ABSTRACT
We have identified a class of azabenzimidazoles as potent and selective JAK1 inhibitors. Investigations into the SAR are presented along with the structural features required to achieve selectivity for JAK1 versus other JAK family members. An example from the series demonstrated highly selective inhibition of JAK1 versus JAK2 and JAK3, along with inhibition of pSTAT3 in vivo, enabling it to serve as a JAK1 selective tool compound to further probe the biology of JAK1 selective inhibitors.
Subject(s)
Imidazoles/pharmacology , Janus Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , STAT3 Transcription Factor/antagonists & inhibitors , Animals , Cell Cycle/drug effects , Cell Death/drug effects , Cell Line, Tumor , Dose-Response Relationship, Drug , Female , Humans , Imidazoles/chemical synthesis , Imidazoles/chemistry , Janus Kinase 1/metabolism , Mice , Mice, Nude , Models, Molecular , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , STAT3 Transcription Factor/metabolism , Structure-Activity RelationshipABSTRACT
The factors that allow self-reactive B cells to escape negative selection and become activated remain poorly defined. Using a BCR knock-in mouse strain, we identify a pathway by which B-cell selection to nucleolar self-antigens is complement dependent. Deficiency in complement component C4 led to a breakdown in the elimination of autoreactive B-cell clones at the transitional stage, characterized by a relative increase in their response to a range of stimuli, entrance into follicles, and a greater propensity to form self-reactive GCs. Using mixed BM chimeras, we found that the myeloid compartment was sufficient to restore negative selection in the autoreactive mice. A model is proposed in which in the absence of complement C4, inappropriate clearance of apoptotic debris promotes chronic activation of myeloid cells, allowing the maturation and activation of self-reactive B-cell clones leading to increased spontaneous formation of GCs.
Subject(s)
B-Lymphocytes/immunology , Complement C4/immunology , Immune Tolerance , Ribonucleoproteins/immunology , Animals , Apoptosis , Autoantigens/immunology , Autoimmunity , B-Lymphocytes/metabolism , Bone Marrow Cells/immunology , Complement C4/deficiency , Complement C4/genetics , Lupus Erythematosus, Systemic/immunology , Lymphocyte Activation , Mice , Mice, Inbred C57BL , Mice, Knockout , Myeloid Cells/immunology , Nucleolus Organizer Region/immunology , Receptors, Antigen, B-Cell/geneticsABSTRACT
The costimulatory receptor CD137 (also known as TNFRSF9 or 4-1BB) sustains effective cytotoxic T-cell responses. Agonistic anti-CD137 cancer immunotherapies are being investigated in clinical trials. Development of the first-generation CD137-agonist monotherapies utomilumab and urelumab was unsuccessful due to low antitumor efficacy mediated by the epitope recognized on CD137 or hepatotoxicity mediated by Fcγ receptors (FcγR) ligand-dependent CD137 activation, respectively. M9657 was engineered as a tetravalent bispecific antibody (mAb2) in a human IgG1 backbone with LALA mutations to reduce binding to FCγRs. Here, we report that M9657 selectively binds to mesothelin (MSLN) and CD137 with similar affinity in humans and cynomolgus monkeys. In a cellular functional assay, M9657 enhanced CD8+ T cell-mediated cytotoxicity and cytokine release in the presence of tumor cells, which was dependent on both MSLN expression and T-cell receptor/CD3 activation. Both FS122m, a murine surrogate with the same protein structure as M9657, and chimeric M9657, a modified M9657 antibody with the Fab portion replaced with an anti-murine MSLN motif, demonstrated in vivo antitumor efficacy against various tumors in wild-type and human CD137 knock-in mice, and this was accompanied by activated CD8+ T-cell infiltration in the tumor microenvironment. The antitumor immunity of M9657 and FS122m depended on MSLN expression density and the mAb2 structure. Compared with 3H3, a murine surrogate of urelumab, FS122m and chimeric M9657 displayed significantly lower on-target/off-tumor toxicity. Taken together, M9657 exhibits a promising profile for development as a tumor-targeting immune agonist with potent anticancer activity without systemic immune activation and associated hepatotoxicity.
