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The thymus prevents autoimmune diseases through mechanisms that operate in the cortex and medulla, comprising positive and negative selection and the generation of regulatory T-cells (Tregs). Egress from the thymus through the perivascular space (PVS) to the blood is another possible checkpoint, as shown by some autoimmune/immunodeficiency syndromes. In polygenic autoimmune diseases, subtle thymic dysfunctions may compound genetic, hormonal and environmental cues. Here, we cover (a) tolerance-inducing cell types, whether thymic epithelial or tuft cells, or dendritic, B- or thymic myoid cells; (b) tolerance-inducing mechanisms and their failure in relation to thymic anatomic compartments, and with special emphasis on human monogenic and polygenic autoimmune diseases and the related thymic pathologies, if known; (c) polymorphisms and mutations of tolerance-related genes with an impact on positive selection (e.g. the gene encoding the thymoproteasome-specific subunit, PSMB11), promiscuous gene expression (e.g. AIRE, PRKDC, FEZF2, CHD4), Treg development (e.g. SATB1, FOXP3), T-cell migration (e.g. TAGAP) and egress from the thymus (e.g. MTS1, CORO1A); (d) myasthenia gravis as the prototypic outcome of an inflamed or disordered neoplastic ‘sick thymus’.

Break et al . (Research Articles, 15 January 2021, eaay5731) conclude that T cell overproduction of interferon-γ causes chronic mucocutaneous candidiasis (CMC), a typical early feature of autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED). This contradicts studies implicating interleukin IL-17 and IL-22 deficiencies as a cause of CMC. We propose that Break et al . have focused on late-arising events rather than more common primary causes of CMC.

Purpose of reviewThymomas appear very rarely after extended thymectomy for early-onset myasthenia gravis (EOMG). We describe 2 such cases that highlight potential early warning signs.Recent findingsIn their 20s, one woman and one man developed EOMG (AChR antibody-positive), requiring extended transsternal removal of hyperplastic thymi at ages 35 and 27, respectively. Their myasthenia gravis was readily controlled for the next 10 and 7 years before deteriorating in both, with appearance of late clinical features and anticytokine autoantibodies suggesting underlying thymomas, namely respiratory infections, genital herpes, chronic candidiasis, and alopecia in the woman and erythroderma and lichen planus in the man, followed by Pseudomonas, Klebsiella, and cytomegalovirus infections plus chronic hepatitis during intensifying immunosuppressive therapy. Type B thymomas were then detected. Despite surgery or radiotherapy, and intensive drug therapy, the patients died 7 and 1 years later.SummaryCertain infections/dermatologic manifestations that associate with long-standing thymomas may herald their late appearance, despite previous thymectomy.

Autoimmune associations in myasthenia gravis (MG)-patients and their relatives have not been re-assessed since their separation into early- or late-onset MG (EOMG, LOMG), or thymoma-associated MG. Here, we analysed 226 EOMG-, 97 LOMG-, and 150 thymoma-patients for autoimmune disorders in themselves and their relatives. From 283 of them sera were tested for different organ- and non-organ-specific autoantibodies (autoAbs) by immunofluorescence test (IFT) and ELISA; genotyping was performed in 213 patients. Relatives with autoimmune disorders were reported by more patients with EOMG (40% of 210) than LOMG (20% of 89; p0.01) than thymomas (8% of 150; p0.001). In 150 genotyped EOMG-females, the known risk allele of the immuno-regulatory PTPN2 2 (R620W) appeared commoner in those with second autoimmune diseases (p ∼ 0.06), or with autoimmune relatives (p ∼ 0.03), than in those without. Organ-specific autoAbs were found in ∼ 30% of all MG-patients, autoAbs to striated muscle only in patients with thymoma-MG (62%) or LOMG (61%). Titers against adrenal cortex were lower in LOMG-patients. Disease-associated autoAbs against systemic targets or 'natural autoAbs' - except of autoAbs to nuclei - were uncommon in all groups (13%). Thus-with rare exceptions in EOMG and LOMG-we found minimal support for the notion that autoimmune patients have wide-ranging autoreactivity that causes disease only if it targets such Achilles' heels as the muscle acetylcholine receptor; even in thymoma-patients the autoAbs are sharply focused on a restricted range of muscle, cytokine and endocrine targets.

Myasthenia gravis (MG) is treated primarily with broad-spectrum immuno-suppressants such as prednisone or azathioprine, which normally require several months to reduce autoantibody titers significantly. This delay may be caused by the resistance of the main antibody-producing cells, the plasma cells, to these drugs. In particular, long-lived plasma cells are resistant to immunosuppressive treatments and can produce (auto-) antibodies for months. Bortezomib is a proteasome inhibitor approved for treating patients with multiple myeloma, a plasma cell malignancy. Recent preclinical studies in cell cultures and animal models, and clinical studies in organ-transplant recipients, have demonstrated that bortezomib can kill non-neoplastic plasma cells within hours. This suggests that proteasome inhibitors could also be used for rapidly reducing autoantibody production in autoimmune diseases. We have begun to assess their potential in MG.

Antibodies to muscle-specific kinase (MuSK) are found in a variable proportion of patients with myasthenia without typical acetylcholine receptor (AChR) antibodies, but their characteristics and pathogenic mechanisms are not fully understood. We discuss the incidence and pathogenicity of MuSK antibodies and how clinical studies, animal models, and cultured cell lines can be used to elucidate their pathogenic mechanisms. Patients without either AChR or MuSK antibodies (seronegative myasthenia) appear to present another disease subtype that is highly similar to that of typical myasthenia gravis. We demonstrate a new method that detects AChR antibodies in these patients and show that these low-affinity AChR antibodies are predominantly IgG1 and can activate complement C3b deposition. Similarly MuSK antibodies, although mainly IgG4, are partially IgG1 and can activate C3b deposition. Overall, these results suggest that complement-activation may be an important pathogenic mechanism even in patients without conventional AChR antibodies.

