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1.
Gene Ther ; 22(5): 391-403, 2015 May.
Article in English | MEDLINE | ID: mdl-25652098

ABSTRACT

Cancer therapy with T cells expressing chimeric antigen receptors (CARs) has produced remarkable clinical responses in recent trials, but also severe side effects. Whereas most protocols use permanently reprogrammed T cells, we have developed a platform for transient CAR expression by mRNA electroporation. This approach may be useful for safe clinical testing of novel receptors, or when a temporary treatment period is desirable. Herein, we investigated therapy with transiently redirected T cells in vitro and in a xenograft mouse model. We constructed a series of CD19-specific CARs with different spacers and co-stimulatory domains (CD28, OX40 or CD28-OX40). The CAR constructs all conferred T cells with potent CD19-specific activity in vitro. Unexpectedly, the constructs incorporating a commonly used IgG1-CH2CH3 spacer showed lack of anti-leukemia activity in vivo and induced severe, partly CD19-independent toxicity. By contrast, identical CAR constructs without the CH2-domain eradicated leukemia in vivo, without notable toxicity. Follow-up studies demonstrated that the CH2CH3-spacer bound soluble mouse Fcγ-receptor I and mediated off-target T-cell activation towards murine macrophages. Our findings highlight the importance of non-signalling CAR elements and of in vivo studies. Finally, the results show that transiently redirected T cells control leukemia in mice and support the rationale for developing an mRNA-CAR platform.


Subject(s)
Leukemia/therapy , Receptors, Antigen, T-Cell/genetics , Receptors, IgG/genetics , T-Lymphocytes/immunology , Animals , Antigens, CD19/genetics , Antigens, CD19/immunology , CD28 Antigens/genetics , CD28 Antigens/immunology , Cell Line, Tumor , Cells, Cultured , Genetic Therapy , HEK293 Cells , Humans , Immunotherapy, Adoptive , Macrophage Activation , Macrophages/immunology , Mice , Mice, Inbred NOD , Receptors, Antigen, T-Cell/immunology , Receptors, IgG/immunology , Receptors, OX40/genetics , Receptors, OX40/immunology , Recombinant Proteins/genetics , Recombinant Proteins/immunology , T-Lymphocytes/transplantation , Xenograft Model Antitumor Assays
2.
Scand J Immunol ; 74(6): 632-9, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21883353

ABSTRACT

We evaluated inflammatory markers in febrile neutropenic lymphoma patients undergoing high-dose chemotherapy with autologous stem cell support. Based on MASCC scores, our patients had a low risk of serious complications and a perspective of a benign initial clinical course of the febrile neutropenia. We also studied the impact of tobramycin given once versus three times daily on these immune markers. Sixty-one patients participating in a Norwegian multicentre prospective randomized clinical trial, comparing tobramycin once daily versus three times daily, given with penicillin G to febrile neutropenic patients, constituted a clinically homogenous group. Four patients had bacteraemia, all isolates being Gram-positive. Thirty-two patients received tobramycin once daily, and 29 patients received tobramycin three times daily. Blood samples were taken at the onset of febrile neutropenia and 1-2 days later. All samples were frozen at -70 °C and analysed at the end of the clinical trial for C-reactive protein (CRP), procalcitonin (PCT), complement activation products, mannose-binding lectin (MBL) and 17 cytokines. We found a mild proinflammatory response in this series of patients. CRP was non-specifically elevated. Ten patients with decreased MBL levels showed the same mild clinical and proinflammatory response. Patients receiving tobramycin once daily showed a more pronounced proinflammatory response compared with patients receiving tobramycin three times daily. Overall, febrile neutropenic cancer patients with a benign clinical course show a mild proinflammatory immune response.


