Browsing by Subject "B-Lymphocytes"
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Item Acyloxyacyl Hydrolase: Studies on Its Regulation and Function in Mus Musculus(2004-01-14) Lu, Mingfang; Munford, Robert S.Acyloxyacyl hydrolase (AOAH) is an enzyme that detoxifies Gram-negative bacterial lipopolysaccharides (LPS) by selectively removing secondary acyl chains from the lipid A moiety. Originally found in neutrophils, it is also produced by monocyte-macrophages and renal proximal tubule cells. In the studies described here, I found that both immature dendritic cells (DCs) of the XS52 cell line and bone marrow-derived DCs produce AOAH. AOAH expression decreased when DCs were incubated with IL-4, IL-1ᬠTNFa and an agonistic CD40 antibody (maturation cocktail), and increased following treatment with microbial agonists that engage 3 distinct Toll-like receptors (LPS, TLR4; CpG oligodeoxynucleotides, TLR9; and a Gram-positive bacterium (Micrococcus luteus), TLR2). Maturation cocktail treatment also diminished, while LPS treatment enhanced or maintained, the cells' ability to kill E. coli, deacylate LPS, and degrade bacterial proteins. Enzymatic deacylation of LPS is thus an intrinsic, regulated mechanism by which DCs may modulate host responses to this potent bacterial agonist. To study the biological functions of AOAH, AOAH-deficient mice were generated by targeted gene disruption. AOAH did not protect mice from lethal doses of LPS or Gram-negative bacterial challenge. In response to subcutaneous injections of LPS, however, AOAH-deficient mice produced significantly higher levels of non-specific (polyclonal) IgM and IgG3 than did wild type mice. Anti-double-stranded DNA and anti-nucleosome IgM and IgG antibody levels were also higher in LPS-immunized AOAH-deficient mice than in wild type control mice. In addition, the partially-deacylated LPS product (dLPS) induced lower polyclonal antibody responses in vivo than did mock-treated LPS, yet the anti-LPS specific responses to dLPS and LPS were equivalent. These results suggest that AOAH may diminish potentially harmful polyclonal antibody responses to Gram-negative infection but maintain the protective anti-LPS specific response. Since B cells do not produce the enzyme, my results also point to an important role for macrophages and DCs in modulating B-cell responses to LPS antigens. In addition, the absence of AOAH did not alter the ability of LPS to function as an adjuvant, indicating that this activity is mechanistically distinct from stimulation of polyclonal antibody production. Finally, the ability of a bacterial lipopeptide to stimulate polyclonal antibody production only in AOAH -/- mice suggests that the enzyme may also regulate immune responses to non-LPS bacterial agonists.Item B Cell Modulation of T Cell Responses in Multiple Sclerosis(2010-01-12) Harp, Christopher Todd; Monson, Nancy L.Until recently, a definitive role for B cells in the pathogenesis of the autoimmune neurological disorder multiple sclerosis (MS), had not been widely accepted, and remains poorly understood. B cells have multiple functions in the immune system and can both positively and negatively modulate immune responses through the production of antibody, cytokine secretion, and/or antigen presentation. Several studies indirectly suggest that B cell-T cell cooperation may be paramount in MS disease pathogenesis, although this interaction has not been well studied in MS. Therefore, the focus of my thesis project was to test the hypothesis that B cells could be efficient neuro-antigen presenting cells in the context of MS. My work has demonstrated that the cerebrospinal fluid (CSF) B cell population in MS shows characteristics of both auto-reactivity and antigen driven selection in a germinal center reaction. These findings suggest that neuro-antigen driven selection had occurred in the periphery and prompted investigation of B cells as neuro-antigen presenting cells. Examination of CD40 ligand (CD40L) and interleukin-4 (IL-4) activated peripheral B cells demonstrated for the first time that B cells could efficiently elicit myelin basic protein (MBP) specific CD8+ and CD4+ T cell proliferation from resting T cells in vitro through a mechanism that was partially dependent on presentation through HLA-DR. Further inquiry into the antigen presentation capacity of specific subpopulations of resting B cells revealed that memory B cells from MS patients (but not healthy donors (HDs)) were significantly better neuro-antigen specific presenting cells than their na?ve counter