ABSTRACT
Antagonism of the Toll-like receptors (TLRs) 7 and TLR8 has been hypothesized to be beneficial to patients suffering from autoimmune conditions. A phenotypic screen for small molecule antagonists of TLR7/8 was carried out in a murine P4H1 cell line. Compound 1 was identified as a hit that showed antagonistic activity on TLR7 and TLR8 but not TLR9, as shown on human peripheral blood mononuclear cells (hPBMCs). It was functionally cross reactive with mouse TLR7 but lacked oral exposure and had only modest potency. Chemical optimization resulted in 2, which showed in vivo efficacy following intraperitoneal administration. Further optimization resulted in 8 which had excellent in vitro activity, exposure and in vivo activity. Additional work to improve physical properties resulted in 15, an advanced lead that had favorable in vitro and exposure properties. It was further demonstrated that activity of the series tracked with binding to the extracellular domain of TLR7 implicating that the target of this series are endosomal TLRs rather than downstream signaling pathways.
Subject(s)
Piperazine/chemistry , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/metabolism , Administration, Oral , Animals , Cell Line , Drug Evaluation, Preclinical , Half-Life , Humans , Interferon-gamma/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/metabolism , Male , Mice , Mice, Inbred BALB C , Piperazine/administration & dosage , Piperazine/pharmacokinetics , Piperazine/pharmacology , Spleen/cytology , Spleen/drug effects , Spleen/metabolism , Structure-Activity Relationship , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 8/antagonists & inhibitorsABSTRACT
Toll-like receptor 7 (Tlr7) has been linked to systemic lupus disease incidence in humans and mice, but how TLR7 potentiates autoimmunity is unclear. We used a Tlr7 transgenic (tg) mouse model to investigate the cellular and molecular events required to induce spontaneous autoimmunity through increased TLR7 activity. We determined that Tlr7 exerts B-cell-intrinsic effects in promoting spontaneous germinal center (GC) and plasmablast B-cell development, and that these B-cell subsets are dependent on T-cell-derived signals through CD40L and SLAM-associated protein (SAP), but not IL-17. Antigen specificity also factored into TLR7-induced disease, as both a restricted T cell receptor (TCR) specificity and MHC haplotype H2(k/k) protected Tlr7tg mice from spontaneous lymphocyte activation and autoantibody production. Inflammatory myeloid cell expansion and autoimmunity did not develop in Tlr7tgIgH(-/-) mice, suggesting either that spontaneous TLR7 activation does not occur in dendritic cells, or, if it does occur, cannot drive these events in the absence of B-cell aid. These data indicate that autoimmune disease in Tlr7tg mice is contingent upon B cells receiving stimulation both through innate pathways and T-cell-derived signals and suggest a codependent relationship between B cells and T cells in the development of autoimmunity.
Subject(s)
Adaptive Immunity/immunology , Autoimmunity/immunology , B-Lymphocytes/immunology , Immunity, Innate/immunology , Lupus Erythematosus, Systemic/immunology , Signal Transduction/immunology , Toll-Like Receptor 7/immunology , Analysis of Variance , Animals , CD40 Ligand/metabolism , Flow Cytometry , Immunohistochemistry , Intracellular Signaling Peptides and Proteins/metabolism , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Real-Time Polymerase Chain Reaction , Receptors, Antigen, T-Cell/metabolism , Signaling Lymphocytic Activation Molecule Associated ProteinABSTRACT
Type I IFNs (IFN-I) are normally produced during antiviral responses, yet high levels of chronic IFN-I expression correlate with autoimmune disease. A variety of viral sensors generate IFN-I in their response, but other than TLRs, it is not fully known which pathways are directly involved in the development of spontaneous immune pathologies. To further explore the link between IFN-I induced by viral pathways and autoimmunity, we generated a new transgenic mouse line containing multiple copies of Ifih1, a gene encoding the cytoplasmic dsRNA sensor MDA5 with proven linkage to diabetes and lupus. We show that MDA5 overexpression led to a chronic IFN-I state characterized by resistance to a lethal viral infection through rapid clearance of virus in the absence of a CD8(+) or Ab response. Spontaneous MDA5 activation was not sufficient to initiate autoimmune or inflammatory pathology by itself, even though every immune cell population had signs of IFN activation. When combined with the lupus-susceptible background of the FcγR2B deficiency, MDA5 overexpression did accelerate the production of switched autoantibodies, the incidence of glomerulonephritis, and early lethality. Thus, MDA5 transgenic mice provide evidence that chronic elevated levels of IFN-I are not sufficient to initiate autoimmunity or inflammation although they might exacerbate an ongoing autoimmune pathology.
