Suppression of allo‐human leucocyte antigen (HLA) antibodies secreted by B memory cells in vitro: intravenous immunoglobulin (IVIg) versus a monoclonal anti‐HLA‐E IgG that mimics HLA‐I reactivities of IVIg

B memory cells remain in circulation and secrete alloantibodies without antigen exposure > 20 years after alloimmunization postpartum or by transplantation. These long‐lived B cells are resistant to cytostatic drugs. Therapeutically, intravenous immunoglobulin (IVIg) is administered to reduce allo‐human leucocyte antigen (HLA) antibodies pre‐ and post‐transplantation, but the mechanism of reduction remains unclear. Recently, we reported that IVIg reacts with several HLA‐I alleles and the HLA reactivity of IVIg is lost after its HLA‐E reactivity is adsorbed out. Therefore, we have generated an anti‐HLA‐E monoclonal antibody that mimics the HLA‐reactivity of IVIg to investigate whether this antibody suppresses IgG secretion, as does IVIg. B cells were purified from the blood of a woman in whose blood the B memory cells remained without antigen exposure > 20 years after postpartum alloimmunization. The B cells were stimulated with cytokines using a well‐defined culture system. The anti‐HLA‐E monoclonal antibody (mAb) significantly suppressed the allo‐HLA class‐II IgG produced by the B cells, and that this suppression was far superior to that by IVIg. These findings were confirmed with HLA‐I antibody secreted by the immortalized B cell line, developed from the blood of another alloimmunized woman. The binding affinity of the anti‐HLA‐E mAb for peptide sequences shared (i.e. shared epitopes) between HLA‐E and other β2‐microglobulin‐free HLA heavy chains (open conformers) on the cell surface of B cells may act as a ligand and signal suppression of IgG production of activated B memory cells. We propose that anti‐HLA‐E monoclonal antibody may also be useful to suppress allo‐HLA IgG production in vivo.     

Anti‐citrullinated glucose‐6‐phosphate isomerase peptide antibodies in patients with rheumatoid arthritis are associated with HLA‐DRB1 shared epitope alleles and disease activity

To identify and characterize anti‐citrullinated glucose‐6‐phosphate isomerase (GPI) peptide antibodies in patients with rheumatoid arthritis (RA). Nine GPI arginine‐bearing peptides in human GPI protein were selected and cyclic citrullinated GPI peptides (CCG‐1–9) were constructed. Samples were obtained from RA (n = 208), systemic lupus erythematosus (SLE) (n = 101), Sjögren's syndrome (SS; n = 101) and healthy controls (n = 174). Antibodies against CCG‐1–9 were measured, and anti‐citrullinated α‐enolase‐1 (CEP‐1), ‐cyclic citrullinated peptides (CCP) and ‐GPI proteins antibodies were also examined. Patients with RA were genotyped for HLA‐DRB1. The numbers of shared epitope (SE) alleles were counted and compared with those of the autoantibodies. Rabbit GPI was citrullinated with rabbit peptidylarginine deiminase and immunoblot analysis of RA sera performed. The levels of autoantibodies were compared before and after treatment with TNF antagonists in 58 RA patients. Anti‐CCG‐2, ‐4 and ‐7 antibodies were detected in 25·5, 33·2 and 37·0% patients with RA, respectively, and these antibodies were very specific for RA (specificity, 98·1–99·7%). Altogether, 44·2, 86·1 and 13·9% of RA sera were positive for anti‐CEP‐1, ‐CCP and ‐GPI protein antibodies, respectively. Anti‐CCG‐2, ‐4 and ‐7 antibodies were correlated with anti‐CCP and anti‐CEP‐1 antibodies and with the presence of HLA‐DRB1 SE alleles. Citrullinated GPI protein was detected using RA sera. Treatment with tumour necrosis factor antagonists reduced significantly the levels of anti‐CCG‐2 and ‐7 but not of anti‐CEP‐1 antibodies. This is the first report documenting the presence of anti‐CCG antibodies in RA. Anti‐CCG‐2 and ‐7 antibodies could be considered as markers for the diagnosis of RA and its disease activity.     

