1. Top of page
  2. Abstract
  6. Acknowledgements


HLA–B27 is capable of forming in vitro a heavy-chain homodimer structure lacking β2-microglobulin. We undertook this study to ascertain if patients with spondylarthritis express β2-microglobulin–free HLA–B27 heavy chains in the form of homodimers and receptors for HLA–B27 homodimers.


Expression of HLA–B27 heavy chains by mononuclear cells was analyzed by fluorescence-activated cell sorter staining, Western blotting with the monoclonal antibody HC-10, and 2-dimensional isoelectric focusing. Fluorescence-labeled tetrameric complexes of HLA–B27 heavy-chain homodimers were constructed in which each dimer comprised one His-tagged heavy chain and one biotinylated heavy chain, and were used to stain patient and control mononuclear cells and transfected cell lines.


Patients with spondylarthritis expressed cell-surface HLA–B27 homodimers. Populations of synovial and peripheral blood monocytes, and B and T lymphocytes from patients with spondylarthritis, and controls carried receptors for HLA–B27 homodimers. Experiments with transfected cell lines demonstrated that KIR3DL1 and KIR3DL2, and immunoglobulin-like transcript 4 (ILT4), but not ILT2, are receptors for HLA–B27 homodimers.


Patients with spondylarthritis express both HLA–B27 heavy-chain homodimers and receptors for HLA–B27 homodimers. This may be of significance with regard to disease pathogenesis.

Possession of HLA–B27 is strongly associated with development of spondylarthritides, a group of related diseases including ankylosing spondylitis (AS) and reactive arthritis (ReA). Despite intensive research, the pathogenic role of HLA–B27 remains unclear (for review, see ref. 1). The natural immunologic function of HLA–B27 is to bind antigenic peptides together with β2-microglobulin (β2m) for presentation to the T cell receptor (TCR) of CD8+ cytotoxic T lymphocytes. However, certain features of disease in HLA–B27 transgenic rat (2) and mouse (3) models of spondylarthritis have suggested a possible pathogenic role for HLA–B27 heavy chains independent of β2m. Thus, murine disease requires expression of HLA–B27 in the absence of murine β2m (mβ2m), and can occur in animals with extremely few CD8+ T cells (3). Furthermore, disease onset is delayed and severity reduced by administration of the monoclonal antibody (mAb) HC-10 (4, 5). HC-10 recognizes free human HLA class I heavy chains (6). These results have led to the suggestion that HLA–B27 heavy chains may be directly involved in disease pathogenesis (3). Disease in the rat requires a high copy number of the HLA–B27 transgene (7), and disease cannot be transferred by CD8+ T cells alone (8).

We recently described the formation of β2m-free disulfide-bonded HLA–B27 heavy-chain homodimers, termed HC-B27 (9). Dimerization in vitro is dependent on the presence of the free cysteine at position 67 of the HLA–B27 heavy-chain α1 helix. The β2m-free HLA–B27 heavy chains could also be detected on the surface of HLA–B27–transfected cells (9). If expressed at the cell surface, HLA–B27 heavy-chain homodimers and multimers could play a role in the pathogenesis of spondylarthritis through interaction with either cell-mediated or humoral receptors.

In addition to direct cognate interactions with the TCR, mature class I complexes have been shown to bind several other immunomodulatory molecules, including members of the killer cell immunoglobulin-like receptor (KIR) family, and the immunoglobulin-like transcripts (ILT; also known as leukocyte Ig-like receptors, or LIR [10]). KIRs are expressed on certain natural killer (NK), T, and NKT cells (for review, see ref. 11). KIRs are polymorphic and demonstrate allele-specific recognition, with the cognate KIR for HLA–B27 being the 3-domain KIR3DL1. ILT/LIRs have a somewhat different expression pattern, with ILT2 expressed on B cells, as well as NK, T cells, and monocyte/macrophages (12). ILT4 is more selectively expressed on dendritic cells, monocytes, and macrophages. ILT2 and ILT4 receptor family members have a broader specificity, with ILT2 recognizing all of the class I alleles previously studied (12). ILT4 binds to most HLA–A and B alleles studied, as well as to the nonclassic HLA–G (13, 14).

Here we show that both HLA–B27 heavy-chain homodimers and receptors for HLA–B27 homodimers are expressed on populations of peripheral blood and synovial monocytes and B and T lymphocytes from patients with spondylarthritis. Control subjects also express receptors for HLA–B27 heavy-chain homodimers. KIR3DL1, KIR3DL2, and ILT4 and at least one additional receptor, but not ILT2, are capable of binding to HLA–B27 heavy-chain homodimers. Such interactions could contribute to joint inflammation and disease pathogenesis in the spondylarthritides.


  1. Top of page
  2. Abstract
  6. Acknowledgements

Patient and control samples.

