PBMCs were plated at 3.75 × 106 cells/ml in RPMI medium (supplemented with 10% human serum, 2 mM glutamine, 100 IU/ml penicillin, 0.1 mg/ml streptomycin) and a LAG-3 antagonist antibody (αLAG-3, Abcam, 17B4, 10 μg/ml) was added to select wells for 30 min. before the addition of PepTivator CMV pp65 and Adv5 hexon (Miltenyi Biotec, 175 ng/ml) and incubated at 37°C for 6 days.
Peptivator cmv pp65
Manufactured by Miltenyi Biotec
Sourced in Germany, United States
PepTivator® CMV pp65 is a peptide pool designed for the stimulation of CMV-specific T cells. It consists of 15-mer peptides spanning the sequence of the cytomegalovirus (CMV) phosphoprotein 65 (pp65).
Automatically generated - may contain errors
Lab products found in correlation
Phytohemagglutinin (pha), by Merck Group (3 mentions)
Facscanto a, by BD (1 mentions)
Cytometric bead array flex set, by BD (1 mentions)
Diva software, by BD (1 mentions)
Human ifn γ elispot kit, by Mabtech (1 mentions)
Biotinylated anti ifn γ antibody, by Mabtech (1 mentions)
Cd3 microbeads, by Miltenyi Biotec (1 mentions)
Texmacs, by Miltenyi Biotec (1 mentions)
Monensin, by Merck Group (1 mentions)
Anti cd107a fitc, by BD (1 mentions)
Macsquant instrument, by Miltenyi Biotec (1 mentions)
Peptivator cmv ie 1, by Miltenyi Biotec (1 mentions)
Peptivator sars cov 2 prot n, by Miltenyi Biotec (1 mentions)
Peptivator sars cov 2 prot s, by Miltenyi Biotec (1 mentions)
Liaison xl analyzer, by DiaSorin (1 mentions)
Clone 1 d1k, by Mabtech (1 mentions)
Streptavidin peroxidase, by Roche (1 mentions)
Penicillin streptomycin, by Euroclone (1 mentions)
3 amino 9 ethylcarbazole substrate, by BD (1 mentions)
Immunospot s6 ultra 5, by Cellular Technology (1 mentions)
10 protocols using peptivator cmv pp65
1
Assessing LAG-3 blockade on CMV/Adv5 response
2
Synthetic Long Peptides for NPM-ALK-Specific T-Cell Responses
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Twenty-five NPM-ALK-derived synthetic long peptides were tested for their capacity to stimulate and detect NPM-ALK-specific T-cell responses. To enable efficient cross-presentation, the selected length of the long peptides was 30 amino acids (aa) with an overlap of 16 aa to include most of the possible CD4+ and CD8+ T-cell epitopes. These peptides were custom synthesized by JPT (JPT Peptide Technologies, Berlin, Germany). The purity of the peptides was above 80%, and their composition was confirmed by mass spectrometry analysis. The lyophilized peptides were dissolved in 50 µl DMSO per peptide. After solubilization, a 5 mM stock solution was prepared using sterile deionized water. The final DMSO concentration did not exceed >1% when dissolved in culture media. Overlapping long peptides used in this study were pooled as described in supplementary data (Supplementary Table 4). The final concentration of NPM-ALK peptide-pools was 1 µM for each peptide.
PepTivator® CMV-pp65 (from Miltenyi Biotec, Bergisch Gladbach, Germany) consists of 15mer peptides with 11 aa overlap, covering the complete sequence of phosphoprotein 65 (pp65) of human CMV. It was used as experimental control to detect pp65-specific T-cells in all patients and healthy donors. CMV (pp65) PepTivator® was used at a final concentration of 0.6 nM of each peptide.
PepTivator® CMV-pp65 (from Miltenyi Biotec, Bergisch Gladbach, Germany) consists of 15mer peptides with 11 aa overlap, covering the complete sequence of phosphoprotein 65 (pp65) of human CMV. It was used as experimental control to detect pp65-specific T-cells in all patients and healthy donors. CMV (pp65) PepTivator® was used at a final concentration of 0.6 nM of each peptide.