Associations of B cell‐activating factor (BAFF) and anti‐BAFF autoantibodies with disease activity in multi‐ethnic Asian systemic lupus erythematosus patients in Singapore

To measure the levels of B cell‐activating factor (BAFF) and endogenous anti‐BAFF autoantibodies in a cohort of multi‐ethnic Asian systemic lupus erythematosus (SLE) patients in Singapore, to determine their correlation with disease activity. Serum samples from 121 SLE patients and 24 age‐ and sex‐matched healthy controls were assayed for BAFF and anti‐BAFF immunoglobulin (Ig)G antibody levels by enzyme‐linked immunosorbent assay (ELISA). The lowest reliable detection limit for anti‐BAFF‐IgG antibody levels was defined as 2 standard deviations (s.d.) from blank. Correlation of serum BAFF and anti‐BAFF IgG levels with disease activity [scored by SLE Activity Measure revised (SLAM‐R)], and disease manifestations were determined in these 121 patients. SLE patients had elevated BAFF levels compared to controls; mean 820 ± 40 pg/ml and 152 pg ± 45/ml, respectively [mean ± standard error of the mean (s.e.m.), P < 0·01], which were correlated positively with anti‐dsDNA antibody levels (r = 0·253, P < 0·03), and SLAM‐R scores (r = 0·627, P < 0·01). In addition, SLE patients had significantly higher levels of anti‐BAFF IgG, which were correlated negatively with disease activity (r = –0·436, P < 0·01), levels of anti‐dsDNA antibody (r = –0·347, P < 0·02) and BAFF (r = –0·459, P < 0·01). The majority of patients in this multi‐ethnic Asian SLE cohort had elevated levels of BAFF and anti‐BAFF antibodies. Anti‐BAFF autoantibody levels correlated negatively with clinical disease activity, anti‐dsDNA and BAFF levels, suggesting that they may be disease‐modifying. Our results provide further information about the complexity of BAFF pathophysiology in different SLE disease populations and phenotypes, and suggest that studies of the influence of anti‐cytokine antibodies in different SLE populations will be required when selecting patients for trials using targeted anti‐cytokine therapies.     

Relationship between natural and infection‐induced antibodies in systemic autoimmune diseases (SAD): SLE,SSc and RA

Infection or vaccine‐induced T cell‐dependent immune response and the subsequent high‐affinity neutralizing antibody production have been extensively studied, while the connection between natural autoantibodies (nAAbs) and disease‐specific antibodies has not been thoroughly investigated. Our goal was to find the relationship between immunoglobulin (Ig)M and IgG isotype nAAbs and infection or vaccine‐induced and disease‐related autoantibody levels in systemic autoimmune diseases (SAD). A previously described indirect enzyme‐linked immunosorbent assay (ELISA) test was used for detection of IgM/IgG nAAbs against citrate synthase (anti‐CS) and F4 fragment (anti‐F4) of DNA topoisomerase I in 374 SAD samples, with a special focus on systemic lupus erythematosus (SLE) (n = 92), rheumatoid arthritis (n = 73) and systemic sclerosis (n = 157) disease groups. Anti‐measles IgG and anti‐dsDNA IgG/IgM autoantibodies were measured using commercial and in‐house indirect ELISA tests. In all SAD groups the anti‐measles IgG‐seropositive cases showed significantly higher anti‐CS IgG titers (P = 0·011). In anti‐dsDNA IgG‐positive SLE patients, we detected significantly higher levels of anti‐CS and anti‐F4 IgG nAAbs (P = 0·001 and < 0·001, respectively). Additionally, we found increased levels of IgM isotypes of anti‐CS and anti‐F4 nAAbs in anti‐dsDNA IgM‐positive SLE patients (P = 0·002 and 0·016, respectively). The association between IgG isotypes of pathogen‐ or autoimmune disease‐related antibodies and the IgG nAAbs may underscore the immune response‐inducible nature of the diseases investigated. The relationship between protective anti‐dsDNA IgM and the IgM isotype of anti‐F4 and anti‐CS may provide immunoserological evidence for the beneficial roles of nAAbs in SLE patients.     

