<p>Several HIV-1 neutralizing antibodies (e.g. 2F5, 4E10) have been shown to react with self-antigens, suggesting that effective humoral responses to HIV-1 may be constrained by the tolerization of HIV-reactive B cells that also recognize self-antigens. I have tracked the development of 2F5-like HIV-1 gp41 membrane proximal external region (MPER)-reactive B cells throughout ontogeny using B-cell tetramer reagents. In BL/6 mice, MPER-binding populations are lost during normal B-cell development and immunization with HIV-1 MPER antigen does not elicit robust humoral responses. I have identified Kynureninase as a self-antigen that is recognized by 2F5 antibody and, therefore, is a molecule that could mediate the developmental loss of B cells reactive to an epitope shared by HIV gp41 and Kynureninase. To recover these MPER-reactive cells, I describe and characterize a stromal-cell independent culture system that efficiently supports pro-B cell to IgM+ B-cell development with near normal levels of IgH and Igkappa diversity. B-cell development in vitro closely follows the patterns of development in vivo with culture derived (CD) B cells demonstrating characteristic patterns of surface antigen expression and gene activation. Immature and transitional B-cell compartments are reduced, due to the induction of tolerance, in the bone marrow of 3H9 IgH knockin mice ; however, cultures of 3H9 IgH knockin pro-B cells yields high frequencies of "forbidden", autoreactive IgM+ B cells. Furthermore, serum IgG autoantibody exceeded that present in autoimmune, C4-/- animals following the reconstitution of RAG-1-/- mice with IgM+ CD cells derived from BL/6 mice. I show that HIV-1 MPER-reactive B cells are recovered from both BL/6 and 2F5 IgH knockin bone marrow using this in vitro culture system. RAG-1-/- mice reconstituted with these culture-derived B and T cells generate strong germinal center and antibody responses to HIV-1 MPER antigens. These data demonstrate that the humoral immune response to this HIV-1 gp41 MPER antigen can be restored in mice when the constraints of B-cell tolerance have been relaxed.</p> / Dissertation

Human T-cell Leukemia Virus type 1 (HTLV-1) is the etiologic agent of the Adult T-cell Leukemia (ATL), an aggressive lymphoproliferation of activated CD4+ T cells. The lack of a reliable small animal model to reproduce in vivo the leukemogenic process associated with HTLV-1 infection has impaired the understanding of the early stages of this process as well as the discovery of effective therapeutic approaches. Recently, improvement in the models of humanized mouse models were achieved allowing the development of a human immune system in mice. Injection of human hematopoietic stem and progenitors cells purified from cord blood into Balb/c Rag2-/-γc-/- newborns allows the de novo production of human dendritic, B and T cells. We infected humanized mice with HTLV-1 producing cell lines resulting in infection of human cells within the mice and the development of lymphomas and leukemias. This infection also results in the alteration of the T-cell development within the thymus pushing the thymocytes toward a more mature phenotype. This small animal model recapitulating in vivo the HTLV-1 infection and its associated pathogenesis gave us the opportunity to study the evolution of the clonality of the virus among human cells in different lymphoid organs. Based on these observations, preliminary results on the use of a new therapeutic approach were obtained. We finally tried to adjust the humanization protocol in order to obtain better engraftment in this model.

