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1.
Oncoimmunology ; 7(2): e1381813, 2018.
Article in English | MEDLINE | ID: mdl-29308301

ABSTRACT

Natural killer (NK) cells serve a critical role in the immune response against microbes and developing tumors. We have demonstrated that NK cells produce stimulatory cytokines (e.g., IFN-γ) in response to potent stimulation via immobilized IgG (to engage Fc receptors) and interleukin (IL)-12. CD25 is a component of the high-affinity IL-2R, which promotes NK cell activation in response to low doses of IL-2 such as those released by activated T cells. We hypothesized that stimulation of NK cells via IgG and IL-12 would enhance CD25 expression and promote NK cell anti-tumor activity in response to low-dose IL-2. It was confirmed that this dual stimulation strategy significantly enhanced NK cell CD25 expression compared to unstimulated cells or cells treated with IgG or IL-12 alone. Dual stimulated NK cells also were more responsive to low-dose IL-2. Dual stimulated NK cells subsequently treated with low-dose IL-2 (10 pg/mL) displayed enhanced intracellular signaling as indicated by increased pSTAT5 levels. IFN-γ production and cytotoxicity against K562 cells by NK cells stimulated with low-dose IL-2 was comparable to that of cells treated with high-dose IL-2 (10 ng/mL). Importantly, cells isolated from head and neck cancer patients receiving the mAb cetuximab and IL-12 on a clinical trial displayed increased CD25 expression following combination therapy compared to baseline. Altogether, these findings suggest that FcR and IL-12R co-stimulation induces expression of the high-affinity IL-2R and promotes NK cell anti-tumor activity.

2.
Vet Immunol Immunopathol ; 145(1-2): 420-30, 2012 Jan 15.
Article in English | MEDLINE | ID: mdl-22244518

ABSTRACT

BACKGROUND: Cancer profoundly affects immunity and causes immunosuppression that contributes to tumor escape, metastases and resistance to therapy. The mechanisms by which cancer cells influence immune cells are not fully known but both innate and adaptive immune cells can be altered by cancer. Myeloid cells are innate immune cells that comprise the mononuclear phagocytic system (MPS) and include monocytes, macrophages, dendritic cells (DCs) and their progenitors. Myeloid cells play important roles in both the promotion and regulation of immune responses. Dysregulated myeloid cells are increasingly being recognized as contributing to cancer-related immunosuppression. This study investigated whether soluble factors produced by canine tumor cells inhibited canine myeloid cell function. METHODS: These studies investigated the utility of using the canine DH82 cell line for assessment of canine myeloid responses to tumor-derived soluble factors (TDSFs). Phenotypic comparisons to canine bone marrow-derived DCs (BM-DCs) and bone marrow-derived macrophages (BM-MΦs) were performed and expression of myeloid cell markers CD11b, CD11c, CD80, and major histocompatibility complex (MHC) class II were evaluated by flow cytometry. Phenotypic and functional changes of DC populations were then determined following exposure to tumor-conditioned media (TCM) from canine osteosarcoma, melanoma and mammary carcinoma cell lines. RESULTS: We found that the canine BM-DCs and the DH82 cell line shared similar CD11b, CD11c and MHC II expression and morphologic characteristics that were distinct from canine BM-MΦs. Myeloid cells exposed to TDSFs showed decreased expression of MHC class II and CD80, had reduced phagocytic activity and suppressed the proliferation of responder immune cells. CONCLUSION: These results show that soluble factors secreted from canine tumor cells suppress the activation and function of canine myeloid cells. Our results suggest that, similar to humans, dysregulated myeloid cells may contribute to immunosuppression in dogs with cancer.


Subject(s)
Dog Diseases/immunology , Immune Tolerance/immunology , Myeloid Cells/immunology , Animals , Bone Neoplasms/immunology , Bone Neoplasms/veterinary , Cell Line , Dendritic Cells/immunology , Dogs , Female , Flow Cytometry/veterinary , Macrophages/immunology , Mammary Neoplasms, Animal/immunology , Melanoma/immunology , Melanoma/veterinary , Osteosarcoma/immunology , Osteosarcoma/veterinary , Phagocytosis/immunology , Tumor Cells, Cultured/immunology
3.
Br J Cancer ; 105(7): 1023-9, 2011 Sep 27.
Article in English | MEDLINE | ID: mdl-21863027

ABSTRACT

BACKGROUND: Expression of microRNAs (miRs) has been shown to be altered in many solid tumours and is being explored in melanoma. The malignant potential of some melanocytic lesions is difficult to predict. We hypothesised that characterisation of miR expression in borderline melanocytic proliferations would lead to the identification of a molecular profile that could be used with known prognostic factors to differentiate lesions with high malignant potential. METHODS: The miR expression profile of melanocytic lesions (benign naevi, malignant melanoma and borderline melanocytic tumours) was evaluated by real-time PCR. RESULTS: PCR analysis revealed primary cutaneous melanomas had an 8.6-fold overexpression of miR-21 and a 7.5-fold overexpression of miR-155 compared with benign naevi (P<0.0001). In situ hybridisation confirmed these results. miR-21 and miR-155 were significantly overexpressed within borderline lesions (P=0.0011 and P=0.0048, respectively). When borderline lesions were categorised by mitotic activity and Breslow thickness, miR-21 was associated with mitotic activity and miR-155 was associated with thickness (P<0.025). Among 14 patients with borderline lesions who underwent sentinel lymph node biopsy (SLNB), positive SLNB was associated with increased miR-21 and miR-155 in the primary lesion compared with lesions with a negative SLNB. CONCLUSION: MicroRNA expression profiles can be used to characterise atypical melanocytic lesions.