parts. This data indicated that a specific peripheral immune response had been generated in response to neuro-antigens in RRMS patients but not HDs. Taken together, these data provide a model where antigen experienced peripheral B cells from MS patients (but not HDs) provide important T cell support through antigen presentation and add to our understanding of the role of B cells in the pathogenesis of this autoimmune disease of the CNS.Item B Cell Signaling and Bioinformatics: Revealing Components of the MHC Class II Antigen Processing and Presentation Pathway(2005-05-11) Lee, Jamie Ann; Scheuermann, Richard H.Stimulation of mature B lymphocytes by extracellular ligands induces phenotypic changes through complex signal transduction pathways. Gene expression is altered as a result of these changes and re-programs the cell to undergo differentiation, activation, effecter function, anergy, and/or apoptosis. Gene expression microarrays are used to determine expression levels of a large number (tens of thousands) of genes simultaneously, resulting in a gene expression profile of the experimental sample. Microarray data must be appended with biological information in order to be interesting, and this field of microarray bioinformatics is rapidly expanding. These studies prompted the development of a bioinformatics tool termed CLASSIFI (Cluster Assignment for Biological Inference), which identifies statistically significant co-clustering of genes with similar Gene Ontology annotation within microarray gene clusters. CLASSIFI was used to analyze microarray results from two B cell projects from the Alliance for Cellular Signaling (AfCS): 1) the BAFF/CD40L project, which evaluates the effects of BAFF and CD40L on primary mouse B cells in long-term cultures, and 2) the B cell single ligand screen project, which evaluates the effects of 32 single ligands on primary mouse B cells in short-term cultures. CLASSIFI was able to identify significant overrepresentation of related genes within gene clusters for both of these data sets and facilitates hypothesis generation as to the biological process affected by a specific ligand. As CLASSIFI is strictly a statistical tool that aids in hypothesis generation, experimental validation of hypotheses was performed. The B cell single ligand screen microarray and CLASSIFI analysis followed by experimental validation revealed a biological process specific to B cell antigen receptor stimulation but not LPS or CD40L stimulation - antigen processing and presentation - and provides the groundwork for new discoveries in this field. As a result, several putative components were identified that are not currently known to play a role in antigen processing and presentation in B cells.Item B-Cell Adapter for Phosphoinositide 3-Kinase Is a Signaling Adapter in the Toll-Like Receptor/Interleukin-1 Receptor Superfamily(2014-02-17) Troutman, Ty Dale; van Oers, Nicolai S. C.; Pasare, Chandrashekhar; Hooper, Lora V.; Chen, Zhijian J.; Krämer, HelmutToll-like receptor (TLR)/Interleukin-1 receptor (IL1R) superfamily members share signaling components and (with the exception of TLR3) depend on the adapter myeloid differentiation primary response gene 88 (MyD88) for engagement of downstream pathways. Signals from the receptor to the adapter are transmitted through homotypic interaction of TIR (Toll-Interleukin-1 receptor) homology domains found in all TLR/IL1R family members and their adapters. The present work defines a novel TLR/IL1R signaling adapter, termed BCAP (B-cell adapter for PI3K), which was identified based on the presence of a cryptic N-terminal TIR domain. I show here that BCAP (B-cell adapter for PI3K) contains a functional TIR domain enabling its participation in the TLR signaling pathway. Through its TIR domain, BCAP associates with the TLR/IL1R signaling adapter MyD88, as well as the TLR signaling adapter toll-interleukin 1 receptor domain containing adapter protein (TIRAP). Importantly, BCAP plays an obligate role in linking TLRs to activation of phosphoinositide 3-kinase (PI3K) through recruitment of PI3K to the signaling complex and relief of inhibitory influences on PI3K activity. Importantly, BCAP selectively mediates TLR signaling towards the PI3K branch without affecting signaling to NFκB nor MAP kinases. In this capacity, BCAP inhibits secretion of inflammatory cytokines and regulates susceptibility to inflammatory colitis. Because the TLR/IL1R family shares signaling components, BCAP may also function in IL1R family signaling. To test this hypothesis, T cells were chosen as