Subject(s)
Antineoplastic Agents/adverse effects , Lymphoma , Neutropenia/drug therapy , Tobramycin/therapeutic use , Adolescent , Adult , Aged , Antineoplastic Agents/therapeutic use , Cytokines/immunology , Female , Humans , Inflammation/immunology , Inflammation/microbiology , Lymphoma/drug therapy , Male , Middle Aged , Neutropenia/chemically induced , Risk Factors , Tobramycin/administration & dosage , Tobramycin/adverse effects , Young Adult
3.
J Exp Med ; 156(4): 1101-14, 1982 Oct 01.
Article in English | MEDLINE | ID: mdl-6961188

ABSTRACT

We demonstrated that the in vitro differentiation of human peripheral blood monocytes to macrophages is dependent on the environment and conditions of monocyte culture. Cultivation of monocytes on glass or microexudate-coated glass gave rise to cells resembling foreign body granuloma macrophages. After an initial rise in Fc receptor- and C3 receptor-mediated phagocytosis, a progressive loss of Fc receptor expression and C3-mediated ingestion were observed. The monocyte surface antigens recognized by the anti-human monocyte monoclonal antibodies 1D5 and 63D3 were lost from the surface of the majority of cells cultured on glass and microexudates. A subpopulation of Fc receptor-positive cells that were 1D5 and 63D3 positive was retained in fully differentiated cell populations. In comparison, monocytes cultivated on collagen matrices gave rise to highly phagocytic cells resembling human resident tissue macrophages. Both Fc- and C3-mediated phagocytosis were enhanced and remained so during the entire length of culture. The surface antigens recognized by the 1D5 antibody, expressed on all freshly seeded monocytes, was maintained on the macrophages. The antigen recognized by the 63D3 antibody was not expressed on mature cells. The present evidence would indicate that variations in expression of phagocytic receptors and the surface antigens 1D5 and 63D3 can be ascribed to the stage of development of the macrophage or its stage of activation, rather than to independent subsets of mononuclear phagocytes.


Subject(s)
Cell Differentiation , Monocytes/physiology , Antibodies, Monoclonal , Collagen , Complement C3/immunology , Culture Techniques , Glass , Histocompatibility Antigens Class II/analysis , Humans , Macrophages/immunology , Monocytes/immunology , Phagocytosis , Receptors, Fc/analysis
4.
J Exp Med ; 161(6): 1569-74, 1985 Jun 01.
Article in English | MEDLINE | ID: mdl-2409204

ABSTRACT

Two T4 cell clones (TLC) specific for antigenic epitopes on Chlamydia trachomatis were studied. Using a panel of allogeneic antigen-presenting cells (APC), both TLC were found to be restricted by HLA class II elements closely associated with, but not identical to the DRw5S specificity, as determined by highly selected alloantisera, a monoclonal antibody (mAb), 109d6, and confirmed on the DNA level by determination of restriction fragment length polymorphisms (RFLP) with a DR beta probe. Furthermore, HLA-DR-specific mAb, including 109d6, but not other HLA class II- or class I-specific antibodies inhibited the two TLC, strongly suggesting that the restriction element is expressed by a DR molecule. Using digestion with Hind III restriction enzyme and a DR beta probe, we found a complete concordance between the appearance of a 9.3 kilobase band and the ability of allogeneic APC to restimulate the T cell clones. Thus, the restriction element for these T cell clones appear to be expressed by DR molecules, but can, at present, only be detected at the genomic level.


Subject(s)
Histocompatibility Antigens Class II/genetics , T-Lymphocytes/immunology , Antibodies, Monoclonal/immunology , Antigen-Presenting Cells/immunology , Antigens, Bacterial/immunology , Chlamydia trachomatis/immunology , Clone Cells/immunology , DNA/genetics , Epitopes/immunology , HLA-DR Antigens , Histocompatibility Antigens Class II/immunology , Humans , T-Lymphocytes/classification
5.
Langmuir ; 26(15): 12592-7, 2010 Aug 03.
Article in English | MEDLINE | ID: mdl-20604580

ABSTRACT

The aim of this work was to develop a novel method of preparation of loaded nanosize capsules based on liquid core encapsulation by biocompatible polyelectrolyte (PE) multilayer adsorption, with or without pegylated outermost layer. Using AOT (docusate sodium salt) as emulsifier, we obtained cores, stabilized by an AOT/PLL (poly-L-lysine hydrobromide) surface complex. These positively charged cores were encapsulated by layer-by-layer adsorption of polyelectrolytes, biocompatible polyanion PGA (poly-L-glutamic acid sodium salt), and biocompatible polycation PLL. We used the saturation method for formation of consecutive layers, and we determined the optimal conditions concerning concentration of surfactant and polyelectrolytes to form stable shells. The average size of the obtained capsules was 60 nm. Pegylated external layer were prepared using PGA-g-PEG (PGA grafted by PEG poly(ethylene glycol)). The capsules were stable for at least a period of 3 months. These nanocapsules were biocompatible when tested for cytotoxicity in a cellular coculture assay and demonstrated no or very low nonspecific binding to peripheral blood mononuclear cells when tested by flow cytometry. In order to study drug effects on leukemia cells, beta-carotene and vitamin A have been encapsulated as model drugs.


