Browsing by Subject "B cells"
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Item Antibody Repertoires in Humanized NOD-scid-IL2Rγnull Mice and Human B Cells Reveals Human-Like Diversification and Tolerance Checkpoints in the Mouse(Public Library of Science, 2012-04-27) Ippolito, Gregory C.; Hoi, Kam Hon; Reddy, Sai T.; Carroll, Sean M.; Ge, Xin; Rogosch, Tobias; Zemlin, Michael; Shultz, Leonard D.; Ellington, Andrew D.; VanDenBerg, Carla L.; Georgiou, GeorgeImmunodeficient mice reconstituted with human hematopoietic stem cells enable the in vivo study of human hematopoiesis. In particular, NOD-scid-IL2Rγnull engrafted mice have been shown to have reasonable levels of T and B cell repopulation and can mount T-cell dependent responses; however, antigen-specific B-cell responses in this model are generally poor. We explored whether developmental defects in the immunoglobulin gene repertoire might be partly responsible for the low level of antibody responses in this model. Roche 454 sequencing was used to obtain over 685,000 reads from cDNA encoding immunoglobulin heavy (IGH) and light (IGK and IGL) genes isolated from immature, naïve, or total splenic B cells in engrafted NOD-scid-IL2Rγnull mice, and compared with over 940,000 reads from peripheral B cells of two healthy volunteers. We find that while naïve B-cell repertoires in humanized mice are chiefly indistinguishable from those in human blood B cells, and display highly correlated patterns of immunoglobulin gene segment use, the complementarity-determining region H3 (CDR-H3) repertoires are nevertheless extremely diverse and are specific for each individual. Despite this diversity, preferential DH-JH pairings repeatedly occur within the CDR-H3 interval that are strikingly similar across all repertoires examined, implying a genetic constraint imposed on repertoire generation. Moreover, CDR-H3 length, charged amino-acid content, and hydropathy are indistinguishable between humans and humanized mice, with no evidence of global autoimmune signatures. Importantly, however, a statistically greater usage of the inherently autoreactive IGHV4-34 and IGKV4-1 genes was observed in the newly formed immature B cells relative to naïve B or total splenic B cells in the humanized mice, a finding consistent with the deletion of autoreactive B cells in humans. Overall, our results provide evidence that key features of the primary repertoire are shaped by genetic factors intrinsic to human B cells and are principally unaltered by differences between mouse and human stromal microenvironments.Item Comparative systemic analysis of human immunoglobulin repertoires(2019-01-31) King, Gregory Ryan; Georgiou, GeorgeThe humoral immune system is majorly composed of B cells producing effector immunoglobulin molecules, the vast diversity of which allow for the neutralization of pathogenic threats never previously seen by the immune system. High-throughput sequencing technology has allowed this vast repertoire to be characterized and quantified, but understanding this complex system requires methods of comparison to identify and differentiate B cell populations. In this thesis, differences between groups of repertoires within individuals are analyzed at both the cellular and proteomic level. Novel experimental techniques and visualization methods will allow for the analyses of several such high-dimensional complex systems, leading to a fuller picture of the B cells’ contribution to the immune system.Item Functional studies of BCL11A: a transcriptional repressor implicated in chromosome 2p13-disrupted malignancy(2002) Liu, Hui; Tucker, Philip W.Many malignancies of mature B lymphocytes are characterized by chromosomal translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32.2, resulting in the deregulation of the translocated proto-oncogene. Although t(2;14)(p13;q32.3) is a rare event in B cell malignancies, gains and amplifications of chromosome 2p13 have been reported in 20% of extranodal B cell non-Hodgkin’s lymphoma (B-NHL) and in Hodgkin’s disease (HD). In previous studies, we have identified a novel Krüppel zinc finger gene BCL11A and showed it to be disrupted and deregulated in four cases of B cell malignancies with t(2;14)(p13;q32.3). Three major alternatively spliced isoforms of BCL11A have been detected: BCL11Axl (~120 kD), BCL11Al (~100 kD) and BCL11As (~30 kD). All three mRNAs were upregulated as a consequence of the t(2;14)(p13;q32). Here we confirm that at the protein level, BCL11A isoforms are overexpressed in malignant cells in which the 2p13 locus is genetically disrupted. We also demonstrate that within normal immune tissues, BCL11A accumulates preferentially in germinal centers. In addition, all three isoforms can physically interact with each other. Both BCL11Axl and BCL11Al can colocalize and physically interact with another human B cell proto-oncogene BCL6. BCL11A isoforms potentiate transcriptional repression activity, which is trichostatin A and BCL6 independent. BCL11Axl and BCL11Al but not BCL11As can induce apoptosis in both lymphoid and nonlymphoid cell lines. The apoptotic effect is independent of BCL6, but can be moderately enhanced by p53, and completely blocked by the overexpression of Bcl-2 or a caspase-9 inhibitor. Potential target genes for BCL11Axl were identified by screening Lymphochip microarrays. 