Immunoglobulin (Ig)G purified from human sera mirrors intravenous Ig human leucocyte antigen (HLA) reactivity and recognizes one's own HLA types,but may be masked by Fab complementarity‐determining region peptide in the native sera

Intravenous immunoglobulin (IVIg) reacted with a wide array of human leucocyte antigen (HLA) alleles, in contrast to normal sera, due possibly to the purification of IgG from the pooled plasma. The reactivity of IgG purified from normal sera was compared with that of native sera to determine whether any serum factors mask the HLA reactivity of anti‐HLA IgG and whether IgG purified from sera can recognize the HLA types of the corresponding donors. The purified IgG, unlike native sera, mirrored IVIg reactivity to a wide array of HLA‐I/‐II alleles, indicating that anti‐HLA IgG may be masked in normal sera – either by peptides derived from soluble HLA or by those from antibodies. A < 3 kDa peptide from the complementarity‐determining region (CDR) of the Fab region of IgG (but not the HLA peptides) masked HLA recognition by the purified IgG. Most importantly, some of the anti‐HLA IgG purified from normal sera – and serum IgG from a few donors – indeed recognized the HLA types of the corresponding donors, confirming the presence of auto‐HLA antibodies. Comparison of HLA types with the profile of HLA antibodies showed auto‐HLA IgG to the donors' HLA antigens in this order of frequency: DPA (80%), DQA (71%), DRB345 (67%), DQB (57%), Cw (50%), DBP (43%), DRB1 (21%), A (14%) and B (7%). The auto‐HLA antibodies, when unmasked in vivo, may perform immunoregulatory functions similar to those of therapeutic preparations of IVIg.     

HLA‐F is a surface marker on activated lymphocytes

Of the three nonclassical class I antigens expressed in humans, HLA‐F has been least characterized with regard to expression or function. In this study, we examined HLA‐F expression focusing on lymphoid cells, where our previous work with homologous cell lines had demonstrated surface HLA‐F expression. HLA‐F protein expression was observed by Western blot analysis in all resting lymphocytes, including B cells, T cells, NK cells, and monocytes, all of which lacked surface expression in the resting state. Upon activation, using a variety of methods to activate different lymphocyte subpopulations, all cell types that expressed HLA‐F intracellularly showed an induction of surface HLA‐F protein. An examination of peripheral blood from individuals genetically deficient for TAP and tapasin expression demonstrated the same activation expression profiles for HLA‐F, but with altered kinetics post‐activation. Further analysis of CD4⁺CD25⁺ Treg showed that HLA‐F was not upregulated on the major fraction of these cells when they were activated, whereas CD4⁺CD25^? T cells showed strong expression of surface HLA‐F when activated under identical conditions. These findings are discussed with regard to possible functions for HLA‐F and its potential clinical use as a marker of an activated immune response.     

Dimorphic HLA‐B signal peptides differentially influence HLA‐E‐ and natural killer cell‐mediated cytolysis of HIV‐1‐infected target cells

As a mechanism of self‐protection, signal peptides cleaved from human leukocyte antigen (HLA) class I products bind to HLA‐E before the complex interacts with the natural killer (NK) cell receptor CD94/NKG2A to inhibit NK‐mediated cell lysis. Two types of the signal peptides differ in their position 2 (P2) anchor residue, with P2‐methionine (P2‐M) having higher HLA‐E binding affinity than P2‐threonine (P2‐T). All HLA‐A and HLA‐C molecules carry P2‐M, whereas HLA‐B products have either P2‐M or P2‐T. Epidemiological evidence suggests that P2‐M is unfavourable in the context of HIV‐1 infection, being associated with accelerated acquisition of HIV‐1 infection in two African cohorts. To begin elucidating the functional mechanism, we studied NK‐mediated killing of CD4⁺ T cells and monocyte‐derived macrophages infected with two laboratory‐adapted HIV‐1 strains and two transmitted/founder (T/F) viruses. In the presence of target cells derived from individuals with the three HLA‐B P2 genotypes (M/M, M/T and T/T), NK‐mediated cytolysis was elevated consistently for P2‐T in a dose‐dependent manner for all cell and virus combinations tested (P = 0·008–0·03). Treatment of target cells with an anti‐HLA‐E monoclonal antibody restored NK‐mediated cytolysis of cells expressing P2‐M. Observations on cell lysis were also substantiated by measurements of HIV‐1 p24 antigen in the culture supernatants. Overall, our experiments indicate that the anti‐HIV‐1 function mediated by NK cells is compromised by P2‐M, corroborating the association of HLA‐B genotype encoding P2‐M with accelerated HIV‐1 acquisition.     