Subject(s)
Nanocapsules/chemistry , Polymers/chemistry , Adsorption , Dioctyl Sulfosuccinic Acid/chemistry , Emulsifying Agents/chemistry , Models, Theoretical , Polyamines/chemistry , Polyelectrolytes , Polyglutamic Acid/chemistry
6.
Scand J Immunol ; 69(4): 319-28, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19284496

ABSTRACT

Most tumour-associated antigens (TAA) are non-mutated self-antigens. The peripheral T cell repertoire is devoid of high-avidity TAA-specific cytotoxic T lymphocytes (CTL) due to self-tolerance. As tolerance is major histocompatibility complex-restricted, T cells may be immunized against TAA presented by a non-self human leucocyte antigen (HLA) molecule and transferred to cancer patients expressing that HLA molecule. Obtaining allo-restricted CTL of high-avidity and low cross-reactivity has, however, proven difficult. Here, we show that dendritic cells transfected with mRNA encoding HLA-A*0201, efficiently present externally loaded peptides from the antigen, Melan-A/MART-1 to T cells from HLA-A*0201-negative donors. CD8(+) T cells binding HLA-A*0201/MART-1 pentamers were detected already after 12 days of co-culture in 11/11 donors. The majority of cells from pentamer(+) cell lines were CTL and efficiently killed HLA-A*0201(+) melanoma cells, whilst sparing HLA-A*0201(+) B-cells. Allo-restricted CTL specific for peptides from the leukaemia-associated antigens CD33 and CD19 were obtained with comparable efficiency. Collectively, the results show that dendritic cells engineered to express defined allo-HLA peptide complexes are highly efficient in generating CTL specifically reacting with tumour-associated antigens.


Subject(s)
Antigens, Neoplasm/immunology , Dendritic Cells/immunology , HLA-A Antigens/immunology , Isoantigens/immunology , T-Lymphocytes, Cytotoxic/immunology , Antigen Presentation/immunology , Cell Line, Tumor , Cytotoxicity, Immunologic , Flow Cytometry , HLA-A Antigens/genetics , HLA-A2 Antigen , Humans , Immunotherapy/methods , Lymphocyte Activation/immunology , Peptides/immunology , Polymerase Chain Reaction , Transfection
7.
J Cell Biol ; 131(1): 261-70, 1995 Oct.
Article in English | MEDLINE | ID: mdl-7559783

ABSTRACT

Peripheral node addressin (PNAd) is a complex mixture of glycoproteins with L-selectin ligand activity that functions in lymphocyte homing. We have investigated the contribution of the sialomucin CD34 relative to other components of PNAd in lymphocyte tethering and rolling in in vitro laminar flow assays. PNAd was isolated with MECA-79 mAb-Sepharose from tonsillar stroma, and the CD34 component (PNAd,CD34+) and CD34-negative component (PNAd,CD34-) separated on CD34 mAb-Sepharose. Lymphocytes on the PNAd,CD34- fraction tether less efficiently, roll faster and are less resistant to shear detachment than on PNAd. The PNAd,CD34+ fraction constitutes about half the total functional activity. These studies show that CD34 is a major functional component of PNAd. Ligand activity in both the PNAd,CD34+ and PNAd,CD34- fractions is expressed on mucin-like domains, as shown with O-sialoglycoprotease. The CD34 component of PNAd has about four times higher tethering efficiency than total tonsillar CD34. CD34 from spleen shows no lymphocyte tethering. Although less efficient than the PNAd,CD34+ fraction from tonsil, CD34 from the KG1a hematopoietic cell line is functionally active as an L-selectin ligand despite lack of reactivity with MECA-79 mAb, which binds to a sulfation-dependent epitope. All four forms of CD34 are active in binding to E-selectin. KG1a CD34 but not spleen CD34 are active as L-selectin ligands, yet both lack MECA-79 reactivity and possess E-selectin ligand activity. This suggests that L-selectin ligands and E-selectin ligands differ in more respects than presence of the MECA-79 epitope.


