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 development and use of a web based human Ig alignment and CDR3 analysis program (JOINSOLVER?) to characterize the human heavy chain CDR3. The CDR3 is the most variable region of immunoglobulin (Ig) and conveys the antigen specificity of the B cell. Much of the CDR3 variability is derived from molecular processing of VH, D and JH segments during VH-D-JH joining which has made identification of D segments and the extent of these molecular mechanisms difficult to assess. JOINSOLVER? uses an intuitive algorithm in which the longest consecutive matching sequence within the VH-JH region is aligned to a database of known human germline D segments. A minimum match of nine nucleotides is required under most circumstances to assign a D segment within 95% confidence limits. From unknown Ig rearrangements, JOINSOLVER? identifies the V, (D) and J genes, mutated nucleotides, N and P nucleotides and quantifies exonuclease activity. We have characterized the molecular mechanisms and impact of positive and negative selection on the normal human CDR3 repertoire. This detailed description of the normal human CDR3 makes it possible to recognize distorted CDR3s that may be characteristic of B cells expressing autoreactive immunoglobulin receptors. Project 2) Repertoire and mutation analysis of B cells from patients with genetic defects in proteins that regulate B cell development and functions: Defective signaling elements that influence developmental pathways, response thresholds and germinal center reactions: HED-ID (IKK-gamma, NEMO), xHIM (CD40 ligand mutation), CD40 deficiency; AID (Activation induced cytidine deaminase) deficiency, the master regulator for somatic hympermutation and isotype switching; Autoimmune Lymphoproliferative Syndrome (Fas mutation) & 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 which is imposed on productive rearrangements. The individual analyses allow us to isolate the contribution of individual 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. 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 immunoglobulin structure. Project 3) B cell repertoire analysis of anergic B cells in patients with active SLE. An unusual population of CD19lowIgD+ cells from active SLE patients were identified and characterized by several biochemical measurements as a population of anergic B cells. Anergy is a mechanism by which autoreactive B cells can become functionally inactive. Therefore, the CD19lowIgD+ subset may represent a population that is enriched with autoreactive B cells. The CD19lowIgD+ subset had an over-representation of B cells expressing VH4 and the repertoire overall was polyclonal rather than oligoclonal. The data suggest anergy or hypo-responsiveness in the CD19loD+ subset may reflect an effort to generally downregulate B cell hyper-reactivity as well as an effort to limit the reactivity of B cells expressiiiiing specific VH4 genes. Project 4) 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. Extensive work has revealed that these cells have a phenotype similar to the T1 B cell subset described in mouse spleen. These B cells are found in bone marrow and peripheral blood of healthy individuals, and elevated proportions of them have been found in peripheral blood of lupus patients and cord blood. The repertoire of these B cells and their functional characteristics cells have been carefully investigated. Project 5) 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. Transgenic mice expressing fully human Ig were investigated to determine how in vivo administration of Protein L (a polyclonal activator of V kappa genes) and SpA (a polyclonal activator of VH3 genes) will impact the peripheral B cell repertoire. Both treatments were shown to deplete the targeted population. To distinguish binding from in vivo deletion, SpA binding studies were performed on human peripheral B cells labeled with biotinylated SpA and sorted into populations of binders and nonbinders, followed by Ig amplification and sequencing of individual B cell Ig receptors. The extent of depletion of VH3+ marginal zone B cells generally correlated with the SpA binding avidity. However, weak binders can escape deletion in the presence of overriding positive selection by another antigen. SpA repertoire analysis has also been conducted on cynamologous macaques, a model for studying nonhuman primate immune responses. Project 6) 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 7) Synergistic Activation of NF-kB by Multiple Kinases. NF-kB is a ubiquitously expressed transcription factor that regulates the expression of more than 100 different genes that are important regulators of inflammation, including cytokines, chemokines and anti-apoptotic proteins. We have found that the coexpression of Raf or Tpl-2 together with NIK in NIH 3T3 murine fibroblasts results in potent, constitutive activation of NF-kB-dependent gene expression. To determine the mechanism underlying the observed synergy, we have studied the nature of the signaling pathways involved and found a role for multiple kinases including p38, mTOR, ERK1/2 and ERK5. Involvement of these kinases was identified using both the expression of dominant interfering mutants as well as specific chemical inhibitors. This suggests the potential for the deregulation of such signaling pathways to bring about chronic NF-kB activation seen in many diseases. They also reveal both the complexity of NF-kB regulation but also the opportunity to therapeutically modify NF-kB activation in inappropriate circumstances such as rheumatoid arthritis and other diseases with underlying chronic inflammation. Other ongoing projects involve: 1. B cell repertoire in bone marrow post-transplantation 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 tonsilar B cell subsets 4. Developmental changes in the human CDR3 5. Role of IL-21 on B cell function 6. Anti-IL-6 receptor therapy trial in SLE
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