Suppression of blastogenesis and proliferation of activated CD4+ T cells: intravenous immunoglobulin (IVIg) versus novel anti‐human leucocyte antigen (HLA)‐E monoclonal antibodies mimicking HLA‐I reactivity of IVIg

Activated CD4⁺ T cells undergo blastogenesis and proliferation and they express several surface receptors, including β2‐microglobulin‐free human leucocyte antigen (HLA) heavy chains (open conformers). Intravenous immunoglobulin (IVIg) suppresses activated T cells, but the mechanism is unclear. IVIg reacts with HLA‐Ia/Ib antigens but its reactivity is lost when the anti‐HLA‐E Ab is adsorbed out. Anti‐HLA‐E antibodies may bind to the peptides shared by HLA‐E and the HLA‐I alleles. These shared peptides are cryptic in intact HLA, but exposed in open conformers. The hypothesis that anti‐HLA‐E monoclonal antibodies (mAbs) that mimic HLA‐I reactivity of IVIg may suppress activated T cells by binding to the shared peptides of the open conformers on the T cell surface was tested by examining the relative binding affinity of those mAbs for open conformers coated on regular beads and for intact HLA coated on iBeads, and by comparing the effects on the suppression of phytohaemagglutinin (PHA)‐activated T cells of three entities: IVIg, anti‐HLA‐E mAbs that mimic IVIg [Terasaki Foundation Laboratory (TFL)‐006 and (TFL)‐007]; and anti‐HLA‐E antibodies that do not mimic IVIg (TFL‐033 and TFL‐037). Suppression of blastogenesis and proliferation of those T cells by both IVIg and the anti‐HLA‐E mAbs was dose‐dependent, the dose required with mAbs 50–150‐fold lower than with IVIg. TFL‐006 and TFL‐007 significantly suppressed blastogenesis and proliferation of activated CD4⁺ T cells, but neither the non‐IVIg‐mimicking mAbs nor control antibodies did so. The suppression may be mediated by Fab‐binding of TFL‐006/TFL‐007 to the exposed shared peptides. The mAb binding to the open conformer may signal T cell deactivation because the open conformers have an elongated cytoplasmic tail with phosphorylation sites (tryosine³²⁰/serine³³⁵).     

Serum concentration of immunoglobulin G‐type antibodies against the whole Epstein–Barr nuclear antigen 1 and its aa35–58 or aa398–404 fragments in the sera of patients with systemic lupus erythematosus and multiple sclerosis

Several studies suggest that infection by Epstein–Barr virus (EBV) might be one of the environmental factors which facilitates the development of autoimmune disorders in genetically susceptible individuals. Recent data indicate that high anti‐Epstein–Barr nuclear antigen 1 (EBNA)‐1 immunoglobulin (Ig)G titre is a strong risk factor for multiple sclerosis (MS) in patients both with and without the main genetic predisposing trait, human leucocyte antigen (HLA)‐DRB1*15:01. Because no similar studies have been published in systemic lupus erythematosus (SLE) patients, we determined the HLA‐DRB1*15:01 carrier state and the serum titres against the whole EBNA‐1 and its small fragments aa35–58 and aa398–404 in 301 SLE patients, 135 MS patients and in 345 healthy controls. The carrier state of the HLA‐DRB1*15:01 allele was deduced from genotyping of a tagSNP (rs3135388) by applying a Taqman‐based assay. The serum concentrations of antibodies to EBNA‐1 and its aa35–58 or aa398–404 fragments were determined using a commercial assay (ETI‐EBNA‐G) and home‐made enzyme‐linked immunosorbent assays, respectively. The serum concentration of anti‐EBNA‐1 antibodies was significantly (P < 0·001) higher both in MS and SLE patients than in controls. Similar significant differences were found both in HLA‐DRB1*15:01 carriers and non‐carriers. Furthermore, titres of antibodies against the aa35–58 EBNA‐1 fragment were elevated both in MS and SLE patients. By contrast, the levels of aa398–404 EBNA‐1 antibodies were elevated significantly only in the SLE patients. These findings indicate that high anti‐EBNA‐1 IgG titres are HLA‐DRB1*15:01‐independent risk factors not only for MS, but also for SLE, while high antibody titres against the aa398–404 fragment are characteristic for SLE.     