Subject(s)
Chemical and Drug Induced Liver Injury , Neoplasms , Humans , Animals , Mice , Mesothelin , Inflammation , Tumor Necrosis Factor Receptor Superfamily, Member 9 , Tumor MicroenvironmentABSTRACT
The discovery of the activating mutation V617F in the JH2 domain of Jak2 and the modulation of oncogenic Stat3 by Jak2 inhibitors have spurred a great interest in the inhibition of the Jak2/Stat pathway in oncology. In this Letter, we communicate the discovery of novel inhibitors of the Jak2/Stat5 axis, the N-(1H-pyrazol-3-yl)pyrimidin-2-amino derivatives. The rationale, synthesis and biological evaluation of these derivatives are reported. Two lead analogs from this series, 6 and 9, displayed prolonged residence time on Jak2, at enzymatic level. Although 6 and 9 exhibited moderate selectivity in a selected kinase panel, we chose to test these inhibitors in vivo as a consequence to their long residence time. However, extended inhibition of Jak2 due to the long residence time, in the form of inhibiting phosphorylation of downstream Stat5, was not recapitulated in an in vivo setting.
Subject(s)
Drug Discovery , Janus Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , STAT5 Transcription Factor/antagonists & inhibitors , Animals , Cell Line, Transformed , Cell Proliferation/drug effects , Dogs , Dose-Response Relationship, Drug , Female , Humans , Janus Kinase 2/genetics , Janus Kinase 2/metabolism , Male , Mice , Mice, Inbred Strains , Models, Molecular , Molecular Conformation , Phosphorylation/drug effects , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Rats , Rats, Wistar , STAT5 Transcription Factor/metabolism , Structure-Activity Relationship , Substrate Specificity , Time FactorsABSTRACT
Synthesis and biological evaluation of a series of 6-aminopyrazolyl-pyridine-3-carbonitriles as JAK2 kinase inhibitors was reported. Biochemical screening, followed by profile optimization, resulted in JAK2 inhibitors exhibiting good kinase selectivity, pharmacokinetic properties, physical properties and pharmacodynamic effects.
Subject(s)
Janus Kinase 2/antagonists & inhibitors , Nitriles/chemical synthesis , Nitriles/pharmacology , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Pyrazoles/chemistry , Pyridines/chemistry , Animals , Cell Line , Cell Proliferation/drug effects , Inhibitory Concentration 50 , Mice , Molecular Structure , Nitriles/chemistry , Nitriles/pharmacokinetics , Protein Kinase Inhibitors/pharmacokinetics , Rats , Structure-Activity RelationshipABSTRACT
Thiazol-2-yl amine was identified as an isosteric replacement for pyrazol-3-yl amine during our efforts to identify potent and selective JAK2 inhibitors. The rationale, synthesis and biological evaluation of several analogs is reported, along with the in vivo evaluation of the lead compounds.
Subject(s)
Amines/chemical synthesis , Antineoplastic Agents/chemical synthesis , Janus Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Pyrazoles/chemistry , Thiazoles/chemistry , Amines/chemistry , Amines/pharmacokinetics , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacokinetics , Cell Line, Tumor , Drug Discovery , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/metabolism , Humans , Janus Kinase 2/metabolism , Mice , Mice, Nude , Microsomes/metabolism , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Rats , Xenograft Model Antitumor AssaysABSTRACT
The design, synthesis and biological evaluation of a series of pyrazol-3-ylamino pyrazines as potent and selective JAK2 kinase inhibitors is reported, along with the pharmacokinetic and pharmacodynamic properties of lead compounds.
