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1.
Cancer Res ; 71(15): 5123-33, 2011 Aug 01.
Article in English | MEDLINE | ID: mdl-21697281

ABSTRACT

Topical application of small molecule Toll-like receptor 7 (TLR7) agonists is highly effective for the treatment of skin tumors, whereas their systemic application has been largely unsuccessful for cancer therapy. One reason may be that repeated systemic application of TLR ligands can induce a state of immune unresponsiveness, termed TLR tolerance. We show here that a single injection of the TLR7 agonist R848 in mice induces a short period of increased response to TLR stimulation followed by a state of hyporesponsiveness lasting several days. This state is characterized by inhibited secretion of the key cytokines interleukin (IL)-12p70 and IL-6 as well as by a block in IFN-α production. We show for the first time that at the cellular level, TLR7 tolerance occurs in both plasmacytoid and myeloid dendritic cells, two cell populations that play a critical role in the initiation and amplification of antitumor immune responses. We further show that TLR7 tolerance in plasmacytoid dendritic cells is accompanied by downregulation of the adaptor protein IL-1 receptor-associated kinase 1. On the basis of these findings, we have designed a novel strategy for the treatment of tumors by using cycles of repeated R848 injections separated by treatment-free intervals. We show in CT26 tumor-bearing mice that this protocol circumvents TLR7 tolerance and improves the efficacy of cancer immunotherapy.


Subject(s)
Carcinoma/drug therapy , Colonic Neoplasms/drug therapy , Imidazoles/therapeutic use , Immune Tolerance/drug effects , Membrane Glycoproteins/agonists , Toll-Like Receptor 7/agonists , Tumor Escape/drug effects , Animals , Carcinoma/immunology , Cells, Cultured/immunology , Colonic Neoplasms/immunology , Cytokines/metabolism , Dendritic Cells/metabolism , Drug Screening Assays, Antitumor , Female , Interferon-alpha/pharmacology , Interleukin-1 Receptor-Associated Kinases/biosynthesis , Interleukin-1 Receptor-Associated Kinases/genetics , Interleukin-12/biosynthesis , Interleukin-12/genetics , Interleukin-6/biosynthesis , Interleukin-6/genetics , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL
2.
J Immunol ; 183(10): 6078-86, 2009 Nov 15.
Article in English | MEDLINE | ID: mdl-19890064

ABSTRACT

RNA oligonucleotides containing immune-activating sequences promote the development of cytotoxic T cell and B cell responses to Ag. In this study, we show for the first time that immunostimulatory RNA oligonucleotides induce a NK cell response that prevents growth of NK-sensitive tumors. Treatment of mice with immunostimulatory RNA oligonucleotides activates NK cells in a sequence-dependent manner, leading to enhanced IFN-gamma production and increased cytotoxicity. Use of gene-deficient mice showed that NK activation is entirely TLR7-dependent. We further demonstrate that NK activation is indirectly induced through IL-12 and type I IFN production by dendritic cells. Reconstitution of TLR7-deficient mice with wild-type dendritic cells restores NK activation upon treatment with immunostimulatory RNA oligonucleotides. Thus, by activating both NK cells and CTLs, RNA oligonucleotides stimulate two major cellular effectors of antitumor immunity. This dual activation may enhance the efficacy of immunotherapeutic strategies against cancer by preventing the development of tumor immune escape variants.


Subject(s)
Adjuvants, Immunologic , Dendritic Cells/immunology , Killer Cells, Natural/immunology , Oligoribonucleotides/immunology , Toll-Like Receptor 7/immunology , Animals , Cell Line, Tumor , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Female , Humans , Interferon Type I/biosynthesis , Interferon Type I/drug effects , Interferon Type I/immunology , Interferon-gamma/biosynthesis , Interferon-gamma/drug effects , Interferon-gamma/immunology , Killer Cells, Natural/drug effects , Killer Cells, Natural/metabolism , Lymphocyte Activation/drug effects , Lymphocyte Activation/immunology , Mice , Mice, Inbred C57BL , Mice, Knockout , Oligodeoxyribonucleotides/immunology , Oligodeoxyribonucleotides/pharmacology , Oligoribonucleotides/pharmacology , Poly A/immunology , Poly A/pharmacology , Toll-Like Receptor 7/genetics , Toll-Like Receptor 7/metabolism
3.
Eur J Immunol ; 37(1): 252-60, 2007 Jan.
Article in English | MEDLINE | ID: mdl-17163454

ABSTRACT

Signals from the pre-B cell receptor (pre-BCR) mediated by the cytoplasmic tails of Ig-alpha/Ig-beta are essential for developing B cells. To analyze the role of Ig-alpha ITAM and non-ITAM tyrosines in pre-BCR signaling, we reconstituted individual tyrosine mutants of Ig-alpha in src homology 2 domain-containing leukocyte protein of 65 kDa (SLP-65)/Ig-alpha double-deficient pre-B cells. We show that the Ig-alpha mutants led to comparable pre-BCR expression on the cell surface, while the pre-BCR-induced tyrosine phosphorylation was different. We further show that the reconstitution of Ig-alpha and the resulting pre-BCR expression led to enrichment of the pre-BCR-expressing cells in vitro irrespective of the introduced Ig-alpha mutation. We show that, even though the enrichment rate increased by lowering the IL-7 concentration, residual amounts of IL-7 were required for optimal enrichment. Our results indicate that surface IL-7 receptor expression is modulated by the pre-BCR, thereby increasing the IL-7 sensitivity of the respective cells. In contrast to the comparable pre-B cell proliferation, however, the Ig-alpha mutants differed in their capacity to induce calcium flux and activate efficient pre-B cell differentiation. Together, our data suggest that ITAM tyrosines and Y204 are required for efficient pre-B cell differentiation but not proliferation.


