• Analysis of in vivo expression and function of Src-family protein tyrosine kinases during lymphocyte development

      Longo, Nancy S.; Abraham, Kristin M. (1999)
      Src-family protein tyrosine kinases (PTKs) are critical components of T and B lymphocyte signaling cascades. To examine the relationship between Src-PTK expression and lymphocyte development, we defined quantitative and qualitative expression patterns of Src-PTKs during lymphocyte ontogeny. A quantitative RT-PCR assay was designed to distinguish transcripts derived from the lck proximal (Type I) vs distal (Type II) promoter elements, and fynT vs fynB isoforms. These assays were then used to measure lck and fyn expression in normal fetal and adult tissue. Changes in Type I and Type II promoter utilization correlate with pre-TCR and alphabetaTCR expression suggesting that as receptor complexes develop they couple to different intracellular signaling cascades that differentially regulate lck expression. The fynT isoform, not fynB, is detected in both lineages, including pre-T and pre-B cells, suggesting a role for Fyn in lymphocyte development before TCRalphabeta or IgM expression. In spite of much lower levels of fynT and lck transcripts in the B cell lineage, compared to lck transcripts in thymocytes; accumulation of lck and fynT correlates temporally with acquisition of immune receptor expression in both lineages. In addition to immune receptors, Src family PTKs are activated by receptors regulating growth and differentiation in thymic stroma which is required for lymphocyte development. To examine the impact of Src-PTK function in thymic stroma on lymphocyte development, a transgenic model system was developed in which the expression of polyoma virus middle T antigen (PymT), a molecule that activates Src-PTKs, was directed to thymic subcapsular epithelial cells using a mutant Class II promoter element. PymT expression constitutively activated endogenous Src-PTKs which phosphorylated PymT, inducing a PymT association with p85, the regulatory subunit of PI 3-kinase. Src-PTK activation was associated with changes in tyrosine phosphorylation of cellular proteins. Transgene expression in subcapsular stroma resulted in upregulation of epidermal growth factor (EGF) receptor and 4 to 5-fold thymic lymphoid hyperplasia. However, normal thymic architecture, proliferation and apoptotic responses, thymocyte differentiation, repertoire selection, and thymic involution were maintained. These findings suggest that the level and/or persistence of signals generated via Src-PTKs in thymic subcapsular epithelium serve as major defining factors influencing homeostatic control of thymic organ size and cellularity.
    • Differential FOXO1 Localization in SLE and Healthy Human Lymphocyte Subsets

      Hritzo, Molly K.; Golding, Amit; 0000-0001-9614-732X (2018)
      Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease characterized by elevated levels of circulating autoantibodies and multi-organ damage. Although SLE is a highly heterogeneous disease, one factor unifies it: lymphocyte hyperactivity driving immunopathogenesis. This involves CD4 helper T cells potentiating autoreactive B cells to produce pathogenic autoantibodies. In healthy individuals, lymphocyte activation is a closely regulated kinetic process controlled by key transcription factors (TF) signaling downstream of the T cell (TCR) and B cell receptor (BCR). Forkhead box O1 (FOXO1) is one such TF that integrates activation and differentiation signals in human lymphocytes. When active, it remains in the nucleus, but upon Akt phosphorylation downstream of TCR or BCR signaling, FOXO1 is inactivated and shuttles to the cytoplasm, linking FOXO1 localization to function. In SLE, both T and B cells are hyperactive, and respond more quickly and strongly to antigen, producing a disproportionate inflammatory response. Thus, we hypothesize that SLE lymphocytes will have altered FOXO1 localization, reflecting altered lymphocyte activation. To address this hypothesis, we first developed a method of examining dynamic native FOXO1 localization in human peripheral lymphocyte subsets using imaging flow cytometry (IFC). IFC combines the quantitative power of flow cytometry with the qualitative images of microscopy and can be performed with many fewer cells than are needed for the more traditional methods. We demonstrated that we can visualize native FOXO1 and detect significant kinetic differences in localization within user-defined subsets of HuT102 cells, a human CD4 T cell line with baseline nuclear FOXO1, as well as primary peripheral human T and B cells. We then used IFC to compare FOXO1 localization in SLE and healthy donor lymphocytes. Interestingly, we found that most T and B cell subsets have nuclear FOXO1 localization in both health and SLE. However, FOXO1 is significantly more cytoplasmic in SLE double negative (DN) atypical memory B cells. Based on our findings, we propose a model by which these DN B cells are highly active in disease flares and may serve as a death-resistant reservoir of autoreactive cells. Future experiments will be aimed at elucidating at how these cells persist in the periphery.
    • Direct Suppression by Healthy and SLE Human Tregs of B cell Immunoglobulin Secretion