Subject(s)
Melanoma/genetics , MicroRNAs/genetics , Mitosis/physiology , Nevus, Epithelioid and Spindle Cell/genetics , Nevus, Pigmented/genetics , Skin Neoplasms/genetics , Humans , In Situ Hybridization , Melanoma/pathology , Mitotic Index , Nevus, Epithelioid and Spindle Cell/pathology , Nevus, Pigmented/pathology , Polymerase Chain Reaction , Sentinel Lymph Node Biopsy , Skin Neoplasms/pathology
4.
Am J Transplant ; 6(8): 1809-19, 2006 Aug.
Article in English | MEDLINE | ID: mdl-16889541

ABSTRACT

Effective cellular immunity to Epstein-Barr virus (EBV), necessary to prevent or cure many post-transplant lymphoproliferative disorders (PTLD), can be inhibited by transforming growth factor-beta (TGF-beta). In vitro, TGF-beta inhibits memory CTL re-stimulation from whole PBMC. We show that the effect of TGF-beta on CTL re-stimulation is not directly on the T cell, but requires an accessory cell (AC) population. Further, pre-treatment of AC with TGF-beta significantly reduces memory CTL re-stimulation and suppresses delayed type hypersensitivity (DTH) responses. Addition of exogenous interferon-gamma to the AC overcomes the effects of TGF-beta. TGF-beta pre-treatment also up-regulates expression of peroxisome-proliferator-activated receptor-gamma (PPAR-gamma) in CD14(+) AC. Importantly, pre-treatment of AC with the PPAR-gamma ligand, ciglitazone, results in significantly reduced memory CTL re-stimulation. Thus, the effects of TGF-beta in this system may be mediated in part via PPAR-gamma, and PPAR-gamma activation could have significant inhibitory effects on memory T-cell responses by affecting AC function. These data have important implications in understanding how memory CTL are re-stimulated and function to prevent disease, especially PTLD.


Subject(s)
Antigen-Presenting Cells/drug effects , Antigen-Presenting Cells/immunology , PPAR gamma/metabolism , T-Lymphocytes, Cytotoxic/drug effects , T-Lymphocytes, Cytotoxic/metabolism , Transforming Growth Factor beta/pharmacology , Antigen-Presenting Cells/metabolism , Cell Line , Gene Expression Regulation , Herpesvirus 4, Human/immunology , Humans , Hypersensitivity, Delayed/immunology , Immunologic Memory/immunology , Interferon-gamma/pharmacology , PPAR gamma/genetics , T-Lymphocytes, Cytotoxic/immunology , Thiazolidinediones/pharmacology
5.
Eur J Immunol ; 31(10): 3016-25, 2001 Oct.
Article in English | MEDLINE | ID: mdl-11592078

ABSTRACT

The Her2/neu (c-erbB-2) oncogene encodes a 185-kDa protein tyrosine kinase which is overexpressed in 20% of breast adenocarcinomas and is recognized by a humanized anti-Her2/neu monoclonal antibody (mAb) (rhu4D5 or Herceptin). Natural killer (NK) cells are capable of mediating antibody-dependent cell cytotoxicity (ADCC) against antibody-coated targets via their expression of a low-affinity receptor for IgG (FcgammaRIII or CD16). NK cells can be expanded in cancer patients via the administration of low-dose interleukin-2 (IL-2) and become potent cytotoxic effectors following exposure to high doses of IL-2. We tested IL-2-activated NK cells against Her2/neu+ (MCF-7Her2/neu) and Her2/neu- (MDA-468) breast cancer cell lines in a 4-h 51Cr-release cytotoxicity assay in the presence or absence of rhu4D5 mAb (effector : target ratio = 10 : 1). Specific lysis of rhu4D5-coated MCF-7Her2/neu and MDA-468 target cells by IL-2-activated NK cells was 35% and 3%, respectively (p < 0.05). Lysis was less than 5% when targets were treated with either the non-humanized mu4D5 mAb or control huIgG. Lysis of rhu4D5-coated MCF-7Her2/neu cells was inhibited by 80 % when NK cells were pre-treated with an anti-Fc receptor antibody prior to use in the cytotoxicity assay. Enhanced ADCC of MCF-7Her2/neu target cells was seen when the effector cells consisted of mononuclear cells obtained from a patient demonstrating significant expansion of NK cells secondary to therapy with low-dose IL-2. Serum from patients receiving infusions of rhu4D5 mAb could substitute for exogenous antibody in the ADCC assay. NK cells activated by rhu4D5-coated tumor cells in the presence of IL-2 also produced large amounts of IFN-gamma with concomitant up-regulation of cell-surface activation markers CD25 and CD69. These results lend support to the concurrent use of rhu4D5 mAb and IL-2 therapy in patients with cancers that express the Her2/neu oncogene.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Breast Neoplasms/therapy , Interleukin-2/pharmacology , Killer Cells, Natural/immunology , Animals , Antibodies, Monoclonal, Humanized , Antibody-Dependent Cell Cytotoxicity , Breast Neoplasms/immunology , Breast Neoplasms/pathology , CD56 Antigen/analysis , Cytokines/biosynthesis , Humans , Killer Cells, Natural/drug effects , Lymphocyte Activation , Mice , Receptor, ErbB-2/analysis , Receptors, IgG/physiology , Trastuzumab , Tumor Cells, Cultured
6.
Blood ; 97(10): 3146-51, 2001 May 15.
Article in English | MEDLINE | ID: mdl-11342442