Subject(s)
Autoimmunity/genetics , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/immunology , Gene Dosage , Interferon Type I/genetics , Virus Diseases/immunology , Animals , Cell Line , Inflammation/etiology , Interferon-Induced Helicase, IFIH1 , Mice , Mice, Transgenic , Viruses/immunologyABSTRACT
Genetic and in vivo evidence suggests that aberrant recognition of RNA-containing autoantigens by Toll-like receptors (TLRs) 7 and 8 drives autoimmune diseases. Here we report on the preclinical characterization of MHV370, a selective oral TLR7/8 inhibitor. In vitro, MHV370 inhibits TLR7/8-dependent production of cytokines in human and mouse cells, notably interferon-α, a clinically validated driver of autoimmune diseases. Moreover, MHV370 abrogates B cell, plasmacytoid dendritic cell, monocyte, and neutrophil responses downstream of TLR7/8. In vivo, prophylactic or therapeutic administration of MHV370 blocks secretion of TLR7 responses, including cytokine secretion, B cell activation, and gene expression of, e.g., interferon-stimulated genes. In the NZB/W F1 mouse model of lupus, MHV370 halts disease. Unlike hydroxychloroquine, MHV370 potently blocks interferon responses triggered by specific immune complexes from systemic lupus erythematosus patient sera, suggesting differentiation from clinical standard of care. These data support advancement of MHV370 to an ongoing phase 2 clinical trial.
Subject(s)
Autoimmune Diseases , Lupus Erythematosus, Systemic , Humans , Mice , Animals , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 7/therapeutic use , Lupus Erythematosus, Systemic/drug therapy , Lupus Erythematosus, Systemic/metabolism , Hydroxychloroquine/pharmacology , Hydroxychloroquine/therapeutic use , InterferonsABSTRACT
Toll-like receptor (TLR) 7 and TLR8 are endosomal sensors of the innate immune system that are activated by GU-rich single stranded RNA (ssRNA). Multiple genetic and functional lines of evidence link chronic activation of TLR7/8 to the pathogenesis of systemic autoimmune diseases (sAID) such as Sjögren's syndrome (SjS) and systemic lupus erythematosus (SLE). This makes targeting TLR7/8-induced inflammation with small-molecule inhibitors an attractive approach for the treatment of patients suffering from systemic autoimmune diseases. Here, we describe how structure-based optimization of compound 2 resulted in the discovery of 34 (MHV370, (S)-N-(4-((5-(1,6-dimethyl-1H-pyrazolo[3,4-b]pyridin-4-yl)-3-methyl-4,5,6,7-tetrahydro-1H-pyrazolo[4,3-c]pyridin-1-yl)methyl)bicyclo[2.2.2]octan-1-yl)morpholine-3-carboxamide). Its in vivo activity allows for further profiling toward clinical trials in patients with autoimmune disorders, and a Phase 2 proof of concept study of MHV370 has been initiated, testing its safety and efficacy in patients with Sjögren's syndrome and mixed connective tissue disease.
ABSTRACT
Inappropriate activation of endosomal TLR7 and TLR8 occurs in several autoimmune diseases, in particular systemic lupus erythematosus (SLE). Herein, the development of a TLR8 antagonist competition assay and its application for hit generation of dual TLR7/8 antagonists are reported. The structure-guided optimization of the pyridone hit 3 using this biochemical assay in combination with cellular and TLR8 cocrystal structural data resulted in the identification of a highly potent and selective TLR7/8 antagonist (27) with in vivo efficacy. The two key steps for optimization were (i) a core morph guided by a TLR7 sequence alignment to achieve a dual TLR7/8 antagonism profile and (ii) introduction of a fluorine in the piperidine ring to reduce its basicity, resulting in attractive oral pharmacokinetic (PK) properties and improved TLR8 binding affinity.