Subject(s)
Drug Discovery , Janus Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Pyrazines/pharmacology , Pyrazoles/pharmacology , Animals , Dogs , Humans , Mice , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Pyrazines/chemical synthesis , Pyrazines/chemistry , Pyrazoles/chemical synthesis , Pyrazoles/chemistry , Rats , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Janus kinases (JAKs) have been demonstrated to be critical in cytokine signaling and have thus been implicated in both cancer and inflammatory diseases. The JAK family consists of four highly homologous members: JAK1-3 and TYK2. The development of small-molecule inhibitors that are selective for a specific family member would represent highly desirable tools for deconvoluting the intricacies of JAK family biology. Herein, we report the discovery of a potent JAK1 inhibitor, 24, which displays â¼1000-fold selectivity over the other highly homologous JAK family members (determined by biochemical assays), while also possessing good selectivity over other kinases (determined by panel screening). Moreover, this compound was demonstrated to be orally bioavailable and possesses acceptable pharmacokinetic parameters. In an in vivo study, the compound was observed to dose dependently modulate the phosphorylation of STAT3 (a downstream marker of JAK1 inhibition).
Subject(s)
Janus Kinase 1/antagonists & inhibitors , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Administration, Oral , Animals , Antineoplastic Agents/administration & dosage , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Biological Availability , Cell Line , Crystallography, X-Ray , Humans , Janus Kinase 1/chemistry , Janus Kinase 1/metabolism , Janus Kinase 2/antagonists & inhibitors , Janus Kinase 2/metabolism , Janus Kinase 3/metabolism , Mice , Phosphorylation/drug effects , STAT3 Transcription Factor/metabolism , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Homologous genes and gene products often have redundant physiological functions. Members of the tumor necrosis factor (TNF) family of cytokines can signal activation, proliferation, differentiation, costimulation, inhibition, or cell death, depending on the type and status of the target cell. TNF, lymphotoxin alpha (LTalpha), and LTbeta form a subfamily of a larger family of TNF-related ligands with their genes being linked within a compact 12-kb cluster inside the major histocompatibility complex locus. Singly TNF-, LTalpha-, and LTbeta-deficient mice share several phenotypic features, suggesting that TNF/LT signaling pathways may regulate overlapping sets of target genes. In order to directly address the issue of redundancy of TNF/LT signaling, we used the Cre-loxP recombination system to create mice with a deletion of the entire TNF/LT locus. Mice with a triple LTbeta/TNF/LTalpha deficiency essentially manifest a combination of LT and TNF single-knockout phenotypes, except for microarchitecture of the spleen, where the disorder of lymphoid cell positioning and functional T- and B-cell compartmentalization is severer than that found in TNF or LT single-knockout mice. Thus, our data support the notion that TNF and LT have largely nonredundant functions in vivo.
Subject(s)
Gene Expression Regulation , Lymphotoxin-alpha/metabolism , Signal Transduction/physiology , Tumor Necrosis Factor-alpha/metabolism , Animals , B-Lymphocytes/physiology , Gene Targeting , Leukocytes/metabolism , Lymphoid Tissue/cytology , Lymphoid Tissue/metabolism , Lymphoid Tissue/pathology , Mice , Mice, Knockout , Mice, Mutant Strains , Multigene Family , Spleen/cytology , Spleen/metabolism , T-Lymphocytes/physiologyABSTRACT
The Wnt pathway is an evolutionarily conserved and tightly regulated signaling network with important roles in embryonic development and adult tissue regeneration. Impaired Wnt pathway regulation, arising from mutations in Wnt signaling components, such as Axin, APC, and ß-catenin, results in uncontrolled cell growth and triggers oncogenesis. To explore the reported link between CK2 kinase activity and Wnt pathway signaling, we sought to identify a potent, selective inhibitor of CK2 suitable for proof of concept studies in vivo. Starting from a pyrazolo[1,5-a]pyrimidine lead (2), we identified compound 7h, a potent CK2 inhibitor with picomolar affinity that is highly selectivity against other kinase family enzymes and inhibits Wnt pathway signaling (IC50 = 50 nM) in DLD-1 cells. In addition, compound 7h has physicochemical properties that are suitable for formulation as an intravenous solution, has demonstrated good pharmacokinetics in preclinical species, and exhibits a high level of activity as a monotherapy in HCT-116 and SW-620 xenografts.
