Mark S. Schlissel - US grants

Two functions of the complement system related to autoimmune diseases are the induction of an inflammatory response and the augmentation of an immune response. Studies of two complement receptors are proposed that address both functions. A group of plasma and membrane proteins sharing a structural motif inhibit the C3/C5 convertase step of the classical and alternative pathways. One member of this family, complement receptor type 1 (CR1; CD35) normally serves as a receptor but is uniquely suited for complement inhibition. CRI binds bivalently to dimers of C3b and C4b, promotes their cleavage by I, dissociates the catalytic subunits from the C3/C5 convertases of both pathways, and is not restricted by alternative pathway activating surfaces. A soluble form of CR1, sCR1 lacking the transmembrane and cytoplasmic domains was prepared to take advantage of these inhibitory activities. The sCRI was at least 100-fold more inhibitory than C4-binding protein and H in vitro and suppressed complement activation and tissue necrosis in vivo in a model of myocardial ischemia/reperfusion injury. These studies will be extended by determining the SCRs required for the inhibitory functions of CRI, preparing soluble CR1/IgG constructs having improved half-lives and tissue distribution and suppressing a complement-dependent model of autoimmune disease, experimental allergic myasthenia gravis. The capacity of complement to enhance the humoral immune response is mediated in part by the B cell receptor, complement receptor type 2 (CR2; CD21) which augments activation of phospholipase C (PLC) by mIgM. CR2 forms a 1:1 complex with CD19, a membrane protein that is expressed at all stages of B cell development except that of the plasma cell, is a member of the immunoglobulin superfamily, has an extended cytoplasmic domain of 247 amino acids, and releases intracellular Ca++ following ligation, all characteristics of a membrane constituent important in the biology of the B cell. In the mature B cell the CR2/CD19 complex may represent a functional signal transducing unit. The proposed studies will demonstrate that CR2 is a ligand binding subunit and CD19 the signal transducing subunit in the complex, that CD19 utilizes a pathway to PLC activation that is distinct from that of mIgM, and that CD19 is coupled to a protein tyrosine kinase. Through the CR2/CD19 complex, complement may trigger B cells by a pathway fundamental to the biology of this cell type.

Two functions of the complement system related to autoimmune diseases are the induction of an inflammatory response and the augmentation of an immune response. Studies of two complement receptors are proposed that address both functions. A group of plasma and membrane proteins sharing a structural motif inhibit the C3/C5 convertase step of the classical and alternative pathways. One member of this family, complement receptor type 1 (CR1; CD35) normally serves as a receptor but is uniquely suited for complement inhibition. CRI binds bivalently to dimers of C3b and C4b, promotes their cleavage by I, dissociates the catalytic subunits from the C3/C5 convertases of both pathways, and is not restricted by alternative pathway activating surfaces. A soluble form of CR1, sCR1 lacking the transmembrane and cytoplasmic domains was prepared to take advantage of these inhibitory activities. The sCRI was at least 100-fold more inhibitory than C4-binding protein and H in vitro and suppressed complement activation and tissue necrosis in vivo in a model of myocardial ischemia/reperfusion injury. These studies will be extended by determining the SCRs required for the inhibitory functions of CRI, preparing soluble CR1/IgG constructs having improved half-lives and tissue distribution and suppressing a complement-dependent model of autoimmune disease, experimental allergic myasthenia gravis. The capacity of complement to enhance the humoral immune response is mediated in part by the B cell receptor, complement receptor type 2 (CR2; CD21) which augments activation of phospholipase C (PLC) by mIgM. CR2 forms a 1:1 complex with CD19, a membrane protein that is expressed at all stages of B cell development except that of the plasma cell, is a member of the immunoglobulin superfamily, has an extended cytoplasmic domain of 247 amino acids, and releases intracellular Ca++ following ligation, all characteristics of a membrane constituent important in the biology of the B cell. In the mature B cell the CR2/CD19 complex may represent a functional signal transducing unit. The proposed studies will demonstrate that CR2 is a ligand binding subunit and CD19 the signal transducing subunit in the complex, that CD19 utilizes a pathway to PLC activation that is distinct from that of mIgM, and that CD19 is coupled to a protein tyrosine kinase. Through the CR2/CD19 complex, complement may trigger B cells by a pathway fundamental to the biology of this cell type.