T lymphocytes play a critical role in adaptive immunity by eliciting and regulating specific immune responses against viral and bacterial pathogens. The development of T cells occurs within the highly specialized thymus and follows a defined set of stage-specific differentiation steps. However, the molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. This was in part due to the inability to obtain substantial numbers of T-lineage cells from hybrid/human fetal thymic organ culture (FTOC) and the inability to recapitulate human T-lymphopoiesis using other systems. To address the molecular and cellular events occurring during early human T-lymphopoiesis, human umbilical cord-blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T-lineage utilizing OP9-DL1 stromal cells. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. In addition, this Thesis demonstrates that in vitro-generated CD34+CD7++ progenitors effectively engrafted the thymus of immunodeficient mice. In addition, two distinct progenitor subsets, CD34+CD45RA+CD7++CD5-CD1a- (proT1) and CD34+CD45RA+CD7++CD5+CD1a- (proT2), were identified with proT2 cells showing a 3-fold enhanced engrafting capacity than the proT1 subset. As proT2 cells exhibit superior engrafting capacity, these cells were tested for their ability to enhance T cell generation following hematopoietic stem cell transplant (HSCT). We observe that when HSCs are coinjected with proT2 cells, a dramatic improvement in HSC-derived T-lymphopoiesis is observed. This Thesis demonstrates that in vitro-derived proT2 cells reorganize the thymus stromal compartment of the host NOD/SCID/γcnull mouse compared to the highly disorganized cortical and medullary compartments in mice not receiving proT cells. This alteration in thymic architecture likely favours the recruitment of BM derived progenitors. Lastly, we address whether functional CD8 T cells can be generated in vitro using hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells and indeed these cells were capable of proliferating, and secreting effector molecules typical of cytotoxic T cells. Taken together, the ability to generate proT cells and mature T cells from Notch-ligand cultures offers a new tool to study human T cell development.

T lymphocytes play a critical role in adaptive immunity by eliciting and regulating specific immune responses against viral and bacterial pathogens. The development of T cells occurs within the highly specialized thymus and follows a defined set of stage-specific differentiation steps. However, the molecular and cellular events occurring at early stages of human T-cell development remain to be fully elucidated. This was in part due to the inability to obtain substantial numbers of T-lineage cells from hybrid/human fetal thymic organ culture (FTOC) and the inability to recapitulate human T-lymphopoiesis using other systems. To address the molecular and cellular events occurring during early human T-lymphopoiesis, human umbilical cord-blood (UCB) hematopoietic stem cells (HSCs) were induced to differentiate to the T-lineage utilizing OP9-DL1 stromal cells. A developmental program involving a sequential and temporally discrete expression of key differentiation markers was revealed. In addition, this Thesis demonstrates that in vitro-generated CD34+CD7++ progenitors effectively engrafted the thymus of immunodeficient mice. In addition, two distinct progenitor subsets, CD34+CD45RA+CD7++CD5-CD1a- (proT1) and CD34+CD45RA+CD7++CD5+CD1a- (proT2), were identified with proT2 cells showing a 3-fold enhanced engrafting capacity than the proT1 subset. As proT2 cells exhibit superior engrafting capacity, these cells were tested for their ability to enhance T cell generation following hematopoietic stem cell transplant (HSCT). We observe that when HSCs are coinjected with proT2 cells, a dramatic improvement in HSC-derived T-lymphopoiesis is observed. This Thesis demonstrates that in vitro-derived proT2 cells reorganize the thymus stromal compartment of the host NOD/SCID/γcnull mouse compared to the highly disorganized cortical and medullary compartments in mice not receiving proT cells. This alteration in thymic architecture likely favours the recruitment of BM derived progenitors. Lastly, we address whether functional CD8 T cells can be generated in vitro using hematopoietic stem cells (HSCs) in coculture with OP9-DL1 cells and indeed these cells were capable of proliferating, and secreting effector molecules typical of cytotoxic T cells. Taken together, the ability to generate proT cells and mature T cells from Notch-ligand cultures offers a new tool to study human T cell development.

No description available.