After informed consent was obtained from all subjects, peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) from patients with spondylarthritis were separated over Ficoll-Hypaque gradients, and were stained on ice or frozen and thawed immediately before use. HLA typing was performed by DNA-based means. We studied 19 HLA–B27+ (18 B*2705 and 1 B*2702) patients with AS (AS 1–19) (14 men, 5 women, mean age 39 years) who fulfilled the modified New York criteria (15) and attended an AS clinic. Nineteen age- and sex-matched healthy laboratory controls were studied. These numbers were chosen to give an 85% power of detecting 1 SD difference between patient and control groups at the 5% significance level. For the experiments shown in Figures 1D and 4C, 2 HLA–B*2705+ patients with postenteritic ReA were studied (1 following confirmed Salmonella enteritidis infection). Ethical permission for this study was obtained from the Central Oxford Research Ethics Committee.

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Figure 1. HLA–B27 heavy-chain expression constructs and dimer tetramer generation. A, Schematic drawing showing different HLA–B27 constructs. B, Construction of HC-B27 tetramers. A mixture of heavy chains containing His tags and biotinylation recognition sequences (BRS) is refolded with peptide by limiting dilution. Purification on a His-binding column followed by biotinylation and tetramerization around streptavidin ensures each homodimer has 1 biotin and 1 His tag. FPLC = fast-pressure liquid chromatography; PE = phycoerythrin; FACS = fluorescence-activated cell sorter.

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Figure 4. HLA–B27 heavy-chain dimers bind KIR3DL1 and KIR3DL2 receptors. A, Left: HC-B27 tetramers, but not HLA–A2/β2-microglobulin (β2m)/peptide tetramers, bind KIR3DL1-transfected baf3 cells. Middle: Binding of HC-B27 tetramers to KIR3DL1 transfectants is blocked by the KIR3DL1-specific monoclonal antibody (mAb) DX9, and also by the HC-10 mAb. Right: HLA–B27/β2m/nucleoprotein (NP) tetramers bind to KIR3DL1 transfectants; binding is inhibited by a 10-fold excess of HC-B27 monomer. B, Left: HC-B27 tetramers, but not HLA–B27/β2m/NP or HLA–A2/β2m/peptide tetramers, bind to KIR3DL2 baf3 transfectants. Right: HC-B27 tetramer binding to KIR3DL2 transfectants is blocked by the KIR3DL2-specific mAb DX31. The HLA–B27/β2m/NP tetramer also binds to ILT4 but at a lower level than HC-B27. C, HC-B27 tetramer staining of synovial fluid lymphocytes from an HLA–B*2705+ patient with postenteric reactive arthritis correlates with staining with the 5.133 mAb. This is a nonblocking mAb, which recognizes KIR3DL1, KIR3DL2, and KIR2DS4 (16). D, HC-B27 tetramer staining of a KIR3DL1- and KIR3DL2-expressing natural killer (NK) cell line derived from a healthy individual (center). The right panel shows inhibition by the DX9 and DX31 mAb in combination. DX9 blocks KIR3DL1 and DX31 KIR3DL2. The left panel shows lack of staining with an HLA–A2/β2m/peptide control tetramer. PE = phycoerythrin; FITC = fluorescein isothiocyanate.

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Western blot analysis and 2-dimensional isoelectric focusing (2D-IEF).

HC-10 Western blotting was performed under standard conditions, using the cell lysate from 107 PBMCs and SFMCs, run on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) with or without 50 mM dithiothreitol. Cells were lysed in 1% (volume/volume) Triton X-100, Tris buffered saline containing 100 ng/ml phenylmethylsulfonyl fluoride, and 10 mM iodoacetamide to block postlysis disulfide rearrangement. For 2D-IEF freshly isolated PBMCs from an HLA–B27+ patient with AS (tissue types HLA–A*201, A*2601, B*1401/4, B*2705, C*01, C*02) were surface labeled with 125I and then immunoprecipitated with HC-10. As a positive control, HLA–B27–transfected class I–deficient C1R cells were used. No 125I surface-labeled heavy chains were immunoprecipitated in nontransfected C1R cells (Bird L, et al: unpublished observations). HLA alleles were first resolved by charge by IEF. Homodimers and monomers of HLA–B27 were then resolved by molecular weight by nonreducing SDS-PAGE in the second dimension.

Generation of tetrameric HC-B27 and HLA–B27/β2m/peptide complexes.