Only around 80% of patients with generalized myasthenia gravis (MG) have serum antibodies to acetylcholine receptor [AChR; acetylcholine receptor antibody positive myasthenia gravis (AChR-MG)] by the radioimmunoprecipitation assay used worldwide. Antibodies to muscle specific kinase [MuSK; MuSK antibody positive myasthenia gravis (MuSK-MG)] make up a variable proportion of the remaining 20%. The patients with neither AChR nor MuSK antibodies are often called seronegative (seronegative MG, SNMG). There is accumulating evidence that SNMG patients are similar to AChR-MG in clinical features and thymic pathology. We hypothesized that SNMG patients have low-affinity antibodies to AChR that cannot be detected in solution phase assays, but would be detected by binding to the AChRs on the cell membrane, particularly if they were clustered at the high density that is found at the neuromuscular junction. We expressed recombinant AChR subunits with the clustering protein, rapsyn, in human embryonic kidney cells and tested for binding of antibodies by immunofluorescence. To identify AChRs, we tagged either AChR or rapsyn with enhanced green fluorescence protein, and visualized human antibodies with Alexa Fluor-labelled secondary or tertiary antibodies, or by fluorescence-activated cell sorter (FACS). We correlated the results with the thymic pathology where available. We detected AChR antibodies to rapsyn-clustered AChR in 66% (25/38) of sera previously negative for binding to AChR in solution and confirmed the results with FACS. The antibodies were mainly IgG1 subclass and showed ability to activate complement. In addition, there was a correlation between serum binding to clustered AChR and complement deposition on myoid cells in patients’ thymus tissue. A similar approach was used to demonstrate that MuSK antibodies, although mainly IgG4, were partially IgG1 subclass and capable of activating complement when bound to MuSK on the cell surface. These observations throw new light on different forms of MG paving the way for improved diagnosis and management, and the approaches used have applicability to other antibody-mediated conditions.

We have studied responses in thymoma patients to interferon-alpha and to the acetylcholine receptor (AChR) in early-onset myasthenia gravis (EOMG), seeking clues to autoimmunizing mechanisms. Our new evidence implicates a two-step process: (step 1) professional antigen-presenting cells and thymic epithelial cells prime AChR-specific T cells; then (step 2) thymic myoid cells subsequently provoke germinal center formation in EOMG. Our unifying hypothesis proposes that AChR epitopes expressed by neoplastic or hyperplastic thymic epithelial cells aberrantly prime helper T cells, whether generated locally or infiltrating from the circulation. These helper T cells then induce antibody responses against linear epitopes that cross-react with whole AChR and attack myoid cells in the EOMG thymus. The resulting antigen-antibody complexes and the recruitment of professional antigen-presenting cells increase the exposure of thymic cells to the infiltrates and provoke local germinal center formation and determinant spreading. Both these and the consequently enhanced heterogeneity and pathogenicity of the autoantibodies should be minimized by early thymectomy.

Murine models for human autoimmune diseases are an essential tool for studying pathogenesis and for identifying new therapeutic targets. Mice are not the natural disease host, and conventional models have proved to be poor predictors of efficacy and safety in recent trials aiming to translate drug and biologic treatments to humans. Evidently, further steps towards recapitulating human diseases are urgently needed, for example using transgenic predisposing human HLA allele(s) plus T-cell receptor(s) implicated in a representative patient's autoimmune disease. The latest development - humanizing most of the immune system by transplanting human hematopoietic stem cells into severely immunodeficient mice - should lead to even better modeling.

Rheumatoid arthritis is a chronic inflammatory disease associated with certain HLA-DR4 subtypes. The target autoantigen(s) is unknown, but type II collagen (CII) is a candidate, with a single immunodominant DR4-restricted 261–273 T cell epitope (CII(261–273)). In the present study, we have prepared HLA-DR4:CII(261–273) tetramers and analyzed peripheral blood, lymph node, and synovial fluid cells from DR4-transgenic mice with early and late collagen-induced arthritis to draw a fuller picture of the role of CII-reactive Th cells in disease development. Their frequencies increased ∼20-fold in blood 1–2 wk postimmunization, and even more in acutely arthritic joints. Our data strongly suggest that CII-specific Th cells are necessary, but not sufficient for collagen-induced arthritis. The CII-specific Th cells displayed an activated proinflammatory Th1 phenotype, and their expansion correlated with onset and severity of arthritis and also with anti-CII Ab levels. Surprisingly, shortly after the first clinical signs of arthritis, activated HLA-DR4:CII tetramer+ cells became undetectable in the synovial fluid and rare in the blood, but persisted in lymph nodes. Consequently, future human studies should focus on patients with early arthritis, and on their synovial cells, to re-evaluate the occurrence and pathogenic importance of CII-specific or other Th cells in rheumatoid arthritis.

Autoimmune polyendocrine syndrome type 1 (APS1) is a childhood-onset monogenic disorder caused by mutations in the autoimmune regulator (AIRE) gene, including the distinctive R139X in Sardinia. Its rarity and great variability in manifestations/onset ages make early diagnosis difficult. To date, very few longitudinal studies of APS1 patients have been reported.The aim of this study was to describe the features and clinical course of APS1 and correlate them with AIRE and HLA class II genotypes in a large homogeneous cohort of Sardinian patients followed for up to 25 yr.Twenty-two pediatric APS1 patients were studied prospectively.This Sardinian series (female/male ratio, 1.44; median current age, 30.7 yr; range, 1.8-46 yr) showed early disease onset (age range, 0.3-10 yr; median, 3.5 yr) and severe phenotype (on average, seven manifestations per patient). Besides the classic triad of chronic mucocutaneous candidiasis, hypoparathyroidism, and Addison's disease, autoimmune hepatitis was a serious and surprisingly common/early/presenting feature (27%; two deaths), with a 5:1 female bias (median age, 6 yr; range, 2.5-11 yr). By contrast, type 1 diabetes was rare (one patient), and hypothyroidism was not seen. Additional disease components (several of them potentially life-threatening) appeared in adulthood. The major nonsense mutation, R139X, was found in 93% of the mutant AIRE alleles. High-titer interferon (IFN)-ω and IFN-α autoantibodies were detected in all patients tested, even preclinically at 4 months of age in one sibling. HLA alleles appear to influence the exact phenotype-the most interesting apparent association being between HLA-DRB1*0301-DQB1*0201, liver-kidney microsome autoantibodies (anti-CYP1A2), and autoimmune hepatitis.APS1 in Sardinia is characterized by severe phenotype, marked clinical heterogeneity, and relative genetic homogeneity. The single AIRE mutation, R139X, and the anti-IFN-ω and IFN-α autoantibodies are helpful for earlier diagnosis, especially when APS1 presents unusually. HLA genotypes can modify the phenotype.

