Dr. David I. Stott,

        Research is being conducted into the immune response in autoimmune diseases (principally systemic lupus erythematosus (SLE) and myositis) and breast cancer.  This includes investigations of the development of ectopic germinal centres and the repertoire of Ig V-genes expressed by B-cells infiltrating the target tissues of autoimmune diseases and the tumour tissue in breast cancer.    We are also investigating the mechanism of somatic hypermutation in a transgenic mouse model.

         Clusters of B-cells and resembling germinal centres are seen in the target tissues of several autoimmune diseases, including the thyroid of patients with Hashimoto’s thyroiditis, the synovial membrane around the inflamed joints of patients with rheumatoid or reactive arthritis, and the salivary glands of patients with Sjögren’s syndrome.  Germinal centres are also found in the thymus of myasthenia gravis patients.  It is not known whether the microenvironment of these cell clusters is sufficient for the induction of a germinal centre response.  We therefore cloned and sequenced rearranged Ig V-genes expressed by clusters of B cells in labial salivary gland biopsies from Sjögren’s 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 centre-type B cell response is taking place within the salivary glands of Sjögren’s syndrome patients.  In view of the recent demonstration of a germinal centre response within the rheumatoid synovial membrane and the existence of similar structures in the target tissues of other autoimmune diseases, we propose that  germinal centre-type responses can be induced in the non-lymphoid target tissues of a variety of autoimmune diseases.

Dominant B-cell clone  (b) present in a cluster of B-cells (a, stained red) in a salivary gland biopsy from a patient with Sjogren's syndrome.

        We have also constructed scFvs using the VH and VL-genes from one of these B-cell clones and  expressed them by phage display.  A Phage-ELISA system has been developed to identify the antigen specificities of the scFvs.  

  Family trees of B-cell clones in clusters of lymphocytes in salivary gland biopsies, based on V-gene sequences.  Numbers at side of arrows show no. of mutations required at each step.  Click on image for larger picture.

    We are studying the mechanism of somatic hypermutation using mice transgenic for the heavy and light chain genes encoding an antibody against hen egg lysozyme (HEL) and a second mouse strain transgenic for a T-cell receptor specific for ovalbumin (OVA).  B-cells and T-cells from the two mouse strains are transferred to an F1 mouse.   Immunisation of these mice with HEL conjugated to OVA stimulates an immune response in which the majority of responding B-cells and germinal centre cells express the transgenic antibody.  This makes it much easier to study the immune response compared with the multitude of antibodies produced in a normal animal.  The project is in two parts:-

1.  In the first part, we are studying the pattern of mutations in transgenic H and L chain V-genes expressed by germinal centre B-cells in response to HEL, by cloning and sequencing the antibody V-genes from microdissected germinal centres.

2.  In the second part, we shall identify non-Ig genes which are expressed in hypermutating B-cells .  This will be done by dissecting out whole germinal centres, the dark zone (the region where B-cells hypermutate) and the light zone, from tissue sections.  Genes which are switched on or off in germinal centre B-cells will be identified by the differential display polymerase chain reaction.  We shall make a cDNA library of the genes that are expressed in B-cells from immunised mice and select, from this library, those genes which we have identified in the hypermutating B-cells. Any novel genes identified in this way will be cloned, sequenced and selected for study in future projects.

Adams, C.L., MacLeod, M.K.L., Milner-White, E.J., Aitken, R., Garside, P. & Stott, D.I. (2003)  “Complete analysis of the B-cell response to a protein antigen, from in vivo germinal center formation to 3-D modelling of affinity maturation”  Immunology 108, 1-14.

        Common variable immunodeficiency syndrome (CVID) is a primary antibody deficiency syndrome in which patients present with recurrent bacterial infections, low or normal numbers of circulating B-lymphocytes and decreased levels of serum immunoglobulins.  It was recently reported that somatic hypermutation of Ig V-genes was defective in 2/6 patients.   Since somatic hypermutation is responsible for affinity maturation of the antibody response, this could explain, not only the origin of the defect in a subset of CVID patients, but also why there is a poor correlation between serum Ig levels and susceptibility to bacterial infections in some patients.  We are therefore investigating somatic hypermutation in memory and naïve B-cells from these patients, using a larger group than in the original study, to determine the proportion of CVID patients with defective somatic hypermutation and the relationship of this defect to other immunological and clinical features of the disease.

        Myasthenia gravis (MG) is an autoimmune disease characterised by weakness of the voluntary muscles and thymic hyperplasia. Large numbers of germinal centres and plasma cells secreting   autoantibodies against the acetyl choline receptor (AChR) are present in the thymus.  These thymic B-cells and plasma cells appear to be an important source of autoantibody, since thymectomy results in a marked improvement of symptoms.  The autoantibodies are known to be pathogenic, causing loss of AChRs and membrane damage at the neuromuscular junction.  Little is known about the genetic and clonal origins of anti-AChR autoantibodies produced in the thymus, or the relationship between specificity and pathogenicity or protection.  Nor is it known why germinal centres and autoantibody-secreting plasma cells develop in the thymus.  We are therefore studying clonal development of B-cells, and somatic hypermutation in their Ig V-genes, within germinal centres in the MG thymus, and the properties of the autoantibodies produced there.

Publications
Sims, G.P., Shiono, H., Willcox, N. and Stott, D.I. (2001)  “Somatic hypermutation and selection of B-cells in thymic germinal centers responding to acetylcholine receptor in myasthenia gravis.”  J. Immunol. 167 , 1935 – 1944.