Subject(s)
Lupus Erythematosus, Systemic/drug therapy , Pyridones/chemistry , Pyridones/pharmacology , Toll-Like Receptor 7/antagonists & inhibitors , Toll-Like Receptor 8/antagonists & inhibitors , Animals , Cells, Cultured , Drug Discovery , Humans , Indazoles/chemistry , Indazoles/pharmacokinetics , Indazoles/pharmacology , Lupus Erythematosus, Systemic/metabolism , Male , Mice, Inbred C57BL , Models, Molecular , Pyridones/pharmacokinetics , Rats, Sprague-Dawley , Toll-Like Receptor 7/chemistry , Toll-Like Receptor 7/metabolism , Toll-Like Receptor 8/chemistry , Toll-Like Receptor 8/metabolismABSTRACT
Class Ia phosphoinositide 3-kinases (PI3Ks) are heterodimers of p110 catalytic and p85 regulatory subunits that mediate a variety of cellular responses to growth and differentiation factors. Although embryonic development is not impaired in mice lacking all isoforms of the p85alpha gene (p85alpha-/- p55alpha-/- p50alpha-/-) or in mice lacking the p85beta gene (p85beta-/-) (D. A. Fruman, F. Mauvais-Jarvis, D. A. Pollard, C. M. Yballe, D. Brazil, R. T. Bronson, C. R. Kahn, and L. C. Cantley, Nat Genet. 26:379-382, 2000; K. Ueki, C. M. Yballe, S. M. Brachmann, D. Vicent, J. M. Watt, C. R. Kahn, and L. C. Cantley, Proc. Natl. Acad. Sci. USA 99:419-424, 2002), we show here that loss of both genes results in lethality at embryonic day 12.5 (E12.5). The phenotypes of these embryos, including subepidermal blebs flanking the neural tube at E8 and bleeding into the blebs during the turning process, are similar to defects observed in platelet-derived growth factor receptor alpha null (PDGFRalpha-/-) mice (P. Soriano, Development 124:2691-2700, 1997), suggesting that PI3K is an essential mediator of PDGFRalpha signaling at this developmental stage. p85alpha-/- p55alpha+/+ p50alpha+/+ p85beta-/- mice had similar but less severe defects, indicating that p85alpha and p85beta have a critical and redundant function in development. Mouse embryo fibroblasts deficient in all p85alpha and p85beta gene products (p85alpha-/- p55alpha-/- p50alpha-/- p85beta-/-) are defective in PDGF-induced membrane ruffling. Overexpression of the Rac-specific GDP-GTP exchange factor Vav2 or reintroduction of p85alpha or p85beta rescues the membrane ruffling defect. Surprisingly, reintroduction of p50alpha also restored PDGF-dependent membrane ruffling. These results indicate that class Ia PI3K is critical for PDGF-dependent actin rearrangement but that the SH3 domain and the Rho/Rac/Cdc42-interacting domain of p85, which lacks p50alpha, are not required for this response.
Subject(s)
Actins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Animals , Cell Membrane/drug effects , Cell Membrane/genetics , Cell Membrane/metabolism , Cell Surface Extensions/drug effects , Embryo Loss/enzymology , Embryo Loss/genetics , Embryo Loss/metabolism , Embryo, Mammalian/embryology , Embryo, Mammalian/enzymology , Embryo, Mammalian/metabolism , Extracellular Signal-Regulated MAP Kinases/metabolism , Isoenzymes/chemistry , Isoenzymes/deficiency , Isoenzymes/genetics , Isoenzymes/metabolism , Mice , Mice, Knockout , Molecular Weight , Phosphatidylinositol 3-Kinases/chemistry , Phosphatidylinositol 3-Kinases/deficiency , Phosphatidylinositol 3-Kinases/genetics , Phosphorylation , Platelet-Derived Growth Factor/pharmacology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , Signal TransductionABSTRACT
Subunit vaccines are typically poorly immunogenic when administered alone, and require adjuvants for robust responses. Triggering TLRs to boost antigen-specific adaptive immunity is an attractive approach to increase the potency and quality of vaccines. However, recent reports suggest that alterations in TLR expression are associated with the pathogenesis of inflammatory and autoimmune diseases. To compare genetic studies with adjuvant studies, we examined whether stimulation through a TLR agonist induces or increases the autoimmune phenotype of healthy or autoimmune mice. C57BL/6, MRL/lpr, and Fcγr2b-deficient mice were dosed i.p. with Poly I:C every other day for 3 weeks, and monitored for signs of autoimmunity over 3 months. A separate group of mice was vaccinated three times i.m. with rPA anthrax antigen with or without Poly I:C with 2 weeks between doses. Immunized groups exhibited robust responses to vaccine and C57BL/6 and MRL/lpr mice showed a statistically significant increase in anti-rPA IgG responses in the presence of Poly I:C. Interestingly, Fcγr2b-/- mice showed increases with the base rPA vaccine, which was not significantly increased when Poly I:C was used as an adjuvant. In the chronically dosed groups, we also observed subtle alterations in levels of total antibody and some autoantibodies. However, there were no statistically significant differences in autoimmune syndrome, as measured by proteinurea, kidney pathology, weight loss, and mortality, with Poly I:C administration in chronic or vaccination mode. Taken together, these results suggest that administration of TLR3 agonists in chronic or vaccination mode does not induce or exacerbate models of systemic lupus erythematosus.
