David M. Tarlinton

Multiple defects in the immune system of Lyn-deficient mice, culminating in autoimmune disease

Mice homozygous for a disruption at the Lyn locus display abnormalities associated with the B lymphocyte lineage and in mast cell function. Despite reduced numbers of recirculating B lymphocytes, Lyn-/- mice are immunoglobulin M (IgM) hyperglobulinemic. Immune responses to T-independent and T-dependent antigens are affected. Lyn-/- mice fail to mediate an allergic response to IgE cross-linking, indicating that activation of LYN plays an indispensable role in Fc epsilon RI signaling. Lyn-/- mice have circulating autoreactive antibodies, and many show severe glomerulonephritis caused by the deposition of IgG immune complexes in the kidney, a pathology reminiscent of systemic lupus erythematosus. Collectively, these results implicate LYN as having an indispensable role in immunoglobulin-mediated signaling, particularly in establishing B cell tolerance.

IL-21 regulates germinal center B cell differentiation and proliferation through a B cell–intrinsic mechanism

Germinal centers (GCs) are sites of B cell proliferation, somatic hypermutation, and selection of variants with improved affinity for antigen. Long-lived memory B cells and plasma cells are also generated in GCs, although how B cell differentiation in GCs is regulated is unclear. IL-21, secreted by T follicular helper cells, is important for adaptive immune responses, although there are conflicting reports on its target cells and mode of action in vivo. We show that the absence of IL-21 signaling profoundly affects the B cell response to protein antigen, reducing splenic and bone marrow plasma cell formation and GC persistence and function, influencing their proliferation, transition into memory B cells, and affinity maturation. Using bone marrow chimeras, we show that these activities are primarily a result of CD3-expressing cells producing IL-21 that acts directly on B cells. Molecularly, IL-21 maintains expression of Bcl-6 in GC B cells. The absence of IL-21 or IL-21 receptor does not abrogate the appearance of T cells in GCs or the appearance of CD4 T cells with a follicular helper phenotype. IL-21 thus controls fate choices of GC B cells directly.

Plasma Cell Ontogeny Defined by Quantitative Changes in Blimp-1 Expression

Plasma cells comprise a population of terminally differentiated B cells that are dependent on the transcriptional regulator B lymphocyte–induced maturation protein 1 (Blimp-1) for their development. We have introduced a gfp reporter into the Blimp-1 locus and shown that heterozygous mice express the green fluorescent protein in all antibody-secreting cells (ASCs) in vivo and in vitro. In vitro, these cells display considerable heterogeneity in surface phenotype, immunoglobulin secretion rate, and Blimp-1 expression levels. Importantly, analysis of in vivo ASCs induced by immunization reveals a developmental pathway in which increasing levels of Blimp-1 expression define developmental stages of plasma cell differentiation that have many phenotypic and molecular correlates. Thus, maturation from transient plasmablast to long-lived ASCs in bone marrow is predicated on quantitative increases in Blimp-1 expression.

The extent of affinity maturation differs between the memory and antibody-forming cell compartments in the primary immune response

Immunization with protein‐containing antigens results in two types of antigen‐specific B cell: antibody forming cells (AFCs) producing antibody of progressively higher affinity and memory lymphocytes capable of producing high affinity antibody upon re‐exposure to antigen. The issue of the inter‐relationship between affinity maturation of memory B cells and AFCs was addressed through analysis of single, antigen‐specific B cells from the memory and AFC compartments during the primary response to a model antigen. Only 65% of splenic memory B cells were found capable of producing high affinity antibody, meaning that low affinity cells persist into this compartment. In contrast, by 28 days after immunization all AFCs produced high affinity antibody. We identified a unique, persistent sub‐population of bone marrow AFCs containing few somatic mutations, suggesting they arose early in the response, yet highly enriched for an identical affinity‐enhancing amino acid exchange, suggesting strong selection. Our results imply that affinity maturation of a primary immune response occurs by the early selective differentiation of high affinity variants into AFCs which subsequently persist in the bone marrow. In contrast, the memory B‐cell population contains few, if any, cells from the early response and is less stringently selected.

FcγRIIb controls bone marrow plasma cell persistence and apoptosis

The survival of long-lived plasma cells, which produce most serum immunoglobulin, is central to humoral immunity. We found here that the inhibitory Fc receptor FcγRIIb was expressed on plasma cells and controlled their persistence in the bone marrow. Crosslinking FcγRIIb induced apoptosis of plasma cells, which we propose contributes to the control of their homeostasis and suggests a method for therapeutic deletion. Plasma cells from mice prone to systemic lupus erythematosus did not express FcγRIIb and were protected from apoptosis. Human plasmablasts expressed FcγRIIb and were killed by crosslinking, as were FcγRIIb-expressing myeloma cells. Our results suggest that FcγRIIb controls bone marrow plasma cell persistence and that defects in it may contribute to autoantibody production.

Loss of the Pro-Apoptotic BH3-only Bcl-2 Family Member Bim Inhibits BCR Stimulation–induced Apoptosis and Deletion of Autoreactive B Cells

During development, the stochastic process assembling the genes encoding antigen receptors invariably generates B and T lymphocytes that can recognize self-antigens. Several mechanisms have evolved to prevent the activation of these cells and the concomitant development of autoimmune disease. One such mechanism is the induction of apoptosis in developing or mature B cells by engagement of the B cell antigen receptor (BCR) in the absence of T cell help. Here we report that B lymphocytes lacking the pro-apoptotic Bcl-2 family member Bim are refractory to apoptosis induced by BCR ligation in vitro. The loss of Bim also inhibited deletion of autoreactive B cells in vivo in two transgenic systems of B cell tolerance. Bim loss prevented deletion of autoreactive B cells induced by soluble self-antigen and promoted accumulation of self-reactive B cells developing in the presence of membrane-bound self-antigen, although their numbers were considerably lower compared with antigen-free mice. Mechanistically, we determined that BCR ligation promoted interaction of Bim with Bcl-2, inhibiting its survival function. These findings demonstrate that Bim is a critical player in BCR-mediated apoptosis and in B lymphocyte deletion.

