Downregulation of endogenous nectin1 in human keratinocytes by herpes simplex virus 1 glycoprotein D excludes superinfection but does not affect NK cell function.

Many viruses downregulate their cognate receptors, facilitating virus replication and pathogenesis via processes that are not yet fully understood. In the case of herpes simplex virus 1 (HSV1), the receptor binding protein glycoprotein D (gD) has been implicated in downregulation of its receptor nectin1, but current understanding of the process is limited. Some studies suggest that gD on the incoming virion is sufficient to achieve nectin1 downregulation, but the virus-encoded E3 ubiquitin ligase ICP0 has also been implicated. Here we have used the physiologically relevant nTERT human keratinocyte cell type - which we have previously shown to express readily detectable levels of endogenous nectin1 - to conduct a detailed investigation of nectin1 expression during HSV1 infection. In these cells, nectin1, but not nectin2 or the transferrin receptor, disappeared from the cell surface in a process that required virus protein synthesis rather than incoming virus, but did not involve virus-induced host shutoff. Furthermore, gD was not only required but was sufficient for nectin1 depletion, indicating that no other virus proteins are essential. NK cells were shown to be activated in the presence of keratinocytes, a process that was greatly inhibited in cells infected with wild-type virus. However, degranulation of NK cells was also inhibited in ΔgD-infected cells, indicating that blocking of NK cell activation was independent of gD downregulation of nectin1. By contrast, a superinfection time-course revealed that the ability of HSV1 infection to block subsequent infection of a GFP-expressing HSV1 was dependent on gD and occurred in line with the timing of nectin1 downregulation. Thus, the role of gD-dependent nectin1 impairment during HSV infection is important for virus infection, but not immune evasion, which is achieved by other mechanisms.

HCMV-secreted glycoprotein gpUL4 inhibits TRAIL-mediated apoptosis and NK cell activation.

Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent.

ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways.

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

The superantigens SpeC and TSST-1 specifically activate TRBV12-3/12-4+ memory T cells.

Severe bacterial or viral infections can induce a state of immune hyperactivation that can culminate in a potentially lethal cytokine storm. The classic example is toxic shock syndrome, a life-threatening complication of Staphylococcus aureus or Streptococcus pyogenes infection, which is driven by potent toxins known as superantigens (SAgs). SAgs are thought to promote immune evasion via the promiscuous activation of T cells, which subsequently become hyporesponsive, and act by cross-linking major histocompatibility complex class II molecules on antigen-presenting cells to particular ß-chain variable (TRBV) regions of αß T cell receptors (TCRs). Although some of these interactions have been defined previously, our knowledge of SAg-responsive TRBV regions is incomplete. In this study, we found that CD4+ and CD8+ T cells expressing TRBV12-3/12-4+ TCRs were highly responsive to streptococcal pyrogenic exotoxin C (SpeC) and toxic shock syndrome toxin-1 (TSST-1). In particular, SpeC and TSST-1 specifically induced effector cytokine production and the upregulation of multiple coinhibitory receptors among TRBV12-3/12-4+ CD4+ and CD8+ memory T cells, and importantly, these biological responses were dependent on human leukocyte antigen (HLA)-DR. Collectively, these data provided evidence of functionally determinative and therapeutically relevant interactions between SpeC and TSST-1 and CD4+ and CD8+ memory T cells expressing TRBV12-3/12-4+ TCRs, mediated via HLA-DR.

Epitope length variants balance protective immune responses and viral escape in HIV-1 infection.

Cytotoxic T lymphocyte (CTL) and natural killer (NK) cell responses to a single optimal 10-mer epitope (KK10) in the human immunodeficiency virus type-1 (HIV-1) protein p24Gag are associated with enhanced immune control in patients expressing human leukocyte antigen (HLA)-B∗27:05. We find that proteasomal activity generates multiple length variants of KK10 (4-14 amino acids), which bind TAP and HLA-B∗27:05. However, only epitope forms ≥8 amino acids evoke peptide length-specific and cross-reactive CTL responses. Structural analyses reveal that all epitope forms bind HLA-B∗27:05 via a conserved N-terminal motif, and competition experiments show that the truncated epitope forms outcompete immunogenic epitope forms for binding to HLA-B∗27:05. Common viral escape mutations abolish (L136M) or impair (R132K) production of KK10 and longer epitope forms. Peptide length influences how well the inhibitory NK cell receptor KIR3DL1 binds HLA-B∗27:05 peptide complexes and how intraepitope mutations affect this interaction. These results identify a viral escape mechanism from CTL and NK responses based on differential antigen processing and peptide competition.

HLA associations in inflammatory arthritis: emerging mechanisms and clinical implications.