ABSTRACT

During the innate immune response to infection, monocyte-derived cytokines (monokines), stimulate natural killer (NK) cells to produce immunoregulatory cytokines that are important to the host's early defense. Human NK cell subsets can be distinguished by CD56 surface density expression (ie, CD56(bright) and CD56(dim)). In this report, it is shown that CD56(bright) NK cells produce significantly greater levels of interferon-gamma, tumor necrosis factor-beta, granulocyte macrophage-colony-stimulating factor, IL-10, and IL-13 protein in response to monokine stimulation than do CD56(dim) NK cells, which produce negligible amounts of these cytokines. Further, qualitative differences in CD56(bright) NK-derived cytokines are shown to be dependent on the specific monokines present. For example, the monokine IL-15 appears to be required for type 2 cytokine production by CD56(bright) NK cells. It is proposed that human CD56(bright) NK cells have a unique functional role in the innate immune response as the primary source of NK cell-derived immunoregulatory cytokines, regulated in part by differential monokine production.


Subject(s)
CD56 Antigen/analysis , Homeostasis , Immunity , Killer Cells, Natural/immunology , Lectins, C-Type , Antigens, CD/analysis , Cell Division , Cells, Cultured , Coculture Techniques , Cytotoxicity, Immunologic , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Humans , Interferon-gamma/biosynthesis , Interferon-gamma/genetics , Interleukin-2/pharmacology , Interleukins/biosynthesis , Ionomycin/pharmacology , Lipopolysaccharides/pharmacology , Lymphotoxin-alpha/biosynthesis , Macrophages/metabolism , Membrane Glycoproteins/analysis , NK Cell Lectin-Like Receptor Subfamily D , RNA, Messenger/analysis , Receptors, IgG/analysis , Receptors, Immunologic/analysis , Receptors, Interleukin-2/analysis , Receptors, Interleukin-2/physiology , Receptors, KIR , Tetradecanoylphorbol Acetate/pharmacology
7.
Blood ; 96(4): 1465-73, 2000 Aug 15.
Article in English | MEDLINE | ID: mdl-10942393

ABSTRACT

The administration of therapeutic doses of recombinant cytokines to patients with malignant disease can be complicated by systemic toxicities, which in their most severe form may present as a systemic inflammatory response. The combination of interleukin (IL)-18 and IL-12 has synergistic antitumor activity in vivo yet has been associated with significant toxicity. The effects of IL-18 plus IL-12 were examined in a murine model, and it was found that the daily, simultaneous administration of IL-18 and IL-12 resulted in systemic inflammation and 100% mortality within 4 to 8 days depending on the strain employed. Mice treated with IL-18 plus IL-12 exhibited unique pathologic findings as well as elevated serum levels of proinflammatory cytokines and acute-phase reactants. The actions of tumor necrosis factor-alpha did not contribute to the observed toxicity, nor did T or B cells. However, toxicity and death from treatment with IL-18 plus IL-12 could be completely abrogated by elimination of natural killer (NK) cells or macrophages. Subsequent studies in genetically altered mice revealed that NK-cell interferon-gamma mediated the fatal toxicity via the signal transducer and activator of transcription pathway of signal transduction. These data may provide insights into methods of ameliorating cytokine-induced shock in humans. (Blood. 2000;96:1465-1473)


Subject(s)
Inflammation/chemically induced , Inflammation/immunology , Interferon-gamma/immunology , Interleukin-12/immunology , Interleukin-12/toxicity , Interleukin-18/immunology , Interleukin-18/toxicity , Killer Cells, Natural/immunology , Signal Transduction/immunology , Animals , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, SCID , Trans-Activators/immunology
9.
J Immunol ; 162(8): 4943-51, 1999 Apr 15.
Article in English | MEDLINE | ID: mdl-10202041

ABSTRACT

The mechanism of cytokine-induced shock remains poorly understood. The combination of IL-2 and IL-12 has synergistic antitumor activity in vivo, yet has been associated with significant toxicity. We examined the effects of IL-2 plus IL-12 in a murine model and found that the daily, simultaneous administration of IL-2 and IL-12 resulted in shock and 100% mortality within 4 to 12 days depending on the strain employed. Mice treated with IL-2 plus IL-12 exhibited NK cell apoptosis, pulmonary edema, degenerative lesions of the gastrointestinal tract, and elevated serum levels of proinflammatory cytokines and acute phase reactants. The actions of TNF-alpha, IFN-gamma, macrophage-inflammatory protein-1alpha, IL-1, IL-1-converting enzyme, Fas, perforin, inducible nitric oxide synthase, and STAT1 did not contribute to the observed toxicity, nor did B or T cells. However, toxicity and death from treatment with IL-2 plus IL-12 could be completely abrogated by elimination of NK cells. These results suggest that the fatal systemic inflammatory response induced by this cytokine treatment is critically dependent upon NK cells, but does not appear to be mediated by the known effector molecules of this cellular compartment. These data may provide insight into the pathogenesis of cytokine-induced shock in humans.