B and T lymphocytes for an interacting system of recognition and effectors cells which protects the animal from infection by a wide variety of microorganisms. Their recognition systems depend on clonotypic cell-surface receptors, immunoglobulin (Ig) on B cells and T cell receptor (TCR) on T cells, of a near-limitless range of specificities. This enormous diversity of receptor specificity is possible because the genes encoding IG and TCR subunits are assembled during lymphocyte development by a novel and highly regulated series of gene-segment rearrangements. The products of these rearranged genes themselves are involved in the regulation of lymphocyte development. This proposal outlines experiments aimed at understanding the role of Ig heavy-chain mu protein in regulating the lymphoid-specific recombinase activity. We propose to use bone marrow from recombinase-deficient Ig gene transgenic mice and fetal liver B cell progenitors isolated on successive days of mouse gestation to help separate the different stage of B cell development from one another. We previously devised techniques which allow us to detect and quantify Ig gene rearrangements, rearrangement reaction intermediates, and relevant gene transcripts using very small numbers of cells. We propose to use these new techniques and purified progenitor B cells to 1) determine the effect of Ig mu and kappa chains and complete IgM on the targeting of he recombinase; 2) elucidate the mechanisms and identify ligands involved in the regulation Ig kappa gene rearrangement by Ig mu protein; 3) test the involvement of the transcription factor NF-kappaB in the activation of Ig kappa gene transcription and rearrangement; and 4) determine the role of surface IgM in eh inactivation of gene rearrangement. Aberrant lymphocyte development can result in disease. For example, several different hematopoietic malignancies involve errors in Ig/TCR gene rearrangement which juxtapose a prot-oncogene with an active Ig or TCR gene. We envision our experiments as providing the ground work for eventual study of the developmental abnormalities of the immune system which lead to immunodeficiency, autoimmunity, and lymphoid malignancy.

DESCRIPTION (Adapted from Investigator's Abstract): Normal B cell development depends on the proper assembly of immunoglobulin (Ig) heavy and light chain genes from their component gene segments by the V(D)J recombinase. Activity of the recombinase depends on the regulated expression of two lymphoid-specific genes, RAG1 and RAG2 and is regulated such that an individual mature B cell expresses a single antigen receptor specificity. This phenomenon, critical for the regulation of the humoral immune response, is known as allelic exclusion. Work from many laboratories, including our own, has uncovered a role for the assembled immunoglobulin chains themselves in regulating the recombinase. In pre-B cells, heavy-chain mu protein assembles with surrogate light-chains and the receptor accessory chains Ig-a and Ig-b to form the pre-B cell receptor. This complex is involved in inhibiting further heavy-chain gene rearrangement and activating kappa light chain gene rearrangement. In this competing renewal application, we propose a series of experiments aimed at understanding how developing B cells regulate the V(D)J recombinase in response to Ig protein expression. Specifically we plan to 1) test the hypothesis that the stochastic and infrequent activation of enhancers regulating the unrearranged Ig k locus contributes to light chain allelic exclusion; 2) identify critical DNA sequences and transcription factors involved in the regulation of RAG1 and RAG2 transcription; 3) determine whether the CH2 and CH3 domains of Ig mu chain are required of activity of the pre-B cell receptor; and 4) identify proteins capable of binding a region of the Ig k 3' enhancer which may be responsible for the lineage specificity and timing of V-to-Jk rearrangement. Abnormal B cell development is associated with immunodeficiency, autoimmune disease, and malignancy. Failure of the pre-B cell receptor to signal results in developmental arrest and decreased B cell production. Failure to delete or energize self-specific B cells can result in autoimmunity. Finally, many lymphoid malignancies are associated with chromosomal translocations involving the aberrant rearrangement of immunoglobulin gene segments and proto-oncogenes. We perform this research in the hope that understanding normal B cell development will provides basic insights into the etiology these diseases leading to their prevention or effective treatment.