a model cell type responding to IL1R family signals. T helper cells utilize IL18 and IL1 (which engage the IL18R or the IL1R respectively, both IL1R family members) cytokines provided by myeloid cells to achieve optimal Th1 and Th17 effector capacities. I show here that BCAP intrinsically regulates differentiation of naïve T cells towards Th1 and Th17 effector lineages by participation in the IL1R family signaling pathways. Further, BCAP intrinsically regulates both T cell proliferation and survival during priming. The significance of this work lies in the revelation of a TLR signaling adapter serving as a node connecting TLRs to PI3K. Further, the findings here will increase the understanding of key signaling pathways involved in disease and inflammation.Item Clinical Diagnostic Potential and Characterization of Distinctly Hypermutated Antibodies in Multiple Sclerosis Patients(2016-08-12) Rounds, William Harold Alexander; Cowell, Lindsay G.; Monson, Nancy L.; Wakeland, Edward K.; Patrie, Steven M.; Ward, E. SallyMultiple sclerosis (MS) diagnosis primarily revolves around the use of brain lesion detection by MRI and the elimination of other possible neurological disorder diagnoses through clinical testing and history. For many patients first experiencing clinical symptoms that could be MS-related, this presents a challenge since diagnostic certainty based on clinical presentation and testing does not always reach a consensus among doctors who evaluate them. With a growing body of evidence for B cell involvement and dysregulation in MS, our group investigated and identified a potential biomarker in the cerebrospinal fluid of patients with MS based on B cell antibody sequencing. This work first identified a distinct mutation pattern in the antibody sequences of CSF-derived B cells, termed the antibody gene signature (AGS), that could be used to identify patients with MS or patients who would convert to MS subsequent to their first onset of clinically detectable symptoms. This thesis project outlines the transition from AGS testing in a laboratory setting to its use and implementation as an additional clinical diagnostic tool for MS (MSPrecise®) using next generation sequencing (NGS). One of its main goals is to thoroughly evaluate the performance of MSPrecise® using the far greater throughput which NGS allows for. Over the course of the project, NGS technology and accuracy optimization methods have advanced significantly. As our laboratory is the first to ever utilize NGS for somatic hypermutation evaluation, we focused strongly on the evaluation of challenges and features associated with NGS use for immune repertoire diversity and somatic hypermutation profiling of clinical samples. In this context, this project also highlights observations on sequence library preparation and post-sequencing data filtering that affect all immune repertoire research that uses these rapidly developing sequencing platforms.Item Dysregulated B Cells in Relapsing Remitting Multiple Sclerosis and Their Impact on T-Cell Function(2015-03-10) Ireland, Sara Jean; Monson, Nancy L.; Farrar, J. David; Davis, Laurie; van Oers, Nicolai S. C.; Kasper, LloydRelapsing-remitting multiple sclerosis (MS) is an autoimmune mediated inflammatory demyelinating disorder of the central nervous system. The role of B cells in the pathoetiology of MS is substantiated by cell depletion therapies but is not well understood. We hypothesized that B cells from MS patients would secrete more pro-inflammatory cytokines and support T cell responses to self-antigens and that the therapeutic agent glatiramer acetate (GA) could modulate these aspects of B cell function. To test this hypothesis we interrogated the ability of memory and naive B cells from healthy donors (HD), MS patients, and GA-treated MS patients (GA-MS) to proliferate and secrete cytokines in vitro. We identified a remarkable loss of IL-10 secretion by B cells from MS patients, but a marked increase in the production of IL-6, particularly from naïve B cells. We also found that memory B cells from MS patients exhibited hyperactive proliferation compared to healthy donors and naïve B cells from MS patients. GA had no measurable impact on any of the B cell functions we tested; however, B cells from GA-treated MS patients had a restored ability to produce IL-10, greatly enhanced immunoglobulin production, altered proliferation capacity and a transient diminishment of IL-6 production for patients on therapy for less than 32 months. To address whether memory or naïve B cells from MS patients