Subject(s)
Interleukins/adverse effects , Killer Cells, Natural/immunology , Shock, Septic/etiology , Shock, Septic/mortality , Animals , Cell Separation , Cytokines/biosynthesis , Cytokines/blood , Drug Therapy, Combination , Female , Interferon-gamma/physiology , Interleukin-1/physiology , Interleukin-12/administration & dosage , Interleukin-12/adverse effects , Interleukin-15/administration & dosage , Interleukin-15/adverse effects , Interleukin-2/administration & dosage , Interleukin-2/adverse effects , Interleukins/administration & dosage , Liver/pathology , Lung/pathology , Macrophage Activation , Macrophages/immunology , Mice , Mice, Inbred BALB C , Mice, Knockout , Mice, SCID , Mice, Transgenic , Monocytes/immunology , Shock, Septic/immunology , Shock, Septic/pathology , Spleen/pathology , Tumor Necrosis Factor-alpha/physiology
10.
Blood ; 92(10): 3647-57, 1998 Nov 15.
Article in English | MEDLINE | ID: mdl-9808558

ABSTRACT

Interleukin-15 (IL-15) is produced by human bone marrow (BM) stromal cells and can induce CD34(+) hematopoietic progenitor cells (HPCs) to differentiate into CD56(+)CD3(-) natural killer (NK) cells in the absence of stromal cells. IL-15 mediates its effects by signaling through the beta and gammac chains of the IL-2/15 receptor (R). The c-kit ligand (KL), also produced by stromal cells, enhances the expansion of NK cells from CD34(+) HPCs in the presence of IL-15, but alone has no ability to differentiate NK cells. Mice deficient in KL do not appear to have a quantitative deficiency in NK cells, suggesting that other stromal cell factors may contribute to NK cell expansion. Flt3 ligand (FL) is also produced by BM stromal cells and has homology with KL. Furthermore, mice with a targeted disruption of the FL gene have reduced numbers of NK cells. We evaluated here the effects of FL on human NK cell development and expansion from CD34(+) HPCs. Like KL, FL significantly enhanced the expansion of NK cells from CD34(+) HPCs in the presence of IL-15, compared with IL-15 alone. However, FL alone had no effect on NK cell differentiation. We therefore explored the mechanism by which FL promotes IL-15-mediated NK cell development. FL was found to induce IL-2/15Rbeta (CD122) expression on CD34(bright) HPCs. The CD34(bright) CD122(+) cell coexpressed CD38, but lacked expression of CD7, CD56, NK cell receptors (NKRs), or cytotoxic activity in the absence of IL-15. Using limiting dilution analysis in the presence of IL-15 alone, we demonstrated that the FL-induced CD34(bright)CD122(+) HPCs had an NK cell precursor frequency 20- to 60-fold higher than the CD34(dim/neg)CD122(-) HPCs and 65- to 235-fold higher than fresh CD34(+) HPCs. KL had similar effects as FL, but induced a significantly lower percentage of CD34(bright)CD122(+) cells (P

Subject(s)
Hematopoietic Stem Cells/drug effects , Interleukin-15/pharmacology , Killer Cells, Natural/cytology , Membrane Proteins/pharmacology , Adult , Animals , Antigens, CD34/analysis , Antigens, Differentiation/analysis , Bone Marrow Cells/cytology , Bone Marrow Cells/drug effects , Bone Marrow Cells/metabolism , CD56 Antigen/analysis , Cell Cycle , Cell Differentiation/drug effects , Cells, Cultured , Cytokines/pharmacology , Drug Synergism , Hematopoietic Stem Cells/cytology , Humans , Interleukin-15/metabolism , Mice , Recombinant Fusion Proteins/pharmacology , Signal Transduction , Stem Cell Factor/pharmacology , Stromal Cells/metabolism
11.
Clin Cancer Res ; 4(9): 2219-28, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9748142

ABSTRACT

IFN-alpha2b has been used to treat patients with malignant melanoma who are at high risk for recurrence after surgical resection. However, its exact mechanism of action is unknown. I hypothesized that IFN-alpha exerts a direct effect on the melanoma cell via the activation of signal transducer and activator of transcription (STAT) proteins. Cell lysates from melanoma cell lines and patient tumor samples stimulated with IFN-alpha were incubated with radiolabeled oligonucleotides representing the high affinity sis-inducible element of c-fos and the IFN stimulated response element and then analyzed for STAT activation using the electrophoretic mobility shift assay. Melanoma cell lines showed no evidence for constitutive STAT activation in the absence of cytokine stimulation but exhibited rapid activation of STAT1 and STAT2 once treated with IFN-alpha. A distinct dose-response curve was noted with maximal STAT activation occurring at approximately 10(5) units/ml IFN-alpha. Genistein, a protein tyrosine kinase inhibitor, completely suppressed IFN-alpha-induced STAT activation. Malignant melanoma tumors obtained from 17 patients exhibited dose-dependent activation of STAT1 and STAT2 in response to treatment with IFN-alpha. Pretreatment of patient melanoma tumor cells with IFN-gamma resulted in a 4 log-fold decrease in the IFN-alpha concentration required for STAT activation and promoted the increased expression of STAT1, STAT2, and p48. In summary, IFN-alpha consistently activated STAT1 and STAT2 proteins in melanoma cell lines and in melanoma tumors obtained directly from patients. This signaling pathway was dramatically sensitized to IFN-alpha by pretreatment of melanoma cells with low concentrations of IFN-gamma. These results provide molecular evidence to support the hypothesis that the clinical response to IFN-alpha may be mediated, in part, by a direct effect on the melanoma cell. These results also suggest a potential role for IFN-gamma in the treatment of malignant melanoma.


