SPPL2a (Signal Peptide Peptidase Like 2a) is an intramembrane aspartyl protease engaged in the function of B-cells and dendritic cells. Despite being an attractive target for modulation of the immune system, selective SPPL2a inhibitors are barely described in the literature. Recently, we have disclosed a selective, small molecular weight agent SPL-707 which confirmed that pharmacological inhibition of SPPL2a leads to the accumulation of its substrate CD74/p8 and as a consequence to a reduction in the number of B-cells as well as myeloid dendritic cells in mice. In this paper we describe the discovery of novel hydroxyethylamine based SPPL2a inhibitors. Starting from a rather lipophilic screening hit, several iterative optimization cycles allowed for its transformation into a highly potent and selective compound 15 (SPL-410) which inhibited in vivo CD74/p8 fragment processing in mice at 10 mg/kg oral dose.

Signal peptide peptidase-like 2a (SPPL2a) is an aspartic intramembrane protease which has recently been shown to play an important role in the development and function of antigen presenting cells such as B lymphocytes and dendritic cells. In this paper, we describe the discovery of the first selective and orally active SPPL2a inhibitor (S)-2-cyclopropyl-N1-((S)-5,11-dioxo-10,11-dihydro-1H,3H,5H-spiro[benzo[d]pyrazolo[1,2-a][1,2]diazepine-2,1'-cyclopropan]-10-yl)-N4-(5-fluoro-2-methylpyridin-3-yl)succinamide 40 (SPL-707). This compound shows adequate selectivity against the closely related enzymes γ-secretase and SPP and a good pharmacokinetic profile in mouse and rat. Compound 40 significantly inhibited processing of the SPPL2a substrate CD74/p8 fragment in rodents at doses ≤10 mg/kg b.i.d. po. Oral dosing of 40 for 11 days at ≥10 mg/kg b.i.d. recapitulated the phenotype seen in Sppl2a knockout (ko) and ENU mutant mice (reduced number of specific B cells and myeloid dendritic cells). Thus, we believe that SPPL2a represents an interesting and druggable pharmacological target, potentially providing a novel approach for the treatment of autoimmune diseases by targeting B cells and dendritic cells.

Emerging approaches to treat immune disorders target positive regulatory kinases downstream of antigen receptors with small molecule inhibitors. Here we provide evidence for an alternative approach in which inhibition of the negative regulatory inositol kinase Itpkb in mature T lymphocytes results in enhanced intracellular calcium levels following antigen receptor activation leading to T cell death. Using Itpkb conditional knockout mice and LMW Itpkb inhibitors these studies reveal that Itpkb through its product IP4 inhibits the Orai1/Stim1 calcium channel on lymphocytes. Pharmacological inhibition or genetic deletion of Itpkb results in elevated intracellular Ca2+ and induction of FasL and Bim resulting in T cell apoptosis. Deletion of Itpkb or treatment with Itpkb inhibitors blocks T-cell dependent antibody responses in vivo and prevents T cell driven arthritis in rats. These data identify Itpkb as an essential mediator of T cell activation and suggest Itpkb inhibition as a novel approach to treat autoimmune disease.

B cell development requires tight regulation to allow for the generation of a diverse repertoire while preventing the development of autoreactive cells. We report, using N-ethyl-N-nitrosourea (ENU)-induced mutagenesis, the identification of a mutant mouse (chompB) with a block in early B cell development. The blockade occurs after the transitional 1 (T1) stage and leads to a decrease in mature B cell subsets and deficits in T cell-dependent antibody responses. Additionally, chompB mice have decreases in myeloid dendritic cells (DCs). The mutation was mapped to the intramembrane protease signal peptide peptidase-like 2a (Sppl2a), a gene not previously implicated in immune cell development. Proteomic analysis identified the invariant chain (CD74) as a key substrate of Sppl2a and suggests that regulated intramembrane proteolysis of CD74 by Sppl2a contributes to B cell and DC survival. Moreover, these data suggest that modulation of Sppl2a may be a useful therapeutic strategy for treatment of B cell dependent autoimmune disorders.

Abstract B cell development requires tight regulation to allow for the generation of a diverse repertoire while preventing the development of autoreactive cells. Here we report, using N-ethyl-N-nitrosourea (ENU)-induced mutagenesis, the identification of a mutant mouse (chompB) with a block in early B cell development. The blockade occurs after the transitional 1 (T1) stage and leads to a decrease in mature B cell subsets and deficits in T-dependent antibody responses. Additionally, chompB mice have decreases in myeloid dendritic cells (DCs). The mutation was mapped to the intramembrane protease signal peptide peptidase-like 2a (Sppl2a), a gene not previously implicated in immune cell development. Proteomic analysis identified the invariant chain (CD74) as a key substrate of Sppl2a and suggests that regulated intramembrane proteolysis (RIP) of CD74 by Sppl2a contributes to B cell and DC survival. Moreover, these data suggest that modulation of Sppl2a may be a useful therapeutic strategy for treatment of B cell dependent autoimmune disorders.