Antigen receptors are assembled from their component gene segments by a series of highly regulated site- specific DNA recombination reactions known as V(D)J recombination. The over-arching goal ofthis research program is to understand in molecular detail the mechanisms underlying the regulation ofthe V(D)J recombinase.Two lymphocyte restricted proteins, RAGl and RAG2 form a complex with one another and recognize pairs ofrecombination signal sequences (RSSs) that flank rearranging gene segments. The RAGs first nick the DNA then introduce dsDNA breaks. Appropriate broken ends are then joined to each other through the action of DNA repair proteins resulting in the formation ofcoding and signal joints. Targeting ofthe recombinase correlates with transcription ofthe unrearranged gene segments and is regulated at the level of RSS accessibility within chromatin structure. Errors in recombinase targeting are associated with chromosomal translocations that result in lymphoid malignancy and defects in V(D)J recombination result in immunodefiency. We propose to 1) test the idea that a gradient of H3K4me3 modified chromatin positioned by the distal germline k promoter determines the efficiency of Igk locus recombination and is important for allelic exclusion; 2) perform a retroviral cDNA library screen for chromatin modifiers that influence recombination; 3) use new high throughput DNA sequencing technologies to extend our studies ofssDNA nicking and RSS end insertion during V(D)J recombination in vivo; 4) examine in detail how a critical transcription factor, E2A, alters chromatin structure to promote recombination in the Igk locus; 5) use gene targeting to delete a CTGF binding site in a DNAse I sensitive region in between the IgHC V and D gene segments in order to test whether it regulates V-to-D rearrangement; and 6) test the h5T)othesis that the interaction between upstream promoters and downstream enhancers is necessary to promote coding joint formation by holding broken coding ends together. V(D)J recombination has been extensively studied using biochemical systems. Our approach is to take what has been learned in vitro and use it to frame and test hypotheses regarding the regulation of recombination in vivo.

DESCRIPTION (adapted from investigator's abstract): Antigen receptor genes are assembled from their component gene segments by a series of site-specific DNA recombination reactions known as V(D)J recombination. Gene segments which undergo this reaction are flanked by conserved DNA elements called recombination signal sequences (RSSs). These elements are recognized in a pairwise fashion by the lymphoid-specific proteins, RAG1 and RAG2, which introduce a pair of double-strand DNA breaks immediately adjacent to the RSSs. The four resultant DNA ends are then joined to form a signal joint and a coding joint. These joining steps utilize components of the dsDNA break repair machinery expressed in all cells. Seven complex genetic loci undergo V(D)J recombination including the immunoglobulin (Ig) mu, kappa, and lambda loci and the T cell receptor (TCR) alpha, beta, gamma, and delta loci. The rearrangement of these loci is regulated in several ways: a) Ig genes fully rearrange only in the B lineage and TCR genes only in the T lineage; b) within each lineage, antigen receptor gene rearrangement is highly ordered, with Ig mu and TCR beta rearrangement preceding Ig kappa and TCR alpha rearrangement for example; and c) an individual B or T cell makes only one productive (in frame) rearrangement at a given locus (allelic exclusion). The aim of this research proposal is to understand how a common V(D)J recombinase recognizing a conserved RSS can generate an exquisitely regulated pattern of gene-segment rearrangement. A wealth of correlative data has led to the hypothesis that accessibility of rearranging gene segments in chromatin determines the targeting of the V(D)J recombinase. The investigator showed recently that recombinant RAG1 and RAG2 when supplemented with nuclear extract could recognize and cleave RSSs in vitro within nuclei from RAG-deficient lymphoid cells. The pattern of cleavage corresponds to the state of development of the nuclei, mimicking the normal pattern of regulation of the recombinase. He went on to show that precisely positioned mononucleosomes can prevent RAG cleavage and that transcriptional enhancers within rearranging loci were critical for accessibility and function of the recombinase. In addition, it was found that nuclear proteins in addition to RAG1 and RAG2 were required for the recombinase to recognize and cleave RSSs within purified genomic DNA substrates. Experiments are proposed to further examine the role of nucleosomes in regulating V(D)J recombination, to purify and molecularly clone factors which help target recombination, to ask whether the V(D)J recombinase has transposase activity in vivo, and to determine the ability of the catalytic domain of RAG2 to complement lymphoid development in a RAG2 mutant mouse.