      Weingartner, Elizabeth; Golding, Amit (2017)
      Regulatory T cells (Tregs) are characterized by their role in maintaining peripheral self-tolerance and immune homeostasis. One such role is regulation of the humoral response, which is most clearly demonstrated by abundant auto-antibody production in Scurfin mice and IPEX patients, both of which lack functional Tregs. Indirect Treg regulation of the humoral response via their influence on helper T cells, in particular in germinal centers, is well established. Prior demonstration of direct Treg inhibition of B cells has also been demonstrated, primarily in mice, but is mostly attributable to Treg killing of B cells. This regulation may be especially relevant in B cell-mediated diseases, such as systemic lupus erythematosus (SLE). SLE is an extremely heterogeneous, autoimmune disease characterized in part by high titers of autoantibodies. We hypothesize SLE Tregs are deficient in their ability to suppress immunoglobulin secretion by B cells relative to Tregs isolated from healthy donors. We addressed this hypothesis by first developing and characterizing a modified suppression assay in which the only variable is the source of Tregs. In this assay, suppression by human Tregs of a homogenous, germinal center like B cell cell line, Ramos, is measured by ELISA for IgM and flow cytometry for cell death. We demonstrate that human Tregs directly suppress IgM by Ramos B cells through a partially contact- and death-independent mechanism. In addition, we demonstrate that pre-stimulation of the Tregs with αCD3/CD28 increases the suppression of IgM secretion but again, is independent of inducing B cell death. After development and characterization of this modified suppression assay, we evaluated the behavior of Tregs isolated from SLE patients in the assay. Interestingly, we found that the average suppression of IgM secretion by SLE Tregs is not statistically different from the average suppression by Tregs isolated from healthy donors. However, we found that SLE Tregs induce death in the Ramos B cells unlike Tregs isolated from healthy donors. Based on our findings, we offer a model by which Treg-induced B cell death might actually promote more SLE disease by further release of auto-antigens. Future experiments will be aimed at elucidating the mechanism and type of cell death.
    • The Evolution of B Cell Selection and Affinity Maturation in Cartilaginous Fishes

      Matz, Hanover Christian; Dooley, Helen, Ph.D.; 0000-0002-0669-1696 (2022)
      Affinity maturation of the B cell immunoglobulin (Ig) repertoire occurs through coordinated somatic hypermutation (SHM) and Darwinian selection of clones in specialized microanatomical structures known as germinal centers (GCs). GCs have only been identified in the endothermic vertebrates, and so it was long presumed that the antigen (Ag)-specific Ig responses of ectothermic vertebrate lineages were “primitive”. However, affinity maturation and immunological memory have subsequently been demonstrated for the oldest extant vertebrate class with Ig-based adaptive immunity, the cartilaginous fishes (Chondrichthyes). In this dissertation, I investigated the cellular model of B cell selection in the nurse shark (Ginglymostoma cirratum) spleen and determined how it influences the dynamics of the Ig repertoire. I found that shark splenic B cell follicles possess many functional analogs of mammalian GCs: (1) segregation of SHM and selection regions by CXCR4/CXCR5 expression in B cells, (2) functional T follicular helper-like cells, (3) presentation of nondegraded Ag, and (4) Ag-driven selection of mutated Ig clones. I also demonstrated that the transcription factor BCL6 likely regulates the shark B cell response. Through a long-term immunization study, I demonstrated that this selection model can generate IgNAR repertoires that are both diverse and of high affinity. In multiple animals immunized by the same methods, I observed uncoupling of the T-dependent isotypes, IgNAR and monomeric IgM. Sharks that produced robust IgNAR titers matured their polyclonal repertoires to subnanomolar binding affinities and generated a diverse pool of memory clones. Together, this suggests that B cell selection in cartilaginous fishes evolved to support both affinity maturation and Ig repertoire diversification, possibly utilizing SHM to anticipate future pathogen variants. Finally, I developed a method of magnetic nanoparticle enrichment to isolate Ag-specific B cell clones directly from the peripheral blood of sharks. Overall, the data presented in this dissertation indicate that all the fundamental components of B cell selection were present at the advent of adaptive immunity in jawed vertebrates. Furthermore, these components were capable of affinity maturation of the B cell repertoire without sacrificing receptor diversity. My findings have many implications for our understanding of the evolution of the B cell response in vertebrate lineages.
    • Molecular identification of a novel seven-transmembrane-domain protein, PB99, expressed at an early stage of B-cell development

