Sandra Gardam

Antigen recognition strength regulates the choice between extrafollicular plasma cell and germinal center B cell differentiation

B cells responding to T-dependent antigen either differentiate rapidly into extrafollicular plasma cells or enter germinal centers and undergo somatic hypermutation and affinity maturation. However, the physiological cues that direct B cell differentiation down one pathway versus the other are unknown. Here we show that the strength of the initial interaction between B cell receptor (BCR) and antigen is a primary determinant of this decision. B cells expressing a defined BCR specificity for hen egg lysozyme (HEL) were challenged with sheep red blood cell conjugates of a series of recombinant mutant HEL proteins engineered to bind this BCR over a 10,000-fold affinity range. Decreasing either initial BCR affinity or antigen density was found to selectively remove the extrafollicular plasma cell response but leave the germinal center response intact. Moreover, analysis of competing B cells revealed that high affinity specificities are more prevalent in the extrafollicular plasma cell versus the germinal center B cell response. Thus, the effectiveness of early T-dependent antibody responses is optimized by preferentially steering B cells reactive against either high affinity or abundant epitopes toward extrafollicular plasma cell differentiation. Conversely, responding clones with weaker antigen reactivity are primarily directed to germinal centers where they undergo affinity maturation.

TRAF2 and TRAF3 Signal Adapters Act Cooperatively to Control the Maturation and Survival Signals Delivered to B Cells by the BAFF Receptor

Tumor necrosis factor receptor-associated factors 2 and 3 (TRAF2 and TRAF3) were shown to function in a cooperative and nonredundant manner to suppress nuclear factor-kappaB2 (NF-kappaB2) activation, gene expression, and survival in mature B cells. In the absence of this suppressive activity, B cells developed independently of the obligatory B cell survival factor, BAFF (B cell-activating factor of the tumor necrosis factor family). However, deletion of either TRAF2 or TRAF3 from the T cell lineage did not promote T cell survival, despite causing extensive NF-kappaB2 activation. This constitutive, lineage-specific suppression of B cell survival by TRAF2 and TRAF3 determines the requirement for BAFF to sustain B cell development in vivo. Binding of BAFF to BAFF receptor reversed TRAF2-TRAF3-mediated suppression of B cell survival by triggering the depletion of TRAF3 protein. This process was TRAF2 dependent, revealing dual roles for TRAF2 in regulating B cell homeostasis.

B Cell Receptor–independent Stimuli Trigger Immunoglobulin (Ig) Class Switch Recombination and Production of IgG Autoantibodies by Anergic Self-Reactive B Cells

In both humans and animals, immunoglobulin (Ig)G autoantibodies are less frequent but more pathogenic than IgM autoantibodies, suggesting that controls over Ig isotype switching are required to reinforce B cell self-tolerance. We have used gene targeting to produce mice in which hen egg lysozyme (HEL)-specific B cells can switch to all Ig isotypes (SWHEL mice). When crossed with soluble HEL transgenic (Tg) mice, self-reactive SWHEL B cells became anergic. However, in contrast to anergic B cells from the original nonswitching anti-HEL × soluble HEL double Tg model, self-reactive SWHEL B cells also displayed an immature phenotype, reduced lifespan, and exclusion from the splenic follicle. These differences were not related to their ability to Ig class switch, but instead to competition with non-HEL–binding B cells generated by VH gene replacement in SWHEL mice. When activated in vitro with B cell receptor (BCR)-independent stimuli such as anti-CD40 monoclonal antibody plus interleukin 4 or lipopolysaccharide (LPS), anergic SWHEL double Tg B cells proliferated and produced IgG anti-HEL antibodies as efficiently as naive HEL-binding B cells from SWHEL Ig Tg mice. These results demonstrate that no intrinsic constraints to isotype switching exist in anergic self-reactive B cells. Instead, production of IgG autoantibodies is prevented by separate controls that reduce the likelihood of anergic B cells encountering BCR-independent stimuli. That bacteria-derived LPS could circumvent these controls may explain the well-known association between autoantibody-mediated diseases and episodes of systemic infection.