Our understanding of the mechanisms underlying HLA associations with inflammatory arthritis continues to evolve. Disease associations have been refined, and interactions of HLA genotype with other genes and environmental risk factors in determining disease risk have been identified. This Review provides basic information on the genetics and molecular function of HLA molecules, as well as general features of HLA associations with disease. Evidence is discussed regarding the various peptide-dependent and peptide-independent mechanisms by which HLA alleles might contribute to the pathogenesis of three types of inflammatory arthritis: rheumatoid arthritis, spondyloarthritis and systemic juvenile idiopathic arthritis. Also discussed are HLA allelic associations that shed light on the genetic heterogeneity of inflammatory arthritides and on the relationships between adult and paediatric forms of arthritis. Clinical implications range from improved diagnosis and outcome prediction to the possibility of using HLA associations in developing personalized strategies for the treatment and prevention of these diseases.

Inhibitory killer cell immunoglobulin-like receptors strengthen CD8+ T cell-mediated control of HIV-1, HCV, and HTLV-1.

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.

Structure of MHC class I-like MILL2 reveals heparan-sulfate binding and interdomain flexibility.

The MILL family, composed of MILL1 and MILL2, is a group of nonclassical MHC class I molecules that occur in some orders of mammals. It has been reported that mouse MILL2 is involved in wound healing; however, the molecular mechanisms remain unknown. Here, we determine the crystal structure of MILL2 at 2.15 Å resolution, revealing an organization similar to classical MHC class I. However, the α1-α2 domains are not tightly fixed on the α3-ß2m domains, indicating unusual interdomain flexibility. The groove between the two helices in the α1-α2 domains is too narrow to permit ligand binding. Notably, an unusual basic patch on the α3 domain is involved in the binding to heparan sulfate which is essential for MILL2 interactions with fibroblasts. These findings suggest that MILL2 has a unique structural architecture and physiological role, with binding to heparan sulfate proteoglycans on fibroblasts possibly regulating cellular recruitment in biological events.

No evidence for a direct role of HLA-B27 in pathological bone formation in axial SpA.

OBJECTIVE: The strong genetic association between HLA-B27 and ankylosing spondylitis has been known for over 40 years. HLA-B27 positivity is possibly associated with severity of ankylosis. We studied the in vitro and in vivo impact of HLA-B27 in models of chondrogenesis and osteogenesis. METHODS: Different in vitro differentiation systems were used to mimic endochondral and direct bone formation. ATDC5 cells and primary human periosteum-derived cells (hPDCs) were transduced with lentiviral vectors expressing HLA-B27 or HLA-B7. These cells and limb bud cells (from HLA-B27 transgenic and wild-type (WT) mice) were cultured in micromasses. To study direct osteogenesis in hPDCs, cells were cultured as monolayers and stimulated with osteogenic media. Chondrogenesis (COL2, ACAN, COL10) and osteogenesis (OSC, ALP, RUNX2) marker expression was studied by quantitative RT-PCR. Colorimetric tests were performed to measure proteoglycans, mineralization and collagens. Collagen antibody-induced arthritis (CAIA) was induced in HLA-B27 transgenic and WT mice. Clinical scoring and µCTs were performed. Statistical analyses were performed by two-way ANOVA. RESULTS: There was no difference in chondrogenesis markers or in colorimetric tests between HLA-B27+ and HLA-B7+ micromasses. Expression of osteogenesis markers and Alizarin red staining was comparable in the HLA-B27+ and the HLA-B7+ hPDCs in monolayers. HLA-B27 transgenic mice showed more severe arthritis compared with WT mice in the CAIA model. µCT analysis showed no increased bone formation in HLA-B27 transgenic mice. CONCLUSION: HLA-B27 seems to enhance joint inflammation in the CAIA model. We could not document a direct effect of HLA-B27 on chondrogenesis or osteogenesis.

The Role of HLA-Class I Heavy-Chain Interactions with Killer-Cell Immunoglobulin-Like Receptors in Immune Regulation.

HLA-class I molecules form trimeric complexes (pMHC) of peptides, class I heavy chains, and ß2microglobulins (ß2m) that regulate immune responses by binding to T cells and other immune receptors. B2m-free class I heavy chains (FHCs) form on cells either as a consequence of the natural turnover of pMHC or, in the case of HLA-F, are expressed without ß2m. Distinct characteristics of certain HLA-class I members, such as HLA-B27 and HLA-F, stabilize these forms facilitating interactions with immune receptors. FHC forms of HLA-class I have been shown to bind to killer-cell immunoglobulin-like receptor (KIR) family members. The binding of FHC forms to KIR3DL2 regulates natural killer (NK) and T-cell functiona and promotes lymphocyte survival. KIR3DL2 binding to B27 FHC dimers has been implicated in the pathogenesis of spondyloarthritis (SpA). KIR3DL2 binding FHC forms could also play a role in immune cell recognition of certain tumors and in regulation of immune homeostasis at the maternal-fetal interface. Here, I review the evidence for the functional interaction of cell surface HLA-class I FHCs with KIR family members. I also discuss the relevance of these interactions in immune homeostasis and immune dysfunction in diseases in which FHC-binding KIRs have been implicated.