Much has been learnt about the mechanisms of thymic self-tolerance induction from work on both the rare autosomal recessive disease autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) and the autoimmune regulator (AIRE) protein mutated in this disease. Normally, AIRE drives low-level expression of huge numbers of peripheral tissue-specific antigens (TSAgs) in medullary thymic epithelial cells (mTECs), leading to the deletion of TSAg-reactive thymocytes maturing nearby. The very recently discovered neutralizing autoantibodies (autoAbs) against Th17-related cells and cytokines in two autoimmunity-related syndromes associated with AIRE-mutant thymi or AIRE-deficient thymomas help to explain the chronic mucocutaneous candidiasis (CMC) seen in both syndromes. The surprising parallels between these syndromes also demand new hypotheses and research into the consequences of AIRE deficiency and the ensuing autoimmunizing pathways, and suggest more appropriate treatment regimens as discussed in this review.

CD248 (endosialin) is a transmembrane glycoprotein that is dynamically expressed on pericytes and fibroblasts during tissue development, tumour neovascularization and inflammation. Its role in tissue remodelling is associated with increased stromal cell proliferation and migration. We show that CD248 is also uniquely expressed by human, but not mouse (C57BL/6), CD8(+) naive T cells. CD248 is found only on CD8(+) CCR7(+) CD11a(low) naive T cells and on CD8 single-positive T cells in the thymus. Transfection of the CD248 negative T-cell line MOLT-4 with CD248 cDNA surprisingly reduced cell proliferation. Knock-down of CD248 on naive CD8 T cells increased cell proliferation. These data demonstrate opposing functions for CD248 on haematopoietic (CD8(+)) versus stromal cells and suggests that CD248 helps to maintain naive CD8(+) human T cells in a quiescent state.

Thymomas are tumors of thymic epithelial cells. They associate more often than any other human tumors with various autoimmune diseases; myasthenia gravis is the commonest, occurring in 10-50% of thymoma patients, depending on the World Health Organization-defined histologic subtype. Most thymomas generate many polyclonal maturing T lymphocytes but in disorganized microenvironments Failure to induce self-tolerance may be a key factor leading to the export of potentially autoreactive CD4 progeny, thus predisposing to autoimmune diseases. Normally, the master Autoimmune Regulator promotes expression of peripheral tissue-restricted antigens such as insulin by medullary thymic epithelial cells and induction of tolerance to them. The failure of approximately 95% of thymomas to express autoimmune regulator is another feature potentially contributing to autoimmunity.

Paraneoplastic autoimmune diseases associate occasionally with small cell lung cancers and gynecologic tumors. However, myasthenia gravis (MG) occurs in at least 30% of all patients with thymomas (usually present at MG diagnosis). These epithelial neoplasms almost always have numerous admixed maturing polyclonal T cells (thymocytes). This thymopoiesis-and export of mature CD4(+)T cells-particularly associates with MG, though there are rare/puzzling exceptions in apparently pure epithelial WHO type A thymomas. Other features potentially leading to inefficient self-tolerance induction include defective epithelial expression of the autoimmune regulator (AIRE) gene and/or of major histocompatibility complex class II molecules in thymomas, absence of myoid cells, failure to generate FOXP3(+) regulatory T cells, and genetic polymorphisms affecting T-cell signaling. However, the strong focus on MG/neuromuscular targets remains unexplained and suggests some biased autoantigen expression, T-cell selection, or autoimmunization within thymomas. There must be further clues in the intriguing serological and cellular parallels in some patients with late-onset MG but without thymomas-and in others with AIRE mutations-and in the contrasts with early-onset MG, as discussed here.

Chronic mucocutaneous candidiasis (CMC) is frequently associated with T cell immunodeficiencies. Specifically, the proinflammatory IL-17A–producing Th17 subset is implicated in protection against fungi at epithelial surfaces. In autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED, or autoimmune polyendocrine syndrome 1), CMC is often the first sign, but the underlying immunodeficiency is a long-standing puzzle. In contrast, the subsequent endocrine features are clearly autoimmune, resulting from defects in thymic self-tolerance induction caused by mutations in the autoimmune regulator (AIRE). We report severely reduced IL-17F and IL-22 responses to both Candida albicans antigens and polyclonal stimulation in APECED patients with CMC. Surprisingly, these reductions are strongly associated with neutralizing autoantibodies to IL-17F and IL-22, whereas responses were normal and autoantibodies infrequent in APECED patients without CMC. Our multicenter survey revealed neutralizing autoantibodies against IL-17A (41%), IL-17F (75%), and/ or IL-22 (91%) in >150 APECED patients, especially those with CMC. We independently found autoantibodies against these Th17-produced cytokines in rare thymoma patients with CMC. The autoantibodies preceded the CMC in all informative cases. We conclude that IL-22 and IL-17F are key natural defenders against CMC and that the immunodeficiency underlying CMC in both patient groups has an autoimmune basis.

Little is known about pathogenesis -- and especially about involvement of CD8(+) T-cells -- in late-onset myasthenia gravis (LOMG). Remarkably, outstanding CD8(+) TCRVbeta-subset expansions were found in 64% and 72% of recent onset LOMG or thymoma-associated MG (vs. 16% with early-onset MG (p

Maternal alloantibodies against the human platelet Ag (HPA)-1a allotype of the platelet β3 integrin GpIIb/IIIa can cause severe fetal or neonatal hemorrhage. Almost all anti-HPA-1a-immune mothers are homozygous for HPA-1b and carry HLA-DR52a (DRB3*0101). The single Pro33 →Leu substitution (HPA-1b→HPA-1a) was previously predicted to create a binding motif for HLA-DR52a that can lead to alloimmunization. We have isolated six CD4+ T cell clones from three such mothers, which all respond to intact HPA-1a+, but not HPA-1b+, platelets. We used them to define the “core” and “anchor” residues of this natural T cell epitope. Molecular modeling based on a recently published crystal structure can explain the preferential presentation of the Leu33 (but not Pro33 variant) by HLA-DR52a rather than the linked HLA-DR3 or the allelic DR52b. The modeling also predicts efficient anchoring at position 33 by several alternative hydrophobic α-amino acids; indeed, a recently identified variant with Val33 is presented well to two clones, and is therefore potentially alloimmunogenic. Finally, these HPA-1a-specific T cell clones use a variety of T cell receptors, but all have a “Th1” (IFN-γ-producing) profile and are suitable for testing selective immunotherapies that might be applicable in vivo.

In early-onset myasthenia gravis (EOMG), the thymus is colonized by lymph node-like infiltrates including T cell areas and germinal centers. Our Group(1) showed (1978) spontaneous anti-acetylcholine receptor (AChR) autoantibody production by EOMG thymic cells. Especially after enzymic dispersal, these are enriched in plasma cells that are evidently autonomous, long-lived, terminally differentiated and radio-resistant. Radiolabeled AChR is highly sensitive both for localizing them in situ and detecting their ongoing antibody production in culture at limiting cell numbers. Thus EOMG thymi are a readily available source of specific autoimmune human plasma cells suitable for studying their biology and testing new therapies.