Altered Migration, Recruitment, and Somatic Hypermutation in the Early Response of Marginal Zone B Cells to T Cell-Dependent Antigen

The early responses of follicular (Fo) and marginal zone (MZ) B cells to T cell-dependent Ag were compared using anti-hen egg lysozyme (HEL+) B cells capable of class switch recombination and somatic hypermutation (SHM). Purified CD21/35intCD23high Fo and CD21/35highCD23low MZ splenic B cells from SWHEL Ig-transgenic mice were transferred into wild-type recipients and challenged with HEL-sheep RBC. Responding HEL+ B cells from both populations switched efficiently to IgG1, generated syndecan-1+ Ab-secreting cells, and exhibited equivalent rates of proliferation. However, the expansion of HEL+ MZ B cells lagged significantly behind that of HEL+ Fo B cells due to less efficient homing to the outer periarteriolar lymphatic sheath and reduced recruitment into the proliferative response. Despite the equivalent rates of class switch recombination, the onset of SHM was delayed in the MZ subset, indicating that these two activation-induced cytidine deaminase-dependent events are uncoupled in the early response of MZ B cells. Migration of HEL+ B cells into germinal centers coincided with the onset of SHM, occurring more rapidly with Fo vs MZ responders. These results are consistent with the concept that Fo and MZ B cells have evolved to specialize in T cell-dependent and T-independent responses respectively.

Increased CD4+Foxp3+ T Cells in BAFF-Transgenic Mice Suppress T Cell Effector Responses

The cytokine B cell activation factor of the TNF family (BAFF) is considered to perform a proinflammatory function. This paradigm is particularly true for B cell-dependent immune responses; however the exact role for BAFF in regulating T cell immunity is ill-defined. To directly assess the effect of BAFF upon T cells, we analyzed T cell-dependent immune responses in BAFF-transgenic (Tg) mice. We found that T cell responses in BAFF-Tg mice are profoundly compromised, as indicated by their acceptance of islet allografts and delayed skin graft rejection. However, purified BAFF-Tg effector T cells could reject islet allografts with a normal kinetic, suggesting that the altered response did not relate to a defect in T cell function per se. Rather, we found that BAFF-Tg mice harbored an increased number of peripheral CD4+Foxp3+ T cells. A large proportion of the BAFF-expanded CD4+CD25+Foxp3+ regulatory T cells (Tregs) were CD62LlowCD103high and ICAM-1high, a phenotype consistent with an ability to home to inflammatory sites and prevent T cell effector responses. Indeed, depletion of the endogenous BAFF-Tg Tregs allowed allograft rejection to proceed, demonstrating that the increased Tregs were responsible for preventing alloimmunity. The ability of BAFF to promote Treg expansion was not T cell intrinsic, as Tregs did not express high levels of BAFF receptor 3, nor did excessive BAFF trigger NF-κB2 processing in Tregs. In contrast, we found that BAFF engendered Treg expansion through an indirect, B cell-dependent mechanism. Thus, under certain conditions, BAFF can play a surprising anti-inflammatory role in T cell biology by promoting the expansion of Treg cells.

Deletion of cIAP1 and cIAP2 in murine B lymphocytes constitutively activates cell survival pathways and inactivates the germinal center response

B cells require signals delivered through B-cell activating factor of the TNF family receptor (BAFF-R) and CD40 to survive and produce antibody responses in vivo. In vitro data indicate that these signals are controlled by the homologous RING finger proteins cIAP1 and cIAP2, in collaboration with TRAF2 and TRAF3. There is also mounting evidence that all 4 of these signaling molecules can act as tumor suppressors in human B-lineage malignancies. However, it has not been possible to identify the roles of cIAP1 and cIAP2 in controlling B-cell physiology because of the absence of an appropriate in vivo model. Here we describe a unique genetically modified mouse in which the linked cIap1 and cIap2 genes can be independently inactivated. Deletion of cIAP1 plus cIAP2 (but not either protein alone) rendered primary B cells independent of BAFF-R for their survival and led to their uncontrolled accumulation in vivo. B cells deficient in cIAP1 and cIAP2 were also incapable of forming germinal centers, a key step in antibody-mediated immunity. These data define a fundamental role for cIAP1/cIAP2 in regulating B-cell survival and responsiveness, show this requires direct binding to TRAF2, and suggest how mutations of TRAF2, TRAF3, and cIAP1/cIAP2 contribute to B-lineage malignancies, such as multiple myeloma.

Non-Canonical NF-κB Signaling Initiated by BAFF Influences B Cell Biology at Multiple Junctures

It has been more than a decade since it was recognized that the nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) transcription factor family was activated by two distinct pathways: the canonical pathway involving NF-κB1 and the non-canonical pathway involving NF-κB2. During this time a great deal of evidence has been amassed on the ligands and receptors that activate these pathways, the cytoplasmic adapter molecules involved in transducing the signals from receptors to nucleus, and the resulting physiological outcomes within body tissues. In contrast to NF-κB1 signaling, which can be activated by a wide variety of receptors, the NF-κB2 pathway is typically only activated by a subset of receptor and ligand pairs belonging to the tumor necrosis factor (TNF) family. Amongst these is B cell activating factor of the TNF family (BAFF) and its receptor BAFFR. Whilst BAFF is produced by many cell types throughout the body, BAFFR expression appears to be restricted to the hematopoietic lineage and B cells in particular. For this reason, the main physiological outcomes of BAFF mediated NF-κB2 activation are confined to B cells. Indeed BAFF mediated NF-κB2 signaling contributes to peripheral B cell survival and maturation as well as playing a role in antibody responses and long term maintenance plasma cells. Thus the importance BAFF and NF-κB2 permeates the entire B cell lifespan and impacts on this important component of the immune system in a variety of ways.