      Xu, Wanping; Berman, Jeffrey E. (1999)
      Seven-transmembrane-domain receptors (STRs) comprise the single largest family of cell surface receptors which transmit signals into cells by coupling with heterotrimeric G-proteins; (hence they are alternatively called G-protein coupled receptors, GPCR), and play versatile functions in the body. Some STRs were demonstrated to be involved in the production and/or migration of B-lymphocytes, the cells of the immune system that mediate humoral immune responses. A novel STR gene, PB99, was isolated and was found to be expressed only in cell lines representing the pre-B stage of B-lymphocyte development, which can be divided into four consecutive stages: pro-B, pre-B, mature B, and plasma cells. Full-length mouse and human PB99 cDNAs were identified and sequenced. Both mouse and human cDNAs encode STRs consisting of a relatively long N-terminal extracellular domain with a leader peptide, followed by a region containing seven transmembrane segments, and ending with a short serine/threonine rich C-terminal cytoplasmic domain. Comparison of the deduced amino, acid sequences showed that human PB99 is 78.6% homologous to mouse PB99. The high conservation during evolution suggests that PB99 performs some critical function. Furthermore, the detection of PB99 protein in bone marrow suggests that the gene is functional in the body. Comparison of PB99 to other STRs indicates that it represents a novel subgroup within a group of STRs which bind peptide ligands. The structural similarity and the sequence identity with GPCR suggest that PB99 may transmit a signal via heterotrimeric G-proteins. PB99 is a cell surface molecule with the N-terminus on the extracellular side, as shown by flow cytometric analysis of HEK293 cells transfected with HA epitope-tagged human PB99. The upregulation of PB99 expression in pre-B cells, first observed in transformed mouse pre-B cell lines, was confirmed in normal cells by quantitative RT-PCR, performed with purified cell populations representing different stages of human B-lymphocyte development. The specific increase in PB99 expression in the early stage of B-cell development suggests that this novel STR plays a signaling role during B-cell lymphopoiesis. Surprisingly, PB99 was also found to be highly expressed in non-B-lineage bone marrow cells, indicating that the gene functions in multiple hematopoietic cell lineages.
    • The Role of Glycolysis and Oxidative Respiration in the Survival and Activation of B cells

      Saltis, Mark; Davidson, Wendy F. (2009)
      The activation of B cells in an immune response places significant metabolic demands on the cell for blast transformation, proliferation, and cytokine and antibody production. It is currently unclear how lymphocytes meet these bioenergetic demands, or conversely, how metabolism influences B cell activation. Evidence suggests that mitochondrial function is critical to the activation and survival of B cells in both resting and stimulated states. While the role of the mitochondria in the signaling of other cell types has been established, it is currently unclear how mitochondrial function and metabolism affect B cell signaling and activation. We have investigated the role of the glycolytic and oxidative pathways, metabolic pathways dependent on mitochondrial function, in events downstream of activation in B cells including cell survival, blast transformation, and proliferation, and determined the mechanisms of regulation of metabolism during different stages of activation. We observed two phases of glycolytic and oxidative activity in B cells stimulated with CpG at early and late stages of activation. We show that CpG-related signaling through the Akt and mTOR pathways are associated with increased metabolism early after activation, and that a more significant increase in metabolism occurs as cell cycle progression occurs that is dependent on activation downstream of cell cycle regulatory CDK proteins, and independent of Akt and mTOR pathways. We provide evidence that disruption of the oxidative pathway and mitochondrial function with rotenone induces rapid cell death. Disruption of glycolysis has indirect effect on oxidative activity and reduces cellular activation, raising the possibility that glycolytic inhibition may reduce availability of pyruvate to feed the citric acid cycle, or induce mitochondrial retrograde signaling resulting in indirect effects on cell activation and oxidative respiration. We observed evidence of increased metabolic activity in B cells from a mouse model of systemic autoimmunity, and a significant decrease in lymphoproliferative disease after glycolytic inhibition in vivo combined with a reduction in B cell subsets in autoimmune mice. In gld mice, a unique population of B cells with an activated phenotype is most significantly reduced, while transitional B cells are most affected B cell population in normal BALB/c mice. While interpretation of these data is complex, it suggests a significant role for glycolysis in the in vivo survival of B cells. In summary, utilization and regulation of energy pathways and mitochondrial function are complex processes that can have profound effects on B cell activation and survival, and that enhanced metabolic activity may play a significant role in the B cell immune response and autoimmunity.