Subject(s)
Adjuvants, Immunologic/administration & dosage , Anthrax/prevention & control , Antibodies, Bacterial/biosynthesis , Lupus Erythematosus, Systemic/immunology , Poly I-C/administration & dosage , Toll-Like Receptor 3/immunology , Animals , Anthrax/immunology , Antigens, Bacterial/administration & dosage , Antigens, Bacterial/immunology , Autoantibodies/biosynthesis , Autoimmunity , Bacterial Toxins/administration & dosage , Bacterial Toxins/immunology , Disease Models, Animal , Female , Gene Expression , Immunization , Injections, Intramuscular , Injections, Intraperitoneal , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/pathology , Mice , Mice, Inbred C57BL , Mice, Inbred MRL lpr , Mice, Transgenic , Receptors, IgG/deficiency , Receptors, IgG/genetics , Receptors, IgG/immunology , Toll-Like Receptor 3/agonists , Toll-Like Receptor 3/geneticsABSTRACT
Phosphoinositide kinase (PI3K) is activated by various receptors on lymphocytes and regulates development, activation, and tolerance. Genetic ablation of PI3K function in T cells leads to the appearance of autoimmune disorders. In B cells, loss of the class IA regulatory subunit p85alpha causes a partial defect in B cell development and proliferation, whereas loss of p85beta alone causes no apparent changes in B cell function. Here we investigate further the consequences of p85beta deletion in B cells, in the presence or absence of p85alpha. We demonstrate that p85beta partially compensates for loss of p85alpha in B cell development and peripheral survival, with greater defects observed when both isoforms are absent. BCR-mediated AKT phosphorylation is partially reduced in p85alpha-deficient B cells and further diminished with concomitant loss of p85beta. Unexpectedly, loss of p85beta results in increased BCR-mediated proliferation and ERK phosphorylation. These results indicate that the p85beta regulatory isoform has partially overlapping functions with p85alpha in B cells as well as a unique role in opposing BCR responses.
Subject(s)
B-Lymphocytes/enzymology , B-Lymphocytes/immunology , Isoenzymes , Phosphatidylinositol 3-Kinases/metabolism , Animals , B-Lymphocytes/cytology , Cell Differentiation , Isoenzymes/genetics , Isoenzymes/metabolism , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Phosphatidylinositol 3-Kinases/genetics , Signal TransductionABSTRACT
Recruitment of PI3K to the cell membrane is an indispensable step in normal lymphocyte proliferation and activation. In this study we identify PI3K as an important signaling molecule for maintaining basal T and B lymphocyte motility and homing in the intact lymph node. Pharmacological inhibition of PI3K catalytic isoforms exerted broad effects on basal lymphocyte motility, including changes in homing kinetics, localization of B cells within the lymph node, and reduced cell velocities. Lymphocytes deficient in either or both of the class IA PI3K regulatory subunits p85alpha and p85beta also exhibited reduced velocities, with the magnitude of reduction depending upon both cell type and isoform specificity. B cells deficient in p85alpha exhibited gross morphological abnormalities that were not evident in cells treated with a PI3K inhibitor. Our results show, for the first time, that class IA PI3Ks play an important role in regulating basal lymphocyte motility and that p85alpha regulatory subunit expression is required to maintain B cell morphology in a manner independent of PI3K catalytic function. Moreover, we demonstrate distinct roles for catalytic domain function and class IA PI3K regulatory domain activity in lymphocyte motility, homing, and homeostatic localization of mature resting B cells.