HLA–B*2705 was expressed and refolded as described previously (9), with the following modifications. The original HLA–B27 construct, carrying a C-terminal biotinylation recognition sequence (B27B), was modified to incorporate a His tag instead of the BirA recognition sequence (B27H; see Figure 1A). The resulting constructs were used to transform recA-BL21(DE3)pLysS(Gold) (Stratagene, Amsterdam, The Netherlands). Homodimer tetramers were generated in which each HLA–B27 homodimer carried only a single biotin tag (shown in Figure 1B). First, a 4:1 mix of B27B and B27H proteins was refolded in the presence of one of the following known viral peptide epitopes: KRWIIMGLNK (HIV gag), RRIYDLIEL (EBV EBNA3C), RRLVVTLQC, or RRLVVTLQCLVLLYA (EBV BCRF1). Refolded protein was biotinylated and then purified on Ni-NTA resin (Fast-Flow; Amersham Pharmacia Biotech, Little Chalfont, UK). Use of a nickel affinity column ensured that all resulting complexes contained a His-tagged B27 molecule. Composition was confirmed by nonreducing and reducing SDS-PAGE. Phycoerythrin (PE)–labeled ExtrAvidin (Sigma, Poole, UK) was then used to generate tetramer complexes. Standard heterodimeric complexes were refolded with β2m and with the peptides listed above or with SRYWAIRTR (influenza nucleoprotein NP383-391, “flu NP”). For these refolds, HLA–B27 with ser67 was used (B27SB, as described previously [9]), and, less efficiently, His-tagged B27cys67 (B27BH; see Figure 1A). To exclude the possibility of dimerization resulting as an artifact of incorporation of the His tag into the HLA–B27 cys67 construct, refolds were also performed with HLA–B27 ser67 His-tagged protein (B27SBH; see Figure 1A). His-tagged B27 ser67 did not form dimers (results not shown). We also studied other HLA molecules for evidence of dimerization in vitro. HLA–A2, B7, and B57 did not form heavy-chain homodimers in vitro (results not shown).

Transfected cell lines, antibodies, and flow cytometry.

Baf3 cells transfected with KIR3DL1, KIR3DL2, or ILT2 receptors, and RBL-2H3 cells transfected with the ILT4 receptor (13) were kind gifts from L. Lanier (University of California, San Francisco) and M. Colonna (St. Louis, MO), respectively. The anti-KIR3DL1 (DX9) and anti-KIR3DL2 (DX31) mAb (both mouse IgG1) were obtained from DNAX Research Institute (Palo Alto, CA). The 5.133 mAb stains (but does not fully block) KIR3DL1, KIR3DL2, and KIR2DS4 (16). HC-10 (IgG2a) (6), a mAb that recognizes HLA class I heavy chains that are either weakly or unassociated with β2m, was a kind gift from Dr. Hidde Pleough (Massachusetts Institute of Technology, Cambridge, MA). W6/32 (IgG2a) is a mAb that recognizes HLA class I molecules associated with β2m. The mAb 27D6 (rat IgM), which recognizes ILT4, and mAb 28C8 (rat IgG1), which recognizes ILT2 and ILT4, have been described previously (14), and were obtained from M. Colonna and L. Lanier, respectively. Fluorescence-activated cell sorter (FACS) staining with mAb was performed under standard conditions on ice in the presence of azide. Tetramer staining was performed at 37°C for 15 minutes using 1 μg of tetramer in the presence of azide, and was analyzed on a FACSCalibur. All stainings were repeated at least 3 times with consistent results.

Generation of an NK cell line expressing KIR3DL1.

NK cells were isolated from PBMCs from a healthy volunteer after depletion of monocytes by adherence to plastic and T cells with anti-CD3 (OKT3)–coated magnetic beads (Dynal, Wirral, UK). Once isolated, cells were stimulated with allogeneic PBMCs and B cell lines, in the presence of 200 units/ml recombinant interleukin-2 (rIL-2) and 100 μg/ml phytohemagglutinin in RPMI 1640 medium (Gibco BRL, Grand Island, NY) supplemented with 15% heat-inactivated human AB serum (Sigma) and 2 mML-glutamine with 10 units/ml penicillin/streptomycin. After 10 days, KIR3DL1 cells were positively selected using anti-mouse Ig magnetic beads (Dynal) after staining with the KIR3DL1-specific mAb (DX9). KIR3DL1-positive NK cells were further CD3 depleted just prior to use. The percentage of KIR3DL1-positive cells was >98% by FACS. NK cell lines were cultured with medium containing 200 units/ml rIL-2, 10 ng/ml rIL-12, and 20 ng/ml rIL-15 for 48 hours before FACS staining with tetramers.


  1. Top of page
  2. Abstract
  6. Acknowledgements

PBMCs and SFMCs from HLA–B27+ patients with spondylarthritis express HLA–B27 homodimers.

Figure 2 shows that HC-10, a mAb with specificity for free HLA class I heavy chains, stained the PBMCs of an HLA–B27+ patient with AS. Significant free cell-surface heavy-chain expression was seen on CD19+ B lymphocytes, CD3+ T cells (Figure 2A), and CD14+ monocytes (Figure 2B). IgG2a isotype control antibody did not stain these cell populations (Figures 2A and B).