In vivo control of B-cell survival and antigen-specific B-cell responses

Summary:  Targeted modification of the mouse genome provides the capability to manipulate complex physiological processes in a precise and controlled manner. Investigation of B‐lymphocyte biology has benefited not only from the targeted modification of genes controlling B‐cell survival and responsiveness, but also from the manipulation of antigen specificity made possible by targeting endogenous immunoglobulin loci. In this review, we discuss recent results obtained from our laboratory using gene‐targeted mouse models to investigate the in vivo regulation of B‐cell survival and responsiveness. The control of BAFF‐dependent survival signals by the TRAF2‐ and TRAF3‐signaling proteins is discussed as is the potential involvement of these molecules in B‐lineage malignancies. We also outline the development and use of the SWHEL model for analyzing antigen‐specific B‐cell responses in vivo. This includes insights into the control of early decision‐making during T‐dependent B‐cell differentiation, the affinity maturation and plasma cell differentiation of germinal center B cells, and the identification of EBI2 as a key regulator of B‐cell migration and differentiation.

Lineage-specific transgene expression in hematopoietic cells using a Cre-regulated retroviral vector.

Transduction of bone marrow stem cells with retroviral expression vectors represents a cheaper and more rapid alternative to conventional transgenesis for studies of in vivo gene function. However, achieving tissue-specific expression of genes inserted into retroviral vectors is notoriously difficult. We have developed a single tri-cistronic retroviral vector (MG(f)I4) that facilitates Cre-dependent, lineage-specific gene expression within hematopoietic cells. Bone marrow stem cells transduced with MG(f)I4 co-express a loxP-flanked (floxed) eGFP cDNA together with truncated human CD4 (hCD4Delta). Open reading frames (ORFs) cloned between these two cDNAs are not constitutively translated but are activated upon Cre-mediated removal of the eGFP cDNA. Mice reconstituted with transduced bone marrow stem cells obtained from Cd19-Cre, Cr2-Cre or Lck-Cre, donors were shown to specifically express an ORF insert in the appropriate lymphocyte subsets. Cells that had activated ORF expression were identifiable by transition from a GFP+, hCD4+ to a GFP(-), hCD4+ phenotype. The use of this novel vector in conjunction with the wide range of well-characterized Cre-transgenic lines will be a versatile tool for exploring gene function within the immune system. In particular, this approach will provide a convenient way to test the functional significance of naturally occurring genetic mutations linked to human disease.

TRAF2 regulates peripheral CD8(+) T-cell and NKT-cell homeostasis by modulating sensitivity to IL-15.

In this study, a critical and novel role for TNF receptor (TNFR) associated factor 2 (TRAF2) is elucidated for peripheral CD8+ T‐cell and NKT‐cell homeostasis. Mice deficient in TRAF2 only in their T cells (TRAF2TKO) show ∼40% reduction in effector memory and ∼50% reduction in naïve CD8+ T‐cell subsets. IL‐15‐dependent populations were reduced further, as TRAF2TKO mice displayed a marked ∼70% reduction in central memory CD8+CD44hiCD122+ T cells and ∼80% decrease in NKT cells. TRAF2TKO CD8+CD44hi T cells exhibited impaired dose‐dependent proliferation to exogenous IL‐15. In contrast, TRAF2TKO CD8+ T cells proliferated normally to anti‐CD3 and TRAF2TKO CD8+CD44hi T cells exhibited normal proliferation to exogenous IL‐2. TRAF2TKO CD8+ T cells expressed normal levels of IL‐15‐associated receptors and possessed functional IL‐15‐mediated STAT5 phosphorylation, however TRAF2 deletion caused increased AKT activation. Loss of CD8+CD44hiCD122+ and NKT cells was mechanistically linked to an inability to respond to IL‐15. The reduced CD8+CD44hiCD122+ T‐cell and NKT‐cell populations in TRAF2TKO mice were rescued in the presence of high dose IL‐15 by IL‐15/IL‐15Rα complex administration. These studies demonstrate a critical role for TRAF2 in the maintenance of peripheral CD8+ CD44hiCD122+ T‐cell and NKT‐cell homeostasis by modulating sensitivity to T‐cell intrinsic growth factors such as IL‐15.