Subject(s)
B-Lymphocytes/immunology , Cell Movement/immunology , Lymph Nodes/immunology , Lymphocyte Activation/immunology , Phosphatidylinositol 3-Kinases/immunology , T-Lymphocytes/immunology , Animals , B-Lymphocytes/enzymology , Catalytic Domain/immunology , Cell Proliferation , Homeostasis/immunology , Isoenzymes/immunology , Isoenzymes/metabolism , Kinetics , Lymph Nodes/enzymology , Mice , Phosphatidylinositol 3-Kinases/metabolism , Protein Structure, Tertiary/physiology , T-Lymphocytes/enzymologyABSTRACT
Nucleic acid-binding innate immune receptors such as Toll-like receptor 7 (TLR7) and TLR9 have been implicated in the development of some autoimmune pathologies. The Y chromosome-linked genomic modifier Yaa, which correlates with a duplication of Tlr7 and 16 other genes, exacerbates lupus-like syndromes in several mouse strains. Here we demonstrated that duplication of the Tlr7 gene was the sole requirement for this accelerated autoimmunity, because reduction of Tlr7 gene dosage abolished the Yaa phenotype. Further, we described new transgenic lines that overexpressed TLR7 alone and found that spontaneous autoimmunity developed beyond a 2-fold increase in TLR7 expression. Whereas a modest increase in Tlr7 gene dosage promoted autoreactive lymphocytes with RNA specificities and myeloid cell proliferation, a substantial increase in TLR7 expression caused fatal acute inflammatory pathology and profound dendritic cell dysregulation. These results underscore the importance of tightly regulating expression of TLR7 to prevent spontaneous triggering of harmful autoreactive and inflammatory responses.
Subject(s)
Autoimmunity/genetics , Dendritic Cells/immunology , Lupus Erythematosus, Systemic/genetics , Membrane Glycoproteins/genetics , Toll-Like Receptor 7/genetics , Animals , Autoantibodies/blood , Autoantibodies/immunology , Autoantigens/immunology , Cell Proliferation , Dendritic Cells/metabolism , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Dosage , Immunohistochemistry , Lupus Erythematosus, Systemic/immunology , Membrane Glycoproteins/biosynthesis , Mice , Mice, Transgenic , RNA/immunology , Reverse Transcriptase Polymerase Chain Reaction , Toll-Like Receptor 7/biosynthesisABSTRACT
The class IA subgroup of phosphoinositide 3-kinase (PI3K) is activated downstream of antigen receptors, costimulatory molecules, and cytokine receptors on lymphocytes. Targeted deletion of individual genes for class IA regulatory subunits severely impairs the development and function of B cells but not T cells. Here we analyze conditional mutant mice in which thymocytes and T cells lack the major class IA regulatory subunits p85alpha, p55alpha, p50alpha, and p85beta. These cells exhibit nearly complete loss of PI3K signaling downstream of the T-cell receptor (TCR) and CD28. Nevertheless, T-cell development is largely unperturbed, and peripheral T cells show only partial impairments in proliferation and cytokine production in vitro. Both genetic and pharmacologic experiments suggest that class IA PI3K signaling plays a limited role in T-cell proliferation driven by TCR/CD28 clustering. In vivo, class IA-deficient T cells provide reduced help to B cells but show normal ability to mediate antiviral immunity. Together these findings provide definitive evidence that class IA PI3K regulatory subunits are essential for a subset of T-cell functions while challenging the notion that this signaling mechanism is a critical mediator of costimulatory signals downstream of CD28.