supported neuro-antigen specific T cell responses, we co-cultured B and T cells in the absence or presence of foreign or neuro-antigens. We found that memory and naïve B cells from MS patients support more CD4+ T cell proliferation and TH1 and TH17 responses to neuro-antigens despite a similar frequency of neuroantigen specific T cells. Together, these data reveal that B cells from MS patients exhibit dysregulated proliferation and cytokine secretion that can be modulated by GA. Furthermore B cells from MS patients support neuroantigen-specific T cell proliferation and pro-inflammatory cytokine production in response to self neuro-antigens.Item Evidence of B Cell Dysregulation in Early Multiple Sclerosis Patients(2017-04-04) Rivas, Jacqueline Ruth; Satterthwaite, Anne B.; Monson, Nancy L.; Stowe, Ann; Cowell, Lindsay G.; Greenberg, Benjamin M.Plasmablasts are a highly differentiated, antibody secreting B cell subset whose prevalence correlates with disease activity in Multiple Sclerosis (MS). For many patients experiencing partial transverse myelitis symptoms, plasmablasts are elevated in the blood and cerebrospinal fluid (CSF) at the first clinical presentation of disease. Plasmablasts are a transient subset, representing the portion of B cells currently participating in an antibody-mediated immune response. However, it has not been investigated whether these cells have the potential to participate in the autoimmune response through the expression of autoreactive receptors. In these studies, we found genetic evidence of B cell dysregulation in early MS patients, likely from a loss of peripheral tolerance, and the development of affinity maturated, autoreactive plasmablasts. Plasmablasts from these early MS patients over-utilize immunoglobulin heavy chain V-region subgroup 4 (VH4) genes, exhibit excess light chain receptor editing, and have increased mutation accumulation in IgG utilizing VH4+ cells. Many highly mutated antibodies utilizing VH4 gene segments from both CSF B cells and peripheral plasmablasts recognize neurons and glial cells. Certain peripheral cells are polyreactive, while those in the CSF are typically specific for central nervous system antigens. Other V gene families have the potential for autoreactivity as well, although the strongest binding was observed in VH4+ antibodies. The peripheral plasmablast response is directed toward cytoplasmic neuronal antigens, and this autoreactivity is detectable in the serum IgG antibody pool. Interestingly, certain mutations in six key codons along the VH4 domain correlate with polyreactivity, neuron reactivity, or glial cell reactivity. Previous work identified that the prevalence of mutations at these codons in CSF B cells predicts conversion to MS, demonstrating their likely role in progression of disease. Plasmablasts may provide a useful biomarker of B cell activation in MS, or may be direct participants in autoimmunity. In either case, the study of plasmablasts provides insight to the development of the autoreactive B cell response in early MS patients.Item The molecular basis of X-linked human primary immunodeficiency disease(1993-06-24) Capra, J. DonaldItem Recent developments in the non-Hodgkins lymphomas(1988-03-03) Smith, R. GrahamItem The Role of Foxo3 in B Cell Tolerance(2017-01-17) Barrios, Evan; Ottens, Kristina; Hinman, Rochelle; Skaug, Brian; Castrillion, Diego; Satterthwaite, AnneB cells secrete antibodies in order to help defend the body against pathogens. Due to the enormous diversity of B cell receptors required to recognize pathogens, some self-reactive receptors are generated. It is important for the immune system to prevent autoimmune cells from attacking one's self. This can be done at the immature B cell stage through receptor editing, deletion of the B cell, or anergy (inactivation) of the B cell. During editing, the receptor recombines its components in order to generate a new receptor, which is tested for auto-reactivity. If the receptor is strongly auto-reactive after editing, the cell dies by apoptosis. Foxo3 is a transcription factor that participates in pro-apoptotic pathways in several cell types. Previous work in our lab showed that apoptosis is reduced in immature B cells from Foxo3-/- mice, and others have observed decreased levels of Foxo3 in B cells from mouse models of lupus (an autoimmune disease in which B cells produce antibodies reactive against the body's own DNA). It is hypothesized that edited cells that remain auto-reactive may survive inappropriately in the absence of Foxo3. B cells that have