<p>T cells and B cells each express unique antigen receptors used to identify, eliminate, and remember pathogens. These receptors are generated through a process known as V(D)J recombination, in which T cell receptor and B cell receptor gene loci undergo genomic recombination. Interestingly, recombination at certain genes is regulated so that a single in-frame rearrangement is present on only one allele per cell. This phenomenon, termed allelic exclusion, requires two steps. First, recombination can occur only on one allele at a time. In the second step, additional recombination must be prevented. Though the mechanism of the second step is well-understood, the first step remains poorly understood.</p><p>The first step of recombination necessitates that alleles rearrange one at a time. This could be achieved either through inefficient recombination or by halting further recombination in the presence of recombination. To separate these mechanisms, we analyzed recombination in nuclei unable to complete recombination. We found that rearrangement events accumulated at antigen receptor loci, suggesting that the presence of recombination does not stop additional rearrangements and asynchronous recombination likely results from inefficient recombination at both alleles.</p><p>Association with repressive subnuclear compartments has been proposed to reduce the recombination efficiency of allelically excluded antigen receptor loci. Of the alleleically excluded loci, <italic>Tcrb</italic> alleles are uniquely regulated during development. Other allelically excluded alleles are positioned at the transcriptionally-repressive nuclear periphery prior to recombination, and relocate to the nuclear interior at the stage in which they recombine. However <italic>Tcrb</italic> alleles remain highly associated with the nuclear periphery during rearrangement. Here we provide evidence that this peripheral subnuclear positioning of <italic>Tcrb</italic> alleles does suppress recombination. We go on to suggest that peripheral localization mediates the first step of allelic exclusion.</p><p>In search of the mechanism by which recombination is suppressed on peripheral <italic>Tcrb</italic> alleles, we investigated the subnuclear localization of a recombinase protein. Two recombinase proteins are required for recombination, one of which is recruited to actively transcribing (and more centrally located) DNA. Here we demonstrate that one recombinase protein is unable to localize to peripheral <italic>Tcrb</italic> alleles, potentially serving as the mechanism by which recombination is suppressed on peripheral alleles.</p> / Dissertation

Thesis (Ph. D.)--Ohio State University, 2007.

Thesis (M.S.)--University of Delaware, 2006. / Principal faculty advisor: Darla J. Sherrier, Dept. of Plant and Soil Science. Includes bibliographical references.

Thesis (Ph. D.)--University of Canterbury, 2006. / Typescript (photocopy). Includes bibliographical references (leaves 109-118). Also available via the World Wide Web.

DNA organization is an intricate and dynamic process. The approximately two meters of DNA in a single cell is wrapped around small proteins called histones. Histones can be compacted into dense coils or loosely distributed along DNA, allowing for cells to control gene expression. This combination of DNA and histones forms chromatin. This work has focused on understanding the role of Polybromo1 (PBRM1), which is a member of a chromatin remodeling complex. PBRM1 is mutated in 3% of all human cancers and is mutated in 40% of renal clear cell carcinomas (ccRCC), the most common type of kidney cancer. Through my work characterizing PBRM1 as a tumor suppressor, we have found PBRM1 acts as a stress sensor. PBRM1 is a member of the Polybromo1 BRG1 associating factors (PBAF) complex which is a subtype of the larger BAF family of chromatin remodelers. Although BAF is essential for cell viability, knockdown of PBRM1 shows minor phenotypic changes in many cell types under standard cell culturing conditions. However, when cells without PBRM1 experience external stress, the reactive oxygen species levels in the cells are elevated and remain high compared to cells with wild type PBRM1. Depending on the cellular environment of the cell, increase in ROS can be growth promoting or growth inhibiting. PBRM1 is a structurally unique protein, containing two bromo-adjacent homologs, a high mobility group and six tandem bromodomains. Due to the multiple reader domains, it is likely PBRM1 acts to target the complex. Taking advantage of a RCCC cell line not expressing PBRM1, we re-expressed full length PBRM1 containing an asparagine to alanine mutation in each bromodomain, disrupting the acetyl-lysine binding. We have found that the bromodomains are cooperative and are facilitating binding of PBAF to chromatin. We found defects in PBRM1’s ability to suppress growth, bind to chromatin, and regulate gene expression when any of the bromodomains were mutated besides the third bromodomain. These results correlated with patient data. Using acetylated histone peptides, we have identified potential combinations of marks that PBRM1 prefers over single marks. Further work needs to be done to characterize how these histone modifications are altered under stress and they contribute to the role of PBRM1 in stress response.