Andrew T Miller

Production of Ins(1,3,4,5)P4 mediated by the kinase Itpkb inhibits store-operated calcium channels and regulates B cell selection and activation.

Antigen receptor–mediated production of inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) in lymphocytes triggers the release of Ca2+ from intracellular stores; this release of Ca2+ results in the opening of store-operated Ca2+ channels in the plasma membrane. Here we report that mice lacking Ins(1,4,5)P3 3-kinase B (Itpkb), which converts Ins(1,4,5)P3 to inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4), had impaired B lymphocyte development and defective immunoglobulin G3 antibody responses to a T lymphocyte–independent antigen. Itpkb-deficient B lymphocytes had the phenotypic and functional features of tolerant B lymphocytes and showed enhanced activity of store-operated Ca2+ channels after B lymphocyte receptor stimulation, which was reversed by the provision of exogenous Ins(1,3,4,5)P4. Our data identify Itpkb and its product Ins(1,3,4,5)P4 as inhibitors of store-operated Ca2+ channels and crucial regulators of B cell selection and activation.

Activin A programs the differentiation of human TFH cells

SUMMARY Follicular helper T (TFH) cells are CD4+ T cells specialized in helping B cells and are associated both with protective antibody responses and autoimmune diseases. The promise of targeting TFH cells therapeutically has been limited by fragmentary understanding of extrinsic signals regulating human TFH cell differentiation. A screen of a human protein library identified activin A as new regulator of TFH cell differentiation. Activin A orchestrated expression of multiple TFH-associated genes, independently or in concert with additional signals. TFH programming by activin A was antagonized by the cytokine IL-2. Activin A’s capacity to drive TFH cell differentiation in vitro was conserved for non-human primates but not mice. Finally, activin A-induced TFH programming was dependent on SMAD2 and SMAD3 signaling and blocked by pharmacological inhibitors.Follicular helper T cells (TFH cells) are CD4+ T cells specialized in helping B cells and are associated both with protective antibody responses and autoimmune diseases. The promise of targeting TFH cells therapeutically has been limited by fragmentary understanding of extrinsic signals that regulate the differentiation of human TFH cells. A screen of a human protein library identified activin A as a potent regulator of TFH cell differentiation. Activin A orchestrated the expression of multiple genes associated with the TFH program, independently or in concert with additional signals. TFH cell programming by activin A was antagonized by the cytokine IL-2. Activin As ability to drive TFH cell differentiation in vitro was conserved in non-human primates but not in mice. Finally, activin-A-induced TFH programming was dependent on signaling via SMAD2 and SMAD3 and was blocked by pharmacological inhibitors.

A Conserved Salt Bridge in the G Loop of Multiple Protein Kinases Is Important for Catalysis and for In Vivo Lyn Function

The glycine-rich G loop controls ATP binding and phosphate transfer in protein kinases. Here we show that the functions of Src family and Abl protein tyrosine kinases require an electrostatic interaction between oppositely charged amino acids within their G loops that is conserved in multiple other phylogenetically distinct protein kinases, from plants to humans. By limiting G loop flexibility, it controls ATP binding, catalysis, and inhibition by ATP-competitive compounds such as Imatinib. In WeeB mice, mutational disruption of the interaction results in expression of a Lyn protein with reduced catalytic activity, and in perturbed B cell receptor signaling. Like Lyn(-/-) mice, WeeB mice show profound defects in B cell development and function and succumb to autoimmune glomerulonephritis. This demonstrates the physiological importance of the conserved G loop salt bridge and at the same time distinguishes the in vivo requirement for the Lyn kinase activity from other potential functions of the protein.

Beyond IP3: roles for higher order inositol phosphates in immune cell signaling.

Nearly 25 years ago the first function of an inositol phosphate, namely Ins(1,4,5)P3, was reported to act as a “second messenger” to mobilize calcium from the endoplasmic reticulum (ER). Since this discovery, many other inositol phosphates and the kinases and phosphatases that generate these inositol phosphates have subsequently been discovered. However, the function of these “higher order” inositol phosphates in biological processes, if any, has remained a mystery. Interest in higher order inositol phosphates, such as Ins(1,3,4,5)P4, was renewed this year following reports of novel roles for these molecules in distinct processes within the immune system ranging from T cell development, B cell development and tolerance induction, as well as neutrophil and mast cell function. In this review, we will touch upon recent advances in inositol phosphate function in mammalian cells. More specifically, we will highlight new studies that have identified novel functions for specific higher order inositol phosphates, such as Ins(1,3,4,5)P4, in the immune system.