ABSTRACT
Treatment of metastatic renal cell carcinoma (mRCC) with agents that block signaling through vascular endothelial growth factor receptor 2 (VEGFR2) induces disease regression or stabilization in some patients; however, these responses tend to be short-lived. Therefore, development of combination therapies that can extend the efficacy of VEGFR antagonists in mRCC remains a priority.We studied murine xenograft models of RCC that become refractory to treatment with the VEGFR tyrosine kinase inhibitor (TKI) sunitinib. Dalantercept is a novel antagonist of Activin receptor-like kinase 1 (ALK1)/Bone morphogenetic protein (BMP) 9 signaling. Dalantercept inhibited growth in the murine A498 xenograft model which correlated with hyperdilation of the tumor vasculature and an increase in tumor hypoxia. When combined with sunitinib, dalantercept induced tumor necrosis and prevented tumor regrowth and revascularization typically seen with sunitinib monotherapy in two RCC models. Combination therapy led to significant downregulation of angiogenic genes as well as downregulation of endothelial specific gene expression particularly of the Notch signaling pathway. We demonstrate that simultaneous targeting of molecules that control distinct phases of angiogenesis, such as ALK1 and VEGFR, is a valid strategy for treatment of mRCC. At the molecular level, combination therapy leads to downregulation of Notch signaling.
Subject(s)
Activin Receptors, Type II/antagonists & inhibitors , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Carcinoma, Renal Cell/drug therapy , Kidney Neoplasms/drug therapy , Neovascularization, Pathologic/prevention & control , Receptors, Vascular Endothelial Growth Factor/antagonists & inhibitors , Activin Receptors, Type II/administration & dosage , Activin Receptors, Type II/genetics , Activin Receptors, Type II/metabolism , Animals , Axitinib , Carcinoma, Renal Cell/blood supply , Carcinoma, Renal Cell/genetics , Cell Line, Tumor , Female , Gene Expression Regulation, Neoplastic/drug effects , Humans , Imidazoles/administration & dosage , Immunoglobulin Fc Fragments/administration & dosage , Indazoles/administration & dosage , Indoles/administration & dosage , Kidney Neoplasms/blood supply , Kidney Neoplasms/genetics , Mice, Nude , Neovascularization, Pathologic/genetics , Neovascularization, Pathologic/metabolism , Protein Kinase Inhibitors/administration & dosage , Pyrroles/administration & dosage , Receptors, Vascular Endothelial Growth Factor/genetics , Receptors, Vascular Endothelial Growth Factor/metabolism , Recombinant Fusion Proteins/administration & dosage , Sunitinib , Tumor Burden/drug effects , Tumor Burden/genetics , Xenograft Model Antitumor AssaysABSTRACT
Programmed cell-death 1 ligand 1 (PD-L1) is a member of the B7/CD28 family of proteins that control T-cell activation. Many tumors can upregulate expression of PD-L1, inhibiting antitumor T-cell responses and avoiding immune surveillance and elimination. We have identified and characterized MEDI4736, a human IgG1 monoclonal antibody that binds with high affinity and specificity to PD-L1 and is uniquely engineered to prevent antibody-dependent cell-mediated cytotoxicity. In vitro assays demonstrate that MEDI4736 is a potent antagonist of PD-L1 function, blocking interaction with PD-1 and CD80 to overcome inhibition of primary human T-cell activation. In vivo MEDI4736 significantly inhibits the growth of human tumors in a novel xenograft model containing coimplanted human T cells. This activity is entirely dependent on the presence of transplanted T cells, supporting the immunological mechanism of action for MEDI4736. To further determine the utility of PD-L1 blockade, an anti-mouse PD-L1 antibody was investigated in immunocompetent mice. Here, anti-mouse PD-L1 significantly improved survival of mice implanted with CT26 colorectal cancer cells. The antitumor activity of anti-PD-L1 was enhanced by combination with oxaliplatin, which resulted in increased release of HMGB1 within CT26 tumors. Taken together, our results demonstrate that inhibition of PD-L1 function can have potent antitumor activity when used as monotherapy or in combination in preclinical models, and suggest it may be a promising therapeutic approach for the treatment of cancer. MEDI4736 is currently in several clinical trials both alone and in combination with other agents, including anti-CTLA-4, anti-PD-1, and inhibitors of IDO, MEK, BRAF, and EGFR.