Subject(s)
Phosphatidylinositol 3-Kinases/metabolism , T-Lymphocytes/physiology , Animals , Cell Proliferation , In Vitro Techniques , Lymphocyte Cooperation , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Mice, Transgenic , Phosphatidylinositol 3-Kinases/classification , Phosphatidylinositol 3-Kinases/deficiency , Phosphoinositide-3 Kinase Inhibitors , Protein Kinases/metabolism , Protein Subunits , Signal Transduction , T-Lymphocytes/cytology , T-Lymphocytes/immunology , TOR Serine-Threonine KinasesABSTRACT
The immune system requires precise regulation of activating and inhibitory signals so that it can mount effective responses against pathogens while ensuring tolerance to self-components. Some of the most potent activation signals are triggered by innate immune molecules, particularly those in the TLR family. Recent studies have shown that engagement of TLRs plays a significant role in both innate and adaptive immunity. This review focuses on the ways that TLR function might contribute to the etiology of lupus-like syndromes in the context of an autoimmune-prone environment. By considering the sources, localization, and expression of both nucleic acids and the molecules that bind them, we discuss several ways that innate immunity can play a role in the development of systemic autoimmunity.
Subject(s)
Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , DNA/immunology , RNA/immunology , Toll-Like Receptors/physiology , Animals , Autoimmune Diseases/metabolism , DNA/metabolism , Humans , Immunity, Innate/genetics , RNA/metabolism , Toll-Like Receptors/genetics , Toll-Like Receptors/metabolismABSTRACT
Antibodies against nuclear self-antigens are characteristic of systemic autoimmunity, although mechanisms promoting their generation and selection are unclear. Here, we report that B cells containing the Y-linked autoimmune accelerator (Yaa) locus are intrinsically biased toward nucleolar antigens because of increased expression of TLR7, a single-stranded RNA-binding innate immune receptor. The TLR7 gene is duplicated in Yaa mice because of a 4-Megabase expansion of the pseudoautosomal region. These results reveal high divergence in mouse Y chromosomes and represent a good example of gene copy number qualitatively altering a polygenic disease manifestation.
Subject(s)
Autoimmunity/genetics , B-Lymphocytes/immunology , Cell Nucleolus/immunology , Gene Duplication , Membrane Glycoproteins/genetics , RNA/immunology , Toll-Like Receptor 7/genetics , Agammaglobulinaemia Tyrosine Kinase , Aminoquinolines/pharmacology , Animals , Antibodies, Antinuclear/biosynthesis , Antibodies, Antinuclear/immunology , Antibody Specificity , Female , Gene Dosage , Imiquimod , In Situ Hybridization, Fluorescence , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Lymphocyte Activation , Male , Membrane Glycoproteins/immunology , Mice , Mice, Inbred C57BL , Mice, Inbred Strains , Oligonucleotide Array Sequence Analysis , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Receptors, Antigen, B-Cell/immunology , Toll-Like Receptor 7/immunology , X Chromosome/genetics , Y Chromosome/geneticsABSTRACT
Sjögren's syndrome (SS) is an autoimmune disease that is characterized by infiltration of exocrine tissues, resulting in xerostomia (dry mouth) and keratoconjunctivitis sicca (dry eyes). Here, we show that mice with T cell-specific loss of class IA phosphoinositide 3-kinase function develop organ-specific autoimmunity that resembles the human disease SS. Most mutant mice aged 3-8 months develop corneal opacity and eye lesions due to irritation and constant scratching. These mice display cardinal signs of primary SS such as marked lymphocytic infiltration of the lacrimal glands, antinuclear antibodies in the serum, and elevated titer of anti-SS-A antibody, in the absence of kidney pathology. Immunofluorescence studies show the presence of numerous CD4+ T cells with a smaller number of CD8+ T cells and B cells in the lacrimal glands. CD4+ T cells from these mice exhibit aberrant differentiation in vitro. These results indicate that aberrant T cells with impaired class IA phosphoinositide 3-kinase signaling can lead to organ-specific autoimmunity. In addition, the mouse model described here represents a tool to study the pathogenesis and treatment of SS.
