The molecular and cellular basis of autoimmunity remains largely unknown. The Repertoire Analysis Group is engaged in several projects aimed at elucidating the mechanisms that contribute to the development of autoimmune disease. Project 1) The use of a web based human immunoglobulin (Ig) alignment and CDR3 analysis program (JOINSOLVER?) to characterize the human heavy chain CDR3. The CDR3, which conveys the antigen specificity of the B cell, is the most variable region of immunoglobulin (Ig) due to extensive molecular processing in the junctions during recombination. This detailed description of the normal human CDR3 makes it possible to recognize distorted CDR3s that may be characteristic of B cells expressing autoreactive Ig receptors. JoinSolver also provides quantitative and qualitative mutation analysis. Project 2) Repertoire and mutation analysis of individual B cells from patients with genetic defects in proteins that regulate B cell development and functions. The following defective signaling elements that influence developmental pathways, response thresholds and germinal center reactions are under investigation: a. HED-ID (IKK-gamma, NEMO mutation), b. X-HIgM (CD40 ligand mutation), c. CD40 deficiency, d. a single nucleotide polymorphism in CD40 that results in complex activation or inhibition in SLE and chronic variable immune deficiency (CVID), e. AID (Activation induced cytidine deaminase) deficiency, f. Autoimmune Lymphoproliferative Syndrome (Fas mutation) & g. Xeroderma Pigmentosa Variant (XPV) defect in the DNA polymerase involved in DNA repair mechanisms. To examine the B cell repertoire in these patients, we amplify and sequence genomic heavy and light chain genes expressed by individual B cells. The technique allows us to analyze molecular mechanisms that are revealed in nonproductive rearrangements as well as the impact of selection that is imposed on productive rearrangements. The individual analyses allow us to isolate the contribution of single elements of the multi-component regulation of somatic hypermutation, class switching, selection, and plasma cell vs memory cell development. IKK-gamma mutations result in the accumulation of B cells with unusually long CDR3s. CD40 & CD40 ligand deficiencies result in an abnormally skewed repertoire enriched with B cells using VH4 rearrangements, a family of genes with a known propensity for autoreactivity. In X-HIgM, mutations are profoundly targeted to C residues characteristic of AID, whereas A/T mutations outside AID targeted mutations in WRC motifs are associated with pol eta activity most likely during replication. The Fas mutation results in overutilization of VH4 genes, failure to develop CD27+ memory B cells and increased survival of B cells with mutations that jeopardize stable Ig structure. Project 3) Phenotypic characterization of human immature B cells. Immature B cells represent a critical stage of B cell development in which autoreactive B cells are tolerized. Elevated proportions of them have been found in peripheral blood of lupus patients. The expression profile of transitional B cells is on-going. Project 4) The role of B cell superantigen in shaping the B cell repertoire. Polyclonal stimulators may alter the mature B cell repertoire by selecting or depleting specific B cells, some of which may become potentially autoreactive. SpA (a polyclonal activator of VH3 genes) administered to transgenic mice expressing fully human Ig caused depletion of VH3+ marginal zone B cells. Depletion generally correlated with the SpA binding avidity, however, weak binders escaped deletion in the presence of overriding positive selection by endogenous antigen. Murine B cells expressing human Ig are also under analysis to examine how self-antigen influences light chain repertoire development. Project 5) The elucidation of the maturational steps of CD4 memory T cells in healthy individuals and patients with a disturbed immunological balance. Three stages of T cell maturation defined by the phenotypic expression of CCR7 and CD27 were identified and used to characterize their functional properties. We observed a definite difference in the maturation and distribution of memory T cells between healthy controls and SLE patients, which could lead to a new potential therapeutic target. Project 6) Synergistic Activation of NF-kB by Multiple Kinases. NF-kB is a ubiquitously expressed transcription factor that regulates the expression of many genes that are important regulators of inflammation. We found coexpression of Raf or Tpl-2 with NIK in NIH 3T3 murine fibroblasts results in potent, constitutive activation of NF-kB-dependent gene expression. Using the expression of dominant interfering mutants as well as specific chemical inhibitors, we found p38, mTOR, ERK1/2 and ERK5are involved. We found that cytoplasmic NIK has the NF-kB-dependent ability to interfere with Tpl-2 activation of the transcription factor AP-1, while nuclear NIK has the same ability, independent of NF-kB. The deregulation of such complex signaling pathways resulting in chronic NF-kB activation is seen in many diseases. Elucidation of NF-kB regulation provides the opportunity to therapeutically modify its inappropriate activation in rheumatoid arthritis and other diseases with underlying chronic inflammation. Project 7) The role of IRTA-2 in B cell responses. IRTA-2 is a B cell-specific member of the FcR homologue family. The translocation of these genes at chromosome 1q21 is associated with abnormalities in Burkitts lymphoma and susceptibility to SLE. We have used specific rtPCR primers and antibodies to examine the expression profile of the soluble, transmembrane and GPI-anchored isoforms of the gene. The presence of ITIM (inhibitory) motifs in the transmembrane form of the molecule suggests it may play an important role in modulating B cell responses. We are currently identifying a functional role for the molecule. Project 8) Repertoire and mutation analysis of anergic CD19lowIgD+ cells in SLE patients. Anergy is a mechanism by which self-reactive B cells may become unresponsive; thus, CD19lowIgD+ cells were analyzed to determine whether this population was oliogoclonal or otherwise enriched with autoreactive B cells. We found the CD19lowIgD+ cells were polyclonal and used many of the same genes utilized by the CD19highIgD+ cells isolated from the same SLE patient. However, a greater representation of B cells expressing VH4 genes were included in the CD19lowIgD+ subset. We concluded that in SLE this form of anergy may be used to generally downregulate B cell hyperactivity, as well as to regulate the activity of specific VH4 B cells. Other ongoing projects: 1. B cell repertoire in bone marrow post-transplantation SLE and anti-TNFalpha treated rheumatoid arthritis patients 2. B cell repertoire analysis in SLE patients who have a single nucleotide polymorphism in the CD40 receptor 3. Repertoire and mutation analysis of follicular-like & marginal zone-like B cells using CD21 and CD23 B cell markers in human spleen, peripheral blood, tonsil and peripheral blood of SLE patients 4. Developmental changes in the human CDR3 5. Repertoire and mutation analysis in SLE patients on trial Anti-IL-6 receptor therapy 6. Repertoire and mutation analysis in identical twins (one with SLE & one without) to compare the B cell repertoire in the presence of a common genetic background 7. SpA repertoire analysis in cynamologous macaques, a model for studying nonhuman primate immune responses 8. Develop JoinSolver for the analysis of heavy and light chain rearrangements with complex mutations, insertions and deletions 9. Pseudo-hybrid formations in heavy chain variable segments as a mechanism of receptor editing 10. Somatic hypermutation analysis of nonproductive rearrangements in tonsil B cell subsets
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