Subject(s)
Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , B7-H1 Antigen/antagonists & inhibitors , Animals , Antibodies, Monoclonal/administration & dosage , Antibody-Dependent Cell Cytotoxicity/drug effects , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , B7-1 Antigen/metabolism , B7-H1 Antigen/metabolism , Binding, Competitive , Colorectal Neoplasms/drug therapy , Colorectal Neoplasms/pathology , Female , Humans , Lymphocyte Activation/drug effects , Lymphocyte Culture Test, Mixed , Melanoma/immunology , Melanoma/pathology , Melanoma/prevention & control , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Inbred NOD , Organoplatinum Compounds/administration & dosage , Oxaliplatin , Pancreatic Neoplasms/immunology , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/prevention & control , Programmed Cell Death 1 Receptor/metabolism , T-Lymphocytes/drug effects , T-Lymphocytes/immunology , Tumor Cells, Cultured , Xenograft Model Antitumor AssaysABSTRACT
Structure based design, synthesis, and biological evaluation of a novel series of 1-methyl-1H-imidazole, as potent Jak2 inhibitors to modulate the Jak/STAT pathway, are described. Using the C-ring fragment from our first clinical candidate AZD1480 (24), optimization of the series led to the discovery of compound 19a, a potent, orally bioavailable Jak2 inhibitor. Compound 19a displayed a high level of cellular activity in hematopoietic cell lines harboring the V617F mutation and in murine BaF3 TEL-Jak2 cells. Compound 19a demonstrated significant tumor growth inhibition in a UKE-1 xenograft model within a well-tolerated dose range.
Subject(s)
Antineoplastic Agents/chemical synthesis , Imidazoles/chemical synthesis , Janus Kinase 2/antagonists & inhibitors , Protein Kinase Inhibitors/chemical synthesis , Animals , Antineoplastic Agents/pharmacology , Dogs , Drug Discovery , Humans , Imidazoles/pharmacology , Mice , Protein Kinase Inhibitors/pharmacology , Rats , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
In this letter, we describe the design, synthesis, and structure-activity relationship of 5-anilinopyrazolo[1,5-a]pyrimidine inhibitors of CK2 kinase. Property-based optimization of early leads using the 7-oxetan-3-yl amino group led to a series of matched molecular pairs with lower lipophilicity, decreased affinity for human plasma proteins, and reduced binding to the hERG ion channel. Agents in this study were shown to modulate pAKT(S129), a direct substrate of CK2, in vitro and in vivo, and exhibited tumor growth inhibition when administered orally in a murine DLD-1 xenograft.
ABSTRACT
The myeloproliferative neoplasms, polycythemia vera, essential thrombocythemia, and idiopathic myelofibrosis are a heterogeneous but related group of hematological malignancies characterized by clonal expansion of one or more myeloid lineages. The discovery of the Jak2 V617F gain of function mutation highlighted Jak2 as a potential therapeutic target in the MPNs. Herein, we disclose the discovery of a series of pyrazol-3-yl pyrimidin-4-amines and the identification of 9e (AZD1480) as a potent Jak2 inhibitor. 9e inhibits signaling and proliferation of Jak2 V617F cell lines in vitro, demonstrates in vivo efficacy in a TEL-Jak2 model, has excellent physical properties and preclinical pharmacokinetics, and is currently being evaluated in Phase I clinical trials.