Subject(s)
Antigens, CD34/physiology , L-Selectin/physiology , Mucins/physiology , Palatine Tonsil/physiology , Antigens, Surface/metabolism , Cell Adhesion/physiology , Endothelium, Vascular/cytology , Humans , Ligands , Lymphocytes/enzymology , Lymphocytes/metabolism , Membrane Proteins , Molecular Weight , Protein Binding/physiology , Receptors, Lymphocyte Homing/metabolism , Rheology , Sialomucins , Spleen/blood supply
8.
Cancer Gene Ther ; 13(10): 905-18, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16710345

ABSTRACT

We have developed an individualized melanoma vaccine based on transfection of autologous dendritic cells (DCs) with autologous tumor-mRNA. Dendritic cells loaded with complete tumor-mRNA may generate an immune response against a broad repertoire of antigens, including unique patient-specific antigens. The purpose of the present phase I/II trial was to evaluate the feasibility and safety of the vaccine, and the ability of the DCs to elicit T-cell responses in melanoma patients. Further, we compared intradermal (i.d.) and intranodal (i.n.) vaccine administration. Twenty-two patients with advanced malignant melanoma were included, each receiving four weekly vaccines. Monocyte-derived DCs were transfected with tumor-mRNA by electroporation, matured and cryopreserved. We obtained successful vaccine production for all patients elected. No serious adverse effects were observed. A vaccine-specific immune response was demonstrated in 9/19 patients evaluable by T-cell assays (T-cell proliferation/interferon-gamma ELISPOT) and in 8/18 patients evaluable by delayed-type hypersensitivity (DTH) reaction. The response was demonstrated in 7/10 patients vaccinated intradermally and in 3/12 patients vaccinated intranodally. We conclude that immuno-gene-therapy with the described DC-vaccine is feasible and safe, and that the vaccine can elicit in vivo T-cell responses against antigens encoded by the transfected tumor-mRNA. The response rates do not suggest an advantage in applying i.n. vaccination.


Subject(s)
Cancer Vaccines/administration & dosage , Cell Transplantation , Dendritic Cells , Melanoma/therapy , RNA, Messenger/genetics , Transfection , Adult , Aged , Animals , Cancer Vaccines/adverse effects , Dogs , Electroporation , Enzyme-Linked Immunosorbent Assay , Humans , Hypersensitivity, Delayed , Immunity, Cellular , Melanoma/immunology , Middle Aged , T-Lymphocytes/immunology
9.
J Natl Cancer Inst ; 80(16): 1322-5, 1988 Oct 19.
Article in English | MEDLINE | ID: mdl-3050139

ABSTRACT

It has been widely assumed that anti-HLA-DR antibodies react with pluripotent stem cells and cannot be used in bone marrow purging. We report a case of non-Hodgkin's lymphoma in which an anti-HLA-DR antibody (AB4) was used for immunomagnetic purging and the subsequent autologous bone marrow transplantation resulted in rapid marrow engraftment with no serious complications. The results indicate that the AB4 antibody, which binds to an antigen encoded by the B3 gene of the DR region, can be safely used in the clinic in the purging of bone marrow from patients with AB4-positive tumors (non-T-cell acute lymphocytic leukemia, non-Hodgkin's lymphoma, and some cases of acute myelogenous leukemia.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Bone Marrow Transplantation , HLA-DR Antigens/immunology , Adult , Female , Humans , Lymphoma, Non-Hodgkin/immunology , Lymphoma, Non-Hodgkin/therapy
10.
Cancer Res ; 40(6): 2054-60, 1980 Jun.
Article in English | MEDLINE | ID: mdl-6966185