Lymphocyte antigen receptor-mediated production of Ins(1,4,5)P3 induces the release of Ca2+ from intracellular stores, resulting in the opening of store-operated Ca2+ (SOC) channels. Mice deficient in inositol(1,4,5)P3-3 kinase B (ITPKb), which converts inositol(1,4,5)P3 (IP3) to inositol(1,3,4,5)P4 (IP4), exhibit a complete block in T cell positive selection. Previous studies demonstrated that IP4 is an inhibitor of SOC channels. To understand the role of ITPKb in mature peripheral lymphocytes, inducible ITPKb-/- mice were generated. Deletion of ITPKb in mature lymphocytes reveals that ITPKb is required for mature T cell function and T-dependent antibody responses. Following antigen receptor activation, the loss of ITPKb leads to enhanced Ca2+ levels and the induction of death effector gene expression resulting in apoptosis. We further demonstrate that IP4 is an inhibitor of open-state Orai1 channels. LMW ITPKb inhibitors were identified using a high-throughput compound screen. Application of ITPKb inhibitors to lymphocytes enhanced Ca2+ responses following antigen receptor stimulation, similar to ITPKb-/- cells. Treatment of mice with ITPKb inhibitors recapitulated the block in T cell development observed in ITPKb-/- mice and inhibited antigen-induced arthritis formation in rats. These data identify ITPKb and IP4 as crucial mediators of lymphocyte development and activation, and suggest that inhibition of ITPKb may provide a novel mechanism to treat autoimmune disease.

Abstract Antigen receptor-mediated production of Ins(1,4,5)P3 induces the release of Ca2+ from intracellular stores, resulting in the opening of store-operated Ca2+ (SOC) channels. We previously reported a mouse line deficient in Inositol(1,4,5)P3-3 kinase B (ITPKb), which converts Ins(1,4,5)P3 (IP3) to Ins(1,3,4,5)P4 (IP4). Mice lacking ITPKb exhibit a complete block in T cell positive selection and possess impaired B cell development and activation. Interestingly, ITPKb-/- cells exhibit enhanced Ag receptor-induced SOC signaling, which can be rescued by adding cell-permeable IP4 to ITPKb-/- cells, suggesting that IP4 serves to inhibit SOC channels. Due to developmental blocks, the role of ITPKb in mature lymphocyte function is unknown. Using the ER-Cre-loxP system, tamoxifen-induced deletion of ITPKb (ITPKbfl/fl) demonstrates a crucial role for ITPKb in mature T cell function. While ITPKbfl/fl mice have normal T-independent Ab responses, T-dependent Ab responses are completely abolished. In vitro, mature ITPKbfl/fl T cells exhibit enhanced SOC entry and reduced proliferative responses to TCR signals. Furthermore, we found that ITPKbfl/fl T cells die rapidly after activation, due in part to enhanced FasL upregulation. Using a HEK293 cell line stably expressing Stim1 and Orai1, we demonstrate that IP4 is an inhibitor of open-state Orai1 channels. These data identify ITPKb and IP4 as essential mediators of lymphocyte development and T cell activation by regulating SOC entry via Orai1.

Foxo transcription factors have a conserved role in the adaptation of cells and organisms to nutrient and growth factor availability. Here we show that Foxo1 has a crucial, nonredundant role in T cells. In naive T cells, Foxo1 controlled the expression of the adhesion molecule L-selectin, the chemokine receptor CCR7 and the transcription factor Klf2, and its deletion was sufficient to alter lymphocyte trafficking. Furthermore, Foxo1 deficiency resulted in a severe defect in interleukin 7 receptor alpha-chain (IL-7R alpha) expression associated with its ability to bind an Il7r enhancer. Finally, growth factor withdrawal induced a Foxo1-dependent increase in Sell, Klf2 and Il7r expression. These data suggest that Foxo1 regulates the homeostasis and life span of naive T cells by sensing growth factor availability and regulating homing and survival signals.