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Figure 2. Patients' expression of free cell-surface HLA class I heavy chains, including HC-B27 homodimers. A, Fluorescence-activated cell sorter staining of peripheral blood B cells and T cells (CD3–peridin chlorophyll protein and CD19–fluorescein isothiocyanate) with monoclonal antibodies HC-10 and W6/32 from an HLA–B27+ patient with ankylosing spondylitis (AS). HC-10 recognizes free class I heavy chains. W6/32 recognizes β2-microglobulin–associated class I molecules. IgG2a isotype control is shown in the solid plots. B, HC-10 staining of monocytes (myelomonocytic gate, CD14+ population) from an HLA–B27+ patient with AS. IgG2a isotype control is shown in the solid plots. C, Expression of HC-B27 homodimers on peripheral blood mononuclear cells (PBMCs) from an HLA–B27+ patient. Material that was 125I surface labeled was immunoprecipitated with HC-10 and analyzed by 2-dimensional isoelectric focusing (IEF). HLA alleles are first resolved by charge by IEF (horizontal axis). Then, homodimers and monomers of HLA–B27 may be resolved by molecular weight by nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) (vertical axis). The lower panel shows positive control HLA–B27–transfected class I–deficient C1R cells. D, HC-10 Western blot of SDS-PAGE of synovial fluid mononuclear cells (SFMCs) and PBMCs from an HLA–B*2705+ patient with postenteric reactive arthritis. HLA class I heavy-chain dimers are detected in the left panel (nonreducing conditions). DTT = dithiothreitol.

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Since HC-10 is not HLA–B27–specific, we next used 2D-IEF to prove that HLA–B27 homodimers were indeed present. In the experiments shown in Figures 2C and D, cells were lysed in the presence of 10 mM iodoacetamide, which prevented postlysis disulfide bond rearrangement under the conditions used in this study (results not shown). Freshly isolated PBMCs from an HLA–B27+ patient were surface labeled with 125I. HC-10 immunoprecipitates were analyzed by nonreducing 2D–gel electrophoresis. First, IEF resolves HLA alleles by charge. Subsequently, homodimers and monomers may be resolved by molecular weight. Figure 2C shows that surface-labeled immunoprecipitated HLA–B27 homodimers were identified running at the same position as HLA–B27–transfected class I–deficient C1R cells (lower panel). The different molecular species with different isoelectric points corresponded to differently sialylated species of HLA–B27. The patient cells expressed a small proportion of HC-10–reactive non–HLA–B27 heavy chains, but these were present exclusively as monomers.

Figure 2D shows that both PBMCs and SFMCs of an HLA–B*2705+ patient with ReA expressed HC-10–reactive disulfide-bonded HLA class I heavy-chain homodimers (molecular weight 90 kd). The right panel shows that these heavy chains run as 45-kd monomers under reducing conditions.

Tetrameric complexes of HLA–B27 heavy-chain homodimers specifically stain monocyte and lymphocyte populations.

To identify potential cellular ligands for HLA–B27 heavy chains, homodimers of recombinant HLA–B27 heavy chain (HC-B27), each carrying a single biotin residue, were tetramerized with ExtrAvidin PE, as shown in Figure 1B. These HC-B27 tetramers were used to stain PBMCs from patients with spondylarthritis. HC-B27 tetramers consistently stained populations of B and T lymphocytes (Figures 3A–C), and also stained almost all CD14+ cells within the myelomonocytic gate (Figure 3D). Low-level (<3%) staining of CD3−,CD56+ NK cells was also seen (data not shown; see below). ExtrAvidin PE controls did not bind significantly (Figure 3, left panels). Specificity of binding was confirmed by blocking with a 10-fold excess of HC-B27 monomer (Figures 3A–C, right panels). In contrast, blocking was not observed with similar concentrations of HLA–A2/β2m/peptide complexes, bovine serum albumin, or irrelevant isotype control antibody (data not shown).

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Figure 3. HC-B27 tetramer fluorescence-activated cell sorter (FACS) staining of PBMC populations from patients with spondylarthritis. The left column shows ExtrAvidin phycoerythrin (PE) control staining, the middle column shows staining with HC-B27 tetramers, and the right column shows blocking by coincubation with a 10-fold excess of untetramerized HC-B27 monomer. A–C, PBMCs from patient 1 (AS) with lymphocytic gate. A, 2-color FACS with CD19–fluorescein isothiocyanate (FITC) and HC-B27 PE. For B and C, 3-color FACS was used, T cells were first gated for CD3 expression. D, Patient 2 (AS) with myelomonocytic gate, 2-color FACS. APC = allophycocyanin; PerCP = peridin chlorophyll protein (see Figure 2 for other definitions).

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HC-B27 binds KIR3DL1 and KIR3DL2.

To define further potential receptors for HLA–B27 homodimers, we studied cell lines transfected with various KIR receptors known to bind to major histocompatibility complex (MHC) class I molecules (11). Among them, KIR3DL1 bound to HLA class I molecules expressing the Bw4 specificity, including HLA–B27 (17). HC-B27 tetramers stained KIR3DL1-transfected cells (Figure 4A, left panel, bold line), while no staining was observed with ExtrAvidin PE (solid line) or HLA–A2 tetramer controls (dotted line). HC-B27 tetramers did not stain nontransfected baf3 cells (data not shown). HC-B27 tetramer staining could be blocked by the KIR3DL1-specific mAb DX9 (Figure 4A, center panel, open line), by the mAb HC-10 (Figure 4A, center panel, dotted line), and by a 10-fold excess of nonbiotinylated HC-B27 monomer (data not shown), consistent with HC-B27 interacting with the KIR3DL1 receptor. In addition, we confirmed that HLA–B27/β2m/peptide complexes bound to KIR3DL1 (Figure 4A, right panel, bold line). This binding could be almost completely inhibited by a 10-fold excess of HC-B27 monomers (Figure 4B, right panel, dotted line).

