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• W2091836639 abstract "Recent reports on functional impairments within CD8 T cell subpopulations [1–5] focused attention on quantitative assessments of functional activity. However, the expansion and propagation of antigen-specific CD8 T cells often requires extensive tissue culture manipulations and frequent re-stimulations. A method that could selectively identify and expand an antigen-specific CD8 population would be of value in these quantitative assessments. Flow cytometric or human leukocyte antigen (HLA) bead sorting of tetrameric-positive virus-specific T cells has proved to be of value [6,7]; however, it requires a flow cytometer, is technically demanding and needs conditions of absolute sterility. We have developed an assay that utilizes anti-phycoerythrin-coated MACS beads (Miltenyi Biotec, Auburn, USA) to select positively phycoerythrin-conjugated tetramer peptide complexes admixed with CD8 T cells to obtain and select enriched specific populations for in-vitro culture. This simplified method can lead to the rapid expansion of an antigen-specific T cell population. To compare the isolation of virus-specific CD8 T cells by anti-phycoerythrin bead separation and flow cytometry, an HLA-A*0201-positive cytomegalovirus (CMV)-seropositive donor with 0.43%, CMV-pp65 (495–503: NLVPMVATV)-specific tetramer-positive CD8 T cells was identified (Fig. 1a). We isolated CMV-pp65 CD8 T cells with anti- phycoerythrin MACS beads by the incubation of 5 × 106 peripheral blood mononuclear cells (PBMC) for 20 min at 37°C with the HLA-A*0201 CMV-pp65-specific tetramer [8]. The PBMC were then incubated with 20 μl anti-phycoerythrin MACS beads (Miltenyi Biotec) at 4°C for 20 min to label the CMV-pp65-specific CD8 T cells. The labelled PBMC were passed through a pre-washed (500 μl of buffer: PBS pH 7.2, 0.5% bovine serum albumin and 2 mM ethylenediamine tetraacetic acid) separation column (Miltenyi Biotec) on a magnet. The column was eluted three times with 500 μl of buffer to remove the unbound CMV-pp65 tetramer-negative PBMC (negative fraction). The separation column containing the phycoerythrin-specific magnetic beads bound to the tetramer-positive T cells was removed from the magnet. The column was washed twice again with 500 μl of buffer then, finally, to ensure all the cells were recovered, a plunger was used to elute the labelled CMV-pp65 tetramer-positive CD8 T cells (positive fraction). Simultaneously, 5 × 106 PBMC were counterstained with a combination of phycoerythrin-conjugated CMV-pp65 tetramer and fluorescein-isothiocyanate-conjugated CD8 monoclonal antibody for flow cytometric isolation, performed using standard methods [9]. The CMV-pp65 tetramer-positive, negative and fluorescence-activated cell sorted (FACS) fractions were cultured in R15-50 medium [RPMI 1640 media supplemented with 15% heat-inactivated fetal calf serum, l-glutamine, hepes, 50 IU/ml IL-2 and penicillin–streptomycin (Biowhittaker, Maryland, USA)] with the addition of pooled irradiated PBMC feeders (at 1 × 106/ml) and anti-CD3 (12F6, Dr J. Wong, Massachusetts, USA).Fig. 1: (a) Dot plot of HLA-A*0201 CMV-pp65-specific CD8 tetramer staining gated on the CD3/CD8 population. From an HLA-A*0201 homozygous individual there were 0.43% tetramer-positive HLA-A*0201 CMV-pp65-specific CD8 T cells represented in the upper right quadrant. (b) Histograms representing the tetramer specificity of the anti-phycoerythrin-MACS bead for the isolation of human leukocyte antigen (HLA) tetrameric complex labelled antigen-specific T cells, after 14 days of stimulation with CMV-pp65 peptide and irradiated peripheral blood mononuclear cell (PBMC) feeders. The cultures were analysed for HLA-A*0201 CMV-pp65-specific CD8 T cells by flow cytometry. The lower panel represents the HLA-A*0201 CMV-pp65 tetramer positively selected population isolated by anti-phycoerythrin beads and the upper panel the ‘negative’ eluted fraction. The HLA-A*0201 CMV-pp65-specific CD8 T cells in the upper panel represent 33.8% of the CD3/CD8 tetramer-positive population. (c) Specific lysis of peptide pulsed B cell lymphoma target by HLA-A*0201 CMV-pp65 tetramer-specific CD8 T cells isolated by anti-phycoerythrin-MACS beads or by sorted using fluorescence-activated cell sorting (FACS) after 14 days of culture. The HLA-A*0201 CMV-pp65 tetramer-`positive’ CD8 T cells and those cultured from the ‘negative’ fraction were assayed in a 51Cr release assay at 1 : 5 or 1 : 10 effector to target (E : T) ratios. In addition, HLA-A*0201 CMV-pp65-specific CD8 T cells isolated by FACS were included, to compare the efficiency of the two methods.After 14 days, the lytic activity of the CMV-pp65-specific CD8 T cell cultures was assessed in a standard 51Cr release assay using CMV-pp65 peptide pulsed HLA-A*0201 B cell lymphoma targets. The CMV-pp65-specific CD8 T cells were added at two effector : target ratios and incubated at 37°C, 5% carbon dioxide for 4 h (Fig. 1c). The CMV-pp65-specific CD8 T cell population was strongly CMV-pp65 tetramer positive after a 14 day in-vitro expansion period (Fig. 1b), whereas the ‘negative’ fraction remained CMV-pp65 tetramer negative (Fig. 1b). From a starting population of 5 × 106 PBMC, we initially isolated approximately 21.5 × 103 tetramer-positive cells (0.43%). This culture was expanded by stimulation with peptide-pulsed irradiated feeders over 14 days, with the result that 33.8% of the CD3/CD8 population were HLA-A*0201 CMV-pp65-specific tetramer positive (Fig. 1b). The 51Cr release assay determined the specificity and function of the antigen-specific CD8 T cells against CMV pp65-peptide pulsed HLA-A*0201 targets. The percentage of specific target cell lysis of target cells was compared for anti-phycoerythrin MACS bead-enriched CMV pp65 antigen-specific CD8 T cell population and the corresponding expanded effector population, isolated by single-cell sorting using phycoerythrin-conjugated tetrameric complexes and FACS (Fig. 1c). The negatively sorted T cell population demonstrated non-significant lysis (< 15%) compared with the positively sorted HLA-A*0201 CMV-pp65-specific CD8 population (> 50%) (Fig. 1c). These data showed that MACS separation with anti-phycoerythrin micro beads is a viable technique for the separation of short-term cell lines and for the cloning of HLA tetrameric complex-labelled antigen-specific T cells. Here we have demonstrated a simple immunomagnetic separation technique that utilizes technology readily available to most immunology laboratories worldwide. The method is specific and represents an inexpensive, reliable alternative to cell sorting by flow cytometry for the isolation of phycoerythrin-conjugated tetramer-positive T cells. Adrian B. McDermotta Hans M. L. Spiegelb Johannes Irschc Graham S. Oggd Douglas F. Nixona" @default.
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• W2091836639 date "2001-04-01" @default.
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• W2091836639 title "A simple and rapid magnetic bead separation technique for the isolation of tetramer-positive virus-specific CD8 T cells" @default.
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• W2091836639 doi "https://doi.org/10.1097/00002030-200104130-00024" @default.
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