ABSTRACT

Mouse peritoneal macrophages were achieved by cocultivation with syngeneic sarcoma cells. The tumor cells died progressively during the cocultivation, leaving highly activated marcophages. Because of great changes in macrophage morphology during the activation, special efforts were made to identify the activated cells as macrophages by their ability to phagocytose latex and to bind opsonized sheep red cells to C3 and Fc receptors and by indirect immunofluorescence with an antimacrophage antiserum. Activation was evaluated by morphology and incorporation of [14C]glucosamine. The activation was found to be independent of the presence of T-cells, B-cells, and immunoglobulin bound to tumor cell surfaces. This was shown by removal of T-cells from the system by treatment with anti-theta and complement and by use of nude mice as the macrophage source and for tumor maintenance. Similarly, B-cells were removed by treatment with anti-immunoglobulin and complement as well as adherence to anti-immunoglobulin-coated plastic dishes. Immunoglobulin bound to tumor cells was removed by trypsinization and by elution at low pH. Culture supernatants from tumor cells and cell-free tumor ascites fluid also induced some activation of the macrophages. This activation differed from the coculture activation in both the extent and kinetics of morphological changes and gave only a small increase in [14C]glucosamine incorporation.


Subject(s)
B-Lymphocytes/immunology , Macrophages/immunology , Sarcoma, Experimental/immunology , T-Lymphocytes/immunology , Animals , Ascitic Fluid/immunology , Cell Communication , Mice
11.
Cancer Res ; 59(6): 1180-3, 1999 Mar 15.
Article in English | MEDLINE | ID: mdl-10096543

ABSTRACT

The therapeutic usefulness of macromolecules, such as in gene therapy, is often limited by an inefficient transfer of the macromolecule to the cytosol and a lack of tissue-specific targeting. The possibility of photochemically releasing macromolecules from endosomes and lysosomes into the cytosol was examined. Endocytosed macromolecules and photosensitizer were exposed to light and intracellular localization and the expression of macomolecules in the cytosol was analyzed. This novel technology, named photochemical internalization (PCI), was found to efficiently deliver type I ribosome-inactivating proteins, horseradish peroxidase, a p21ras-derived peptide, and a plasmid encoding green fluorescent protein into cytosol in a light-dependent manner. The results presented here show that PCI can induce efficient light-directed delivery of macromolecules into the cytosol, indicating that PCI may have a variety of useful applications for site-specific drug delivery, e.g., in gene therapy, vaccination, and cancer treatment.


Subject(s)
Cytosol/metabolism , Drug Delivery Systems/methods , Photosensitizing Agents/chemistry , Endocytosis , Endosomes/metabolism , Humans , Light , Lysosomes/metabolism , Macromolecular Substances , Photochemistry/methods , Tumor Cells, Cultured
12.
Leukemia ; 6(8): 845-52, 1992 Aug.
Article in English | MEDLINE | ID: mdl-1379314

ABSTRACT

Immunomagnetic beads are well suited for positive selection of CD34+ cells. However, both unspecific binding of beads to cells as well as the effectiveness of detachment of beads from cells may represent significant problems. We used an anti-Fab antiserum (DETACHaBEAD, Dynal) for rapid and effective detachment of immunomagnetic beads from the positively selected cells. By this detachment technique, the cells remained phenotypically unaltered. To reduce unspecific binding, we have coated various anti-CD34 monoclonal antibodies directly to paramagnetic beads M450 (Dynal). Use of beads coated with BI-3C5 was found to be optimal with regard to yield and purity of the isolated cells. The yield was on average 1.5% (range 0.5-2.5%) of bone marrow mononuclear cells and the purity was usually greater than 95% CD34+ cells of the isolated cells. Subpopulations of the cells expressed myeloid markers (CD13, CD33, and to a lesser extent CD15 and CD14) or early B-lineage markers (CD19 and CD10). Most of the cells expressed CD38, and a majority of the cells also expressed CD41. In general, most of the CD34+ cells with low forward scatter expressed B-lineage markers, as was also the case for the few contaminating CD34- cells which were found to be predominantly CD37+ mature B cells. Reactivity with antibodies against T-lineage markers (CD2, CD3, CD4, CD7, and CD8) was generally detected only on 1-2% of the cells or less. Isolated cells responded to interleukin 3, granulocyte-macrophage colony-stimulating factor, mast cell growth factor, and/or granulocyte colony-stimulating factor alone or in combinations in short-term liquid cultures. The cells were also markedly enriched for granulocyte-macrophage colony-forming units as well as for early progenitor cells capable of forming blast colonies on preformed stromal feeder layers. Moreover, the CD34- population was depleted of 70-80% of CFU-GM and cells capable of blast colony formation. Thus, we conclude that the isolated cells are phenotypically unaltered after isolation, and show a normal response in various in vitro assays.