Unexpectedly, KIR3DL2-transfected cells could also be stained by HC-B27 tetramers, and staining could be blocked by the KIR3DL2-specific mAb DX31 (Figure 4B shows representative experiments). This receptor was previously reported to bind to HLA–A3 and HLA–A11, for example (16, 18). As expected, standard HLA–B27/β2m/peptide and HLA–A2/β2m/peptide tetramers did not stain KIR3DL2 transfectants (Figure 4B, left panel, dotted and open lines).

We next studied patient T lymphocytes to determine if the ex vivo staining observed with HC-B27 tetramers could be explained by interaction with KIR3DL1 and/or KIR3DL2. We first looked for evidence that tetramer-staining cells expressed these KIRs. The anti-KIR3DL1 + KIR3DL2 + KIR2DS4 antibody 5.133 did not block staining of HC-B27 tetramers (data not shown) and was therefore used to costain PBMCs and SFMCs. Although this mAb stained only a small proportion of the total cells within the lymphocyte gate, 30% of the HC-B27 tetramer-staining cells were also 5.133+ (Figure 4C), the majority being CD8+ (data not shown). This finding was consistent with the partial inhibition of HC-B27 staining by a combination of DX9 and DX31 blocking mAb (data not shown).

We next derived a KIR3DL1-expressing NK cell line from a healthy donor. One hundred percent of the line expressed KIR3DL1 and 50% expressed KIR3DL2 (data not shown). This line was strongly stained by the HC-B27 tetramer (Figure 4D, central panel), but not by an irrelevant HLA–A2/β2m/peptide tetramer (Figure 4D, left panel). HC-B27 staining was blocked with the DX9 and DX31 mAb in combination (Figure 4D, right panel). These mAb, used singly, gave partial blocking (data not shown). These results confirm that HC-B27 can bind to KIR3DL1 and KIR2DL2 expressed on NK cells. Substantially lower levels of staining were observed on unstimulated NK cells (data not shown), which may partly explain the low levels of NK cell staining observed ex vivo.

HC-B27 binds ILT4 but not ILT2, and binding of HC-B27 to monocytes is partially inhibited by antibodies to ILT4.

Since ILT2 is expressed on B, T, NK cells, and monocytes and has been reported to bind to most HLA class I molecules (12), we looked for evidence of binding of HC-B27 tetramers to ILT2-transfected cells. In repeated experiments using HC-B27 tetramers refolded with a variety of peptides, no binding to ILT2 was observed, although HLA–B27/β2m/peptide tetramers invariably bound (Figure 5A). However, HC-B27 tetramers consistently bound to cell lines transfected with ILT4 (Figure 5B, bold line). Binding was greater than or equal to that observed for HLA–B27/β2m/peptide tetramers (Figure 5B and data not shown). Inhibition of binding with the 27D6 mAb (Figure 5B, dotted line), which recognizes ILT4, confirmed that binding to these transfectants was due to interaction with ILT4.

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Figure 5. HLA–B27 heavy-chain dimers bind immunoglobulin-like transcript 4 (ILT4), but not ILT2. Binding to monocytes, but not B lymphocytes, is inhibited by an ILT2- and ILT4-specific mAb. A, HLA–B27/β2m/NP tetramers, but not HC-B27 tetramers, bind ILT2-transfected baf3 cells. B, HC-B27 tetramers bind ILT4-transfected RBL-2H3 cells; binding is blocked by the 27D6 mAb, which recognizes ILT4. C, Upper panels: mAb 28C8, which binds to ILT2 and ILT4 receptors, does not inhibit HC-B27 tetramer staining of B cells from a patient with ankylosing spondylitis (AS). Lower panels: mAb 28C8 partially blocks binding of control HLA–B27/β2m/peptide tetramers to B lymphocytes (same experiment). D, The mAb 28C8 partially blocks binding of both HC-B27 (left) and control HLA–B27/β2m/peptide (right) tetramers to monocytes from a patient with AS. APC = allophycocyanin (see Figure 4 for other definitions).

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Consistent with the above experiments showing that HLA–B27 heavy chains do not interact with ILT2 on transfected cells, HC-B27 staining of patient peripheral blood CD19+ B lymphocytes could not be blocked with blocking antibodies recognizing ILT2 (Figure 4C and data not shown). In a parallel control experiment, the 28C8 mAb inhibited HLA–B27/β2m/peptide tetramer binding to B cells by >50% (Figure 4C, lower panels). Since ILT4 is expressed on monocytes (13), we determined whether mAb to ILT4 could block HC-B27 tetramer binding to monocytes in patients with spondylarthritis. HC-B27 staining of monocytes could be only partially inhibited with mAb 28C8 (which recognizes both ILT2 and ILT4; Figure 5D, left panel) and 27D6 (data not shown). Control HLA–B27/β2m/peptide tetramers stained monocytes at a lower level than HC-B27 tetramers, and staining could largely be blocked with the 28C8 mAb (Figure 5D, right panel). These results suggested that ILT4 contributes to the binding of HC-B27 tetramers to monocytes, but that an additional receptor or receptors for HC-B27 is also present on B cells and monocytes.