Subject(s)
Antineoplastic Agents/pharmacology , DNA-Binding Proteins/drug effects , DNA-Binding Proteins/physiology , Interferon-alpha/pharmacology , Melanoma/therapy , Trans-Activators/drug effects , Trans-Activators/physiology , Adult , Electrophoresis , HeLa Cells , Histocompatibility Antigens Class I/biosynthesis , Humans , Interferon alpha-2 , Interferon-gamma/pharmacology , Kinetics , Male , Melanoma/metabolism , Protein-Tyrosine Kinases/antagonists & inhibitors , Protein-Tyrosine Kinases/physiology , Recombinant Proteins , Signal Transduction/drug effects , Signal Transduction/physiology , Tumor Cells, Cultured , Up-Regulation/drug effects
12.
J Am Coll Surg ; 187(2): 171-7, 1998 Aug.
Article in English | MEDLINE | ID: mdl-9704964

ABSTRACT

BACKGROUND: Paget's disease (PD) of the breast is an uncommon condition traditionally treated with mastectomy. To assess the natural history and treatment options in PD we have reviewed the experience at the Roswell Park Cancer Institute (RPCI). STUDY DESIGN: The medical records of patients treated for PD at RPCI between 1963 and 1996 were reviewed. RESULTS: Eighty patients were treated in the period reviewed. The median follow up was 61 months. Sixty-eight of 80 patients had nipple changes consistent with PD including 30 who had an associated breast mass (group 1), and 38 who had no associated mass (group 2). Of the 68 patients with clinical PD, 58 (85%) were treated with mastectomy while the remainder were treated with some form of breast conserving therapy. Breast cancer (BC) was found in 56 of 68 patients (82%) with clinical PD, including 28 of 30 patients (93%) in group 1 and 28 of 38 patients (74%) in group 2. Breast cancer was centrally located (within 2 cm of the areolar margin) in 61% of patients, including 71% in group 1 and 50% in group 2. Of the 28 patients with an associated BC in group 1, 21 (75%) had invasive cancer, 6 (21%) had ductal carcinoma in situ (DCIS), and 16 (57%) had pathologic axillary nodes. Of the 28 patients with an associated BC in group 2, 10 (36%) had invasive cancer, 18 (64%) had DCIS, and 6 (21%) had positive axillary nodes. The median survival was significantly shorter for group 1 (42 months) than for group 2 (126 months; p = 0.007). CONCLUSION: Most patients with PD have an associated BC (82%) that was centrally located (61%). In those patients without an associated mass, a significant number (26% in this series) may not have an underlying BC and may be overtreated with mastectomy. On the other hand, a significant number of patients with PD and no associated breast mass will be found to have a peripherally located tumor (29% in this series). These non-central BC masses could potentially be missed by a wide local excision that includes the areolar complex.


Subject(s)
Breast Neoplasms/surgery , Paget's Disease, Mammary/surgery , Breast Neoplasms/pathology , Breast Neoplasms, Male/pathology , Breast Neoplasms, Male/surgery , Female , Humans , Lymph Node Excision , Male , Mastectomy , Mastectomy, Segmental , Mastectomy, Simple , Middle Aged , Paget's Disease, Mammary/pathology , Retrospective Studies
13.
Blood ; 90(9): 3647-53, 1997 Nov 01.
Article in English | MEDLINE | ID: mdl-9345049

ABSTRACT

The administration of low dose interleukin-2 (IL-2) results in a selective expansion of natural killer (NK) cells in vivo, and promotes the differentiation of NK cells from hematopoietic precursor cells in vitro. We have previously shown that stem cell factor (SCF ), the ligand to the c-kit tyrosine kinase receptor, enhances IL-2-induced NK cell proliferation and differentiation in vitro. Here, we investigated the effects of SCF plus IL-2 delivered to mice in vivo. Eight-week-old C57BL/6 mice were treated with a continuous subcutaneous infusion of IL-2 (1 x 10(4) IU/d) plus a daily intraperitoneal dose of SCF (100 microg/kg/d), IL-2 alone, SCF alone, or vehicle alone for 8 weeks. The in vivo serum concentration of IL-2 ranged between 352 +/- 12.0 pg/mL and 606 +/- 9.0 pg/mL, achieving selective saturation of the high affinity IL-2 receptor, while the peak SCF serum concentration was 296 +/- 13.09 ng/mL. Alone, the daily administration of SCF had no effect on the expansion of NK cells. The continuous infusion of IL-2 alone did result in a significant expansion of NK1.1+CD3- cells compared to mice treated with placebo or SCF. However, mice treated with both SCF and IL-2 showed an increase in the absolute number of NK cells that was more than twofold that seen with IL-2 alone, in the spleen (P