In vivo identification of an HLA‐G complex as ubiquitinated protein circulating in exosomes

The nonclassical human leukocyte antigen‐G (HLA‐G) is a tolerogenic molecule that can be released to the circulation by expressing cells. This molecule can form dimers but some other complexed HLA‐G forms have been proposed to be present in vivo. Here, we further characterized these other complexed HLA‐G forms in vivo. Ascitic and pleural exudates from patients were selected based on positivity for HLA‐G by ELISA. Complexed HLA‐G was detected in exosomes, which indicates an intracellular origin of these forms. 2D‐PAGE analysis of exudates and isolated exosomes showed that these high molecular weight complexes were more heterogeneous than the HLA‐G1 expressed by cell cultures. Treatment with deglycosylating enzymes did not change the molecular weight of HLA‐G complexes. Immunoblot analysis of exudates and exosomes with an anti‐ubiquitin antibody showed that at least some of these structures correspond to ubiquitinated HLA‐G. HLA‐G ubiquitination could be reproduced in vitro in HLA‐G1‐transfected cell lines, although with a lower modified/nonmodified protein proportion than in exudates. In summary, we demonstrate new circulating HLA‐G forms in vivo that open a new perspective in the study of HLA‐G function and analysis.     

Epitopes and motifs of the HLA‐B*14 allele family products and related HLA‐B14 cross‐reactive specificities

The split specificities of HLA‐B14 (B64, B65) are assigned to the B*14:01 (B64) and B*14:02 (B65) products only. Of the further 50 B*14 expressed products, only B*14:03 and B*14:06 are officially designated as HLA‐B14. The B*14:08 product differs from B64 by a single amino acid substitution of W97R, while the B*14:53 specificity (which is a “short” B14 and neither B64 nor B65) differs from B64 by three residues (W97S, Y113H and F116Y). Comprehensive testing of B*14:08:01 cells (using 49 alloantisera with B64 or B64, B65 specificities, and five monoclonal antibodies with B65 or B64, B65 activity) showed that the B*14:08 specificity is, like the B*14:53 product, neither B64 nor B65 and appears as a “short” B14 specificity. To help understand the serological reactivity of the B*14:08 and B*14:53 products, and B64 and B65, we identified seven published epitopes (11AV, 97W, 61ICT, 116F, 131S+163T, 170RH and 420) and, by inspection, 29 motifs, that encompass one or more of B64, B65 and various HLA‐B14 cross‐reactive group specificities. We then considered the possession of these epitopes and motifs by the products of B*14:01 to B*14:06, B*14:08 and B*14:53. Seventeen of the 29 motifs fully complied with the one‐/two‐patch functional epitope concept for amino acid proximity, as determined by Cn3D software, the remainder partially complied. The nature and patterns of epitopes and motifs possessed by both B*14:08 and B*14:53 specificities supported their designation as HLA‐B14 but non‐B64/B65. Also that epitope 97W, with 11S or 11A, is critical for serological B64 and B65 reactivity. And conversely, that epitope 116F, and several identified motifs, are probably unimportant for HLA‐B14 antibody reactivity. The previous submission that the B*14:03 specificity is HLA‐B65 was compatible with its epitope/motif pattern. B*14:04 cells would also be expected to react as B65, based on its epitope/motif pattern, and not as B64 as previously implied. Also, from their epitope/motif patterns, and external serological information, it is probable that the B*14:05 and B*14:06 specificities will both appear as “short” HLA‐B14, non‐B64/B65. Several epitopes and motifs encompassed a range of HLA‐B specificities included in the serological HLA‐B14 cross‐reactive group, thus supporting these original serological findings.     

Deciphering allogeneic antibody response against native and denatured HLA epitopes in organ transplantation

Anti‐HLA donor‐specific antibodies are deleterious for organ transplant survival. Class I HLA donor‐specific antibodies are identified by using the Luminex single antigen beads (LSAB) assay, which also detects anti‐denatured HLA antibodies (anti‐dHLAs). Anti‐dHLAs are thought to be unable to recognize native HLA (nHLA) on the cell surface and therefore to be clinically irrelevant. Acid denaturation of nHLA on LSAB allows anti‐dHLAs to be discriminated from anti‐nHLAs. We previously defined a threshold for the ratio between mean fluorescence intensity against acid‐treated (D for denaturation) and nontreated (N) LSAB, D ≥ 1.2 N identifying the anti‐dHLAs. However, some anti‐dHLAs remained able to bind nHLA on lymphocytes in flow cytometry crossmatches, and some anti‐nHLAs conserved significant reactivity toward acid‐treated LSAB. After depleting serum anti‐nHLA reactivity with HLA‐typed cells, we analyzed the residual LSAB reactivity toward nontreated and acid‐treated LSABs, and then evaluated the ability of antibodies to recognize nHLA alleles individually. We observed that sera can contain mixtures of anti‐nHLAs and anti‐dHLAs, or anti‐nHLAs recognizing acid‐resistant epitopes, all possibly targeting the same allele(s). Therefore, the anti‐HLA antibody response can be highly complex and subtle, as is the accurate identification of pathogenic anti‐HLA antibodies in human serum.     