28 out of 39 target genes were down-regulated by BCL11Axl. A number of genes functioning in signal transduction, cell cycle control and apoptosis were identified, including Akt2, ATR, Trio, NFκB, and MDM2. Based on the data cited above, we propose that BCL11A induces apoptosis through blockage of the PI3K-Akt cell survival pathway. Although the mechanism by which deregulated BCL11A causes B cell malignancies remains elusive, we suggest that the inhibition of cell cycle checkpoint pathways and Rac-Rho signaling pathways via down regulation of ATR and Trio might play critical roles in BCL11A tumorigenesis.Item Identification and characterization of a positive regulatory region for activation induced cytidine deaminase mediated gene conversion in chicken B cells(2009-12) Kim, Yonghwan, 1975-; Tian, Ming, Ph. D.; Tucker, Philip W.; Paull, Tanya T.; Iyer, Vishwanath; Yin, WhitneyB cells have unique machinery to make up a large pool of antibody repertoire. After V(D)J recombination in early B cell development, the rearranged immunoglobulin genes are further diversified by somatic hypermutation (SHM), gene conversion (GC) and class switch recombination (CSR). Acitvation induced cytidine deaminase (AID) is a key initiating factor for SHM, GC and CSR. A majority of research data supports the model that AID modifies Ig genes at the DNA level by deaminating cytosines to uracils. The mutagenic activity of AID is largely restricted to Ig genes to avoid genomic instability in general. The specificity cannot be attributed to the primary sequence of the Ig genes since unrelated DNA is mutated by AID in the context of Ig genes. A clue to this problem is that AID function is dependent on transcription. Since not all transcribed genes are mutated by AID, there must be something special about the transcription of Ig genes, and the reasoning has prompted extensive analysis of Ig promoters and enhancers. We addressed this question in chicken B cell line DT40. We identified a 2.4-kilobase regulatory region which is important for AID function both within and outside of Ig locus. This regulatory region contains binding sites for multiple transcription factors. Mutation of these binding sites impairs AID mediated gene conversion. In addition, ablation of NF-κB family member, c-Rel and p50, reduces the AID targeting function of this regulatory region. Since the implicated transcription factors have been reported to associate with histone acetylases, the regulatory region may function by facilitating the access of AID to target DNA. To test this hypothesis, we used the I-SceI endonuclease and dam methylase as probes for chromatin structure. We found that the regulatory region does not increase chromatin accessibility to these probes. In fact, the regulatory region appears to interfere with the cleavage of target DNA by I-SceI. Another possible role of the regulatory region could be direct recruitment of AID to Ig genes. To test this hypothesis, we utilized Dam identification method. Surprisingly, we found that the regulatory region facilitates AID targeting to the Igλ locus.Item Structure-functional analyses of Bright, a B cell regulator of immunoglobulin heavy chain transcription(2004) Kim, Dongkyoon; Tucker, Philip W.Bright is a B cell specific trans-activator that regulates IgH gene transcription by binding promoter and enhancer-associated MARs within the IgH gene locus. Domains important for Bright function include the ARID, which mediates sequence-specific binding to DNA and REKLES, a highly conserved but less well understood region within Bright and its two paralogues, Bdp (Bright and dri-like protein) and Bright-like. This thesis further explores features of Bright with respect to the role of these domains in cellular localization, protein-protein interactions, and transcriptional activation. Unexpectedly for a transcription factor, Bright accumulates to significant levels within the cytoplasm as well as the nucleus. Bright appears to be actively exported in a CRM-1 dependent manner. The nuclear localization of Bright is mediated by residues in the N-terminus of the REKLES domain (REKLESa), whereas nuclear export is mediated by the C-terminal region of RECKLES (REKLESb) along with the C-terminal 19 amino acids. Bright accumulates in the nucleus as a result of the loss of its nuclear export activity. This nuclear accumulation also correlates with the cell cycle arrest at G2/M phase. The REKLESb domain also mediates self-association of Bright or heteromeric association of Bright and Bdp. Point mutations in the REKLESb domain abolish the DNA-binding and nuclear export activity of Bright. In addition, Bdp appears to interact with Bright and to facilitate the nuclear localization and retention Bright. DNA binding and trans-activation studies indicate that the promoter-associated MARs repress IgH transcription in non-B cells, and Bright alleviates this effect. Promoter-MAR mediated Bright trans-activation is antagonized by direct competition of MAR DNA binding by the ubiquitously expressed repressor complex NF-mNR. However, it was found that NF-mNR includes Bright in B cells but not in non-B cells. The binding activity of NF-mNR and Bright in B cells is reciprocally altered during the cell division cycle and by the B cell mitogen LPS. LPS treatment had no effect on Bright localization but increased the total amount of Bright in the nucleus and cytoplasm. The increased level of Bright appears to displace NF-mNR from the MARs and to facilitate IgH gene transcription.