Subject(s)
Interleukin-2/administration & dosage , Killer Cells, Natural/cytology , Stem Cell Factor/administration & dosage , Administration, Cutaneous , Animals , Cell Division/drug effects , Cells, Cultured , Drug Synergism , Female , Injections, Intraperitoneal , Interleukin-2/pharmacokinetics , Killer Cells, Natural/drug effects , Mice , Mice, Inbred C57BL , Stem Cell Factor/pharmacokinetics
14.
J Clin Invest ; 99(5): 937-43, 1997 Mar 01.
Article in English | MEDLINE | ID: mdl-9062351

ABSTRACT

Resting lymphocyte survival is dependent upon the expression of Bcl-2, yet the factors responsible for maintaining lymphocyte Bcl-2 protein expression in vivo are largely unknown. Natural killer (NK) cells are bone marrow-derived lymphocytes that constitutively express the beta and common gamma(c) subunits of the IL-2 receptor (R) as a heterodimer with intermediate affinity for IL-2. IL-15 also binds to IL-2Rbeta gamma(c) and is much more abundant in normal tissues than IL-2. Mice that lack the IL-2 gene have NK cells, whereas mice and humans that lack IL-2R gamma(c) do not have NK cells. Further, treatment of mice with an antibody directed against IL-2Rbeta results in a loss of the NK cell compartment. These data suggest that a cytokine other than IL-2, which binds to IL-2Rbeta gamma(c), is important for NK cell development and survival in vivo. In the current report, we show that the recently described IL-15R(alpha) subunit cooperates with IL-2Rbeta gamma(c) to transduce an intracellular signal at picomolar concentrations of IL-15. We demonstrate that resting human NK cells express IL-15R(alpha) mRNA and further, that picomolar amounts of IL-15 can sustain NK cell survival for up to 8 d in the absence of serum. NK cell survival was not sustained by other monocyte-derived factors (i.e., TNF-alpha, IL-1beta, IL-10, IL-12) nor by cytokines known to use gamma(c) for signaling (i.e., IL-4, IL-7, IL-9, IL- 13). One mechanism by which IL-15 promotes NK cell survival may involve the maintenance of Bcl-2 protein expression. Considering these functional properties of IL-15 and the fact that it is produced by bone marrow stromal cells and activated monocytes, we propose that IL-15 may function as an NK cell survival factor in vivo.


Subject(s)
Cell Survival , Interleukin-15/pharmacology , Killer Cells, Natural/metabolism , Killer Cells, Natural/physiology , Receptors, Interleukin/biosynthesis , Receptors, Interleukin/physiology , Animals , Apoptosis , Biological Assay , Blotting, Northern , Blotting, Western , DNA/metabolism , Flow Cytometry , Gene Expression Regulation , Humans , Interleukins/pharmacology , Mice , Propidium , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Messenger/analysis , RNA, Messenger/biosynthesis , Receptors, Interleukin/genetics , Receptors, Interleukin-2/genetics , Receptors, Interleukin-2/physiology , Signal Transduction , Transcription, Genetic , Transfection , Tumor Cells, Cultured , Tumor Necrosis Factor-alpha/pharmacology
15.
Eur J Immunol ; 27(2): 354-60, 1997 Feb.
Article in English | MEDLINE | ID: mdl-9045904

ABSTRACT

Natural killer (NK) cells are bone marrow-derived large granular lymphocytes that express the CD56 surface antigen. The CD56bright NK subset represents approximately 10% of all NK cells and is thought to be the least differentiated NK cell component in blood. The most mature NK cell expresses CD56 at low density and CD16 (FcR gammaIII) at high density, whereas CD56bright NK cells either lack CD16 (CD56bright CD16-) or express it at low density (CD56bright CD16dim). c-kit is a tyrosine kinase receptor which is expressed on both CD34+ hematopoietic precursor cells and CD56bright NK cells. In the current study, we characterize interleukin (IL)-2 receptor (IL-2R) and c-kit expression in each of the CD56bright subsets. Both the CD56bright CD16- and CD56bright CD16dim NK subsets express the high-affinity IL-2R and the c-kit receptor when isolated from fresh blood. However, each CD56bright NK cell subset has distinct functional responses to IL-2, the c-kit ligand (KL), or both. Activation of the high-affinity IL-2R on CD56bright CD16- NK cells induces a proliferative response that is significantly weaker than that observed in the CD56bright CD16dim NK cell subset. Incubation of the CD56bright CD16 NK cell subset with KL significantly enhances IL-2-induced proliferation, while KL has no such effect on the CD56bright CD16dim NK subset. Activation of the high-affinity IL-2R in both CD56bright subsets induces lymphokine-activated killer (LAK) activity, but the addition of KL has no effect on LAK activity. Co-stimulation of either CD56bright subset with IL-12 and concentrations of IL-2 that only saturate the high-affinity IL-2R induces substantial interferon (IFN)-gamma production. The addition of KL to this co-stimulatory signal enhances IFN-gamma production in both CD56bright NK subsets. The distinct functional responses to IL-2 and KL seen in the CD56bright CD16- and CD56bright CD16dim NK subsets provide insight into IL-2R signaling and suggest that each phenotype identifies a discrete stage of NK cell differentiation.


