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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2019)

B cells are essential players in the humoral immune response, generating both short- and long-lived antibody responses against a wide range of microbes. Rearrangement of the immunoglobulin locus, via V(D)J recombination, leads to vast combinatorial diversity in the B cell receptor (BCR) repertoire. As a result, the precursor frequency of any particular BCR specificity is low, and B cells must clonally expand in order to properly mount an immune response. Despite efficient central tolerance mechanisms, autoreactive B cells persist into the periphery. Thus, B cells must both scale their responses to foreign antigen, and simultaneously prevent inappropriate activation by self-antigens. The molecular mechanisms which restrict B cell responses to self-antigens while facilitating such scaling are incompletely understood.

Nr4a1-3 are primary response genes whose expression is induced by mitogenic stimuli in a variety of cell types. The Nr4a genes encode a small family of orphan nuclear hormone receptors that function, in T cells, to mediate antigen-induced cell death as well as other immunoregulatory roles. Although the Nr4a family are among the most highly induced genes in B cells following BCR stimulation, their function in this context is unknown. In this thesis, we examine the expression and functional role of Nr4a1 (Nur77) in B cell responses following both chronic and acute BCR stimulation.

We utilize two different models of B cell autoimmunity, the IgHEL/sHEL dTg and VH3H9 models, to study the role of Nur77 in the context of chronic antigen stimulation. In both model systems, endogenous Nur77 expression is elevated in autoreactive B cells. Furthermore, through use of the Nur77-eGFP reporter in which eGFP is placed under the control of the regulatory regions of the Nr4a1 gene, we find that Nur77-eGFP expression correlates with the induction of central and peripheral tolerance mechanisms. Although Nur77 expression was dispensable for receptor editing, central deletion, follicular exclusion, IgM downregulation and anergic BCR signal transduction, we identified a specific role for Nur77 in mediating antigen-induced cell death of self-reactive B cells in the periphery. Nur77-deficient anergic B cells exhibited a survival advantage in vitro and in vivo that was dependent upon antigen and a limiting supply of the B cell survival factor BAFF. Thus, we identified a role for Nur77 in regulating competitive fitness of anergic B cells in the periphery. In the VH3H9 model, loss of Nur77 expression leads to an expansion in the overall size of the B cell compartment, and is sufficient to break tolerance.

In the context of acute antigen receptor signaling, we identify a role for Nur77 in restraining the survival and proliferation of B cells which receive BCR stimulation (signal 1) alone, thus enforcing reliance upon costimulatory signals (signal 2). Here, we find that endogenous Nur77 is rapidly and robustly induced following BCR stimulation, and its induction scales with both the amount and affinity of antigen. Through the use of mixed in vitro competitive co-cultures, we find that Nr4a1-/- B cells out compete Nr4a1+/+ B cells in response to BCR stimulation alone. However, this competitive advantage is lost in the presence of costimulatory signals, alone or in conjunction with BCR stimulation. Accordingly, we find increased titers of antigen specific antibody in Nur77-deficient mice immunized with TI-II but not TI-I or T-dependent immunizations. Finally, we identify a competitive advantage of Nr4a1-/- B cells in vivo in response to T-dependent immunizations in contexts where antigen is limiting. Thus, we find that Nur77 acts to restrain B cell responses when B cells receive BCR signaling alone, rendering B cells reliant upon “signal 2”, and also acts to restrain B cells in situations where antigen and access to signal 2 is limiting.