Subject(s)
Hematopoietic Stem Cells/cytology , Antigens, CD , Antigens, CD34 , Cell Division , Cell Separation/methods , Hematopoietic Stem Cells/immunology , Humans , Immunoglobulin Fab Fragments/immunology , Immunologic Techniques , In Vitro Techniques , Magnetics
13.
Article in English | MEDLINE | ID: mdl-1673156

ABSTRACT

The effect of lymphokines on the replication of HIV-1 has previously been investigated using HIV-1-infected cell lines or PBMCs infected in vitro with HIV-1. We have examined the effect of rIFN alpha 2, rFIN beta, and rIFN gamma and recombinant tumor necrosis factor-alpha (rTNF alpha) on the replication of HIV-1 in vitro in naturally HIV-1-infected CD4+ T cells from asymptomatic HIV-1-seropositive individuals. rIFN alpha 2 inhibited the replication of HIV-1 effectively at concentrations that can be achieved in vivo. The inhibitory activity was most efficacious when rIFN alpha 2 was added as the CD4+ T cells were being activated, and less but still considerable when rIFN alpha 2 was added 4-96 h after CD4+ T-cell activation. rIFN alpha 2 exerted a suppressive effect on the proliferation of the CD4+ T cells, but this effect was small at concentrations that caused 90% inhibition of the replication of HIV-1. rIFN beta, rIFN gamma, and rTNF alpha had no effect on the replication of HIV-1, but rIFN beta and rTNF alpha had a costimulatory effect on CD4+ T-cell proliferation. Activated CD8+ T cells secrete a HIV-1-inhibitory soluble factor. Blocking experiments using an IFN alpha 2-neutralizing MAb showed that this HIV-1-inhibitory factor is not IFN alpha 2.


Subject(s)
Cytokines/pharmacology , HIV-1/drug effects , Interferon Type I/pharmacology , Interferon-gamma/pharmacology , Tumor Necrosis Factor-alpha/pharmacology , Antibodies, Monoclonal/immunology , CD4-Positive T-Lymphocytes/drug effects , CD4-Positive T-Lymphocytes/microbiology , Cell Line , Cytokines/metabolism , Dose-Response Relationship, Immunologic , HIV-1/growth & development , Humans , Interferon-gamma/metabolism , Lymphocyte Activation/drug effects , Recombinant Proteins , Solubility , T-Lymphocytes, Regulatory/metabolism , Tumor Necrosis Factor-alpha/metabolism , Virus Replication/drug effects
14.
J Immunol Methods ; 43(3): 251-9, 1981.
Article in English | MEDLINE | ID: mdl-6972976

ABSTRACT

The bacterium Brucella melitensis has been shown to bind selectively to human B-lymphocytes. The specificity of this binding can be exploited in a rapid technique for the determination of human B-lymphocytes and monocytes by use of rhodamine-labeled Brucella melitensis. This fluorescent labeled regent is simple to use and provides highly specific identification of human B-lymphocytes and monocytes, as demonstrated in a series of experiments characterizing known cellular markers on T- and B-lymphocytes, their separated cellular subpopulations and lymphoblastoid cell lines. The use of fluorescence conjugated bacteria greatly simplifies the application of this highly specific technique and makes its use more practical in routine screening for B-cell and monocyte populations.


Subject(s)
B-Lymphocytes/metabolism , Brucella/metabolism , Monocytes/metabolism , Antigens , B-Lymphocytes/immunology , Blood Cell Count , Cell Line , Cell Separation , Humans , Lymphocytes/classification , Phagocytes , Rhodamines , Time Factors
15.
J Immunol Methods ; 90(2): 179-87, 1986 Jun 24.
Article in English | MEDLINE | ID: mdl-3088118

ABSTRACT

A monoclonal antibody of the IgM isotype, ITI-5C2, which binds with high affinity to CD8 molecules, was directly conjugated to the monosized magnetic microspheres M-450. This permits selective removal of the CD8+ T cell subset (T8) from peripheral blood mononuclear cell suspensions in a rapid one-step procedure. With a low ratio of microspheres to cells (2:1), functionally active T8 cells can be recovered. In vitro experiments involving such positively selected T8 cells or recombinations of isolated T8 and T4 subsets, demonstrate that the presence of M-450 microspheres coated with ITI-5C2 do not interfere with the immunological functions of the positively selected cells. The method has possible application in the isolation of all cell populations where high avidity mAbs of appropriate specificity are available.