Summary In sporadic autoimmune disorders, dendritic cells are increasingly being incriminated as agents provocateurs. However, the mechanisms and any ‘danger signals’ that induce them to autoimmunize remain enigmatic. Here, we focus on unexpected clues from two prototypic/ highly informative autoimmune syndromes, acquired thymoma-associated myasthenia gravis and the monogenic autoimmune polyendocrine syndrome type-1 (APS1), caused by mutations in the AutoImmune Regulator (AIRE). Both involve the thymus, and in both we find early, persistent, highly prevalent and high-titre neutralizing autoantibodies against type-I interferons, regardless of the exact AIRE genotype or the characteristically variable clinical phenotype in APS1. Thus these key innate↔adaptive immune intermediaries are now implicated in APS1 and paraneoplastic myasthenia as well as in systemic lupus erythematosus and other sporadic autoimmune disorders. The currently accepted notion that autoimmunization proceeds automatically (by ‘default’) does not explain how, when or where autoimmune responses are initiated against which targets in APS1, or whether exogenous or internal danger signals are involved, or predict whether the primary auto-immunogenic targets are AIRE-dependent. As the parallels between these syndromes must hold novel clues to these puzzles, they demand explanations. To unify these and other findings, we propose that autoimmunization occurs centrally in aberrant thymic environments rendered ‘dangerous’ by AIRE-deficiency (possibly by excess undegraded nucleic acids/dead cell debris). The ensuing autoreactivity focuses early on the locally abundant type I interferons and then on other peripheral tissue autoantigens that are still expressed despite the absence of AIRE. These ideas raise numerous questions that others may already have the materials to address.

In early-onset myasthenia gravis, the thymus contains lymph node-type infiltrates with frequent acetylcholine receptor (AChR)-specific germinal centers. Our recent evidence/two-step hypothesis implicates hyperplastic medullary thymic epithelial cells (expressing isolated AChR subunits) in provoking infiltration and thymic myoid cells (with intact AChR) in germinal center formation. To test this, we screened for complement attack in a wide range of typical generalized myasthenia patients. Regardless of the exact serology, thymi with sizeable infiltrates unexpectedly showed patchy up-regulation of both C5a receptor and terminal complement regulator CD59 on hyperplastic epithelial cells. These latter also showed deposits of activated C3b complement component, which appeared even heavier on infiltrating B cells, macrophages, and especially follicular dendritic cells. Myoid cells appeared particularly vulnerable to complement; few expressed the early complement regulators CD55, CD46, or CR1, and none were detectably CD59+. Indeed, when exposed to infiltrates, and especially to germinal centers, myoid cells frequently labeled for C1q, C3b (25 to 48%), or even the terminal C9, with some showing obvious damage. This early/persistent complement attack on both epithelial and myoid cells strongly supports our hypothesis, especially implicating exposed myoid cells in germinal center formation/autoantibody diversification. Remarkably, the similar changes place many apparent AChR-seronegative patients in the same spectrum as the AChR-seropositive patients.

Myasthenia gravis is an autoimmune disease affecting the neuromuscular junction, which produces fatiguable weakness. Treatment of this condition includes cholinesterase inhibitors, immunosuppressive therapy and/or thymectomy. Optimal perioperative management of thymectomy is crucial in achieving favourable outcomes postoperatively and in the control of myasthenia. This article reviews the basic principles of thymectomy and its perioperative care with respect to myasthenia.

Thymomas are thymic epithelial neoplasms, associated with a variety of autoimmune disorders (especially myasthenia gravis), that apparently result from aberrant intra-tumourous thymopoiesis and export of inefficiently tolerized T-cells to the periphery. The autoimmune regulator (AIRE) drives the expression of self-antigens in the thymic medulla and plays an essential role in ‘central’ tolerance in both humans and mice. However, while inactivating AIRE mutations result in the ‘autoimmune polyendocrinopathy syndrome type 1’ (APS-1), its major features are not well reproduced in AIRE-knock-out mice. Therefore, alternative human disease scenarios with concomitant AIRE deficiency may be valuable tools to test conclusions drawn from mouse models. Here we show, in a large series, that ∼95% of thymoma patients are ‘chimeric’; expression of AIRE and major AIRE-related autoantigens (eg insulin) were undetectable in their tumours but maintained in their remnant thymic tissue and lymph nodes. Notably, despite the AIRE-deficient thymopoiesis in thymomas, disorders and autoantibodies typical of APS-1 were distinctly uncommon in these patients. The one striking similarity was in the recently observed neutralizing anti-type I interferon (IFN) antibodies, which are found at diagnosis in 100% of patients with APS-1 and in ∼60% of patients with thymomas, as we show here. We conclude that APS-1 type autoantigens must be protected from autoimmunity by mechanisms that do not extend to the muscle autoantigens so frequently targeted in thymoma patients but so rarely recognized in APS-1. Thus our findings argue strongly for a tolerogenic function of AIRE beyond its role in negative T-cell selection in human thymopoiesis, and/or for specific autoimmunization against muscle in thymomas. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

In generalized myasthenia gravis (MG) patients without detectable acetylcholine receptor (AChR) antibodies (SNMG), the thymus is often reported as "normally involuted." We analyzed thymic compartments in 67 patients with generalized MG, with AChR antibodies (AChR+, n = 23), with muscle-specific kinase (MuSK) antibodies (MuSK+, n = 14) or with neither (MuSK-, n = 30), and in 11 non-MG controls. Four of 14 MuSK+ thymi had rare small germinal centers, but overall they were not different from age-matched controls. However, approximately 75% MuSK- samples showed lymph node-type infiltrates similar to those in AChR+ patients, but with fewer germinal centers. These variations may explain some apparent differences in responses to thymectomy in SNMG.