    Cover page: Negative regulation of B cell responses by Nur77 (Nr4a1)
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2019)
    It has long been appreciated that highly autoreactive BCRs are actively removed from the developing B cell repertoire by Ag-dependent receptor editing and deletion. However, there is persistent debate about whether mild autoreactivity is simply tolerated or positively selected into the mature B cell repertoire as well as at what stage, to what extent, under what conditions, and into which compartments this occurs. In this study, we describe two minor, trackable populations of B cells in B1-8i Ig transgenic mice that express the VH186.2 H chain and recognize a common foreign Ag (the hapten 4-hydroxy-3-nitrophenylacetyl) but differ in L chain expression. We use the Nur77-eGFP reporter of BCR signaling to define their reactivity toward endogenous Ags. The less autoreactive of these two populations is strongly counterselected during the development of mature B1a, follicular, and marginal zone B cells. By genetically manipulating the strength of BCR signal transduction via the titration of surface CD45 expression, we demonstrate that this B cell population is not negatively selected but instead displays characteristics of impaired positive selection. We demonstrate that mild self-reactivity improves the developmental fitness of B cell clones in the context of a diverse population of B cells, and positive selection by endogenous Ags shapes the mature B cell repertoire.
      Cover page: Optimal Development of Mature B Cells Requires Recognition of Endogenous Antigens.
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2021)
      B cell clones compete for entry into and dominance within germinal centers (GCs), where the highest-affinity B cell receptors (BCRs) are selected. However, diverse and low-affinity B cells can enter and reside in GCs for extended periods. To reconcile these observations, we hypothesize that a negative feedback loop may operate within B cells to preferentially restrain high-affinity clones from monopolizing the early GC niche. Here, we report a role for the nuclear receptor NUR77/Nr4a1 in this process. We show that NUR77 expression scales with antigen stimulation and restrains B cell expansion. Although NUR77 is dispensable for regulating GC size when GCs are elicited in a largely clonal manner, it serves to curb immunodominance under conditions where diverse clonal populations must compete for a constrained niche. We propose that this is important to preserve early clonal diversity in order to limit holes in the post-immune repertoire and to optimize GC selection.
        Cover page: Negative feedback by NUR77/Nr4a1 restrains B cell clonal dominance during early T-dependent immune responses
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2017)
        B-1a cells are a unique population of innate-like B cells with a highly restricted and self-reactive BCR repertoire. Preimmune "natural" IgM produced by B-1a-derived plasma cells is essential for homeostatic clearance of cellular debris and forms a primary layer of protection against infection. In this study, we take advantage of a fluorescent reporter of BCR signaling to show that expression of the orphan nuclear hormone receptor Nur77 is upregulated under steady-state conditions in self-reactive B-1a cells in response to chronic Ag stimulation. Nur77-deficient mice exhibit elevated natural serum IgM (but not IgG) and marked expansion of IgM plasma cells of B-1a origin. Moreover, we show that Nur77 restrains the turnover of B-1a cells and the accumulation of immature IgM plasma cells. Thus, we identify a new critical negative-regulatory pathway that serves to maintain B-1a cells in a quiescent state in the face of chronic endogenous Ag stimulation.
          Cover page: Nur77 Is Upregulated in B-1a Cells by Chronic Self-Antigen Stimulation and Limits Generation of Natural IgM Plasma Cells
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2019)
          Nur77 (Nr4a1) belongs to a small family of orphan nuclear receptors that are rapidly induced by BCR stimulation, yet little is known about its function in B cells. We have previously characterized a reporter of Nr4a1 transcription, Nur77-eGFP, in which GFP expression faithfully detects Ag encounter by B cells in vitro and in vivo. In this study, we report that Nur77 expression correlates with the degree of self-reactivity, counterselection, and anergy among individual B cell clones from two distinct BCR transgenic mouse models but is dispensable for all of these tolerance mechanisms. However, we identify a role for Nur77 in restraining survival of self-reactive B cells in the periphery under conditions of competition for a limited supply of the survival factor BAFF. We find that Nur77 deficiency results in the progressive accumulation of self-reactive B cells in the mature repertoire with age and is sufficient to break B cell tolerance in VH3H9 H chain transgenic mice. We thus propose that Nur77 is upregulated in self-reactive B cells in response to chronic Ag stimulation and selectively restricts the survival of these cells, gradually pruning self-reactivity from the mature repertoire to impose a novel layer of peripheral B cell tolerance.
            Cover page: Nur77 Links Chronic Antigen Stimulation to B Cell Tolerance by Restricting the Survival of Self-Reactive B Cells in the Periphery
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2020)
            Antigen stimulation (signal 1) triggers B cell proliferation and primes B cells to recruit, engage and respond to T cell help (signal 2). Failure to receive signal 2 within a defined time window results in B cell apoptosis, yet the mechanisms that enforce dependence on co-stimulation are incompletely understood. Nr4a1-3 encode a small family of orphan nuclear receptors that are rapidly induced by B cell antigen receptor stimulation. Here, we show that Nr4a1 and Nr4a3 play partially redundant roles to restrain B cell responses to antigen in the absence of co-stimulation and do so, in part, by repressing the expression of BATF and, consequently, MYC. The NR4A family also restrains B cell access to T cell help by repressing expression of the T cell chemokines CCL3 and CCL4, as well as CD86 and ICAM1. Such NR4A-mediated regulation plays a role specifically under conditions of competition for limiting T cell help.
              Cover page: NR4A nuclear receptors restrain B cell responses to antigen when second signals are absent or limiting
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