ABSTRACT
We have shown that interleukin-12 (IL-12) generated a strong, albeit transient, anti-tumor response, mostly mediated by natural killer (NK) cell. T cell participation, in addition to NK cells, was essential for persistence of the anti-tumor response. Ligation of 4-1BB, a co-stimulatory receptor expressed on activated T cells, is known to amplify T cell-mediated immunity. In this study, we compared the effect of a systemically delivered agonistic anti-4-1BB monoclonal antibody (anti-4-1BB mAb) with intra-tumoral adenoviral-mediated gene transfer of the 4-1BB ligand (ADV/4-1BBL) to liver metastases in a syngeneic animal model of breast cancer. Both treatments induced a dramatic regression of pre-established tumor. When combined with intra-tumoral delivery of the IL-12 gene, both anti-4-1BB mAb and ADV/4-1BBL were synergistic and led to survival rates of 87% and 78%, respectively. The anti-tumor immunity is mainly mediated by CD4+ T cells in IL-12 plus 4-1BB ligand-treated animals, and CD8+ T cells in IL-12 plus anti-4-1BB mAb-treated animals. However, only long-term survivors after treatment with IL-12 and 4-1BBL genes have showed significantly potent, systemic, and tumor-specific T cell-mediated immunity.
Subject(s)
Breast Neoplasms/therapy , Genetic Therapy/methods , Immunotherapy/methods , Liver Neoplasms/secondary , Liver Neoplasms/therapy , Adenoviridae/genetics , Animals , Antibodies, Monoclonal/therapeutic use , Antigens, CD , Breast Neoplasms/immunology , Combined Modality Therapy , Female , Genetic Vectors/administration & dosage , Genetic Vectors/genetics , Immunity, Cellular , Injections, Intralesional , Interleukin-12/administration & dosage , Liver Neoplasms/immunology , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Receptors, Nerve Growth Factor/immunology , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/immunology , T-Lymphocytes/immunology , Tumor Necrosis Factor Receptor Superfamily, Member 9ABSTRACT
Immunoreceptor tyrosine-based inhibitory motifs (ITIM) have been implicated in the negative modulation of immunoreceptor signaling pathways. The IL-4R alpha-chain (IL-4Ralpha) contains a putative ITIM in the carboxyl terminal. To determine the role of ITIM in the IL-4 signaling pathway, we ablated the ITIM of IL-4Ralpha by deletion and site-directed mutagenesis and stably expressed the wild-type (WT) and mutant hIL-4Ralpha in 32D/insulin receptor substrate-2 (IRS-2) cells. Strikingly, 32D/IRS-2 cells expressing mutant human (h)IL-4Ralpha were hyperproliferative in response to IL-4 compared with cells expressing WT hIL-4Ralpha. Enhanced tyrosine phosphorylation of Stat6, but not IRS-2, induced by hIL-4 was observed in cells expressing mutant Y713F. Using peptides corresponding to the ITIM of hIL-4Ralpha, we demonstrate that tyrosine-phosphorylated peptides, but not their nonphosphorylated counterparts, coprecipitate SH2-containing tyrosine phosphatase-1, SH2-containing tyrosine phosphatase-2, and SH2-containing inositol 5'-phosphatase. The in vivo association of SH2-containing inositol 5'-phosphatase with IL-4Ralpha was verified by coimmunoprecipitation with anti-IL-4Ralpha Abs. These results demonstrate a functional role for ITIM in the regulation of IL-4-induced proliferation.