HC-B27 staining of synovial fluid T cells is greater than that seen for peripheral blood.

The staining levels in patients with AS did not differ significantly from those of controls. Two of the AS patients studied developed peripheral joint involvement during the study period, and we were able to study synovial fluid lymphocytes. Figure 6A shows a representative staining, in which HC-B27 tetramers bound at greater levels to synovial fluid lymphocytes than peripheral blood lymphocytes. We also wished to determine if patients with spondylarthritis expressed greater levels of HC-B27 ligands in their peripheral blood than controls. We therefore studied 19 patients with AS and 19 age- and sex-matched healthy controls. Figure 6B shows that HC-B27 tetramers stained up to 6% of patient PBMC CD3+,CD8+ T cells (mean ± SD 3.14 ± 2.26). This was not significantly greater than that observed for control PBMCs (mean ± SD 2.28 ± 1.235; P = 0.076 by Student's t-test). Lower levels of staining were observed for patient CD4+ T cells, which did not differ significantly from controls. Higher levels of staining were seen for patient B cells, but these did not differ significantly from controls (Figure 6B). Almost all monocytes from both patients and controls were stained (for representative staining, see Figures 3D and 5D).

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Figure 6. A, Comparison of HC-B27 tetramer staining of synovial fluid and peripheral blood T cells from a patient with ankylosing spondylitis and peripheral joint synovitis. B, Comparison of HC-B27 tetramer staining of peripheral blood lymphocytes from spondylarthritis patients and controls. Top, CD8 and CD4 T cells. Bottom, CD19+ B cells. Specified values are the means; bars show the EX-PE = ExtrAvidin phycoerythrin; FITC = fluorescein isothiocyanate; PerCP = peridin chlorophyll protein.

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  1. Top of page
  2. Abstract
  6. Acknowledgements

We have shown that patients with spondylarthritis express cell-surface HLA–B27 heavy-chain homodimers (HC-B27) and also cellular receptors for HC-B27. A role for free HLA–B27 heavy chains in the pathogenesis of spondylarthritis has previously been suggested following studies of HLA–B27–transgenic β2m-deficient mice (3). HLA–B27+, β2m−/− murine peripheral blood lymphocytes (PBLs) express cell-surface HLA–B27 heavy chains following concanavalin A treatment (3), and heavy-chain expression can also be detected in the thymus and PBLs of B27+, mβ2m−/− huβ2m+ mice (4). Furthermore, disease onset can be delayed by administration of HC-10, an antibody with specificity for HLA–B27 heavy chains, but not by ME1, an mAb that recognizes HLA–B27 associated with β2m and peptide (4, 5). HLA–B27 heavy chains have been found to preferentially form disulfide-bonded homodimers in vitro (9), and β2m-free HLA–B27 heavy chains can be detected at the cell surface of certain HLA–B27–transfected cell lines with defects in antigen presentation (9).

Here we have shown that patients with spondylarthritis express HC-10–reactive class I heavy chains on monocytes and lymphocytes ex vivo, and that HC-B27 homodimers are expressed on PBMCs. We have also shown that some heavy chains in SFMCs as well as PBMCs are expressed in the form of homodimers. Expression of β2-free heavy chains has been described previously (19), and activated human lymphocytes are known to express conformationally distinct free heavy chains on the cell surface (20), where clustering has been observed (21). Thus, cell-surface heavy-chain expression is not confined to patients with spondylarthopathy or indeed to HLA–B27. However, cell-surface expression of HC-B27 homodimers and subsequent interaction with heavy-chain receptors could differ quantitatively or qualitatively from other class I molecules and contribute to the pathogenesis of the spondylarthropathies. The ability of HLA–B27 to form disulfide bonds through cys 67 may be important in this respect.

We have used fluorescent tetrameric complexes of HC-B27 homodimers to show that patients with spondylarthritis express HC-B27 receptors on the cell surface of populations of monocytes, lymphocytes, and NK cells. Although numbers of HC-B27 tetramer-staining cells in peripheral blood did not differ significantly between patients and controls, we found evidence of increased staining levels for patient synovial fluid T lymphocytes. HLA–B27 homodimers were constructed using a mixture of His-tagged and biotin-tagged heavy chains followed by 2 purification steps. This not only minimized the possibility of contamination with bacterial proteins, but also ensured consistent and optimum tetramerization, since each heavy-chain dimer carried only a single biotin residue. If heavy-chain dimers bearing 2 biotin sites were used, variable interactions with streptavidin could occur. Two biotin residues on a single homodimer could occupy 2 of the 4 binding sites on a single avidin molecule, substantially reducing the avidity of the tetramer reagent, and preventing detection of low-affinity interactions. Alternatively, 2 biotin residues on a single homodimer could bind to different streptavidin molecules, generating multimolecular aggregates capable of artifactual binding.