Subject(s)
Phosphatidylinositol 3-Kinases/deficiency , Sjogren's Syndrome/enzymology , Sjogren's Syndrome/immunology , T-Lymphocytes/enzymology , T-Lymphocytes/immunology , Animals , Autoantibodies/blood , Cell Differentiation , Disease Models, Animal , Humans , In Vitro Techniques , Lacrimal Apparatus/enzymology , Lacrimal Apparatus/immunology , Lacrimal Apparatus/pathology , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Mutant Strains , Mice, Transgenic , Phosphatidylinositol 3-Kinases/classification , Signal Transduction , Sjogren's Syndrome/pathology , T-Lymphocytes/pathology , T-Lymphocytes, Helper-Inducer/enzymology , T-Lymphocytes, Helper-Inducer/immunology , T-Lymphocytes, Helper-Inducer/pathology , T-Lymphocytes, Regulatory/enzymology , T-Lymphocytes, Regulatory/immunology , T-Lymphocytes, Regulatory/pathologyABSTRACT
Cells of the immune system carry out diverse functions that are controlled by surface receptors for antigen, costimulatory molecules, cytokines, chemokines, and other ligands. A shared feature of signal transduction downstream of most receptors on immune cells, as in nonhematopoietic cell types, is the activation of phosphoinositide 3-kinase (PI3K). The mechanism by which this common signaling event is elicited by distinct receptors and contributes to unique functional outcomes is an intriguing puzzle. Understanding how specificity is achieved in PI3K signaling is of particular significance because altered regulation of this pathway is observed in many disease states, including leukemia and lymphoma. Here we review recent advances in the understanding of PI3K signaling mechanisms in different immune cells and receptor systems. We emphasize the concept that PI3K and its products are components of complex networks of interacting proteins and second messengers, rather than simple links in linear signaling cascades.
Subject(s)
Lymphocyte Activation/immunology , Lymphocytes/enzymology , Models, Immunological , Phosphatidylinositol 3-Kinases/immunology , Signal Transduction/immunology , Animals , Enzyme Activation/immunology , Humans , Lymphocytes/immunologyABSTRACT
Phosphoinositide 3-kinase activation is important for lymphocyte proliferation and survival. Disrupting the gene that encodes the major phosphoinositide 3-kinase regulatory isoform p85alpha impairs B cell development and proliferation. However, T cell functions are intact in the absence of p85alpha. In this study, we test the hypothesis that the related isoform p85beta is an essential regulatory subunit for T cell signaling. Unexpectedly, T cells lacking p85beta showed a marked increase in proliferation and decreased death when stimulated with anti-CD3 plus IL-2. Both CD4(+) and CD8(+) T cells completed more cell divisions. Transcriptional profiling revealed reduced levels of caspase-6 mRNA in p85beta-deficient T cells, which was paralleled by reduced caspase-6 enzyme activity. Increased T cell accumulation was also observed in vivo following infection of p85beta-deficient mice with mouse hepatitis virus. Together, these results suggest a unique role for p85beta in limiting T cell expansion.
Subject(s)
Lymphocyte Activation , Phosphatidylinositol 3-Kinases/physiology , T-Lymphocytes/immunology , Animals , B-Lymphocytes/physiology , Calcium/metabolism , Caspase 6 , Caspases/physiology , Interleukin-2/pharmacology , Mice , Mice, Inbred C57BL , Phosphatidylinositol 3-Kinases/chemistry , Phospholipase C gamma , Protein Subunits , T-Lymphocytes/physiology , Type C Phospholipases/physiologyABSTRACT
BCR-ABL and v-ABL are oncogenic forms of the Abl tyrosine kinase that can cause leukemias in mice and humans. ABL oncogenes trigger multiple signaling pathways whose contribution to transformation varies among cell types. Activation of phosphoinositide 3-kinase (PI3K) is essential for ABL-dependent proliferation and survival in some cell types, and global PI3K inhibitors can enhance the antileukemia effects of the Abl kinase inhibitor imatinib. Although a significant fraction of BCR-ABL-induced human leukemias are of B-cell origin, little is known about PI3K signaling mechanisms in B-lineage cells transformed by ABL oncogenes. Here we show that activation of class I(A) PI3K and downstream inactivation of FOXO transcription factors are essential for survival of murine pro/pre-B cells transformed by v-ABL or BCR-ABL. In addition, analysis of mice lacking individual PI3K genes indicates that products of the Pik3r1 gene contribute to transformation efficiency by BCR-ABL. These findings establish a role for PI3K signaling in B-lineage transformation by ABL oncogenes.