Subject(s)
Antibodies, Monoclonal , Antigens, Surface/immunology , T-Lymphocytes, Cytotoxic/immunology , T-Lymphocytes/immunology , Antigens, Differentiation, T-Lymphocyte , Cell Separation/methods , Cytotoxicity, Immunologic , Humans , Magnetics , Microspheres
16.
J Immunol Methods ; 118(2): 251-5, 1989 Mar 31.
Article in English | MEDLINE | ID: mdl-2466905

ABSTRACT

We have developed a simple method for isolation of functionally active T cell receptor (TCR)-gamma delta positive cells from human peripheral blood, using immunomagnetic separation techniques. After culture with feeder cells and interleukin-2, cells thus isolated showed a CD3+ TCR-alpha beta- phenotype, and stained with antibodies against the TCR-gamma delta complex. The TCR-gamma delta+ cells were functionally active, responding with DNA synthesis when stimulated via their CD3 and CD2 molecules in concert, or when interleukin-2 was added.


Subject(s)
Cell Separation , Receptors, Antigen, T-Cell , T-Lymphocytes/classification , Adult , Antibodies, Monoclonal , Cell Line , Cell Separation/methods , Flow Cytometry , Humans , Leukocyte Count , Magnetics , Microspheres , Phenotype , Receptors, Antigen, T-Cell/analysis , Staining and Labeling , T-Lymphocytes/analysis , T-Lymphocytes/physiology
17.
J Immunol Methods ; 114(1-2): 95-9, 1988 Nov 10.
Article in English | MEDLINE | ID: mdl-2972783

ABSTRACT

A simple, one-step quantitative assay for the detection of biologically active interleukin-2 (IL-2) is described. It is based on culture of pure CD3+ T cells which have been positively selected from blood mononuclear cells by particle (M450)-bound anti-CD3 monoclonal antibody (mAb). During culture, activation of the T cells via CD3 will occur, leading to expression of IL-2 receptors but not IL-2 production. By adding IL-2 a proliferative response is evoked, giving a linear dose-response curve for IL-2 concentrations between 0.01-20 U/ml. The cells were unresponsive to IL-1, IL-4, tumor necrosis factor alpha (TNF-alpha), interferon-alpha (IFN-alpha), IFN-gamma, phytohemagglutinin and concanavalin A. The responsiveness to IL-2 was enhanced by TNF-alpha and inhibited by IFN-alpha, while the other tested lymphokines and mitogens did not influence the proliferative response. Antibodies to IL-2 and to IL-2 receptor suppressed the IL-2 response in a dose-dependent manner. The method is both simple and specific, and obviates the necessity for keeping assay cells in long term culture.


Subject(s)
Interleukin-2/analysis , Lymphocyte Activation , T-Lymphocytes/immunology , Antibodies, Monoclonal , Antigens, Differentiation, T-Lymphocyte/immunology , CD3 Complex , Cell Separation , Cells, Cultured , Dose-Response Relationship, Immunologic , Humans , Interleukin-2/physiology , Microspheres , Mitogens , Phenotype , Receptors, Antigen, T-Cell/immunology , T-Lymphocytes/classification
18.
Transplantation ; 43(3): 366-71, 1987 Mar.
Article in English | MEDLINE | ID: mdl-3103274

ABSTRACT

A new technique for depletion of T cells from bone marrow is presented. Bone marrow cells (BMC) were rosetted with magnetic monosized polystyrene microspheres coated with monoclonal antibodies (MAbs) specific for T cell CD2 and CD3 antigens. Rosetted T cells were subsequently removed from non-T cells with the aid of a magnet. This immunomagnetic separation procedure was carried out in less than 40 min and reproducibly removed T cells, leaving a maximum of 0.025% sheep-red-blood-cell (SRBC) rosette-forming cells and less than 0.02% T cells as detected by a T cell limiting dilution assay. The efficacy of the depletion procedure was further shown by flow cytometry data, by effective removal of cells from a T cell line added to the BMC prior to immunomagnetic separation, and by abrogation of interleukin 2 (IL-2)-producing capacity in T-cell-depleted BMC (BMC-T). The T cell depletion procedure provided a 43-74% recovery of non-T cells present in the Isopaque-Ficoll-isolated bone marrow mononuclear cell fraction and did not disturb the growth potential of stem cells, as assayed by hematopoietic stem cell assays.