Subject(s)
CD56 Antigen/analysis , Interleukin-2/pharmacology , Killer Cells, Natural/drug effects , Killer Cells, Natural/immunology , Stem Cell Factor/pharmacology , Adenocarcinoma , Animals , Humans , Killer Cells, Lymphokine-Activated/drug effects , Killer Cells, Lymphokine-Activated/immunology , Killer Cells, Lymphokine-Activated/metabolism , Killer Cells, Natural/metabolism , Mast-Cell Sarcoma , Mice , Receptors, IgG/analysis , Tumor Cells, Cultured
17.
Blood ; 88(10): 3887-93, 1996 Nov 15.
Article in English | MEDLINE | ID: mdl-8916954

ABSTRACT

The allogeneic mixed lymphocyte reaction (MLR) is a complex in vitro assay of T-cell recognition and responsiveness in which interleukin-2 (IL-2) plays a central role. We have previously demonstrated that c-kit ligand (KL) can enhance IL-2-induced proliferation in a subset of human natural killer cells expressing the c-kit tyrosine kinase receptor. In the present study, we asked whether KL could enhance IL-2-mediated T-cell proliferation in the allogeneic MLR. We demonstrate that the vast majority of activated human T-cell clones express the c-kit mRNA transcript. Binding studies performed on activated T cells with radioiodinated KL were consistent with the expression of a single class of c-kit receptors. The addition of exogenous KL to the MLR led to an increase in tritiated thymidine (3[H]-TdR) incorporation in the absence of other exogenous cytokines, and did so in a dose-dependent fashion. A reproducible increase in 3[H]-TdR incorporation was noted at concentrations of KL, which approximate those normally found in vivo. Antibody blocking of KL binding to c-kit, T-cell depletion and sorting experiments suggest that the action of KL is mediated at least in part by a direct effect on both CD4+ and CD8+ T-cells. KL's enhancement of the MLR also requires the binding of IL-2 to its high-affinity IL-2 receptor. Given the abundance of KL normally found in human serum, these data suggest that this cytokine may have a role during T-cell activation in vivo.


Subject(s)
Lymphocyte Culture Test, Mixed , Stem Cell Factor/pharmacology , T-Lymphocyte Subsets/immunology , Animals , DNA Replication , Histocompatibility , Humans , Immunity, Cellular , Interleukin-2/physiology , Isoantigens/immunology , Lymphocyte Activation , Mice , Proto-Oncogene Proteins c-kit/biosynthesis , Proto-Oncogene Proteins c-kit/classification , RNA, Messenger/biosynthesis , Receptors, Interleukin-2/drug effects , Receptors, Interleukin-2/physiology , Stem Cell Factor/biosynthesis , Stem Cell Factor/genetics , T-Lymphocyte Subsets/metabolism
18.
J Biol Chem ; 271(24): 13968-75, 1996 Jun 14.
Article in English | MEDLINE | ID: mdl-8662928

ABSTRACT

The cytoplasmic domain of the receptor for interleukin 10 (IL-10R) contains two box 3 sequence motifs that have been identified in the signal-transducing receptor subunits for IL-6-type cytokines and noted to be required for activating STAT3 and inducing transcription through IL-6-responsive elements. To determine whether the IL-10R has signaling functions similar to IL-6R in cells normally expressing these receptors, leukocytes of the B-, T-, and NK-cell lineages were treated with either cytokine. Both cytokines activated factors that bound to the sis-inducible element and included STAT1 and STAT3. The cell response to IL-10 characteristically differed from that to IL-2/IL-15, IL-4, and interferon gamma. The signaling capabilities of the IL-10R for activating specific STAT proteins and inducing gene transcription were defined by reconstitution of receptor functions in transfected tissue culture cells. COS-1 cells, co-expressing the human IL-10R and individual STAT proteins, confirmed a preference of the IL-10R for STAT3 and STAT1. Unlike many hematopoietin receptors, the IL-10R did not detectably activate STAT5. The IL-10R, together with reporter gene constructs containing different IL-6-responsive gene elements, reconstituted in hepatoma cells an induction of transcription by IL-10 that was comparable to that by IL-6. This regulation could not be appreciably modified by enhanced expression of STAT proteins. The similar actions of IL-10R and IL-6R on the induction of endogenous IL-6-responsive genes were demonstrated in hepatoma cells stably expressing the IL-10R. These receptor functions required the presence of the box 3 motifs, as shown by the analysis of the mouse IL-10R constructs containing progressively truncated cytoplasmic domains. The data demonstrate that the IL-10R, unlike other members of the interferon receptor family, is highly effective in recruiting the signaling pathways of IL-6-type cytokine receptors.


