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? (JS). The CDR3, which conveys the antigen specificity of the B cell, is the most variable region of Ig due to extensive molecular processing in the junctions during recombination. Detailed description of the normal human CDR3 has made it possible to recognize distorted CDR3s that may be characteristic of B cells expressing autoreactive Ig receptors. JS?s detailed mutation analysis provides quantitative and qualitative assessments of mechanistic elements of somatic hypermutation. Continuous development of JS software provides sophisticated repertoire & 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. X-HIgM (CD40 ligand mutation), b. CD40 deficiency, c. a single nucleotide polymorphism in CD40 that results in complex activation or inhibition in SLE and chronic variable immune deficiency (CVID), d. AID (Activation induced cytidine deaminase) deficiency, e. Autoimmune Lymphoproliferative Syndrome (Fas mutation) & g. Xeroderma Pigmentosa Variant (XPV) defect in the DNA polymerase involved in DNA repair mechanisms. 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. CD40 ligand deficiencies result in an abnormally skewed repertoire enriched with B cells using VH4 rearrangements that have a propensity for autoreactivity. 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. Mutations are 20-fold decreased in AID deficiency as well as total loss of mutational targeting. 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. We identified genes that are either differentially regulated or only expressed in T1 B cells by Affymetrix microarray technology, validating our previously published phenotyping data. These genes include members of the TNF receptor superfamily and genes associated with development and apoptosis in other cell types. We are currently validating the expression of these genes. Project 4) 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 5) 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. IRTA-2 is upregulated by BCR ligation and a variety of other stimuli. An antibody that specifically targets the transmembrane isoform can partially inhibit anti-IgM/anti-CD40 induced proliferation, but does not affect IL-21-induced plasma cell differentiation and immunoglobulin secretion. Project 6) 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 expressingVH4 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. Project 7) Vitamin D modulation of B cell responses. Previously, it has been shown that Vitamin D can modulate T cells and APC function and thereby indirectly effect B cell function. We have shown that Vitamin D also has direct effects on B cells. We have previously shown that IL-21 is a very potent inducer of plasma cell differentiation. Vitamin D can partially block IL-21-induced class-switch recombination, plasma cell production and immunoglobulin secretion. We have been examining the effects of Vitamin D on B cell function as a prelude for its use as a therapy for SLE patients. Other ongoing projects:? 1. B cell repertoire in bone marrow post-transplantation SLE and anti-TNFalpha treated rheumatoid arthritis patients? 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. Repertoire and mutation analysis in SLE patients on trial Anti-IL-6 receptor therapy? 5. 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? 6. Develop JoinSolver for the analysis of heavy and light chain rearrangements with complex mutations, insertions and deletions ? 7. Pseudo-hybrid formations in heavy chain variable segments as a mechanism of receptor editing? 8. Somatic hypermutation analysis of nonproductive rearrangements in tonsil B cell subsets
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