Tonic BCR signaling represses receptor editing via Raf- and calcium-dependent signaling pathways.

Light chain receptor editing is an important mechanism that prevents B cell self-reactivity. We have previously shown that tonic signaling through the BCR represses RAG expression at the immature B cell stage, and that initiation of light chain rearrangements occurs in the absence of these tonic signals in an in vitro model of B cell development. To further test our hypothesis we studied the effect of itpkb deficiency (itpkb(-/-) mice) or Raf hyper-activation (Raf-CAAX transgenic mice), two mutations that enhance BCR signaling, on receptor editing in an in vivo model. This model relies on transferring bone marrow from wild-type or mutant mice into mice expressing an anti-kappa light chain transgene. The anti-kappa transgene induces receptor editing of all kappa light chain expressing B cells, leading to a high frequency of lambda light chain expressing B cells. Anti-κ transgenic recipients of bone marrow from itpkb(-/-) or Raf-CAAX mice showed lower levels of editing to λ light chain than did non-transgenic control recipients. These results provide evidence in an in vivo model that enhanced BCR signaling at the immature B cell stage of development suppresses light chain receptor editing.

Topical SMIP-7.7, a toll-like receptor 7 agonist, protects against genital herpes simplex virus type-2 disease in the guinea pig model of genital herpes

Background: Development of more effective therapies for genital herpes simplex virus type-2 (HSV-2) infections remains a priority. The toll-like receptors (TLR) are attractive targets for the immunomodulation of primary and recurrent genital herpes infection. The guinea pig model of genital HSV-2 disease was therefore used to evaluate the efficacy of a new TLR-7 agonist, SMIP-7.7. Methods: The effects of SMIP-7.7 at concentrations between 0.90% and 0.09% were compared to the vehicle control or Aldara® (3M Health Care Limited, Northridge, CA, USA) as treatment for genital HSV-2 infections. Following intravaginal inoculation of Hartley guinea pigs with 106 pfu HSV-2 (MS strain), animals were treated intravaginally beginning at 36 h post-infection. Animals were evaluated for acute disease, acute virus replication, recurrent disease and shedding, as well as infection of the dorsal root ganglia. Results: Treatment with SMIP-7.7 significantly decreased mean total lesion scores during primary infection (all doses, P<0.01 compared with vehicle control, and similar to Aldara®). Vaginal virus titres were reduced in treated animals compared with vehicle control (P<0.001 for each treatment versus vehicle control on day 4). Treatment with SMIP-7.7 also significantly decreased the number of recurrent lesion days, the number of days with recurrent virus shedding and the infection of the dorsal root ganglia compared to the vehicle control, and was similar to Aldara®. As opposed to Aldara®, SMIP-7.7 did not induce fever or weight loss during treatment. Conclusions: SMIP-7.7 improves the outcome of primary and recurrent HSV-2 disease comparable to Aldara® but without some of the side effects associated with Aldara®.

Inhibition of the Inositol Kinase Itpkb Augments Calcium Signaling in Lymphocytes and Reveals a Novel Strategy to Treat Autoimmune Disease

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.

Stable nanoemulsions for the delivery of small molecule immune potentiators

Adjuvants are required to enhance immune responses to typically poorly immunogenic recombinant antigens. Toll-like receptor agonists (TLRa) have been widely evaluated as adjuvants because they activate the innate immune system. Currently, licensed vaccines adjuvanted with TLRa include the TLR4 agonist monophosphoryl lipid, while additional TLRa are in clinical development. Unfortunately, naturally derived TLRa are often complex and heterogeneous entities, which brings formulation challenges. Consequently, the use of synthetic small-molecule TLRa has significant advantages because they are well-defined discrete molecules, which can be chemically modified to modulate their physicochemical properties. We previously described the discovery of a family of TLR7 agonists based on a benzonaphthyridine scaffold. In addition, we described how Alum could be used to deliver these synthetic TLRa. An alternative adjuvant approach with enhanced potency over Alum are squalene containing oil-in-water emulsions, which have been included in licensed influenza vaccines, including Fluad (MF59 adjuvanted) and Pandemrix (AS03 adjuvanted). Here, we describe how to enable the co-delivery of a TLR7 agonist in a squalene-based oil-in-water emulsion, for adjuvant evaluation.