Summary Previous work has shown that mature B cells depend upon survival signals delivered to the cells by their antigen receptor (BCR). To identify the molecular nature of this survival signal, we have developed a genetic approach in which ablation of the BCR is combined with the activation of specific, BCR dependent signaling cascades in mature B cells in vivo. Using this system, we provide evidence that the survival of BCR deficient mature B cells can be rescued by a single signaling pathway downstream of the BCR, namely PI3K signaling, with the FOXO1 transcription factor playing a central role.

ORAI1 is a pore subunit of the store-operated Ca 2؉ release-activated Ca 2؉ (CRAC) channel. To examine the physiological consequences of ORAI1 deficiency, we generated mice with targeted disruption of the Orai1 gene. The results of immunohistochemical analysis showed that ORAI1 is expressed in lymphocytes, skin, and muscle of wild-type mice and is not expressed in Orai1 ؊/؊ mice. Orai1 ؊/؊ mice with the inbred C57BL/6 background showed perinatal lethality, which was overcome by crossing them to outbred ICR mice. Orai1 ؊/؊ mice were small in size, with eyelid irritation and sporadic hair loss resembling the cyclical alopecia observed in mice with keratinocyte-specific deletion of the Cnb1 gene. T and B cells developed normally in Orai1 ؊/؊ mice, but B cells showed a substantial decrease in Ca 2؉ influx and cell proliferation in response to B-cell receptor stimulation. Naïve and differentiated Orai1 ؊/؊ T cells showed substantial reductions in store-operated Ca 2؉ entry, CRAC currents, and cytokine production. These features are consistent with the severe combined immunodeficiency and mild extraimmunological symptoms observed in a patient with a missense mutation in human ORAI1 and distinguish the ORAI1-null mice described here from a previously reported Orai1 gene-trap mutant mouse which may be a hypomorph rather than a true null.Ca 2ϩ is a universal second messenger that regulates a multitude of cellular functions, including secretion, muscle contraction, ion channel function, and gene expression (5). In many nonexcitable cells, Ca 2ϩ influx occurs through "storeoperated" Ca 2ϩ channels which open in response to depletion of endoplasmic reticulum (ER) Ca 2ϩ stores (40). Physiologically, this occurs when ligand binds to receptors, such as G protein-coupled receptors, immunoreceptors, and receptor tyrosine kinases, that are coupled to the activation of phospholipase C. The resulting production of inositol trisphosphate leads to efflux of Ca 2ϩ from the ER through inositol trisphosphate receptors and decreased Ca 2ϩ concentration in the ER lumen. This decrease directly regulates the opening of storeoperated Ca 2ϩ channels in the plasma membrane (26). In lymphocytes and other immune system cells, the major route of Ca 2ϩ influx is through store-operated Ca 2ϩ releaseactivated Ca 2ϩ (CRAC) channels. CRAC currents (I CRAC ) were first identified in T cells and mast cells (20,21,27,53), and Ca 2ϩ influx through CRAC channels is known to be essential for T-cell activation (8, 25). Mutant Jurkat tumor T-cell lines lacking functional CRAC channels cannot be activated properly (7); moreover, T cells obtained from three independent families of patients with hereditary severe combined immunodeficiency (SCID) were shown to be severely deficient in store-operated Ca 2ϩ entry and the CRAC channel current, I CRAC (10,13,23,36). T-cell responses, particularly proliferation and cytokine production in vitro in response to T-cell receptor stimulation, were strongly impaired in patients from two of these families, explaining their SC...

Germinal centers (GC) are sites of intense B cell proliferation, central for T cell dependent antibody responses. However, the role of MYC, a key cell cycle regulator, in this process has been questioned. Here, we identified MYC positive B cell subpopulations in immature and mature GCs, and show through genetic ablation of Myc that they play indispensable roles in GC formation and maintenance. The identification of these functionally critical cellular subsets has important implications for human B cell lymphomagenesis, which mostly originates from GC B cells and frequently involves MYC chromosomal translocations. As these translocations are generally dependent on transcription of the recombining partner loci, the MYC positive GC subpopulations may be at a particularly high risk for malignant transformation.