Subject(s)
B-Lymphocyte Subsets/cytology , CD79 Antigens/physiology , Cell Differentiation/immunology , Cell Proliferation , Stem Cells/cytology , Stem Cells/immunology , Tyrosine , Adaptor Proteins, Signal Transducing/biosynthesis , Adaptor Proteins, Signal Transducing/genetics , Amino Acid Motifs/genetics , Animals , B-Lymphocyte Subsets/immunology , B-Lymphocyte Subsets/metabolism , CD79 Antigens/deficiency , CD79 Antigens/genetics , Cell Differentiation/genetics , Cells, Cultured , Membrane Glycoproteins/biosynthesis , Membrane Glycoproteins/genetics , Membrane Glycoproteins/physiology , Mice , Mice, Inbred BALB C , Mutagenesis, Site-Directed , Phosphorylation , Pre-B Cell Receptors , Receptors, Antigen, B-Cell/biosynthesis , Receptors, Antigen, B-Cell/genetics , Receptors, Antigen, B-Cell/physiology , Signal Transduction/genetics , Signal Transduction/immunology , Stem Cells/metabolism , Tyrosine/genetics
4.
Immunol Res ; 34(2): 143-55, 2006.
Article in English | MEDLINE | ID: mdl-16760574

ABSTRACT

B cell development is characterized by a coordinated progression through defined stages that are controlled at several checkpoints. Signals from the pre-B cell receptor (pre-BCR) are essential for regulated transition from the pre-B cell stage. The adaptor protein SLP-65 plays a key role in this signaling pathway. Recent findings indicate an additional function of SLP-65 as a tumor suppressor that regulates pre-B cell proliferation. We will discuss here the potential mechanisms by which SLP-65 controls the pre-B cell checkpoint.


Subject(s)
Adaptor Proteins, Signal Transducing/physiology , B-Lymphocytes/cytology , Carrier Proteins/physiology , Phosphoproteins/physiology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/physiopathology , Signal Transduction , Tumor Suppressor Proteins/physiology , Agammaglobulinaemia Tyrosine Kinase , Animals , B-Lymphocytes/physiology , Cell Differentiation , Cell Proliferation , Humans , Membrane Glycoproteins/metabolism , Pre-B Cell Receptors , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/immunology , Protein-Tyrosine Kinases , Receptors, Antigen, B-Cell
5.
Nat Immunol ; 6(2): 204-10, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15654340

ABSTRACT

Membrane recruitment of adaptor proteins is crucial for coupling antigen receptors to downstream signaling events. Despite the essential function of the B cell adaptor SLP-65, the mechanism of its recruitment to the plasma membrane is not yet understood. Here we show that a highly conserved leucine zipper in the SLP-65 N terminus is responsible for membrane association. Alterations in the N terminus abolished SLP-65 membrane localization and activity, both of which were restored by replacement of the N terminus with a myristoylation signal. The N terminus is an autonomous domain that confers specific localization and function when transferred to green fluorescent protein or the adaptor protein SLP-76. Our data elucidate the mechanism of SLP-65 membrane recruitment and suggest that leucine zipper motifs are essential interaction domains of signaling proteins.


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/metabolism , Cell Membrane/metabolism , Leucine Zippers , Phosphoproteins/chemistry , Phosphoproteins/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Animals , Carrier Proteins/genetics , Cell Line , Humans , Mice , Molecular Sequence Data , Mutation/genetics , Myristic Acid/metabolism , Phosphoproteins/genetics , Protein Binding , Protein Transport , Receptors, Antigen, B-Cell/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Sequence Alignment
6.
Nature ; 423(6938): 452-6, 2003 May 22.
Article in English | MEDLINE | ID: mdl-12761551

ABSTRACT

Acute lymphoblastic leukaemia (ALL) is the commonest form of childhood malignancy, and most cases arise from B-cell clones arrested at the pre-B-cell stage of differentiation. The molecular events that arrest pre-B-cell differentiation in the leukaemic pre-B cells have not been well characterized. Here we show that the differentiation regulator SLP-65 (an adaptor protein also called BLNK or BASH) inhibits pre-B-cell leukaemia in mice. Reconstitution of SLP-65 expression in a SLP-65-/- pre-B-cell line led to enhanced differentiation in vitro and prevented the development of pre-B-cell leukaemia in immune-deficient mice. Tyrosine 96 of SLP-65 was required for this activity. The murine SLP-65-/- pre-B-cell leukaemia resembles human childhood pre-B ALL. Indeed, 16 of the 34 childhood pre-B ALL samples that were tested showed a complete loss or drastic reduction of SLP-65 expression. This loss is probably due to the incorporation of alternative exons into SLP-65 transcripts, leading to premature stop codons. Thus, the somatic loss of SLP-65 and the accompanying block in pre-B-cell differentiation might be one of the primary causes of childhood pre-B ALL.


Subject(s)
Carrier Proteins/metabolism , Phosphoproteins/deficiency , Phosphoproteins/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Adaptor Proteins, Signal Transducing , Adolescent , Adult , Agammaglobulinaemia Tyrosine Kinase , Animals , B-Lymphocytes/metabolism , Base Sequence , Binding Sites , Carrier Proteins/genetics , Cell Differentiation , Cell Division , Cells, Cultured , Child , Child, Preschool , Exons/genetics , Gene Deletion , Gene Expression Regulation, Neoplastic , Humans , Infant , Mice , Mice, Knockout , Mutation , Phosphoproteins/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/pathology , Protein-Tyrosine Kinases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Spleen/pathology
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