undergone receptor editing are more likely to express the Igλ light chain and to have undergone a process called "RS recombination." Foxo3-/- mice were found by flow cytometry to exhibit increased Igλ+ B cells in the immature B cells of the bone marrow, as well as in the transitional B cells of the spleen. PCR demonstrated increased RS recombination in Igλ+ B cells from Foxo3-/- mice compared to wild-type mice. Anti-double-stranded DNA ELISAs were run on supernatants from total B cells stimulated by LPS to secrete antibodies. These showed no difference in auto-reactivity between wild-type mice and Foxo3-/- mice. Thus, there appears to be an increase in receptor editing in the knockout mice, but not an overall increase in the auto-reactivity of the total population of B cells. This suggests that the reduced apoptosis of Foxo3-/- immature B cells allows cells that were originally auto-reactive a longer window of time in which to edit their receptors away from auto-reactivity.Item Role of Nlc Cells in Murine Models of T Cell-Dependent Responses(2007-12-17) Jennings, Paula Alessandra; Yuan, DorothyNK cells are part of the innate immune system, yet they can also modulate the acquired immune response. Activated NK cells, for instance, can increase antigen specific IgG2a production in response to T independent responses, mostly through IFN-gamma secretion. Previous experiments examining the effect of NK cells on T cell- dependent antigens in various laboratories have yielded inconsistent conclusions. Therefore attempts were made to further investigate this question. Whereas depletion of NK cells had no detectable effect on the response to a TD antigen in Ribi adjuvant injected intraperitoneally, the secondary IgG1 response can be significantly reduced. This result suggested a role for NK cells on the generation of memory T cells. Therefore experiments were initiated to investigate the effect of NK cells on T cell proliferation. The absence of NK cells during immunization was found to reduce primary T cell proliferation. Such effect was not observed when B cell antigen presentation was absent, which is known to be important for memory T cell generation. A direct effect of NK cells on B cell antigen presentation was assessed in vitro. These experiments showed that NK cells can upregulate B cell antigen presentation of ovalbumin to na?T cells expressing a transgene specific to the ovalbumin derived peptide, OVAp. This increase is contact dependent and can occur in the absence of IFN-gamma . Moreover, the NK cell enhancement of B cell presentation of intact protein was greater than the presentation of OVAp, which requires no processing. These experiments suggest that the upregulation involves both processing and presentation of antigen. These experiments show that NK cells have a direct effect on B cell antigen presentation and provide a mechanistic basis for the role of NK cells in modulating in vivo T cell- dependent antibody responses.Item Using B Cell Characteristics as Predictors of Multiple Sclerosis in Clinically Isolated Syndrome Patients(2009-06-15) Cameron, Elizabeth; Monson, Nancy L.Clinically isolated syndrome (CIS) is the diagnosis of patients who have experienced a single event due to nerve demyelination of the white matter of the central nervous system. This can be due to numerous causes, both autoimmune and infectious. We hypothesized that CIS patients with B cell characteristics like those seen in multiple sclerosis (MS) patients would develop clinically definite MS. We have determined that, like MS patients, several CIS patients have an increased frequency of VH4-expressing CD19\super +\nosupersub B cells in their cerebrospinal fluid (CSF) compared to peripheral B cells from healthy donors (HCPB) or CSF B cells from patients with neurological diseases not related to MS. However, VH4 bias was a moderate predictor for conversion to MS. Nevertheless, detailed analysis of antibody V-gene repertoires revealed eight codons that are significantly more mutated in the MS CSF than HCPB VH4-expressing B cells. This MS-specific antibody signature includes 25% of all mutations within the repertoires of CSF-derived B cells from MS patients. We then used the prevalence of this signature to predict if CIS patients converted to MS within two years of repertoire sampling. Indeed, we accurately predicted conversion to MS in 10 of 11 CIS patients. The B cell VH4 antibody signature can potentially be used as a diagnostic and prognostic tool for MS.Item [UT Southwestern Medical Center News](2013-02-24) Carlton, Jeffrey