Subject(s)
Antigens, CD/physiology , DNA-Binding Proteins/metabolism , Interleukin-10/pharmacology , Interleukin-6/pharmacology , Receptors, Interleukin/physiology , Signal Transduction , T-Lymphocytes/physiology , Trans-Activators/metabolism , Transcription, Genetic , Acute-Phase Proteins/metabolism , Amino Acid Sequence , Animals , Antigens, CD/biosynthesis , Base Sequence , Binding Sites , Carcinoma, Hepatocellular , Cell Line , Chlorocebus aethiops , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/blood , Leukemia, Lymphocytic, Chronic, B-Cell/immunology , Leukocytes/metabolism , Liver Neoplasms , Mice , Molecular Sequence Data , Oligodeoxyribonucleotides , Receptors, Interleukin/biosynthesis , Receptors, Interleukin-10 , Receptors, Interleukin-6 , Recombinant Proteins/metabolism , STAT3 Transcription Factor , Signal Transduction/drug effects , T-Lymphocytes/immunology , Transcription, Genetic/drug effects , Transfection , Tumor Cells, Cultured
19.
J Clin Invest ; 96(6): 2578-82, 1995 Dec.
Article in English | MEDLINE | ID: mdl-8675621

ABSTRACT

Natural killer (NK) cells are large granular lymphocytes that constitutively express functional IL-2 receptors. We have shown that recombinant human IL-15 uses the IL-2 receptor to activate human NK cells and can synergize with recombinant human IL-12 to stimulate NK cell production of IFN-gamma in vitro. IFN-gamma production by NK cells is critical in the prevention of overwhelming infection by obligate intracellular microbial pathogens in several experimental animal models. Herein, we demonstrate that human monocytes produce IL-15 protein within 5 h of activation with LPS. Using an IL-15-neutralizing antiserum in a coculture of LPS-activated monocytes and NK cells, we demonstrate that monocyte-derived IL-15 is critical for optimal NK cell production of IFN-gamma. Endogenous IL-15 activates NK cells through the IL-2 receptor, and with endogenous IL-12, regulates NK cell IFN-gamma after monocyte activation by LPS. These in vitro studies are the first to characterize a function for endogenous IL-15, and as such, suggest an important role for IL-15 during the innate immune response. IL-15 may be an important ligand for the NK cell IL-2 receptor in vivo.


Subject(s)
Interferon-gamma/biosynthesis , Interleukins/biosynthesis , Interleukins/pharmacology , Killer Cells, Natural/immunology , Lymphocyte Activation , Monocytes/immunology , Receptors, Interleukin-2/physiology , Animals , Antibodies, Monoclonal , Blotting, Western , Cells, Cultured , Coculture Techniques , Drug Synergism , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Humans , Immunohistochemistry , Interleukin-12/pharmacology , Interleukin-15 , Interleukins/analysis , Killer Cells, Natural/drug effects , Kinetics , Lipopolysaccharides/pharmacology , Monocytes/drug effects , Recombinant Proteins/pharmacology
20.
Blood ; 85(12): 3577-85, 1995 Jun 15.
Article in English | MEDLINE | ID: mdl-7540068

ABSTRACT

Human natural killer (NK) cells are large granular lymphocytes that constitutively express functional forms of the interleukin-2 receptor (IL-2R) and lyse tumor and virally infected cells without prior sensitization. NK cells with high density expression of CD56 (CD56bright) express the high affinity IL-2R and proliferate in response to low (picomolar) concentrations of IL-2. CD56dim NK cells express the intermediate affinity IL-2R and demonstrate enhanced cytotoxic activity without proliferation in response to high (nanomolar) concentrations of IL-2. In the present study, we characterized IL-10R expression on human NK cells and the functional consequences of IL-10 binding directly to highly purified subsets of CD56bright and CD56dim NK cells. Binding studies using 125I-IL-10 indicated that resting human NK cells constitutively express the IL-10 receptor protein at a surface density of approximately 90 receptor sites per cell, with a kd of approximately 1 nmol/L. Alone, IL-10 did not induce proliferation of CD56bright or CD56dim NK cell subsets. However, at low concentrations (0.5 to 5 ng/mL), IL-10 significantly augmented IL-2-induced proliferation of the CD56bright NK cell subset mediated via the high-affinity IL-2R. In the absence of IL-2, IL-10 was able to induce significant NK cytotoxic activity against NK-resistant tumor cell targets in both subsets of NK cells in a dose-dependent fashion. Furthermore, the combination of IL-10 and IL-2 had an additive effect on NK cytotoxic activity, whereas that of IL-10 and IL-12 did not. Production of interferon-gamma, tumor necrosis factor-alpha, and granulocyte-macrophage colony-stimulating factor by IL-2-activated NK cells was also significantly enhanced by IL-10. Neither resting nor activated human NK cells appear to produce human IL-10 protein. In summary, NK cells constitutively express the IL-10R protein in low density, and the functional consequences of IL-10 binding directly to human NK cell subsets appear to be stimulatory and dose-dependent. In contrast to its direct effects on human T cells and monocytes/macrophages, IL-10 potentiates cytokine production by human NK cells.


Subject(s)
Interleukin-10/metabolism , Killer Cells, Natural/metabolism , Receptors, Interleukin/biosynthesis , Antigens, CD/biosynthesis , Antigens, Differentiation, T-Lymphocyte/biosynthesis , CD56 Antigen , Cell Division/drug effects , Cells, Cultured , Humans , Interleukin-10/pharmacology , Interleukin-2/pharmacology , Killer Cells, Natural/immunology , RNA, Messenger/analysis , Radioligand Assay , Receptors, Interleukin-10
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