goat-anti-human biotinylated cd27 57703  (R&D Systems)

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses

doi: 10.1007/s00262-009-0711-x

Figure Lengend Snippet: Examples of acellular aAPC systems are schematised. a Acellular aAPCs were developed by coating streptavidin-labelled microbeads with biotinylated HLA/peptide monomers along with biotinylated anti-CD28 co-stimulatory antibody [22, 32, 36]. b In some cases, the recombinant co-stimulatory molecules, such as B7.1 and ICAM-1 were non-covalently immobilised onto the beads [28]. c HLA/peptide molecules have also been conjugated to an immunoglobulin heavy chain, producing a dimeric HLA/Immunoglobulin fusion protein. These fusion proteins were then immobilised onto beads along with anti-CD28 co-stimulatory antibody [26]. d In this study, soluble antigen-presenting complexes were developed by cross-linking biotinylated HLA/peptide complexes with biotinylated co-stimulatory antibody onto a streptavidin core molecule

Article Snippet: Goat-anti-human biotinylated CD27 (clone 57703), CD28 (clone 37407) and CD40L (clone 40804) antibodies (R&D, Minneapolis, USA) were resuspended in the same buffer as the HLA/peptide monomers (20 mM Tris–Hcl pH 8, 150 mM NaCl) and added to the biotinylated HLA/peptide monomers at a theoretical 1:3 molar ratio.

Techniques: Recombinant

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses

doi: 10.1007/s00262-009-0711-x

Figure Lengend Snippet: The HLA/peptide-costimulatory antibody complexes (sAPCs) were tested for the presence of both refolded HLA class I molecules and costimulatory antibody (CD27, CD28, CD40L and IgG isotype control) in a modified ELISA assay as described in “Materials and methods”. Serial dilutions (1 in 2) of sAPCs and negative controls, including HLA/peptide tetramers and soluble costimulatory antibody, were assessed in parallel. Both Fc regions of the costimulatory antibodies and HLA/peptide complexes were detected in all the sAPCs, above the background level of detection obtained with conventional HLA/peptide tetramers or soluble co-stimulatory antibodies

Article Snippet: Goat-anti-human biotinylated CD27 (clone 57703), CD28 (clone 37407) and CD40L (clone 40804) antibodies (R&D, Minneapolis, USA) were resuspended in the same buffer as the HLA/peptide monomers (20 mM Tris–Hcl pH 8, 150 mM NaCl) and added to the biotinylated HLA/peptide monomers at a theoretical 1:3 molar ratio.

Techniques: Modification, Enzyme-linked Immunosorbent Assay

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses

doi: 10.1007/s00262-009-0711-x

Figure Lengend Snippet: Representative HLA/tetramer staining of CMV-specific CD8+ T cells stimulated with sAPCs. PBMCs derived from an HLA-A*0201 CMV-seropositive donor (a) and from an HLA-A*0201 CMV-seronegative donor (b) were incubated without antigen (Medium) stimulated with HLA/CMV-IgG tetramer (CMV Tet/IgG), sAPC-CD27/28, sAPC-CD27/28.40L or HLA/tetramer plus soluble co-stimulatory antibodies. Activated T cells were stained with HLA-A2/CMV Allophycocyanin Tetramer and analysed by flow cytometry. Cells are gated on CD3+ T cells and the percentage of CMV specific CD8+ T cells are shown in each upper right gate

Article Snippet: Goat-anti-human biotinylated CD27 (clone 57703), CD28 (clone 37407) and CD40L (clone 40804) antibodies (R&D, Minneapolis, USA) were resuspended in the same buffer as the HLA/peptide monomers (20 mM Tris–Hcl pH 8, 150 mM NaCl) and added to the biotinylated HLA/peptide monomers at a theoretical 1:3 molar ratio.

Techniques: Staining, Derivative Assay, Incubation, Flow Cytometry

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses

doi: 10.1007/s00262-009-0711-x

Figure Lengend Snippet: The proliferation and differentiation kinetic of CMV-specific T cells stimulated with sAPCs is dependent on the costimulatory signal provided. a PBMCs derived from an HLA-A*0201 CMV-seropositive donor were labelled with CFSE prior to the second and to the fourth stimulation as described in material and method and shown in Fig. 4. A negative control (Medium alone) and positive control (polyclonal stimulus via PHA) were also included. Activated T cells were harvested three days after the second (two stimulations) and fourth stimulations (four stimulations), stained with HLA-A2/CMV Allophycocyanin tetramer and acquired by flow cytometry. Cells were gated on CD3+ CD8+ CMV tetramer+ cells, and analysed for the loss of CFSE fluorescence. b Activated T cells were harvested 3 days after the fourth stimulation and stained with HLA/CMV Allophycocyanin tetramer, CD27 and CD45RO cell surface markers. Lymphocytes were acquired by flow cytometry and gated on CD3+ CD8+ CMV tetramer+ cells. The frequencies of naïve (CD27+CD45RO−), memory (CD27+CD45RO+), effector (CD27−CD45RO+) and terminal effector (CD27−CD45RO−) CMV specific CD8+ T cells is shown for the different stimulation conditions

Article Snippet: Goat-anti-human biotinylated CD27 (clone 57703), CD28 (clone 37407) and CD40L (clone 40804) antibodies (R&D, Minneapolis, USA) were resuspended in the same buffer as the HLA/peptide monomers (20 mM Tris–Hcl pH 8, 150 mM NaCl) and added to the biotinylated HLA/peptide monomers at a theoretical 1:3 molar ratio.

Techniques: Derivative Assay, Negative Control, Positive Control, Staining, Flow Cytometry, Fluorescence

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Soluble HLA/peptide monomers cross-linked with co-stimulatory antibodies onto a streptavidin core molecule efficiently stimulate antigen-specific T cell responses

doi: 10.1007/s00262-009-0711-x

Figure Lengend Snippet: BCR/ABL-specific T cells were generated from HLA-A*0301 healthy donors following stimulation with sAPCs. PBMCs derived from five HLA-A*0301 healthy donors were stimulated with sAPC-CD27, sAPC-CD27/28 or sAPC-CD27/28/40L. In parallel, PBMCs were incubated without antigen (Medium), or stimulated with either autologous peptide pulsed-irradiated DCs (DCs-peptide), or with HLA/CML tetramers (CML tetramer) plus soluble co-stimulatory antibodies (Tet + sol). Activated T cells were harvested three days after the fourth stimulation and stained with HLA-A3/CML or HLA-B8/CML (control) Allophycocyanin Tetramers and analysed by flow cytometry. The frequency of tetramer positive cells is expressed as a percentage of CD3+CD8+ T cells and the mean frequency values are shown for each condition (a). b Representative HLA/tetramer dot plots of BCR/ABL-specific T cells

Article Snippet: Goat-anti-human biotinylated CD27 (clone 57703), CD28 (clone 37407) and CD40L (clone 40804) antibodies (R&D, Minneapolis, USA) were resuspended in the same buffer as the HLA/peptide monomers (20 mM Tris–Hcl pH 8, 150 mM NaCl) and added to the biotinylated HLA/peptide monomers at a theoretical 1:3 molar ratio.

Techniques: Generated, Derivative Assay, Incubation, Irradiation, Staining, Flow Cytometry