Membrane-bound Fas ligand only is essential for Fas-induced apoptosis

Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family and its receptor, Fas, are critical for shutdown of chronic immune responses1-3 and prevention of autoimmunity4,5. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice6,7 and humans8,9. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding10,11. We generated gene-targeted mice that selectively lack either secreted FasL (ΔsFasL) or membrane-bound FasL (ΔmFasL) to resolve which of these forms is required for cell killing and to explore their hypothetical non-apoptotic activities. Mice lacking sFasL (FasLΔs/Δs) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasLΔm/Δm) could not kill cells through Fas activation. FasLΔm/Δm mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasLgld/gld mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, (on a C57BL/6 background) FasLΔm/Δm mice succumbed to SLE-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and considerably later in FasLgld/gld mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.Fas ligand (FasL), an apoptosis-inducing member of the TNF cytokine family, and its receptor Fas are critical for the shutdown of chronic immune responses and prevention of autoimmunity. Accordingly, mutations in their genes cause severe lymphadenopathy and autoimmune disease in mice and humans. FasL function is regulated by deposition in the plasma membrane and metalloprotease-mediated shedding. Here we generated gene-targeted mice that selectively lack either secreted FasL (sFasL) or membrane-bound FasL (mFasL) to resolve which of these forms is required for cell killing and to explore their hypothesized non-apoptotic activities. Mice lacking sFasL (FasLΔs/Δs) appeared normal and their T cells readily killed target cells, whereas T cells lacking mFasL (FasLΔm/Δm) could not kill cells through Fas activation. FasLΔm/Δm mice developed lymphadenopathy and hyper-gammaglobulinaemia, similar to FasLgld/gld mice, which express a mutant form of FasL that cannot bind Fas, but surprisingly, FasLΔm/Δm mice (on a C57BL/6 background) succumbed to systemic lupus erythematosus (SLE)-like autoimmune kidney destruction and histiocytic sarcoma, diseases that occur only rarely and much later in FasLgld/gld mice. These results demonstrate that mFasL is essential for cytotoxic activity and constitutes the guardian against lymphadenopathy, autoimmunity and cancer, whereas excess sFasL appears to promote autoimmunity and tumorigenesis through non-apoptotic activities.

The generation of antibody-secreting plasma cells

The regulation of antibody production is linked to the generation and maintenance of plasmablasts and plasma cells from their B cell precursors. Plasmablasts are the rapidly produced and short-lived effector cells of the early antibody response, whereas plasma cells are the long-lived mediators of lasting humoral immunity. An extraordinary number of control mechanisms, at both the cellular and molecular levels, underlie the regulation of this essential arm of the immune response. Despite this complexity, the terminal differentiation of B cells can be described as a simple probabilistic process that is governed by a central gene-regulatory network and modified by environmental stimuli.

Early appearance of germinal center–derived memory B cells and plasma cells in blood after primary immunization

Immunization with a T cell–dependent antigen elicits production of specific memory B cells and antibody-secreting cells (ASCs). The kinetic and developmental relationships between these populations and the phenotypic forms they and their precursors may take remain unclear. Therefore, we examined the early stages of a primary immune response, focusing on the appearance of antigen-specific B cells in blood. Within 1 wk, antigen-specific B cells appear in the blood with either a memory phenotype or as immunoglobulin (Ig)G1 ASCs expressing blimp-1. The memory cells have mutated VH genes; respond to the chemokine CXCL13 but not CXCL12, suggesting recirculation to secondary lymphoid organs; uniformly express B220; show limited differentiation potential unless stimulated by antigen; and develop independently of blimp-1 expression. The antigen-specific IgG1 ASCs in blood show affinity maturation paralleling that of bone marrow ASCs, raising the possibility that this compartment is established directly by blood-borne ASCs. We find no evidence for a blimp-1–expressing preplasma memory compartment, suggesting germinal center output is restricted to ASCs and B220+ memory B cells, and this is sufficient to account for the process of affinity maturation.

The development and fate of follicular helper T cells defined by an IL-21 reporter mouse

Germinal centers require CD4+ follicular helper T cells (TFH cells), whose hallmark is expression of the transcriptional repressor Bcl-6, the chemokine receptor CXCR5 and interleukin 21 (IL-21). To track the development and fate of TFH cells, we generated an IL-21 reporter mouse by introducing sequence encoding green fluorescent protein (GFP) into the Il21 locus; these mice had expression of IL-21–GFP in CD4+CXCR5+PD-1+ TFH cells. IL-21–GFP+ TFH cells were multifunctional helper cells that coexpressed several cytokines, including interferon-γ (IFN-γ), IL-2 and IL-4. TFH cells proliferated and gave rise to transferrable memory cells with plasticity, which differentiated after recall into conventional effector helper T cells and TFH cells. Thus, we demonstrated that TFH cells were not terminally differentiated but instead retained the flexibility to be recruited into other helper T cell subsets and nonlymphoid tissues.