Differences in tetramer generation may explain the discrepancy between our results for normal PBMCs and those of a recent study using tetramers of HLA–B27 heavy chains, where the only significant binding observed was to monocytes (22). In this study we did not include patients with spondylarthritis, where we have seen particularly strong staining of synovial fluid T cells (see Figure 4C). We have shown that HC-B27 tetramers can bind to KIR3DL1, KIR3DL2, and ILT4 receptors, and almost certainly an additional receptor or receptors expressed on monocytes and lymphocytes, but not to ILT2. Although KIR3DL1 is known to bind to HLA–B27/β2m/peptide complexes (14), it was somewhat unexpected that a β2m-free HLA heavy-chain structure could bind to its cognate KIR. We believe that this interaction is likely to be functionally relevant in vivo, since binding of HLA–B27/β2m heterodimers to KIR-3DL1–transfected cells could be blocked by HC-B27. KIR3DL1 recognition is dependent on residues 77–83 of the class I α1 helix (23). Our data thus suggest that the HC-B27 structure closely resembles that of HLA–B27/β2m/peptide complexes in this region (see below).

Binding of HC-B27 to KIR3DL2 was unexpected but consistently observed in repeated experiments. The level of staining of a transfected cell line was comparable with that observed for DX31, a KIR3DL2-specific mAb. KIR3DL2 did not bind to a number of different HLA–B27/β2m/peptide complexes, consistent with previous studies showing specificity for HLA–A allotypes (18). We suggest that a conformational change upon HC-B27 dimerization permits binding to KIR3DL2. Interestingly, KIR3DL2 is itself usually expressed as a disulfide-bonded homodimer (P140) (18).

HC-B27 tetramers bound to both CD4+ and CD8+ T cells ex vivo. Our results suggest that this binding, as well as that seen at lower levels for NK cells, was due to interaction with KIR3 and related receptors rather than with the TCR. Thus, HC-B27 binding to T cells correlated with KIR expression, and mAb against KIR3DL1 and KIR3DL2 blocked HC-B27 staining of a KIR3DL1-expressing NK line. Interestingly, HC-B27 tetramers stained only a limited proportion of the total number of KIR3DL1/2-expressing cells ex vivo. This finding is consistent with the observation that binding of standard tetrameric complexes to such receptors on lymphocytes is not commonly observed (Bowness P, et al: unpublished observations). HC-B27 FACS staining of a KIR3DL1-expressing NK cell line was, however, increased following stimulation with IL-12 or IL-15. It is likely that binding is dependent on activation-induced changes in KIR expression, possibly including conformational change or a local aggregation of KIR. Similar phenomena have been described for other tetrameric complexes, with HLA–G tetramer staining of CD14+,ILT4+ monocytes significantly enhanced by the presence of an ILT4-binding mAb (14). Recently, virus-specific CD8+ T lymphocytes whose ability to bind cognate MHC/peptide tetrameric complexes varies without any change in the level of cell-surface TCR have also been described (24). There is evidence that this phenomenon may be due to association in lipid rafts (25).

Taken together with these findings, our results also suggest that HLA–B27 homodimers and multimers might be more potent than standard HLA complexes in their interactions with cells expressing non-TCR HLA receptors. We believe that alternative explanations for our data, such as changes in expression of different allelic forms of KIR3DLl (26), are unlikely, although it is possible that we observed stronger staining of purified cell populations because of a reduction in competition between multiple receptors for the same ligand (HC-B27) with a mixed population. Finally, the failure of mAb to KIR3DL1 and KIR3DL2 to block HC-B27 binding to T cells suggests that some of the binding observed was due to expression of an additional heavy-chain receptor or receptors such as LIR-6 (22).

Tetrameric complexes of HC-B27 heavy-chain homodimers bound to T cells of both patients with spondylarthritis and controls, although binding to CD8+ T lymphocytes was not significantly higher in the small group of patients with AS studied. What might be the functional consequences of binding of HLA–B27 heavy chains to KIR receptors expressed on T cells? Although the role of KIR expression on T cells is not yet clearly defined, there is accumulating evidence that KIR expression on CD8+ memory T cells is associated with increased T cell survival (27), and that in vitro engagement of KIRs on T cells can inhibit activation-induced cell death (28). In the context of spondylarthritis, one attractive and testable hypothesis is that engagement by HLA–B27 heavy chains of KIRs on T cells at sites of inflammation, such as the joints, could promote the survival of proinflammatory T cell clones. There is some evidence for KIR expressing CD8+ T cell clones recognizing self-peptides. In this respect, engagement of both KIR3DL1 and KIR3DL2 by HC-B27 could promote the survival of not only T cells expressing the cognate KIR for heterodimers of HLA–B27 but also T cells expressing the KIR for HLA–A3 and HLA–A11.