Subject(s)
Janus Kinase 2/antagonists & inhibitors , Pyrazoles/chemical synthesis , Pyrimidines/chemical synthesis , STAT Transcription Factors/physiology , Animals , Cell Line, Tumor , Crystallography, X-Ray , Dogs , Female , Humans , In Vitro Techniques , Janus Kinase 2/chemistry , Mice , Mice, Nude , Microsomes, Liver/metabolism , Models, Molecular , Phosphorylation , Protein Conformation , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Pyrimidines/pharmacokinetics , Pyrimidines/pharmacology , Rats , STAT Transcription Factors/metabolism , STAT3 Transcription Factor/metabolism , STAT5 Transcription Factor/metabolism , Signal Transduction , Stereoisomerism , Structure-Activity Relationship , Xenograft Model Antitumor AssaysABSTRACT
Persistent activation of Stat3 is oncogenic and is prevalent in a wide variety of human cancers. Chronic cytokine stimulation is associated with Stat3 activation in some tumors, implicating cytokine receptor-associated Jak family kinases. Using Jak2 inhibitors, we demonstrate a central role of Jaks in modulating basal and cytokine-induced Stat3 activation in human solid tumor cell lines. Inhibition of Jak2 activity is associated with abrogation of Stat3 nuclear translocation and tumorigenesis. The Jak2 inhibitor AZD1480 suppresses the growth of human solid tumor xenografts harboring persistent Stat3 activity. We demonstrate the essential role of Stat3 downstream of Jaks by inhibition of tumor growth using short hairpin RNA targeting Stat3. Our data support a key role of Jak kinase activity in Stat3-dependent tumorigenesis.
Subject(s)
Janus Kinase 2/antagonists & inhibitors , Prostatic Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Pyrazoles/pharmacology , Pyrimidines/pharmacology , STAT3 Transcription Factor/antagonists & inhibitors , Signal Transduction/drug effects , Cell Line, Tumor , Humans , Male , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/pathology , STAT3 Transcription Factor/metabolismABSTRACT
Nonhomologous end-joining (NHEJ) repairs DNA double-strand breaks (DSBs) during V(D)J recombination in developing lymphocytes and during immunoglobulin (Ig) heavy chain (IgH) class switch recombination (CSR) in peripheral B lymphocytes. We now show that CD21-cre-mediated deletion of the Xrcc4 NHEJ gene in p53-deficient peripheral B cells leads to recurrent surface Ig-negative B lymphomas ("CXP lymphomas"). Remarkably, CXP lymphomas arise from peripheral B cells that had attempted both receptor editing (secondary V[D]J recombination of Igkappa and Iglambda light chain genes) and IgH CSR subsequent to Xrcc4 deletion. Correspondingly, CXP tumors frequently harbored a CSR-based reciprocal chromosomal translocation that fused IgH to c-myc, as well as large chromosomal deletions or translocations involving Igkappa or Iglambda, with the latter fusing Iglambda to oncogenes or to IgH. Our findings reveal peripheral B cells that have undergone both editing and CSR and show them to be common progenitors of CXP tumors. Our studies also reveal developmental stage-specific mechanisms of c-myc activation via IgH locus translocations. Thus, Xrcc4/p53-deficient pro-B lymphomas routinely activate c-myc by gene amplification, whereas Xrcc4/p53-deficient peripheral B cell lymphomas routinely ectopically activate a single c-myc copy.
Subject(s)
B-Lymphocytes , Cell Transformation, Neoplastic/immunology , DNA-Binding Proteins/immunology , Gene Rearrangement, B-Lymphocyte , Immunoglobulin Class Switching , Recombination, Genetic , Amino Acid Sequence , Animals , B-Lymphocytes/immunology , B-Lymphocytes/physiology , Base Sequence , DNA Damage , DNA Repair , DNA-Binding Proteins/genetics , Genes, Immunoglobulin Heavy Chain , Humans , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Heavy Chains/immunology , Lymphoma/genetics , Lymphoma/immunology , Lymphoma/pathology , Lymphoma, B-Cell/genetics , Lymphoma, B-Cell/immunology , Mice , Mice, Knockout , Molecular Sequence Data , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/immunology , Sequence Alignment , Translocation, Genetic , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/immunologyABSTRACT
Spontaneous loss-of-function mutations in the protein-tyrosine phosphatase Shp1 cause the motheaten phenotype, characterized by widespread inflammation and autoimmunity. Because Shp1 is expressed in all hematopoietic cells, it has been unclear which aspects of the motheaten phenotypes are primary effects of Shp1 deficiency. We generated mice (Ptpn6(f/f);CD19-cre) that delete Shp1 specifically in B cells. Analysis of these mice indicates that the increase in B-1a cells in motheaten mice is a cell-autonomous consequence of Shp1 deficiency. Shp1-deficient B-1a cells could be derived from adult bone marrow and had N-nucleotide additions, consistent with an adult origin. Shp1 deficiency altered calcium response evoked by B cell antigen receptors and impaired CD40-evoked proliferation. Young Ptpn6(f/f);CD19-cre mice exhibited elevated serum immunoglobulins and impaired antibody responses to immunization, whereas older Ptpn6(f/f);CD19-cre mice developed systemic autoimmunity, characterized by DNA antibodies and immune complex glomerulonephritis. Thus, Shp1 deficiency in B cells alone perturbs B cell development and causes autoimmune disease.
