David I. Stott

Antigen-driven clonal proliferation of B cells within the target tissue of an autoimmune disease, the salivary glands of patients with Sjögren's syndrome

Structures resembling germinal centers are seen in the salivary glands of patients with Sjögrens syndrome, but it is not known whether the microenvironment of these cell clusters is sufficient for the induction of a germinal center response. Therefore, we cloned and sequenced rearranged Ig V genes expressed by B cells isolated from sections of labial salivary gland biopsies from two Sjögrens syndrome patients. Rearranged V genes from B cells within one cell cluster were polyclonal and most had few somatic mutations. Two adjacent clusters from another patient each contained one dominant B cell clone expressing hypermutated V genes. None of the rearranged V genes was found in both clusters, suggesting that cells are unable to migrate out into the surrounding tissue and seed new clusters. The ratios of replacement to silent mutations in the framework and complementarity determining regions suggest antigen selection of high-affinity mutants. These results show that an antigen-driven, germinal center-type B cell response is taking place within the salivary glands of Sjögrens syndrome patients. In view of the recent demonstration of a germinal center response within the rheumatoid synovial membrane and the existence of similar structures in the target tissues of other autoimmune diseases, we propose that germinal center- type responses can be induced in the nonlymphoid target tissues of a variety of autoimmune diseases.

Immunoblotting and dot blotting.

A variety of methods has been available for many years for the analytical separation of mixtures of proteins into their component parts by electrophoresis in a gel, usually agarose or polyacrylamide. Popular techniques are: zonal electrophoresis in agarose gel or on cellulose acetate membranes; discontinuous electrophoresis in polyacrylamide gel (PAGE); SDS-polyacrylamide gel electrophoresis (SDS-PAGE); isoelectric focusing (IEF); and two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), which is capable of resolving complex mixtures of proteins containing hundreds or even thousands of components. All of these methods require some means of identifying particular proteins of interest. In some cases it is possible to do this simply on the basis of mobility, molecular weight (MW) (SDS-PAGE) or by using selective stains, e.g., for enzyme activity. These methods of identification are severely limited in applicability and, for this reason, antibodies have been used as highly specific probes for electrophoretically separated proteins, ever

Somatic hypermutation and selection of B cells in thymic germinal centers responding to acetylcholine receptor in myasthenia gravis

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.

Systemic autoimmune disease induced by dendritic cells that have captured necrotic but not apoptotic cells in susceptible mouse strains

Systemic lupus erythematosus (SLE) is an autoimmune disorder of a largely unknown etiology. Anti‐double‐stranded (ds) DNA antibodies are a classic hallmark of the disease, although the mechanism underlying their induction remains unclear. We demonstrate here that, in both lupus‐prone and normal mouse strains, strong anti‐dsDNA antibody responses can be induced by dendritic cells (DC) that have ingested syngeneic necrotic (DC/nec), but not apoptotic (DC/apo), cells. Clinical manifestations of lupus were evident, however, only in susceptible mouse strains, which correlate with the ability of DC/nec to release IFN‐γ and to induce the pathogenic IgG2a anti‐dsDNA antibodies. Injection of DC/nec not only accelerated disease progression in the MRL/MpJ‐lpr/lpr lupus‐prone mice but also induced a lupus‐like disease in the MRL/MpJ‐+/+ wild‐type control strain. Immune complex deposition was readily detectable in the kidneys, and the mice developed proteinuria. Strikingly, female MRL/MpJ‐+/+ mice that had received DC/nec, but not DC/apo, developed a ‘butterfly’ facial lesion resembling a cardinal feature of human SLE. Our study therefore demonstrates that DC/nec inducing a Th1 type of responses, which are otherwise tightly regulated in a normal immune system, may play a pivotal role in SLE pathogenesis.

Scenarios for Autoimmunization of T and B Cells in Myasthenia Gravis

Abstract: We have studied responses in thymoma patients to interferon‐α 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.