Anti‐transglutaminase antibodies in non‐coeliac children suffering from infectious diseases

Anti‐transglutaminase antibodies are the diagnostic markers of coeliac disease. A role is suggested for infectious agents in the production of anti‐transglutaminase antibodies. The aim was to measure positive anti‐transglutaminase antibody levels in children with infectious diseases and to compare immunological and biological characteristics of the anti‐transglutaminase antibodies derived from these children with that from coeliac patients. Two hundred and twenty‐two children suffering from infectious diseases were enrolled prospectively along with seven biopsy‐proven coeliacs. Serum samples were tested for anti‐transglutaminase antibodies and anti‐endomysium antibodies; positive samples were tested for coeliac‐related human leucocyte antigen (HLA)‐DQ2/8 and anti‐viral antibodies. Purified anti‐transglutaminase antibodies from the two study groups were tested for urea‐dependent avidity, and their ability to induce cytoskeletal rearrangement and to modulate cell‐cycle in Caco‐2 cells, using phalloidin staining and bromodeoxyuridine incorporation assays, respectively. Nine of 222 children (4%) tested positive to anti‐transglutaminase, one of whom also tested positive for anti‐endomysium antibodies. This patient was positive for HLA‐DQ2 and was diagnosed as coeliac following intestinal biopsy. Of the eight remaining children, two were positive for HLA‐DQ8. Levels of anti‐transglutaminase returned to normal in all subjects, despite a gluten‐containing diet. Purified anti‐transglutaminase of the two study groups induced actin rearrangements and cell‐cycle progression. During an infectious disease, anti‐transglutaminase antibodies can be produced temporarily and independently of gluten. The infection‐triggered anti‐transglutaminase antibodies have the same biological properties as that of the coeliacs, with the same in‐vivo potential for damage.     

Guanine polynucleotides are self‐antigens for human natural autoantibodies and are significantly reduced in the human genome

In the course of investigating anti‐DNA autoantibodies, we examined IgM and IgG antibodies to poly‐G and other oligonucleotides in the sera of healthy persons and those diagnosed with systemic lupus erythematosus (SLE), scleroderma (SSc), or pemphigus vulgaris (PV); we used an antigen microarray and informatic analysis. We now report that all of the 135 humans studied, irrespective of health or autoimmune disease, manifested relatively high amounts of IgG antibodies binding to the 20‐mer G oligonucleotide (G20); no participants entirely lacked this reactivity. IgG antibodies to homo‐nucleotides A20, C20 or T20 were present only in the sera of SLE patients who were positive for antibodies to dsDNA. The prevalence of anti‐G20 antibodies led us to survey human, mouse and Drosophila melanogaster (fruit fly) genomes for runs of T20 and G20 or more: runs of T20 appear > 170 000 times compared with only 93 runs of G20 or more in the human genome; of these runs, 40 were close to brain‐associated genes. Mouse and fruit fly genomes showed significantly lower T20/G20 ratios than did human genomes. Moreover, sera from both healthy and SLE mice contained relatively little or no anti‐G20 antibodies; so natural anti‐G20 antibodies appear to be characteristic of humans. These unexpected observations invite investigation of the immune functions of anti‐G20 antibodies in human health and disease and of runs of G20 in the human genome.     

Impact and production of Non‐HLA‐specific antibodies in solid organ transplantation

Organ transplantation is an effective way to treat end‐stage organ disease. Extending the graft survival is one of the major goals in the modern era of organ transplantation. However, long‐term graft survival has not significantly improved in recent years despite the improvement of patient management and advancement of immunosuppression regimen. Antibody‐mediated rejection is a major obstacle for long‐term graft survival. Donor human leucocyte antigen (HLA)‐specific antibodies were initially identified as a major cause for antibody‐mediated rejection. Recently, with the development of solid‐phase‐based assay reagents, the contribution of non‐HLA antibodies in organ transplantation starts to be appreciated. Here, we review the role of most studied non‐HLA antibodies, including angiotensin II type 1 receptor (AT₁R), K‐α‐tubulin and vimentin antibodies, in the solid organ transplant, and discuss the possible mechanism by which these antibodies are stimulated.     