Inositol 1,4,5-trisphosphate 3-kinase B (or Itpkb) converts inositol 1,4,5-trisphosphate to inositol 1,3,4,5-tetrakisphosphate upon Ag receptor activation and controls the fate and function of lymphocytes. To determine the role of Itpkb in B cell tolerance, Itpkb−/− mice were crossed to transgenic mice that express a BCR specific for hen egg lysozyme (IgHEL). B cells from Itpkb−/− IgHEL mice possess an anergic phenotype, hypoproliferate in response to cognate Ag, and yet they exhibit enhanced Ag-induced calcium signaling. In IgHEL transgenic mice that also express soluble HEL, lack of Itpkb converts anergy induction to deletion. These data establish Itpkb as a negative regulator of BCR signaling that controls the fate of developing B cells and tolerance induction.

Foxo transcription factors play an evolutionary conserved role in cellular and organismal adaptation to nutrients and growth factor availability. Here we show that, in accord with its preferential expression in lymphocytes, the transcription factor Foxo1 plays a non-redundant role in peripheral naïve T cell homeostasis. Our experiments demonstrate that in naïve T cells Foxo1 controls the expression of the homing receptors L-Selectin and Ccr7 as well as the transcription factor Klf2, and its deletion is sufficient to alter lymphocyte trafficking in vivo. In addition, Foxo1 deficiency is associated with a severe defect in IL-7Ra expression, correlated with the ability of Foxo1 to directly bind an evolutionarily conserved region upstream of the Il7ra transcriptional start site. Finally, in agreement with the regulatory mechanisms controlling Foxo activity, we show that growth factor withdrawal induces a specific Foxo1-dependent increase in L-selectin, Klf2 and IL7ra expression. Overall, these data support a model where the regulation of Foxo1 activity depending on growth factor availability regulates optimal homing and detection of survival signals.

Foxo transcription factors regulate cell cycle progression, cell survival and DNA-repair pathways. Here we demonstrate that deficiency in Foxo3 resulted in greater expansion of T cell populations after viral infection. This exaggerated expansion was not T cell intrinsic. Instead, it was caused by the enhanced capacity of Foxo3-deficient dendritic cells to sustain T cell viability by producing more interleukin 6. Stimulation of dendritic cells mediated by the coinhibitory molecule CTLA-4 induced nuclear localization of Foxo3, which in turn inhibited the production of interleukin 6 and tumor necrosis factor. Thus, Foxo3 acts to constrain the production of key inflammatory cytokines by dendritic cells and to control T cell survival.

FOXO1 is a transcription factor, a member of a class of evolutionary conserved forkhead genes that also includes Foxo3a, Foxo4, and Foxo6. FOXOs regulate the expression of apoptotic, cell cycle progression, and detoxification genes. In response to growth factor signaling, FOXOs are phosphorylated and excluded from the nucleus to prevent their transcriptional activity. FOXOs have been extensively studied and shown to be important in longevity and cancer. FOXO1 is highly expressed in humans and mice; however, its function in T cells has not been examined. Foxo1 was conditionally deleted in T cells, Foxo1 KO mice displayed a higher proportion of activated T cells. In addition, Foxo1 KO mice had an increased number of CD4+ but not CD8+ T cells in lymph node and spleen. Upon activation, FOXO1 KO T cells, proliferated, yet failed to accumulate due to impaired survival. This accumulation defect after activation was also observed in FOXO1 KO TCR transgenic T cells. When we examined the role of FOXO1 in survival and cytokine rescue in activated CD4+ T cells, we observed that FOXO1 KO CD4+ T cells were less sensitive to growth factor withdrawal induced apoptosis. This FOXO1‐mediated survival effect can be partially attributed to decreased levels of Bim expression. These findings demonstrate that FOXO1 plays a very important role in regulating T cell activation and survival.

T cells enigmatically require caspase-8, an inducer of apoptosis, for antigen-driven expansion and effective antiviral responses, and yet the pathways responsible for this effect have been elusive. A defect in caspase-8 expression does not affect progression through the cell cycle but causes an abnormally high rate of cell death that is distinct from apoptosis and does not involve a loss of NFκB activation. Instead, antigen or mitogen activated Casp8-deficient T cells exhibit an alternative type of cell death similar to programmed necrosis that depends on receptor interacting protein (Ripk1). The selective genetic ablation of caspase-8, NFκB, and Ripk1, reveals two forms of cell death that can regulate virus-specific T cell expansion.

Caspase-8 is a cysteine protease that is an essential initiator of death receptor-mediated apoptosis. Paradoxically, it has been shown that T cells require Caspase-8 for proliferation following antigenic or mitogenic stimulation. We have generated a T cell-specific conditional deletion of Caspase-8, and found that deletion of Caspase-8 causes profound hypo-proliferation in T cells, both in vitro and in an immune response to LCMV. Further characterization has revealed that T cells deficient in Caspase-8 do not have a defect in cell cycle progression, but rather experience an abnormally high rate of cell death that does not exhibit hallmarks of classic apoptosis. Contrary to published work, the loss of cell viability does not result from a defect in NFkB activation. Instead, we find Caspase-8 deficient T cells die by an alternative form of cell death.