Matthews, I., Sims, G.P., Ledwidge, S., Plested, P., Stott, D.I., Willcox, N. and Vincent, A. (2002) “Fetal specificity of antibodies to human acetylcholine receptor cloned from the thymus of two multiparous women.”  Lab Invest. 82, 1407-1417.

Shiono, H., Roxanis, I., Zhang, W., Sims, G., Meager, A., Jacobson, L.W., Liu, J.-L., Matthews, I., Wong, Y.-L., Bonifati, M., Micklem, K., Stott, D.I., Todd, J.A., Beeson, D., Newsom-Davis, J., Vincent, A., and Willcox, N. (2002)  “Scenarios for autoimmunization of T and B cells in myasthenia gravis.” Annals New York Academy of Sciences, in press.

Sims, G.P., Willcox, N. & Stott, D.I. (2002)  “The autoantibody repertoire in myasthenia gravis.” in “Frontiers in Autoimmunity”, Zouali, M. (ed.), IOS Press, Amsterdam, The Netherlands, in press.

        Breast cancer is the most common type of cancer, affecting 1/12 females. Although CD8+ T-cells and NK cells are thought to be the main effector cells in killing tumour cells, they are ineffective once a cancer has become established.  There has recently been renewed interest in antibodies against tumour antigens for diagnosis, imaging of metastases and immunotherapy, due to developments in genetic engineering of antibodies and identification of tumour-specific antigens.  Little is known about the origins and specificity of the lymphocytes infiltrating the tissues in breast cancer.  We propose to determine the V-gene repertoire and antigen specificity of infiltrating B-cells in ductal breast carcinoma and to compare them with B-cells in germinal centres of the draining lymph node and PBL to determine whether they are a selected subset of these cells.  Their antigen receptors will be reconstituted by phage display and used to identify the tumour antigens recognised by the infiltrating B-cells.

Publications
Nzula, S., Going, J.J. & Stott, D.I.  “Antigen-driven clonal proliferation, somatic hypermutation and selection of B lymphocytes infiltrating human ductal breast carcinomas.”  Submitted for publication.

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6. Generation Of Pathogenic Antibodies And Somatic Hypermutation In SLE

Collaborator: Dr. Max Field, Centre for Rheumatic Diseases, Glasgow Royal Infirmary

        The origins of the self-reactive B cells in systemic lupus erythematosus (SLE) are unknown, but clusters of B-cells, T-cells and dendritic cells, which may act as foci for presentation of self-antigens, are seen in skin and vasculitic lesions of SLE patients.  The objectives of this project are to identify the germline Ig V-genes expressed by B cells in these clusters and in lymph node and splenic germinal centres; to determine whether the B-cell repertoire at these centres of autoantibody production differs from that in peripheral blood; to determine the effects of somatic hypermutation on the specificity and affinity of these autoantibodies; and whether the autoantibody repertoire is shaped by receptor editing and revision.

Publications

Stott, D.I. (1990) "Lessons about autoantibody specificity in SLE from animal models".  Clin. Exp. Immunol., 81, 1-4.

Stott, D.I., Brighouse, G., Gyotoku, Y., and Lambert, P.-H. (1990). Analysis of the autoimmune response in lupus mice: The behaviour and lifespan of anti-DNA-secreting B-cell clones.  Autoimmunity 8, 159-168.

Stott, D.I. (1992) "Spectrotypes of anti-DNA antibodies show that anti-DNA-secreting B-cell clones of SLE patients are restricted in number, stable and long lived".  Autoimmunity, 12, 249-258.

Huang, F.-P. and  Stott, D.I. (1993) "Restoration of an early, progressive defect in responsiveness to T-cell activation in lupus mice by exogenous IL-2".  Autoimmunity, 15, 19-29.

Huang, F.-P. and Stott, D.I. (1995) "Dual inhibitory and stimulatory activities in serum from SLE patients and lupus mice that regulate the proliferation of an IL-2 dependent T-cell line".  Lupus, 4, 297-303.

Huang, F.-P., Feng, G.-J., Lindop, G., Stott, D.I. and Liew, F.Y. (1996) "The role of interleukin-12 and nitric oxide in the development of spontaneous autoimmune disease in MRL-lpr/lpr mice."  J. Exp. Med., 183 , 1447-1460.

Huggins, M.L., Huang, F.-P., Xu, D. Lindop, G. and Stott, D.I. (1997)  "Modulation  of the autoimmune response in lupus mice by oral administration of attenuated  Salmonella typhimurium expressing the IL-2 and TGF-b genes.  Annals N.Y. Acad. Sci.  815, 499-502.

Niedbala, W.G. and Stott, D.I. (1998)  "A comparison of three methods of production of human monoclonal antibodies."  Hybridoma, 17 , 299-304.

Huggins, M.L., Huang, F.-P., Xu, D., Lindop, G., and Stott, D.I. (1999). Modulation of autoimmune disease in the MRL-lpr/lpr  mouse by IL-2 and TGF-b1 gene therapy using attenuated Salmonella typhimurium  as gene carrier.  Lupus 8, 29-38.

Fraser, N.L.W., Rowley, G., Field, M. and Stott, D.I. (2003)  “Development of the Immunoglobulin V-gene Repertoire in the Autoimmune Disease, Systemic Lupus Erythematosus.”  Arthritis Research & Therapy 5, R114-R121.

Stott, D.I. (2000) "Immunoblotting, Dot Blotting and ELISPOT Assays.  Principles, Methods and Applications."  Journal of Immunoassay, 21, 273-296.