HC-B27 heavy-chain tetramers were also found to stain CD19+ B lymphocytes in repeated experiments. Because HC-B27 does not bind to ILT2 transfectants, this cannot be explained by binding to ILT2 receptors, which are known to be expressed on B cells and bind all mature HLA molecules studied previously (12). Furthermore, HC-B27 staining of CD19+ B cells ex vivo could not be blocked with an excess of mAb recognizing ILT2. This result is consistent with those recently obtained using HLA–B27 heavy-chain tetramers constructed differently and ILT2 transiently expressed in 293 T cells (22). A likely explanation for our results is that a subpopulation of CD19+ cells expresses a different ILT family member capable of binding HC-B27. Transcription of a number of ILT/LIRs has been demonstrated in B cells (and monocytes; see below), some of which are potentially stimulatory (12, 29). Although the physiologic role of ILT/LIR receptors is not well understood, ILT2 ligation has been shown to inhibit both NK- and T cell–mediated cytotoxicity. Thus, it is possible that, in the presence of stimulatory interactions with other heavy-chain receptors, the failure of HC-B27 to interact with ILT2 could have an overall proinflammatory action.

Since ILT2 is thought to recognize regions on the class I α3 domain (30, 31), loss of recognition by ILT2 of HC-B27 suggests that HC-B27 has a different α3 domain conformation to standard HLA–B27/β2m/peptide complexes (in contrast to the similar interaction with KIR3DL1, suggesting a similar α1/α2 conformation).

We have also shown that HC-B27 homodimers bind to ILT4, an inhibitory receptor expressed on monocytes, macrophages, and dendritic cells (13). ILT4 is known to bind to a number of classic MHC molecules and also the nonclassic molecule HLA–G (13, 14), and is thought to transduce a negative signal (13). Interestingly, HC-B27 binding to monocytes could be only partially inhibited using an mAb recognizing ILT4. A likely explanation for our findings is that HC-B27 binds both to ILT4 and to an additional receptor or receptors expressed on monocytes.

How might HC-B27 homodimers trigger or perpetuate spondylarthritis? One possibility is that infection (for example, with intracellular bacteria known to trigger ReA) could increase cellular expression of HLA–B27 homodimers. Under these circumstances, the balance between normal HLA–B27 heterodimer and homodimer expression could be important in determining disease outcome. ILT2 is broadly expressed on the majority of CD4+ and CD8+ T cells, and receptor ligation can inhibit T cell activation (12). Since homodimers did not bind to ILT2, lack of interaction with this receptor could contribute to pathogenesis by reducing the threshold required for T cell activation, particularly if more homodimer were expressed at sites of inflammation. Alternatively, interaction of HC-B27 with an immunoreceptor that does not recognize standard HLA–B27 heterodimers, such as KIR3DL2 (expressed on both T and NK cells), could have an immunomodulatory action.

Could the interaction of free MHC heavy chains with MHC receptors explain the development of arthritis in animal models of spondylarthropathy? Support for such a general mechanism comes from a preliminary report showing that the introduction into T cells of NKB1, an allelic form of KIR3DL1, produces particularly severe arthritis in HLA–B27–transgenic mice (32). Although rodents do not have KIR receptors, they express a variety of MHC receptors, including the paired inhibitory receptors (33, 34), orthologs of the human ILT/LIR family. Finally, either murine or human free heavy chains expressed at high levels could adopt a related conformation to cell-surface B27 homodimers and interact with similar receptors. This could explain the observations that certain β2m mice strains can develop arthritis even in the absence of HLA–B27 (35) and that transgenic rats expressing the ser67 mutant of HLA–B27 still develop arthritis albeit at a reduced frequency (36). Moreover, our results do not exclude, but rather complement, existing hypotheses such as the role of “arthritogenic peptides” and “ER misfolding” in disease and suggest an additional level at which molecular characteristics of HLA–B27 may contribute to inflammation through immunoreceptor recognition.

In conclusion, our data show that patients with spondylarthritis express cell-surface HLA–B27 heavy-chain homodimers (HC-B27). We also show that HC-B27 homodimers can interact with class I receptors such as KIR and ILT, and that such receptors are expressed on T and B lymphocytes, NK cells, and myelomonocytic cells. We propose that dimerization may promote the creation of a conformation of HLA–B27 capable of binding to an overlapping but distinct repertoire of immunoreceptors compared with the standard trimeric complex of HLA–B27, β2m, and peptide. These findings raise the novel possibility that interaction of HLA–B27 heavy chains with immunoreceptors on cells of the myelomonocytic cell lineage or lymphocytes might be involved in the pathogenesis of spondylarthritis.


  1. Top of page
  2. Abstract
  6. Acknowledgements

We are grateful to Chen Au Peh and Frances Hall for ideas and discussion, to Abigail King, Louise Jones, and Graham Ogg for control HLA–A2 tetramers, and to Kati de Gleria for peptide synthesis.


  1. Top of page
  2. Abstract
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