Immunoglobulin A anti‐phospholipid antibodies in Swedish cases of systemic lupus erythematosus: associations with disease phenotypes,vascular events and damage accrual

Immunoglobulin (Ig) G‐ and IgM‐class anti‐cardiolipin antibodies (aCL) and lupus anti‐coagulant (LA) are included in the 1997 update of the American College of Rheumatology (ACR‐97) systemic lupus erythematosus (SLE) criteria. Despite limited evidence, IgA‐aCL and IgA anti‐β₂‐glycoprotein‐I (anti‐β₂GPI) were included in the 2012 Systemic Lupus International Collaborating Clinics criteria. The present study aimed to evaluate IgG‐/IgA‐/IgM‐aCL and anti‐β₂GPI occurrence in relation to disease phenotype, smoking habits, pharmacotherapy, anti‐phospholipid syndrome (APS) and organ damage among 526 Swedish SLE patients meeting ACR‐97. Patients with rheumatoid arthritis (n = 100), primary Sjögren’s syndrome (n = 50) and blood donors (n = 507) served as controls. Anti‐phospholipid antibodies (aPL) were analysed by fluoroenzyme‐immunoassays detecting aCL/anti‐β₂GPI. Seventy‐six (14%) SLE cases fulfilled the Sydney APS‐criteria, and ≥ 1 aCL/anti‐β₂GPI isotype (IgG/IgA/IgM) occurred in 138 SLE patients (26%). Forty‐five (9%) of the SLE cases had IgA‐aCL, 20 of whom (4%) lacked IgG‐/IgM‐aCL. Seventy‐four (14%) tested positive for IgA anti‐β₂GPI, 34 (6%) being seronegative regarding IgG/IgM anti‐β₂GPI. Six (1%) had APS manifestations but were seropositive regarding IgA‐aCL and/or IgA anti‐β₂GPI in the absence of IgG/IgM‐aPL and LA. Positive LA and IgG‐aPL tests were associated with most APS‐related events and organ damage. Exclusive IgA anti‐β₂GPI occurrence associated inversely with Caucasian ethnicity [odds ratio (OR) = 0·21, 95% confidence interval (CI) = 0·06–0·72) and photosensitivity (OR = 0·19, 95% CI = 0·05–0·72). Nephritis, smoking, LA‐positivity and statin/corticosteroid‐medication associated strongly with organ damage, whereas hydroxychloroquine‐medication was protective. In conclusion, IgA‐aPL is not rare in SLE (16%) and IgA‐aPL analysis may have additional value among SLE cases with suspected APS testing negative for other isotypes of aPL and LA.     

Clinical implication of human leukocyte antigen (HLA)‐F expression in breast cancer

Human leukocyte antigen (HLA)‐F is one of the non‐classical HLA class I molecules that protects the fetus in pregnancy. HLA‐F expression was immunohistochemically examined and the association between clinical parameters and HLA‐F expression was analyzed. Cancerous HLA‐F and stromal HLA‐F‐positive infiltrating cells were detected in 91 (40.0%) and 186 (81.6%) cases, respectively. HLA‐F positivity in cancer cells was significantly associated with tumor size (P < 0.05). There was a weak correlation between HLA‐F positivity of cancer cells and HLA‐F positive infiltrative cells (P < 0.01, r = 0.11). HLA‐F positivity did not affect patients' survival in 209 breast cancer. However, confined to stage II breast cancer, the HLA‐F positive group showed significantly poorer outcomes than the HLA‐F negative group (P < 0.05). The present study provides the first evidence that HLA‐F positivity in breast carcinoma affects clinicopathological factors and could be selected as a prognostic marker for limited clinical stage.     

