Sarah E. Bell

A Crucial Role for the p110δ Subunit of Phosphatidylinositol 3-Kinase in B Cell Development and Activation

Mice lacking the p110δ catalytic subunit of phosphatidylinositol 3-kinase have reduced numbers of B1 and marginal zone B cells, reduced levels of serum immunoglobulins, respond poorly to immunization with type II thymus-independent antigen, and are defective in their primary and secondary responses to thymus-dependent antigen. p110δ−/− B cells proliferate poorly in response to B cell receptor (BCR) or CD40 signals in vitro, fail to activate protein kinase B, and are prone to apoptosis. p110δ function is required for BCR-mediated calcium flux, activation of phosphlipaseCγ2, and Brutons tyrosine kinase. Thus, p110δ plays a critical role in B cell homeostasis and function.

Impaired Development of Th2 Cells in IL-13-Deficient Mice

We report that Th2 cell cultures generated using T cells or splenocytes from IL-13-deficient mice produce significantly reduced levels of IL-4, IL-5, and IL-10 compared with wild-type. In contrast, IL-4 and IL-5 production by mast cells stimulated in vitro with PMA, ionomycin, or IgE cross-linking are unaffected. In vitro Th2 cell differentiation cannot be rescued by the addition of exogenous factors, but in vivo antigen challenge and administration of IL-13 can increase Th2-like cytokine responses as can infection with the parasitic nematode Nippostrongylus brasiliensis. IL-13-deficient mice also have lower basal levels of serum IgE and biased antigen-specific immunoglobulin responses. Thus, IL-13 is an important regulator of Th2 commitment and may therefore play a central role in atopy and infectious diseases.

Signal transduction through Vav-2 participates in humoral immune responses and B cell maturation.

B and T lymphocytes develop normally in mice lacking the guanine nucleotide exchange factor Vav-2. However, the immune responses to type II thymus-independent antigen as well as the primary response to thymus-dependent (TD) antigen are defective. Vav-2–deficient mice are also defective in their ability to switch immunoglobulin class, form germinal centers and generate secondary immune responses to TD antigens. Mice lacking both Vav-1 and Vav-2 contain reduced numbers of B lymphocytes and display a maturational block in the development of mature B cells. B cells from Vav-1−/−Vav-2−/− mice respond poorly to antigen receptor triggering, both in terms of proliferation and calcium release. These studies show the importance of Vav-2 in humoral immune responses and B cell maturation.

Phosphoinositide 3-Kinase Activity in T Cells Regulates the Magnitude of the Germinal Center Reaction

The generation of high-affinity Abs is essential for immunity and requires collaboration between B and T cells within germinal centers (GCs). By using novel mouse models with a conditional deletion of the p110δ catalytic subunit of the PI3K pathway, we established that p110δ is required in T cells, but not in B cells, for the GC reaction. We found the formation of T follicular helper (TFH) cells to be critically dependent on p110δ in T cells. Furthermore, by deleting phosphatase and tensin homolog deleted on chromosome 10, which opposes p110δ in activated T cells, we found a positive correlation between increased numbers of TFH cells and GC B cells. These results are consistent with the hypothesis that T cell help is the limiting factor in the GC reaction. P110δ was not required for the expression of B cell lymphoma 6, the downregulation of CCR7, or T cell entry into primary follicles. Instead, p110δ was the critical catalytic subunit for ICOS downstream signaling and the production of key TFH cytokines and effector molecules. Our findings support a model in which the magnitude of the GC reaction is controlled by the activity of the PI3K pathway in TFH cells.

Deletion of the RNA-binding proteins ZFP36L1 and ZFP36L2 leads to perturbed thymic development and T lymphoblastic leukemia.

ZFP36L1 and ZFP36L2 are RNA-binding proteins (RBPs) that interact with AU-rich elements in the 3′ untranslated region of mRNA, which leads to mRNA degradation and translational repression. Here we show that mice that lacked ZFP36L1 and ZFP36L2 during thymopoiesis developed a T cell acute lymphoblastic leukemia (T-ALL) dependent on the oncogenic transcription factor Notch1. Before the onset of T-ALL, thymic development was perturbed, with accumulation of cells that had passed through the β-selection checkpoint without first expressing the T cell antigen receptor β-chain (TCRβ). Notch1 expression was higher in untransformed thymocytes in the absence of ZFP36L1 and ZFP36L2. Both RBPs interacted with evolutionarily conserved AU-rich elements in the 3′ untranslated region of Notch1 and suppressed its expression. Our data establish a role for ZFP36L1 and ZFP36L2 during thymocyte development and in the prevention of malignant transformation.