Acetylcholine receptor (AChR) antibodies are present in around 85% of patients with myasthenia gravis (MG) as measured by the conventional radioimmunoprecipitation assay. Antibodies that block the fetal form of the AChR are occasionally present in mothers who develop MG after pregnancy, especially in those whose babies are born with arthrogryposis multiplex congenita. The antibodies cross the placenta and block neuromuscular transmission, leading to joint deformities and often stillbirth. In these mothers, antibodies made in the thymus are mainly specific for fetal AChR and show restricted germline origins, suggesting a highly mutated clonal response; subsequent spread to involve adult AChR could explain development of maternal MG in those cases who first present after pregnancy. In the 15% of "seronegative" MG patients without AChR antibodies (SNMG), there are serum factors that increase AChR phosphorylation and reduce AChR function, probably acting via a different membrane receptor. These factors are not IgG and could be IgM or even non-Ig serum proteins. In a proportion of SNMG patients, however, IgG antibodies to the muscle-specific kinase, MuSK, are present. These antibodies are not found in AChR antibody-positive MG and are predominantly IgG4. MuSK antibody positivity appears to be associated with more severe bulbar disease that can be difficult to treat effectively.

In humans, interleukin-12 (IL-12) and interferon-alpha (IFN-alpha) normally favor IFN-gamma-producing "Th1" T cell responses. Myasthenia gravis (MG) patients with thymomas frequently have high-titer neutralizing autoantibodies against these cytokines, but not against IFN-gamma. Because they occasionally develop intractable (even fatal) infections, we have tested effects of their sera on the generation of IFN-gamma responses by healthy adult T cells to autologous lipopolysaccharide (LPS)-treated dendritic cells (DC). Anti-IL-12(+) sera consistently reduced IFN-gamma responses substantially, whether assessed by intracellular staining or ELISA. Therefore, thymoma patients with intractable infections might benefit from cautious IFN-gamma therapy. We discuss wider implications of the surprising rarity of clear clinical hazards-or benefits-of these autoantibodies.

We have screened for spontaneous anticytokine autoantibodies in patients with infections, neoplasms and autoimmune diseases, because of their increasingly reported co-occurrence. We tested for both binding and neutralizing autoantibodies to a range of human cytokines, including interleukin-1alpha (IL-1alpha), IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, interferon-alpha2 (IFN-alpha2), IFN-omega, IFN-beta, IFN-gamma, tumour necrosis factor alpha (TNF-alpha), transforming growth factor beta-1 (TGF-beta1) and granulocyte-macrophage colony stimulating factor (GM-CSF), in plasmas or sera. With two notable exceptions described below, we found only occasional, mostly low-titre, non-neutralizing antibodies, mainly to GM-CSF; also to IL-10 in pemphigoid. Strikingly, however, high-titre, mainly IgG, autoantibodies to IFN-alpha2, IFN-omega and IL-12 were common at diagnosis in patients with late-onset myasthenia gravis (LOMG+), thymoma (T) but no MG (TMG-) and especially with both thymoma and MG together (TMG+). The antibodies recognized other closely related type I IFN-alpha subtypes, but rarely the distantly related type I IFN-beta, and never (detectably) the unrelated type II IFN-gamma. Antibodies to IL-12 showed a similar distribution to those against IFN-alpha2, although prevalences were slightly lower; correlations between individual titres against each were so modest that they appear to be entirely different specificities. Neither showed any obvious correlations with clinical parameters including thymoma histology and HLA type, but they did increase sharply if the tumours recurred. These antibodies neutralized their respective cytokine in bioassays in vitro; although they persisted for years severe infections were surprisingly uncommon, despite the immunosuppressive therapy also used in most cases. These findings must hold valuable clues to autoimmunizing mechanisms in paraneoplastic autoimmunity.

The weakness in myasthenia gravis (MG) is mediated by autoantibodies against adult muscle acetylcholine receptors (AChR) at the neuromuscular junction; most of these antibodies also bind to fetal AChR, which is present in the thymus. In rare cases, babies of mothers with MG, or even of asymptomatic mothers, develop a severe developmental condition, arthrogryposis multiplex congenita, caused by antibodies that inhibit the ion channel function of the fetal AChR while not affecting the adult AChR. Here we show that these fetal AChR inhibitory antibodies are significantly more common in females sampled after pregnancy than in those who present before pregnancy, suggesting that they may be induced by the fetus. Moreover, we were able to clone high-affinity combinatorial Fab antibodies from thymic cells of two mothers with MG who had babies with arthrogryposis multiplex congenita. These Fabs were highly specific for fetal AChR and did not bind the main immunogenic region that is common to fetal and adult AChR. The Fabs show strong biases to VH3 heavy chains and to a single Vkappa1 light chain in one mother. Nevertheless, they each show extensive intraclonal diversification from a highly mutated consensus sequence, consistent with antigen-driven selection in successive steps. Collectively, our results suggest that, in some cases of MG, initial immunization against fetal AChR is followed by diversification and expansion of B cells in the thymus; maternal autoimmunity will result if the immune response spreads to the main immunogenic region and other epitopes common to fetal and adult AChR.

The autoimmune myasthenias are prototypes of an emerging group of autoantibody-mediated neurological diseases, mostly with very well defined target molecules, whether receptors or ion channels (or both). At the neuromuscular junction, these include presynaptic calcium channels in the Lambert–Eaton myasthenic syndrome (LEMS) and postsynaptic acetyl-choline receptors (AChR) in myasthenia gravis (MG). These disorders are particularly informative because of the distinct aetiological factors in the several patient subgroups, including characteristic associations with small cell lung cancers in the LEMS or with thymomas in MG; also – but mainly in other subgroups – with certain immuno-genetic variants, both in the HLA region and at other immuno-regulatory loci. Here, I briefly summarise current knowledge in these areas and discuss its implications. Keywords: myasthenia gravis; Lambert–Eaton myasthenic syndrome; HLA and disease; immunogenetics; humoral autoimmunity; autoantibody; thymoma; thymic hyperplasia; germinal centre

Bortezomib is a potent inhibitor of proteasomes currently used to eliminate malignant plasma cells in multiple myeloma patients. It is also effective in depleting both alloreactive plasma cells in acute Ab-mediated transplant rejection and their autoreactive counterparts in animal models of lupus and myasthenia gravis (MG). In this study, we demonstrate that bortezomib at 10 nM or higher concentrations killed long-lived plasma cells in cultured thymus cells from nine early-onset MG patients and consistently halted their spontaneous production not only of autoantibodies against the acetylcholine receptor but also of total IgG. Surprisingly, lenalidomide and dexamethasone had little effect on plasma cells. After bortezomib treatment, they showed ultrastructural changes characteristic of endoplasmic reticulum stress after 8 h and were no longer detectable at 24 h. Bortezomib therefore appears promising for treating MG and possibly other Ab-mediated autoimmune or allergic disorders, especially when given in short courses at modest doses before the standard immunosuppressive drugs have taken effect.