Subject(s)
Interleukin-4/physiology , Lymphocyte Activation/immunology , Phosphoric Monoester Hydrolases/metabolism , Receptors, Immunologic/metabolism , Receptors, Interleukin-4/metabolism , Tyrosine/metabolism , src Homology Domains/immunology , Amino Acid Motifs/genetics , Amino Acid Sequence , Cell Line , Cytoplasm/enzymology , Cytoplasm/genetics , Cytoplasm/immunology , Enzyme Activation/genetics , Enzyme Activation/immunology , Humans , Insulin Receptor Substrate Proteins , Intracellular Signaling Peptides and Proteins , Janus Kinase 1 , Lymphocyte Activation/genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoproteins/metabolism , Phosphoric Monoester Hydrolases/physiology , Phosphorylation , Protein Phosphatase 1 , Protein Phosphatase 2 , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatase, Non-Receptor Type 6 , Protein Tyrosine Phosphatases/metabolism , Protein-Tyrosine Kinases/metabolism , Receptors, Immunologic/genetics , Receptors, Immunologic/physiology , Receptors, Interleukin-4/genetics , Receptors, Interleukin-4/physiology , SH2 Domain-Containing Protein Tyrosine Phosphatases , STAT6 Transcription Factor , Sequence Deletion , Signal Transduction/genetics , Signal Transduction/immunology , Trans-Activators/metabolism , Tyrosine/geneticsABSTRACT
The SH2-containing inositol 5'-phosphatase (SHIP) is tyrosine-phosphorylated in response to cytokines such as interleukin (IL)-3, granulocyte-macrophage colony-stimulating factor, and macrophage colony-stimulating factor. SHIP has been shown to modulate negatively these cytokine signalings; however, a potential role in IL-4 signaling remains uncharacterized. It has been recently shown that IL-4 induces tyrosine phosphorylation of SHIP, implicating the phosphatase in IL-4 processes. Tyrosine kinases, Jak1 and Jak3, involved in IL-4 signaling can associate with SHIP, yet only Jak1 can tyrosine-phosphorylate SHIP when co-expressed. In functional studies, cells overexpressing wild type SHIP are found to be hyperproliferative in response to IL-4 in comparison to parental cells. In contrast, cells expressing catalytically inactive form, SHIP(D672A), show reduced proliferation in response to IL-4. These changes in IL-4-induced proliferation correlate with alterations in phosphatidylinositol 3,4,5-triphosphate levels. However, no differential activation of STAT6, Akt, IRS-2, or p70(S6k), in response to IL-4, was observed in these cells. These data suggest that the catalytic activity of SHIP acts in a novel manner to influence IL-4 signaling. In addition, these data support recent findings that suggest there are uncharacterized signaling pathways downstream of phosphatidylinositol 3,4,5-triphosphate.
Subject(s)
Interleukin-4/pharmacology , Phosphoric Monoester Hydrolases/physiology , Signal Transduction , Cell Division/physiology , Cell Line , Gene Expression , Humans , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , src Homology DomainsABSTRACT
IL-4 plays an important role in regulating immune responses. Distinct signaling pathways, including those for gene activation and cell differentiation and those for cell proliferation and protection from apoptosis, are initiated from the receptor complex for IL-4 following ligand-receptor engagement. Several advances have been made in our understanding of how distinct functions of IL-4 are mediated. Most of these studies employed artificial mutations of the IL-4-receptor alpha chain using site-directed mutagenesis and/or deletional mutation. In addition, naturally occurring mutations of the IL-4-receptor alpha chain have been identified and implicated as a genetic predisposition for allergic disorders. The results of these studies suggest a modular organization of the receptor and an independent regulation of gene activation and cell growth.
Subject(s)
Receptors, Interleukin-4/genetics , Animals , Humans , Mutation , Receptors, Interleukin-4/metabolism , Signal TransductionSubject(s)
Interleukin-4/metabolism , Intracellular Signaling Peptides and Proteins , Receptors, Interleukin-4/metabolism , Repressor Proteins , Signal Transduction/physiology , Animals , Carrier Proteins/metabolism , Humans , Insulin Receptor Substrate Proteins , Macromolecular Substances , Models, Biological , Phosphatidylinositol-3,4,5-Trisphosphate 5-Phosphatases , Phosphoproteins/metabolism , Phosphoric Monoester Hydrolases/metabolism , Phosphotransferases/metabolism , Protein Serine-Threonine Kinases , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-pim-1 , Receptors, Interleukin-4/chemistry , STAT6 Transcription Factor , Suppressor of Cytokine Signaling 1 Protein , Suppressor of Cytokine Signaling Proteins , Trans-Activators/metabolismABSTRACT
The bias favoring deletion over inversion in DH-JH rearrangement has been known for years, but the underlying mechanism has yet to be fully defined. It has been suggested that the ratio of deletion/inversion is determined by the combined effect of two factors: (i) the relative strengths of 5' and 3' recombination signal sequences (RSS) of a DH segment, and (ii) the efficiency with which the deletional product (one joint) forms relative to the inversional product (two joints). In this study, we analyzed for the first time the effect of factor 1 alone on the biased 3' RSS utilization in DH-JH joining by using deletional plasmids in an extrachromosomal substrate V(D)J recombination assay. It was found that the 3' RSS and associated coding end (12 bp) mediate recombination more efficiently than the 5' RSS/coding end DH-JH plasmids. These results demonstrate that the effect of the RSS/coding end alone can account, at least partially, for the predominant deletion in DH-JH recombination. The potential effect of the relative strength of RSS and associated coding end on the ordered rearrangement of DH-JH followed by VH to DH-JH was also assessed. When recombination frequencies of D-->J (3' DH to J3) were compared with frequencies of V-->D (VHPJ14 to 3' DH or VHOX2 to 3' DH), it was found that V-->D joining was, if anything, more efficient than D-->J joining. Therefore, if all three segments were accessible, RSS/coding end effects would not contribute to the ordered rearrangement of the IgH locus.