The RNA binding protein Zfp36l1 is required for normal vascularisation and post-transcriptionally regulates VEGF expression

The Zfp36l1 gene encodes a zinc finger‐containing mRNA binding protein implicated in the posttranscriptional control of gene expression. Mouse embryos homozygous for a targeted mutation in the Zfp36l1 locus died mid‐gestation and exhibited extraembryonic and intraembryonic vascular abnormalities and heart defects. In the developing placenta, there was a failure of the extraembryonic mesoderm to invaginate the trophoblast layer. The phenotype was associated with an elevated expression of vascular endothelial growth factor (VEGF)‐A in the embryos and in embryonic fibroblasts cultured under conditions of both normoxia and hypoxia. VEGF‐A overproduction by embryonic fibroblasts was not a consequence of changes in Vegf‐a mRNA stability; instead, we observed enhanced association with polyribosomes, suggesting Zfp36l1 influences translational regulation. These data implicate Zfp36l1as a negative regulator of Vegf‐a gene activity during development. Developmental Dynamics 235:3144–3155, 2006.

Immunological Function in Mice Lacking the Rac-Related GTPase RhoG

ABSTRACT RhoG is a low-molecular-weight GTPase highly expressed in lymphocytes that activates gene transcription and promotes cytoskeletal reorganization in vitro. To study the in vivo function of RhoG, we generated mice homozygous for a targeted disruption of the RhoG gene. Despite the absence of RhoG, the development of B and T lymphocytes was unaffected. However, there was an increase in the level of serum immunoglobulin G1 (IgG1) and IgG2b as well as a mild increase of the humoral immune response to thymus-dependent antigens. In addition, B- and T-cell proliferation in response to antigen receptor cross-linking was slightly increased. Although RhoG deficiency produces a mild phenotype, our experiments suggest that RhoG may contribute to the negative regulation of immune responses. The lack of a strong phenotype could indicate a functional redundancy of RhoG with other Rac proteins in lymphocytes.

The RNA-binding protein HuR is essential for the B cell antibody response

Post-transcriptional regulation of mRNA by the RNA-binding protein HuR (encoded by Elavl1) is required in B cells for the germinal center reaction and for the production of class-switched antibodies in response to thymus-independent antigens. Transcriptome-wide examination of RNA isoforms and their abundance and translation in HuR-deficient B cells, together with direct measurements of HuR-RNA interactions, revealed that HuR-dependent splicing of mRNA affected hundreds of transcripts, including that encoding dihydrolipoamide S-succinyltransferase (Dlst), a subunit of the 2-oxoglutarate dehydrogenase (α-KGDH) complex. In the absence of HuR, defective mitochondrial metabolism resulted in large amounts of reactive oxygen species and B cell death. Our study shows how post-transcriptional processes control the balance of energy metabolism required for the proliferation and differentiation of B cells.

PLCγ2 regulates Bcl-2 levels and is required for survival rather than differentiation of marginal zone and follicular B cells

B cells from phospholipase C (PLC)γ2‐deficient mice express reduced levels of the pro‐survival protein Bcl‐2 and show a defect in the development of transitional T3 and marginal zone (MZ) B cells that reflects reduced B cell survival. Introduction of a bcl‐2 transgene restored the numbers of MZ, T3 and follicular B cells in PLCγ2–/– mice. Restricting the B cell repertoire in PLCγ2‐deficient mice by the introduction of a BCR transgene resulted in a striking reduction in the number of IgM‐positive B cells and a paucity of IgD‐expressing cells in the spleen which was also rescued by the bcl‐2 transgene. BCR‐stimulated ERK and IκBα phosphorylation were PLCγ2 dependent, while calcium flux was reduced, but not abrogated, in the absence of PLCγ2, suggesting an ancillary role for PLCγ1. The bcl‐2 transgene rescued development of PLCγ2–/– B cells and serum IgM levels but did not restore BCR‐mediated signaling, proliferation or serum IgG3 levels. These data suggest that PLCγ2 performs a critical role in B cell development through regulation of survival rather than differentiation.

Impaired B-1 and B-2 B cell development and atypical splenic B cell structures in IL-7 receptor-deficient mice

The cytokine IL‐7 and its receptor are essential for normal B and T lymphopoiesis. We have analyzed the role of this receptor in B cell development throughout ontogeny in IL‐7 receptor α‐deficient mice. We demonstrate that the IL‐7 receptor becomes progressively more important with age. B lymphopoiesis takes place, albeit at reduced levels, in fetal liver and bone marrow of young mice, but is arrested in adults. The outcome is a severe reduction, from an early age, in peripheral B cells including follicular, marginal zone and B‐1 B cells as well as perturbed splenic B cell structures, which are restored after adoptive transfer of normal spleen cells. We conclude that in the absence of the IL‐7 receptor, the residual B lymphopoiesis occurring early in ontogeny must be facilitated by another component, whereas the IL‐7 receptor is the key factor in adults. The impairment of marginal zone and B‐1 B cells in IL‐7 receptor‐ but not IL‐7‐deficient mice suggests non‐redundant functions for the IL‐7 receptor ligands, IL‐7 and thymic stromal lymphopoietin.