Patients with the autoimmune polyendocrine syndrome type I (APS-I), caused by mutations in the autoimmune regulator (AIRE) gene, and myasthenia gravis (MG) with thymoma, show intriguing but unexplained parallels. They include uncommon manifestations like autoimmune adrenal insufficiency (AI), hypoparathyroidism, and chronic mucocutaneous candidiasis plus autoantibodies neutralizing IL-17, IL-22, and type I IFNs. Thymopoiesis in the absence of AIRE is implicated in both syndromes. To test whether these parallels extend further, we screened 247 patients with MG, thymoma, or both for clinical features and organ-specific autoantibodies characteristic of APS-I patients, and we assayed 26 thymoma samples for transcripts for AIRE and 16 peripheral tissue-specific autoantigens (TSAgs) by quantitative PCR. We found APS-I–typical autoantibodies and clinical manifestations, including chronic mucocutaneous candidiasis, AI, and asplenia, respectively, in 49 of 121 (40%) and 10 of 121 (8%) thymoma patients, but clinical features seldom occurred together with the corresponding autoantibodies. Both were rare in other MG subgroups (n = 126). In 38 patients with APS-I, by contrast, we observed neither autoantibodies against muscle Ags nor any neuromuscular disorders. Whereas relative transcript levels for AIRE and 7 of 16 TSAgs showed the expected underexpression in thymomas, levels were increased for four of the five TSAgs most frequently targeted by these patients’ autoantibodies. Therefore, the clinical and serologic parallels to APS-I in patients with thymomas are not explained purely by deficient TSAg transcription in these aberrant AIRE-deficient tumors. We therefore propose additional explanations for the unusual autoimmune biases they provoke. Thymoma patients should be monitored for potentially life-threatening APS-I manifestations such as AI and hypoparathyroidism.

Background: Patients with MG often have other autoantibodies in addition to those against the acetylcholine receptor (AChR). It has been suggested that antibodies to the muscle protein titin may be diagnostic of a thymoma, but they have also been found in patients with late-onset MG. Antibodies to certain cytokines have also been detected in patients with MG and thymoma, and it is not clear whether these antibodies could be more useful clinically. The authors measured antibodies against titin and the cytokines interferon α (IFNα) and interleukin 12 (IL12) in patients with MG and thymoma or thymoma recurrence, and in patients with MG but without thymoma presenting before (early-onset MG) or after (late-onset MG) 40 years of age. Method: Levels of titin, IFNα, and IL12 antibodies were determined by radioimmunoassay in 191 patients with MG and 82 controls. Results: As previously reported, titin antibodies were uncommon in patients with early-onset MG. However, in patients with late-onset MG, titin antibodies had similar prevalence and levels to those in patients with MG and thymoma, although the antibodies were uncommon in patients between 40 and 60 years of age presenting without a tumor. By contrast, cytokine antibodies were more common in patients with thymoma than in patients without thymoma, and cytokine antibodies typically increased substantially if the thymoma recurred. Conclusions: Measurement of titin antibodies has limited use in predicting the presence of a tumor, unless the patient is less than 60 years of age, but measurement of IFNα and IL12 antibodies may be helpful in identifying patients with a thymoma recurrence, particularly when mediastinal imaging is equivocal.

The muscle weakness in myasthenia gravis (MG) is mediated by autoantibodies against the nicotinic acetylcholine receptor (AChR) at the neuromuscular junction. Production of these pathogenic autoantibodies is believed to be associated with germinal centers (GC) and anti-AChR-secreting plasma cells in the hyperplastic thymus of patients with early onset MG (EOMG). Here, we describe the repertoire of rearranged heavy chain V genes and their clonal origins in GC from a typical EOMG patient. Three hundred fifteen rearranged Ig VH genes were amplified, cloned, and sequenced from sections of four thymic GC containing AChR-specific B cells. We found that thymic GC contain a remarkably heterogeneous population of B cells. Both naive and circulating memory B cells undergo Ag-driven clonal proliferation, somatic hypermutation, and selection. Numerous B cell clones were present, with no individual clone dominating the response. Comparisons of B cell clonal sequences from different GC and known anti-AChR Abs from other patients showed convergent mutations in the complementarity determining regions. These results are consistent with AChR driving an ongoing GC response in the thymus of EOMG patients. This is the first detailed analysis of B cell clones in human GC responding to a defined protein Ag, and the response we observed may reflect the effects of chronic stimulation by autoantigen.

Dendritic cells (DC) are crucial for the induction of immune responses and thus an inviting target for modulation by pathogens. We have previously shown thatPlasmodium falciparum-infected erythrocytes inhibit the maturation of DCs. IntactP. falciparum-infected erythrocytes can bind directly to CD36 and indirectly to CD51. It is striking that these receptors, at least in part, also mediate the phagocytosis of apoptotic cells. Here we show that antibodies against CD36 or CD51, as well as exposure to early apoptotic cells, profoundly modulate DC maturation and function in response to inflammatory signals. Although modulated DCs still secrete tumor necrosis factor-α, they fail to activate T cells and now secrete IL-10. We therefore propose that intactP. falciparum-infected erythrocytes and apoptotic cells engage similar pathways regulating DC function. These findings may have important consequences for the treatment of malaria and may suggest strategies for modulating pathological immune responses in autoimmune diseases.

We have analyzed a series of HLA region markers in 207 UK Caucasoids with early-onset myasthenia gravis (EOMG, onset before age 40), where there is a strong female bias. The well known associations with HLA-DR3 and -B8 have now proved to be significantly stronger in the 165 females than in the 42 males. In patients (of either sex) lacking -DR3, there was also a significant increase in HLA-DR2. Although the muscle weakness in EOMG is clearly mediated by autoantibodies, the associations are consistently stronger with HLA-B8 (in class I) than with HLA-DR3 (in class II), as confirmed here. We therefore typed 87–137 cases for polymorphisms at four loci in the intervening class III region, and also at three in the adjacent stretch of class I. At each locus, one allele tended to co-occur with HLA-B8 and showed strong and highly significant associations in the patients. There appeared to be a region of maximal susceptibility extending from HSP70 (in class III) past HLA-B and HLA-C at least 600kb telomerically into the class I region, which is now being mapped in detail. Any candidate genes here that act shortly after puberty may allow more precise localization of susceptibility.

