Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides-6

<p><b>Copyright information:</b></p><p>Taken from "Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides"</p><p>BMC Immunology 2005;6():24-24.</p><p>Published online 5 Dec 2005</p><p>PMCID:PMC1325046.</p><p>Copyright © 2005 Moro et al; licensee BioMed Central Ltd.</p>roduction to different doses of p39 peptide. 10T cells are incubated for 48 hours with 5 × 10HLA-DR*1101LCL cells and the indicated doses of p39 peptide. IFN-γ in the supernatant is measured by ELISA. (b) Staining of a MAGE-3-specific T cell clone. The cells are stained at day 19 from re-stimulation with 10 μg of either CLIP-loaded or p39-loaded HLA-DR*1101-Bir tetramers for 2 hours at 37°C

Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides-1

<p><b>Copyright information:</b></p><p>Taken from "Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides"</p><p>BMC Immunology 2005;6():24-24.</p><p>Published online 5 Dec 2005</p><p>PMCID:PMC1325046.</p><p>Copyright © 2005 Moro et al; licensee BioMed Central Ltd.</p>ipitation of HLA-DR*1101-Ig and HLA-DR*1101-Bir molecule with 30 μl of L243-sepharose beads from 1 ml of S2 cells culture supernatant after CuSO4 induction. The lanes contain the following material: 1. sup, 30 μl of culture supernatant before immunoprecipitation; 2. bound, the immunoprecipitated soluble recombinant HLA-DR*1101; and 3. not bound, 30 μl of culture supernatant after immunoprecipitation. Proteins were separated on SDS-page, transferred to filter and revealed with anti his-tag antibody. (b) Densitometric analysis of the protein bands displayed in panel (a), showing the elative efficiency of immunoprecipitation of the two soluble recombinant HLA-DR*1101 proteins with L243-Sepharose beads, as an indirect indicator of the percentage of correctly folded molecule. (c) Size exclusion chromatography of HLA-DR*1101-Ig molecules, after purification of ProtA affinity chromatography. The elution profile of the molecule from a Superdex 200 gel filtration column is shown. Inset shows the dot-blot analysis performed on the protein contained in the indicated elution peaks. Spotted proteins are probed with either anti-His tag antibody, to verify the presence of the HLA-DR*1101 molecule, or L243 mAb (Anti-HLA-DR) to verify the correct conformation of the molecule. (d) Elution profile from Superdex 200 gel filtration column and dot-blot analysis on eluted peaks of HLA-DR11-Bir molecules, performed as described in (c). (e) Calibration profile of the Superdex 200 gel filtration column

Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides-4

<p><b>Copyright information:</b></p><p>Taken from "Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides"</p><p>BMC Immunology 2005;6():24-24.</p><p>Published online 5 Dec 2005</p><p>PMCID:PMC1325046.</p><p>Copyright © 2005 Moro et al; licensee BioMed Central Ltd.</p>lation with p2 and APCs. The histograms in the upper row display the amount of surface TCR (mfi) expressed by the T cells at the time of tetramer staining, as determined by anti-CD3 staining. The histograms in the lower row show the staining with the p2-loaded HLA-DR*1101-Bir tetramers (mfi) performed at the indicated time after restimulation

Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides-5

<p><b>Copyright information:</b></p><p>Taken from "Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides"</p><p>BMC Immunology 2005;6():24-24.</p><p>Published online 5 Dec 2005</p><p>PMCID:PMC1325046.</p><p>Copyright © 2005 Moro et al; licensee BioMed Central Ltd.</p> from a HLA-DR*1101 healthy donor after one round of stimulation with 5 μM of p2-peptide and 40 u/ml of IL-2. (b) Intracellular production of IFN-γ by T cells contained in the p2-enriched PBMC. T cells were stimulated for 6 hours with p2 in the presence of HLA-DR*1101LCL cells at 37°C. Brefeldin A was added after the first hour of stimulation. The cells were then fixed, permeabilised and stained with anti-IFN-γ mAb. Cells unstimulated (nil) and stimulated policlonally with PMA-Ionomycin (PMA/ionomycin) are shown as controls. Numbers in quadrants indicate the percentage of T cells stained with anti-IFN-γ mAb (c) Staining of PBMCs from HLA-DR*1101 healthy donor with HA-loaded HLA-DR*1101-Bir tetramers. T cells were after three rounds of stimulation with 1 μg/ml of HA peptide and 40 u/ml of IL-2. The staining is performed at the indicated days after the third stimulation. Numbers in quadrants indicate the percentage of T cells stained by HLA-DR tetramers. (d) Intracellular production of IFN-γ by CD4T cells contained in the HA-specific T cell line at day 8 from the third stimulation. Activation and staining of CD4T cells was performed as described in (b)

Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides-3

<p><b>Copyright information:</b></p><p>Taken from "Generation of functional HLA-DR*1101 tetramers receptive for loading with pathogen or tumour derived synthetic peptides"</p><p>BMC Immunology 2005;6():24-24.</p><p>Published online 5 Dec 2005</p><p>PMCID:PMC1325046.</p><p>Copyright © 2005 Moro et al; licensee BioMed Central Ltd.</p>aded HLA-DR*1101-Bir tetramers at different temperature. (a) The relative affinity for the p2-HLA-DR*1101 displayed by the two TCC clones 162 and 51 is determined in an IFN-γ-releasing assay following production in response to different doses of p2 peptide. 10T cells are incubated with 5 × 10HLA-DR*1101 LCL cells and the indicated doses of p2 peptide. After 48 hours, the concentration of IFN-γ in the culture supernatant is measured by ELISA. Indicated are the concentration of p2 peptide required to elicit half-maximum release of IFN-γ in the two TCC. (b) Staining of TCC162, TCC51 and the irrelevant TCC with either p2-loaded HLA-DR*1101-Ig or p2-loaded HLA-DR*1101-Bir tetramers. Staining is performed for 2 hours at the indicated temperatures with 10 μg of tetramer per sample. The tetramer staining on CD3CD4gated cells is shown. The amount of surface TCR, obtained by staining with anti-CD3 mAb, expressed by the TCC in the different conditions is shown by the mean fluorescent intensity (CD3 mfi)