Antibodies to acetylcholine receptor in parous women with myasthenia: Evidence for immunization by fetal antigen

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 Vκ1 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.

Modulation of autoimmune disease in the MRL-lpr/lpr mouse by IL-2 and TGF-β1 gene therapy using attenuated Salmonella typhimurium as gene carrier

We have investigated the effects of interleukin-2 (IL-2) and transforming growth factor-b (TGF-b) gene therapy on the progress of autoimmune disease in MRL-lpr/lpr mice, a murine model of systemic lupus erythematosus (SLE). These mice have uncontrolled proliferation of T cells, an impaired response to T cell mitogen and produce autoantibodies against nuclear antigens, including DNA. Immune complexes formed by these autoantibodies are believed to cause glomerulonephritis and vasculitis in lupus mice and human SLE. Since there is an imbalance of cytokine production in both SLE patients and lupus mice, we examined the effects of cytokine gene therapy on the progression of autoimmune disease in MRL-lpr/lpr mice. The mice were treated orally with a non-pathogenic strain of Salmonella typhimurium bearing the aroA-aroD- mutations and carrying the murine genes encoding IL-2 and TGF-b. The bacteria synthesise and slowly release the cytokines in vivo. Our results show that, contrary to expectation, TGF-b gene therapy produced no improvement in pathology and generally had opposite effects to those of IL-2. IL-2 gene therapy restored the defective T cell proliferative response to mitogen and suppressed the autoantibody response, glomerulonephritis and growth of lymphoid tumours.

Biosynthesis and assembly of IgM. Addition of J chain to intracellular pools of 8S and 19S IgM.

The addition of J chain to intracellular and secreted IgM has been studied in mitogen-stimulated mouse spleen cells and two mouse myelomas (MOPC 104E and Y5781). The cells of Y5781 contain a large pool of intracellular 19S IgM and it is shown that this IgM is assembled by two pathways, one via the addition of J chain to the 8S IgM subunit and the second by direct addition of J chain to 19S IgM. The absence of J chain on the intracellular IgMs of MOPC 104E and mitogen-stimulated spleen cells suggests that IgM may be assembled in these cells predominantly by the second pathway. The detection in Y5781 cells of a population of 19S IgM molecules without J chain, to which J chain is added shortly before or during secretion, demonstrates that J chain is not necessary for the polymerisation of IgM in the intact cell.

The incidence of monoclonal gammopathy in a population over 45 years old determined by isoelectric focusing

Summary. Immuno‐isoelectric focusing (IIEF), a technique previously shown to be sensitive for the detection of paraproteinaemia, was used to test 200 individuals over the age of 45, without history of B cell neoplasm, for the presence of serum paraproteinaemia. 11% of these individuals had evidence of paraproteinaemia detectable by IIEF compared with only 2% by zonal and immunoelectrophoresis. A further 12% had oligoclonal immunoglobulins and the remainder had no qualitative abnormality of the immunoglobulin profile. These results are discussed with particular reference to the aetiology, diagnosis and monitoring of potential B cell neoplasm in high risk individuals or groups.

VH replacement in rearranged immunoglobulin genes

Examples suggesting that all or part of the VH segment of a rearranged VHDJH may be replaced by all or part of another VH have been appearing since the 1980s. Evidence has been presented of two rather different types of replacement. One of these has gained acceptance and has now been clearly demonstrated to occur. The other, proposed more recently, has not yet gained general acceptance because the same effect can be produced by polymerase chain reaction artefact. We review both types of replacement including a critical examination of evidence for the latter. The first type involves RAG proteins and recombination signal sequences (RSS) and occurs in immature B cells. The second was also thought to be brought about by RAG proteins and RSS. However, it has been reported in hypermutating cells which are not thought to express RAG proteins but in which activation‐induced cytidine deaminase (AID) has recently been shown to initiate homologous recombination. Re‐examination of the published sequences reveals AID target sites in VH‐VH junction regions and examples that resemble gene conversion.