Subject(s)
Autoimmune Diseases/enzymology , Autoimmune Diseases/genetics , B-Lymphocyte Subsets/enzymology , B-Lymphocyte Subsets/immunology , Cell Differentiation/immunology , Gene Deletion , Protein Tyrosine Phosphatase, Non-Receptor Type 6/genetics , Animals , Antigen-Antibody Complex/metabolism , Autoimmune Diseases/pathology , B-Lymphocyte Subsets/cytology , Cell Differentiation/genetics , Cells, Cultured , Glomerulonephritis/enzymology , Glomerulonephritis/genetics , Glomerulonephritis/immunology , Glomerulonephritis/pathology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Protein Tyrosine Phosphatase, Non-Receptor Type 6/deficiencyABSTRACT
The FDC-specific molecular signals required in the formation of FDC networks, B cell follicles, and germinal centers (GCs) have remained poorly understood. We used FDC-specific gene targeting to investigate the function of p55TNFR and IKK2 in lymphoid organ structure and function. Here we show that FDC-specific expression of p55TNFR is necessary and sufficient to promote FDC network and B cell follicle formation, restore the expression of CXCL13 and VCAM-1/ICAM-1 in FDCs, and lead to productive GCs. Notably, FDC-specific disruption of IKK2 does not affect formation of FDC networks. Yet, after antigen engagement or immune complex (IC) deposition, FDCs lacking IKK2 fail to upregulate VCAM-1 and ICAM-1, and GCs remain sterile. These findings demonstrate that IKK2-independent function of p55TNFR on FDCs is sufficient to support the development of FDC networks and GCs, while FDC-specific IKK2 is indispensable for the generation of efficient humoral immune responses.
Subject(s)
Antibody Formation , Dendritic Cells, Follicular/immunology , I-kappa B Kinase/metabolism , Receptors, Tumor Necrosis Factor, Type I/metabolism , Animals , Antibody Formation/genetics , Apoptosis , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Cell Adhesion Molecules/metabolism , Chemokine CXCL13 , Chemokines/genetics , Chemokines/metabolism , Chemokines, CXC/genetics , Chemokines, CXC/metabolism , Dendritic Cells, Follicular/cytology , Dendritic Cells, Follicular/metabolism , Gene Targeting , I-kappa B Kinase/genetics , Immunoglobulin G/immunology , Intercellular Adhesion Molecule-1/genetics , Intercellular Adhesion Molecule-1/metabolism , Lymphoid Tissue/cytology , Lymphoid Tissue/metabolism , Mice , Mice, Transgenic , Receptors, Complement 3d/genetics , Receptors, Tumor Necrosis Factor, Type I/genetics , Transcription Factors/metabolism , Up-Regulation , Vascular Cell Adhesion Molecule-1/genetics , Vascular Cell Adhesion Molecule-1/metabolismABSTRACT
We previously showed that type I interferon-induced, Cre-mediated ablation of surface BCR expression in mature B cells through Ig-heavy chain deletion results in apoptosis of these cells. This led to the hypothesis that survival signals from the BCR are vital for mature B cells. Here, we test two critical assumptions of this model. First, we demonstrate loss of mature B cells upon induced mutation of a signaling module of the BCR, not precluding BCR surface expression. Second, we show that the cells are also lost upon BCR inactivation in the absence of an exogenous inducer like interferon, excluding that cell death depends on previous cellular activation by the latter. Kinetic data demonstrate that BCR-less mature B cells have a severely reduced lifespan, with a half-life of 3-6 days. Together these results establish that BCR signaling is required to keep resting mature B cells alive in vivo.