Data showing non-conventional HLA-B27 expression in axial joints and gut tissue from B27 transgenic rats, and in frozen and paraffin-fixed synovial SpA tissue.

Data is presented showing expression of non-conventional (NC) heavy chain forms of B27 in synovial tissues from SpA patients. Data is presented showing the expression patterns of NC-B27 in joint, gastrointestinal and lymphoid tissues from B27 transgenic (TG(1)) rats with M. tuberculosis-induced SpA. Expression of NC-B27 was determined by immunohistochemistry and flow cytometry using HC10 and HD6 antibodies. These data are the extension of the data presented and discussed in "Non-conventional forms of HLA-B27 are expressed in Spondyloarthritis joints and gut tissue" (O. Rysnik, K. McHugh, L. M. van Duivenvoorde, M. N. van Tok, G. Guggino, J. D. Taurog, S. Kollnberger, F. Ciccia, D. L. Baeten, P. Bowness, 2016) [1].

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

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

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

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

Silencing or inhibition of endoplasmic reticulum aminopeptidase 1 (ERAP1) suppresses free heavy chain expression and Th17 responses in ankylosing spondylitis.

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.

HLA-B27-Homodimer-Specific Antibody Modulates the Expansion of Pro-Inflammatory T-Cells in HLA-B27 Transgenic Rats.

OBJECTIVES: HLA-B27 is a common genetic risk factor for the development of Spondyloarthritides (SpA). HLA-B27 can misfold to form cell-surface heavy chain homodimers (B272) and induce pro-inflammatory responses that may lead to SpA pathogenesis. The presence of B272 can be detected on leukocytes of HLA-B27+ Ankylosing spondylitis (AS) patients and HLA-B27 transgenic rats. We characterized a novel B272-specific monoclonal antibody to study its therapeutic use in HLA-B27 associated disorders. METHODS: The monoclonal HD5 antibody was selected from a phage library to target cell-surface B272 homodimers and characterized for affinity, specificity and ligand binding. The immune modulating effect of HD5 was tested in HLA-B27 transgenic rats. Onset and progression of disease profiles were monitored during therapy. Cell-surface B272 and expansion of pro-inflammatory cells from blood, spleen and draining lymph nodes were assessed by flow cytometry. RESULTS: HD5 bound B272 with high specificity and affinity (Kd = 0.32 nM). HD5 blocked cell-surface interaction of B272 with immune regulatory receptors KIR3DL2, LILRB2 and Pirb. In addition, HD5 modulated the production of TNF from CD4+ T-cells by limiting B272 interactions in vitro. In an HLA-B27 transgenic rat model repetitive dosing of HD5 reduced the expansion of pro-inflammatory CD4+ T-cells, and decreased the levels of soluble TNF and number of cell-surface B272 molecules. CONCLUSION: HD5 predominantly inhibits early TNF production and expansion of pro-inflammatory CD4+ T-cells in HLA-B27 transgenic rats. Monoclonal antibodies targeting cell-surface B272 propose a new concept for the modulation of inflammatory responses in HLA-B27 related disorders.

The Leukocyte Immunoglobulin-Like Receptor Family Member LILRB5 Binds to HLA-Class I Heavy Chains.

OBJECTIVE: The leukocyte immunoglobulin-like receptor (LILR) family includes inhibitory and stimulatory members which bind to classical and non-classical HLA-class I. The ligands for many LILR including LILRB5 have not yet been identified. METHODS: We generated C-terminal eGFP and N-terminal FLAG-tagged fusion constructs for monitoring LILR expression. We screened for LILR binding to HLA-class I by tetramer staining of 293T cells transfected with LILRA1, A4, A5 A6 and LILRB2 and LILRB5. We also studied HLA class I tetramer binding to LILRB5 on peripheral monocyte cells. LILRB5 binding to HLA-class I heavy chains was confirmed by co-immunoprecipitation. RESULTS: HLA-B27 (B27) free heavy chain (FHC) dimer but not other HLA-class I stained LILRB5-transfected 293T cells. B27 dimer binding to LILRB5 was blocked with the class I heavy chain antibody HC10 and anti-LILRB5 antisera. B27 dimers also bound to LILRB5 on peripheral monocytes. HLA-B7 and B27 heavy chains co-immunoprecipitated with LILRB5 in transduced B and rat basophil RBL cell lines. CONCLUSIONS: Our findings show that class I free heavy chains are ligands for LILRB5. The unique binding specificity of LILRB5 for HLA-class I heavy chains probably results from differences in the D1 and D2 immunoglobulin-like binding domains which are distinct from other LILR which bind to ß2m-associated HLA-class I.

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

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

Critical role of endoplasmic reticulum aminopeptidase 1 in determining the length and sequence of peptides bound and presented by HLA-B27.

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.

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

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

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

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