Hansasuta, P., T. Dong, H. Thananchai, M. Weekes, C. Willberg, H. Aldemir, authors have shown increased expression of HLA-class I H chains S. Rowland-Jones, and V. M. Braud. 2004. Recognition of HLA-A3 and HLA- A11 by KIR3DL2 is peptide-specific. Eur. J. Immunol. 34: 1673–1679. and B27 FHC species on the surface of monocyte populations in 15. Stewart-Jones, G. B., K. di Gleria, S. Kollnberger, A. J. McMichael, E. Y. Jones, B27+ SpA and PsA patients (5, 7, 28, 29, 34). and P. Bowness. 2005. Crystal structures and KIR3DL1 recognition of three How could KIR3DL2 bind to HLA-class I H chains? One immunodominant viral peptides complexed to HLA-B*2705. Eur. J. Immunol. 35: 341–351. possibility is that KIR3DL2 binds to shared features of HLA-class I 16. Kollnberger, S., A. Chan, M. Y. Sun, L. Y. Chen, C. Wright, K. di Gleria, weakly in a similar way to KIR3DL1 (25, 35). Because the D0 A. McMichael, and P. Bowness. 2007. Interaction of HLA-B27 homodimers with by guest on October 5, 2021 domain of KIR3DL1 binds to a conserved region of all HLA-class KIR3DL1 and KIR3DL2, unlike HLA-B27 heterotrimers, is independent of the sequence of bound peptide. Eur. J. Immunol. 37: 1313–1322. I, it is possible that the D0 domain of KIR3DL2 could bind to 17. Chan, A. T., S. D. Kollnberger, L. R. Wedderburn, and P. Bowness. 2005. Ex- a similar region in HLA-B27. Disulfide-dependent dimerization pansion and enhanced survival of natural killer cells expressing the killer immunoglobulin-like receptor KIR3DL2 in spondylarthritis. Arthritis Rheum. and/or multimerization of B27 H chains could increase the avidity 52: 3586–3595. of HLA-B27 for KIR3DL2. 18. Bowness, P., A. Ridley, J. Shaw, A. T. Chan, I. Wong-Baeza, M. Fleming, How could these interactions be involved in arthritic disease? We F. Cummings, A. McMichael, and S. Kollnberger. 2011. Th17 cells expressing KIR3DL2+ and responsive to HLA-B27 homodimers are increased in ankylosing propose that KIR3DL2 binding to B27 FHC could promote the spondylitis. J. Immunol. 186: 2672–2680. survival and differentiation of proinflammatory leukocytes in AS. 19. Fernandes, R. A., C. Yu, A. M. Carmo, E. J. Evans, P. A. van der Merwe, and One possibility is that KIR3DL2 ligation could divert the pro- S. J. Davis. 2010. What controls T cell receptor phosphorylation? Cell 142: 668– 669. duction of cytokines in inflammation from Th1 to Th17. Indeed, 20. Giles, J., J. Shaw, C. Piper, I. Wong-Baeza, K. McHugh, A. Ridley, D. Li, KIR3DL2 ligation by B27 FHC inhibits production of IFN-g I. Lenart, A. N. Antoniou, K. DiGleria, et al. 2012. HLA-B27 homodimers and by NK cells and can promote survival of Th17 cells under some free H chains are stronger ligands for leukocyte Ig-like receptor B2 than classical HLA class I. J. Immunol. 188: 6184–6193. circumstances (16, 18). 21. Peh, C. A., S. R. Burrows, M. Barnden, R. Khanna, P. Cresswell, D. J. Moss, and In this study, we show that B27 FHC species, including B27 J. McCluskey. 1998. HLA-B27-restricted antigen presentation in the absence of tapasin reveals polymorphism in mechanisms of HLA class I peptide loading. dimers, bind more strongly to the NK receptor KIR3DL2 than Immunity 8: 531–542. conventional HLA-class I. Stronger binding of B27 FHC to 22. Bird, L. A., C. A. Peh, S. Kollnberger, T. Elliott, A. J. McMichael, and KIR3DL2 compared with other HLA-class I could promote the P. Bowness. 2003. Lymphoblastoid cells express HLA-B27 homodimers both intracellularly and at the cell surface following endosomal recycling. Eur. J. survival and differentiation of inflammatory leukocytes in SpA. Immunol. 33: 748–759. 23. Perosa, F., G. Luccarelli, M. Prete, E. Favoino, S. Ferrone, and F. Dammacco. 2003. Beta 2-microglobulin-free HLA class I heavy chain epitope mimicry Acknowledgments by monoclonal antibody HC-10-specific peptide. J. Immunol. 171: 1918– 1926. We thank Alison Han, Rebecca Horsfall, Alistair Waugh, and Kati diGleria 24. Seitz, C., B. Uchanska-Ziegler, A. Zank, and A. Ziegler. 1998. The monoclonal for purification of Abs and peptides used in this study. antibody HCA2 recognises a broadly shared epitope on selected classical as well as several non-classical HLA class I molecules. Mol. 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