The malaria parasite Plasmodium falciparum is one of the most successful human pathogens. Specific virulence factors remain poorly defined, although the adhesion of infected erythrocytes to the venular endothelium has been associated with some of the syndromes of severe disease. Immune responses cannot prevent the development of symptomatic infections throughout life, and clinical immunity to the disease develops only slowly during childhood. An understanding of the obstacles to the development of protective immunity is crucial for developing rational approaches to prevent the disease. Here we show that intact malaria-infected erythrocytes adhere to dendritic cells, inhibit the maturation of dendritic cells and subsequently reduce their capacity to stimulate T cells. These data demonstrate both a novel mechanism by which malaria parasites induce immune dysregulation and a functional role beyond endothelial adhesion for the adhesive phenotypes expressed at the surface of infected erythrocytes.

No immunodominant T-cell epitopes have yet been reported in the human acetylcholine receptor (AChR), the target of the pathogenic autoantibodies in myasthenia gravis (MG). We have selected and characterized T cells from MG patients by restimulation in culture with recombinant human AChR to alpha, gamma and epsilon subunits; the gamma and epsilon distinguish the fetal and adult AChR isoforms, respectively. We obtained clones specific for the epsilon, rather than the alpha or gamma, subunit in 3 of the first 4 early-onset MG cases tested. They all responded to peptide epsilon201-219 and to low concentrations of adult but not fetal AChR. Moreover, although using different T-cell receptor genes, they were all restricted to HLA-DR52a (DRB3*0101), a member of the strongly predisposing HLA-A1-B8-DR3 haplotype. This apparently immunodominant epsilon201-219 epitope (plus DR52a) was also recognized by clones from an elderly patient whose MG had recently been provoked by the drug D-penicillamine. In all 4 cases, however, the serum antibodies reacted better with fetal than adult AChR and may thus be end products of determinant spreading initiated by adult AChR-specific T cell responses. Furthermore, as these T cells had a pathogenic Th1 phenotype, with the potential to induce complement-activating antibodies, they should be important targets for selective immunotherapy.

In myasthenia gravis (MG), antibodies to the muscle acetylcholine receptor (AChR) cause muscle weakness. Experimental autoimmune myasthenia gravis (EAMG) can be induced by immunisation against purified AChR; the main immunogenic region (MIR) is a conformation-dependent site that includes alpha 67-76. EAMG can also occur after immunisation against extracellular AChR sequences, but this probably involves intramolecular determinant spreading. In MG patients, thymic hyperplasia and germinal centres are found in about 50%, and thymoma in 10-15%. The heterogeneous, high affinity, IgG anti-AChR antibodies appear to be end-products of germinal centre responses, and react mainly with the MIR or a site on fetal AChR; the latter contains a gamma subunit and is mainly expressed on myoid cells in the thymic medulla. T cells cloned against recombinant AChR subunits recognise principally two naturally processed epitopes: epsilon 201-219 derived from adult AChR which is expressed in muscle, and sometimes in thymic epithelium, and alpha 146-160, common to fetal and adult AChR. Since AChR is not normally co-expressed with class II, it is unclear how CD4+ responses to AChR alpha and epsilon subunits are initiated, and how and where these spread to induce antibodies against fetal AChR. Various possibilities, including upregulation of class II on muscle/myoid cells and involvement of CD8+ responses to AChR and other muscle antigens, are discussed.

Using an acetylcholine receptor-specific T-cell clone (TCC) from a myasthenia gravis patient, we have compared the induction of unresponsiveness by three agents: an anti-Vβ monoclonal antibody (mAb), complexes of MHC class II with specific peptide (DR4:peptide) and free peptide. Pretreatment with each agent without antigen-presenting cells specifically rendered the TCC unresponsive to a subsequent optimal stimulus. A substantial proportion of the DR4:peptide pretreated cells underwent apoptosis and the remainder were profoundly unresponsive. Apoptosis was less prominent after pretreatment with free peptide and was not significant with anti-Vβ mAb; with both, the unresponsiveness was transient in the survivors. These diverse effects of engaging the T-cell receptor in the absence of costimulation suggest that these agents act via different mechanisms, and give insights into the potential for specific immunotherapy.

Late-onset myasthenia gravis (LOMG) has become the largest MG subgroup, but the underlying pathogenetic mechanisms remain mysterious. Among the few etiological clues are the almost unique serologic parallels between LOMG and thymoma-associated MG (TAMG), notably autoantibodies against acetylcholine receptors, titin, ryanodine receptor, type I interferons or IL-12. This is why we checked LOMG patients for two further peculiar features of TAMG – its associations with the CTLA4 high/gain-of-function +49A/A genotype and with increased thymic export of naive T cells into the blood, possibly after defective negative selection in AIRE-deficient thymomas. We analyzed genomic DNA from 116 Caucasian LOMG patients for CTLA4 alleles by PCR/restriction fragment length polymorphism, and blood mononuclear cells for recent thymic emigrants by quantitative PCR for T cell receptor excision circles. In sharp contrast with TAMG, we now find that: i) CTLA4 low +49G(+) genotypes were more frequent ( p = 0.0029) among the 69 LOMG patients with age at onset ≥60 years compared with 172 healthy controls; ii) thymic export of naive T cells from the non-neoplastic thymuses of 36 LOMG patients was lower ( p = 0.0058) at diagnosis than in 77 age-matched controls. These new findings are important because they suggest distinct initiating mechanisms in TAMG and LOMG and hint at aberrant immuno-regulation in the periphery in LOMG. We therefore propose alternate defects in central thymic or peripheral tolerance induction in TAMG and LOMG converging on similar final outcomes. In addition, our data support a 60-year-threshold for onset of ‘true LOMG’ and an LOMG/early-onset MG overlapping group of patients between 40 and 60.

Almost all patients with autoimmune polyendocrine syndrome (APS)-I have high titer neutralizing autoantibodies to type I interferons (IFN), especially IFN-ω and IFN-α2, whatever their clinical features and onset-ages. About 90 % also have antibodies to interleukin (IL)-17A, IL-17F and/or IL-22; they correlate with the chronic mucocutaneous candidiasis (CMC) that affects ~90 % of patients. Our aim was to explore how early the manifestations and endocrine and cytokine autoantibodies appear in young APS-I patients. That may hold clues to very early events in the autoimmunization process in these patients.Clinical investigations and autoantibody measurements in 13 APS-I patients sampled before age 7 years, and 3 pre-symptomatic siblings with AIRE-mutations in both alleles.Antibody titers were already high against IFN-α2 and IFN-ω at age 6 months in one sibling-8 months before onset of APS-I-and also against IL-22 at 7 months in another (still unaffected at age 5 years). In 12 of the 13 APS-I patients, antibody levels were high against IFN-ω and/or IL-22 when first tested, but only modestly positive against IFN-ω in one patient who had only hypo-parathyroidism. Endocrine organ-specific antibodies were present at age 6 months in one sibling, and as early as 36 and 48 months in two of the six informative subjects.This is the first study to collate the onset of clinical features, cytokine and endocrine autoantibodies in APS-I infants and siblings. The highly restricted early autoantibody responses and clinical features they show are not easily explained by mere loss of broad-specific self-tolerance inducing mechanisms, but hint at some more sharply focused early event(s) in autoimmunization.