A peptide mimic blocks the cross‐reaction of anti‐DNA antibodies with glomerular antigens

Anti‐DNA antibodies play a pivotal role in the pathogenesis of lupus nephritis by cross‐reacting with renal antigens. Previously, we demonstrated that the binding affinity of anti‐DNA antibodies to self‐antigens is isotype‐dependent. Furthermore, significant variability in renal pathogenicity was seen among a panel of anti‐DNA isotypes [derived from a single murine immunoglobulin (Ig)G3 monoclonal antibody, PL9‐11] that share identical variable regions. In this study, we sought to select peptide mimics that effectively inhibit the binding of all murine and human anti‐DNA IgG isotypes to glomerular antigens. The PL9‐11 panel of IgG anti‐DNA antibodies (IgG1, IgG2a, IgG2b and IgG3) was used for screening a 12‐mer phage display library. Binding affinity was determined by surface plasmon resonance. Enzyme‐linked immunosorbent assay (ELISA), flow cytometry and glomerular binding assays were used for the assessment of peptide inhibition of antibody binding to nuclear and kidney antigens. We identified a 12 amino acid peptide (ALWPPNLHAWVP, or ‘ALW’) which binds to all PL9‐11 IgG isotypes. Preincubation with the ALW peptide reduced the binding of the PL9‐11 anti‐DNA antibodies to DNA, laminin, mesangial cells and isolated glomeruli significantly. Furthermore, we confirmed the specificity of the amino acid sequence in the binding of ALW to anti‐DNA antibodies by alanine scanning. Finally, ALW inhibited the binding of murine and human lupus sera to dsDNA and glomeruli significantly. In conclusion, by inhibiting the binding of polyclonal anti‐DNA antibodies to autoantigens in vivo, the ALW peptide (or its derivatives) may potentially be a useful approach to block anti‐DNA antibody binding to renal tissue.     

Antibodies to age‐β2glycoprotein I in patients with anti‐phospholipid antibody syndrome

Anti‐phospholipid antibody syndrome (APS) is a systemic autoimmune disease characterized clinically by arterial and/or venous thromboses, recurrent abortions or fetal loss and serologically by the presence of ‘anti‐phospholipid antibodies’ (aPL). The main target antigen of the antibodies is β₂glycoprotein I (β₂GPI). Post‐translational oxidative modifications of the protein have been widely described. In this study we aimed to analyse sera reactivity to glucose‐modified β₂GPI (G‐β₂GPI). Sera collected from 43 patients with APS [15 primary APS (PAPS) and 28 APS associated with systemic lupus erythematosus (SLE) (SAPS)], 30 with SLE, 30 with rheumatoid arthritis (RA) and 40 healthy subjects were analysed by an enzyme‐linked immunosorbent assay (ELISA) using a G‐β₂GPI. Nine of 15 consecutive PAPS out‐patients (60%) and 16 of 28 SAPS (57.1%) showed serum antibodies [immunoglobulin (Ig)G class] against G‐β₂GPI (anti‐G‐β₂GPI) by ELISA. The occurrence of anti‐G‐β₂GPI was significantly higher in APS patients compared to patients suffering from SLE. No RA patients or control healthy subjects resulted positive for anti‐G‐β₂GPI. Of note, aG‐β₂GPI prompted to identify some APS patients (four PAPS and seven SAPS), who were negative in the classical anti‐β₂GPI test. Moreover, in APS patients, anti‐G‐β₂GPI titre was associated significantly with venous thrombosis and seizure in APS patients. This study demonstrates that G‐β₂GPI is a target antigen of humoral immune response in patients with APS, suggesting that β₂GPI glycation products may contain additional epitopes for anti‐β₂GPI reactivity. Searching for these antibodies may be useful for evaluating the risk of clinical manifestations.     

Clinical application of anti double-stranded and single-stranded DNA antibodies measurements by enzyme-linked immunosorbent assay (ELISA) using E. coli plasmid DNA]

Anti double-stranded (ds) and single-stranded (ss) DNA IgG antibodies were detected in patients with various connective tissue diseases by RECOMBIGEN ELISA Anti ds-DNA Kit and ELISA Anti ss-DNA Kit (Nippon DPC Co) using E. coli plasmid DNA. High levels of anti ds-DNA and anti ss-DNA antibodies were mostly found and distributed in patients with systemic lupus erythematosus(SLE) and allied disorders. The sensitivity of Anti ds-DNA Kit was higher than that of current ELISA kit using calf thymus DNA antigen. In SLE patients with anti DNA antibodies sixty per cent of sera were reacted with both ds- and ss-DNA antigens, and forty per cent of sera were reacted only with ss-DNA antigen; there was no sample reacted positively with ds-DNA antigen alone. RECOMBIGEN ELISA Anti ds-DNA Kit and ELISA Anti ss-DNA Kit are highly sensitive and useful methods to detect anti ds- and/or ss-DNA IgG antibodies in patients with SLE.