Subject(s)
Lymphocyte Depletion , T-Lymphocytes/immunology , Antibodies, Monoclonal/immunology , Antigens, Differentiation, T-Lymphocyte , Antigens, Surface/immunology , Bone Marrow Cells , Cell Line , Colony-Forming Units Assay , Humans , Interleukin-2/biosynthesis , Isoantigens/immunology , Lectins/pharmacology , Lymphocyte Activation/drug effects , Magnetics , Microspheres , Polystyrenes , Rosette Formation , T-Lymphocytes/metabolism
19.
Biotechniques ; 30(5): 972-5, 2001 May.
Article in English | MEDLINE | ID: mdl-11355359

ABSTRACT

In this study, we have applied automated constant denaturant capillary electrophoresis (ACDCE) for the detection of KRAS exon 1 mutations. Samples from 191 sporadic colon carcinomas previously analyzed for KRAS mutations with allele-specific PCR (ASPCR), temporal temperature gradient electrophoresis (TTGE), and constant denaturant capillary electrophoresis (CDCE) were analyzed. In ACDCE, an unmodified ABI Prism 310 genetic analyzer with constant denaturant conditions separated fluorescein-labeled PCR products. Temperature in combination with a chemical denaturant was used for separation. The optimal separation conditions for PCR-amplified KRAS exon 1 fragments were determined by adjusting the temperature before electrophoresis. In the ACDCE analysis, the sequence of a mutant was determined by comparing the electropherogram of the fragment to that of known mutations followed by mixing the sample with control mutations before reanalysis. In a titration experiment mixing mutant and wild-type alleles, the sensitivity for mutation detection was shown to be 0.6% in this automated CDCE technique. The automation of CDCE allowed rapid analysis of a large number of test samples over as short period of time and with a commercially available apparatus.


Subject(s)
Autoanalysis , Colorectal Neoplasms/genetics , Electrophoresis, Capillary/methods , Exons , Genes, ras/genetics , Mutation , Codon , Cryopreservation , DNA Mutational Analysis , Hot Temperature , Humans , Polymerase Chain Reaction , Protein Denaturation , Sensitivity and Specificity , Sodium Dodecyl Sulfate , Tumor Cells, Cultured
20.
Thromb Haemost ; 57(2): 212-6, 1987 Apr 07.
Article in English | MEDLINE | ID: mdl-3299855

ABSTRACT

A method is described for the identification of antigens by monoclonal antibodies. This is applicable whenever precipitating antibodies to the same antigens from a different species are available. The method is based upon: Separation and immunoprecipitation of cellular proteins with a polyspecific antiserum in crossed immunoelectrophoresis in the presence of the non-denaturing detergent Triton X-100 and the monoclonal antibody. Coprecipitation of the monoclonal antibody with its antigen. Subsequent passive transfer of the monoclonal antibody in the antibody-antigen complex onto a nitrocellulose membrane. Visualization of the blotted antibody using an enzyme-linked secondary antibody and a chromogenic substrate. Identification of the corresponding antigen by comparisons to the immunoprecipitate pattern of the original immunoplate. To test this method we have analyzed the detection of the antigens recognized by six previously described monoclonal antibodies against platelet membrane proteins and von Willebrand factor. Specific immunoblots were obtained in each case using small amounts of monoclonal antibodies. Thus, the technique provides an alternative when epitopes are denatured by SDS, and avoids the use of radioactively labelled monoclonal antibodies.


Subject(s)
Antibodies, Monoclonal/immunology , Antigens/immunology , Blood Platelets/immunology , Immunoelectrophoresis , Humans , Immunoelectrophoresis/methods , Immunologic Techniques
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