DESCRIPTION (provided by applicant): B cell development generates a large and diverse repertoire of mono-specific and self-tolerant B cells expressing surface immunoglobulin (Ig) as the recognition component of their antigen receptor. Ig diversity depends upon a tightly regulated novel site-specific DNA recombination reaction known as V(D)J recombination which assembles the variable exons of the Ig heavy-chain and light-chain genes from their component gene-segments. A pair of lymphoid-specific proteins, RAG1 and RAG2, form a complex with one another that recognizes pairs of rearranging gene segments and helps catalyze their joining. This process is regulated during lymphoid development with respect to lineage (Ig genes fully rearrange in B but not T cells), order within the lineage (heavy-chain rearranges before light-chain), and the use of alleles (an individual cell expresses only one functional Ig molecule on its surface, a phenomenon known as allelic exclusion). Previous work has shown that targeting of the recombinase depends upon the accessibility of gene- segments within chromatin structure and that transcription of unrearranged gene segments correlates with their accessibility to the recombinase. This competing renewal of a long-standing program of research on the regulation of B cell development aims to understand the mechanisms which regulate V(D)J recombination during generation of the primary B cell repertoire through the pursuit of four specific aims. 1) To identify the cis-acting DNA sequences and trans-acting factors which are required for the transcriptional regulation of the RAG1 and RAG2 genes;2) To determine how the Ig kappa locus is activated for transcription and rearrangement in pre-B cells in such a fashion that only one allele is functionally rearranged;3) To determine what role if any the transcription factor NF-kB plays in the regulation of receptor editing and positive selection of the B cell repertoire;and 4) to test hypotheses regarding the mechanisms of lineage specificity and allelic exclusion of IgHC V-to-DJ rearrangement. These studies are significant because defective V(D)J recombination can lead to profound immunodeficiency, because mistakes in targeting the recombinase are associated with genomic instability, chromosomal translocations, and malignancy, and because appropriate regulation of the recombinase is necessary to avoid autoimmune disease.

DESCRIPTION (provided by applicant): Developing B cells undergo regulated cell division and apoptosis in response to their success in assembling the genes encoding their antigen receptors. In addition, the specificity of these receptors is monitored and developing cells with the potential for self-specificity are signaled to either edit their receptors or undergo apoptosis. This research proposal focuses on a protein tyrosine kinase, c-Abl, which our preliminary data and the experiments of others lead us to believe may be involved in these key developmental processes, c-Abl is the cellular homologue of v-Abl, the transforming gene of the Abelson Murine Leukemia Virus (A-MuLV) which causes acute B cell leukemia in mice. In humans, c-Abl is involved in a disease-associated chromosomal translocation which generates the BCR-Abl fusion protein in several forms of leukemia. We propose to test hypotheses regarding the biological functions of the Abl kinase in developing B cells and its role in transformation through pursuit of two specific aims. First, we will perform experiments aimed at understanding how v-Abl disrupts signaling pathways, blocks differentiation, and prevents the apoptosis of leukemic murine pro-B cell lines. These experiments will use a newly available specific inhibitor of the Abl tyrosine kinase, STI-571 (Gleevec), DNA microarrays, and a recently-developed retroviral cDNA cloning strategy called CPR. Second, we will test hypotheses regarding the role of c-Abl in the regulation of cell proliferation, viability, gene expression, pre-BCR signaling, allelic exclusion, receptor editing and clonal deletion during normal murine B cell development. We will attempt to identify the critical targets of the Abl kinase involved in these processes. These experiments will take advantage of primary cell culture systems and STI-571, as well as available null mutations in ARG (Abl-related gene) and c-Abl. These studies are significant because of the involvement of c-Abl in the etiology of chronic myelogenous leukemia and acute lymphocytic leukemia in humans and the growing use of the Abl inhibitor STI-571 in the clinical treatment of various malignancies.