Caspase-8 is an essential component of death receptor-mediated apoptosis. Along with Fas-associated death domain protein, it is also essential for T cell proliferation in response to antigenic or mitogenic stimuli. To determine whether caspase-8 is also required for B cell proliferation, we generated mice with a B cell-specific Casp8 deficiency. Unlike T cells, caspase-8 was not required for Ag receptor-driven proliferation or Ab formation. Rather, Casp8-deficient B cells failed to proliferate in response to dsRNA and LPS, ligands for TLR3 and TLR4, respectively, but responded normally to the TLR9 agonist CpG DNA. Similarly, Ab production to trinitrophenol-LPS was selectively reduced in B cell-specific Casp8-deficient mice. The activation of NF-kappa B or IFN regulatory factor 3 was found to be unaffected by the loss of caspase-8, implicating it in a novel pathway important for some forms of innate immunity mediated by B cells.

Fas-associated death domain (FADD) is a death domain containing cytoplasmic adapter molecule required for the induction of apoptosis by death receptors. Paradoxically, FADD also plays a crucial role in the development and proliferation of T cells. Using T cells from mice expressing a dominant negative form of FADD (FADDdd), activation with anti-TCR Ab and costimulation or exogenous cytokines is profoundly diminished. This is also seen in wild-type primary T cells transduced with the same transgene, demonstrating that FADD signaling is required in normally differentiated T cells. The defective proliferation does not appear to be related to the early events associated with TCR stimulation. Rather, with a block in FADD signaling, stimulated T cells exhibit a high rate of cell death corresponding to the initiation of cell division. Although CD4 T cells exhibit a moderate deficiency, this effect is most profound in CD8 T cells. In vivo, the extent of this defective accumulation is most apparent; lymphocytic choriomenigitis virus-infected FADDdd-expressing mice completely fail to mount an Ag-specific response. These results show that, in a highly regulated fashion, FADD, and most likely caspases, can transduce either a signal for survival or one that leads directly to apoptosis and that the balance between these opposing outcomes is crucial to adaptive immunity.

B cell development is a tightly controlled pro­ cess that promotes the survival and expansion of B cells with affinity for invading organisms while eliminating autoreactive B cells and thereby preventing autoimmunity (Browning, 2006; Allman and Pillai, 2008). The recent clinical success of B cell targeted therapies in a multi­ tude of autoimmune disorders has highlighted the potential role of B cells in disease pathology. Work in the last several decades has also revealed that B cells have alternative effector functions in addition to antibody production, and can play a role in the pathology of a multitude of human diseases and provide additional thera­ peutic intervention points (Browning, 2006; Gürcan et al., 2009). Although much has been discovered about lymphocyte development and activation through the use of traditional gene targeting approaches, it also has its limitations. These include require­ ments for a gene in embryonic development, the development of a particular cell type, or a hy­ pothesis that a gene is involved in the function of a particular cell type or pathway. Forward ge­ netic approaches have classically proven to be valuable tools in many model organisms, and work from the last 10 years has shown that it can also be a useful tool in mice (Beutler et al., 2006; Cook et al., 2006). Random mutagenesis also provides the opportunity to identify mutants with reduced or increased activity as opposed to full ablation, which can provide insight into how a particular molecule is functioning. To this end, we used a forward genetic screen using N­ethyl­N­nitrosourea (ENU)– induced mutagenesis in mice to identify novel regulators of immune function. We discovered a CORRESPONDENCE Michael Cooke: mcooke@gnf.org The intramembrane protease Sppl2a is required for B cell and DC development and survival via cleavage of the invariant chain B cell development requires tight regulation to allow for the generation of a diverse repertoire while preventing the development of autoreactive cells. We report, using N-ethyl-N-nitrosourea (ENU)–induced mutagenesis, the identification of a mutant mouse (chompB) with a block in early B cell development. The blockade occurs after the transitional 1 (T1) stage and leads to a decrease in mature B cell subsets and deficits in T cell–dependent antibody responses. Additionally, chompB mice have decreases in myeloid dendritic cells (DCs). The mutation was mapped to the intramembrane protease signal peptide peptidase-like 2a (Sppl2a), a gene not previously implicated in immune cell development. Proteomic analysis identified the invariant …