The cytokine TNF family member B cell-activating factor (BAFF; also termed BLyS) is essential for B cell generation and maintenance. Three receptors have been identified that bind to BAFF: transmembrane activator, calcium modulator, and cyclophilin ligand interactor (TACI); B cell maturation Ag (BCMA); and BAFF-R. Recently, it was shown that A/WySnJ mice, which contain a dramatically reduced peripheral B cell compartment due to decreased B cell life span, express a mutant BAFF-R. This finding, together with normal or enhanced B cell generation in mice deficient for BCMA or TACI, respectively, suggested that the interaction of BAFF with BAFF-R triggers signals essential for the generation and maintenance of mature B cells. However, B cells in mice deficient for BAFF differ phenotypically and functionally from A/WySnJ B cells. Residual signaling through the mutant BAFF-R could account for these differences. Alternatively, dominant-negative interference by the mutant receptor could lead to an overestimation of the importance of BAFF-R. To resolve this issue, we generated BAFF-R-null mice. Baff-r−/− mice display strongly reduced late transitional and follicular B cell numbers and are essentially devoid of marginal zone B cells. Overexpression of Bcl-2 rescues mature B cell development in Baff-r−/− mice, suggesting that BAFF-R mediates a survival signal. CD21 and CD23 surface expression are reduced on mature Baff-r−/− B cells, but not to the same extent as on mature B cells in BAFF-deficient mice. In addition, we found that Baff-r−/− mice mount significant, but reduced, Ag-specific Ab responses and are able to form spontaneous germinal centers in mesenteric lymph nodes. The reduction in Ab titers correlates with the reduced B cell numbers in the mutant mice.

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The maintenance of mature B cells hinges on signals emitted from the BAFF-R cell-surface receptor, but the nature of these signals is incompletely understood. Inhibition of canonical NF-kappaB transcription factor activity through ablation of the essential scaffold protein NEMO arrests B cell development at the same stage as BAFF-R deficiency. Correspondingly, activation of this pathway by constitutively active IkappaB Kinase2 renders B cell survival independent of BAFF-R:BAFF interactions and prevents proapoptotic PKCdelta nuclear translocation. In addition, canonical NF-kappaB activity mediates differentiation and proper localization of follicular and marginal zone B cells in the absence of BAFF-R, but not CD19. By replacing BAFF-R signals, constitutive canonical NF-kappaB signaling, a hallmark of various B cell lymphomas, causes accumulation of resting B cells and promotes their proliferation and survival upon activation, but does not per se induce lymphomagenesis. Therefore, canonical NF-kappaB activity can substitute for BAFF-R signals in B cell development and pathogenesis.

ORAI1 is the pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel, which is responsible for store-operated Ca2+ entry in lymphocytes. A role for ORAI1 in T cell function in vivo has been inferred from in vitro studies of T cells from human immunodeficient patients with mutations in ORAI1 and Orai1−/− mice, but a detailed analysis of T cell-mediated immune responses in vivo in mice lacking functional ORAI1 has been missing. We therefore generated Orai1 knock-in mice (Orai1KI/KI) expressing a nonfunctional ORAI1-R93W protein. Homozygosity for the equivalent ORAI1-R91W mutation abolishes CRAC channel function in human T cells resulting in severe immunodeficiency. Homozygous Orai1KI/KI mice die neonatally, but Orai1KI/KI fetal liver chimeric mice are viable and show normal lymphocyte development. T and B cells from Orai1KI/KI mice display severely impaired store-operated Ca2+ entry and CRAC channel function resulting in a strongly reduced expression of several key cytokines including IL-2, IL-4, IL-17, IFN-γ, and TNF-α in CD4+ and CD8+ T cells. Cell-mediated immune responses in vivo that depend on Th1, Th2, and Th17 cell function were severely attenuated in ORAI1-deficient mice. Orai1KI/KI mice lacked detectable contact hypersensitivity responses and tolerated skin allografts significantly longer than wild-type mice. In addition, T cells from Orai1KI/KI mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease. These findings reaffirm the critical role of ORAI1 for T cell function and provide important insights into the in vivo functions of CRAC channels for T cell-mediated immunity.

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