In autoimmune disorders, inactivation of pathogenic antigen-specific T cells, rather than global immunosuppression, would be highly desirable. One way to achieve this would be to deliver the first antigen-specific signal to the T cell in the absence of the second costimulatory signal. Myasthenia gravis (MG) is a well-characterized autoimmune disease in which T cell-dependent autoantibodies are directed against the acetylcholine receptor (A ChR) at the neuromuscular junction. AChR-specific T cells have been cloned from MG patients, and in this study, we have induced long-lasting tolerance in vitro in one particular clone (PM-A1) with a known peptide epitope (alpha 144-163) and MHC class II restriction (DR4 Dw14.2 or 4.2) by using soluble MHC-class II peptide complexes. Preincubation of PM-A1 T cells with such complexes induced death by apoptosis in < or = 40-50% of the AChR-specific cells. Surviving cells remained refractory to stimulation with AChR-derived synthetic peptides or recombinant polypeptides for < or = 38 d after complex treatment. These effects were highly specific, dose-dependent and required > 2 h preincubation. The T cells could be protected from the tolerizing effects of complex by coincubation with DR-matched or -mismatched antigen-presenting cells. This work shows that antigen-specific T cells can be selectively killed or anergized using soluble MHC class II: peptide complexes. Such an antigen-specific therapy offers a rational approach to the immunotherapy of autoimmune or allergic disease in vivo.

Lymph node-type T- and B-cell infiltrates with germinal centers are characteristic features of the hyperplastic thymus in early onset myasthenia gravis (EOMG).Epstein-Barr virus (EBV) infection confers survival advantages on B cells, and has recently been implicated in tertiary lymphoid tissue formation in EOMG. We evaluated the frequency of intrathymic EBV-infected B-lineage cells and antiviral immune responses in treatment-naive patients with EOMG. Real-time polymerase chain reaction was performed to quantify the content of genomic EBV DNA (BamHI-W repeat region) in thymic cell suspensions. Serial paraffin sections of EOMG thymi were analyzed for the presence of EBV-encoded RNA by in situ hybridization and for viral gene expression by immunohistochemistry. Humoral and cellular immune responses to viral antigens were quantified by enzyme-linked immunosorbent assay and flow cytometry-based intracellular cytokine staining. We detected minimal levels of viral DNA-corresponding to single viral genomes-in only 6 of 16 hyperplastic EOMG thymi, indicating extreme rarity of viral copy numbers in the investigated thymic samples. That was confirmed by similar rarity of EBV-encoded RNA and viral proteins identified in thymic sections. Furthermore, EBV-specific T- and B-cell responses were unchanged in patients with EOMG. These findings do not support an etiologic role for EBV in the initiation of EOMG.

Patients with autoimmune polyendocrine syndrome type I (APS I) or acquired thymoma-associated myasthenia gravis (MG) surprisingly share several common features, including defective expression of the transcription factor AIRE and autoantibodies against type I interferons. Here, we have adapted and validated the radioligand-binding assay we recently developed against (35)S-Met-interferon-ω, for rapid and specific screening for autoantibodies against interferons-α2 and -α8. We then investigated their potential for diagnosis and for predicting clinical manifestations in patients with APS I and different subgroups of MG. Autoantibodies against interferons-ω, -α2, and -α8 occurred more often in patients with APS I (100%) and MG with thymoma (73%) than in late-onset MG (39%) and early-onset MG (5%). These autoantibodies showed preferences for interferon-ω in APS I and for the interferon-αs in MG, hinting at thymic aberrations in both groups. The exact profile of type I interferon antibodies may indicate MG subtype and may hint at thymoma recurrence.

Purpose Approximately one half of patients with Lambert-Eaton myasthenic syndrome (LEMS) have small-cell lung carcinomas (SCLC), aggressive tumors with poor prognosis. In view of its profound impact on therapy and survival, we developed and validated a score to identify the presence of SCLC early in the course of LEMS. Patients and Methods We derived a prediction score for SCLC in LEMS in a nationwide cohort of 107 Dutch patients, and validated it in a similar cohort of 112 British patients. A Dutch-English LEMS Tumor Association Prediction (DELTA-P) score was developed based on multivariate logistic regression. Results Age at onset, smoking behavior, weight loss, Karnofsky performance status, bulbar involvement, male sexual impotence, and the presence of Sry-like high-mobility group box protein 1 serum antibodies were independent predictors for SCLC in LEMS. A DELTA-P score was derived allocating 1 point for the presence of each of the following items at or within 3 months from onset: age at onset ≥ 50 years, smoking at diagnosis, weight loss ≥ 5%, bulbar involvement, erectile dysfunction, and Karnofsky performance status lower than 70. The area under the curve of the receiver operating curve was 94.4% in the derivation cohort and 94.6% in the validation set. A DELTA-P score of 0 or 1 corresponded to a 0% to 2.6% chance of SCLC, whereas scores of 4, 5, and 6 corresponded to chances of SCLC of 93.5%, 96.6%, and 100%, respectively. Conclusion The simple clinical DELTA-P score discriminated patients with LEMS with and without SCLC with high accuracy early in the course of LEMS.

The nicotinic acetylcholine receptor (AChR) is both the best-characterized transmitter receptor-ion channel and the target for the pathogenic antibodies in the human autoimmune disease myasthenia gravis (MG). In cloning and sequencing its components in man, we found that the $\alpha $-subunit was transcribed in two isoforms, with (P3A$^{+}$) or without (P3A$^{-}$) a 75 base pair exon that had not been described in other species. While studying the human T lymphocyte response to recombinant AChR, we found that part of this P3A insert was recognized by one T cell line (from an MG patient), whereas another line only recognized the uninterrupted insertion site. To establish whether this exon is also translated in normal human muscle, we initially raised anti-peptide antibodies to the relevant amino acid sequences, but these failed to bind native AChR (affinity-purified from muscle on $\alpha $-neurotoxin columns). We therefore exploited the great sensitivity and specificity of these T cells to detect the two isoforms after unfolding by antigen-presenting cells, and have been able to show that both are expressed in affinity-purified human muscle AChR.