3
Cytokine Profiling of Antigen-Specific T-Cell Activation
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PBMCs from healthy donors (0.2×106 cells/well in round-bottom 96-well plate, at 1×106/mL in complete RPMI+10% fetal calf serum) incubated with a pool of peptides covering the sequence of cytomegalovirus (CMV) pp65 in triplicate (PepTivator CMV pp65 form Miltenyi Biotec), with or without efti and/or an antagonist anti-PD-1 antibody (clone EH12.1, BD Biosciences) as indicated in figure 2 legend in complete RPMI. After 2 days, cells and/or supernatant were collected and cytokines/chemokines were assessed by Cytometric Bead Array flex set (BD Biosciences) to measure concentrations of cytokines in the supernatant. In other series of experiments, PBMCs were stimulated as above and cells were collected to be stained with a mix of the following conjugated antibodies to determine expression of activation markers. Anti-CD14-APC (clone MφP9), Anti-CD4-PerCPCy5.5 (clone L200), anti-CD8-PE (clone RPA-T8), anti-CD25-PECy7 (clone M-251), anti-CD69-APCCy7 (clone FN50) were purchased from BD Biosciences. Antibody staining was performed at 4°C during 30 min in phosphate buffered saline (PBS), bovine serum albumine (BSA) 0.5%, azide 0.1%. Data were collected on a FACS Canto A and CD14−CD8+ and CD14−CD4+ gated populations were analyzed on DIVA software (BD Biosciences). Comparisons of combined cell treatment to treatment with single agents were analyzed by Student’s t-test.
4
IFN-γ ELISpot Assay for CMV-Specific T Cells
5
Expansion of CMV-specific CTLs from PBMCs
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PBMCs were obtained from blood donors at the Etablissement Français du Sang (EFS) with informed consent (Blood products transfer agreement relating to biomedical research protocol 97/5-B—DAF 03/4868). CMV+ donor PBMCs were resuspended in the serum-free culture medium TexMacs (Miltenyi-Biotech) at 107 cells/mL and at a density of 5×106 cells/cm2. Then, 20 µL PepTivator-CMVpp65 (Miltenyi-Biotech; 130-093-435), 30 nmol of each peptide, and approximately 140 peptides were added per mL of cell suspension. The final concentration of PepTivator-CMVpp65 was 0.6 nmol (approximately 1 µg of each peptide/mL). With these stimulation conditions, PBMCs were incubated for 4 hr and then transferred to culture flasks at 106 cells/mL in RPMI medium with 8% human serum (EFS Pays de la Loire, France) and 50 IU IL-2/mL. CTL was maintained in culture for 25 days.
6
CMV pp65-Mediated NK Cell Degranulation
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The final product was incubated for 4 h at 37°C with 10% CO2 in the presence of monensin (5 ng/mL, Sigma-Aldrich, Saint Louis, Missouri, USA) and anti-CD107a FITC (BD Biosciences PHArmingen) with PepTivator® CMV pp65 (Miltenyi). Stimulation with PHA (Sigma-Aldrich) was used as a positive control for degranulation. Unstimulated cells were used as a negative control for degranulation. Data acquisition was performed using a MACSQuant instrument (Miltenyi Biotech). Data analysis was performed using FlowJo software v10.
7
SARS-CoV-2 and HCMV Peptide Stimulation Assay
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Peptide pools of overlapping 15-mer sequences covering the complete SARS-CoV-2 nucleocapsid (PepTivator® SARS-CoV-2 Prot_N, Miltenyi Biotec), immunodominant sequence domains of SARS-CoV-2 spike (PepTivator® SARS-CoV-2 Prot_S, Miltenyi Biotec) protein, HCMV IE1 (PepTivator® CMV IE-1, Miltenyi Biotec) and pp65 (PepTivator® CMV pp65, Miltenyi Biotec) proteins were used for stimulation of peripheral blood T cells. Within the first 24h after blood collection, 800µL heparinized whole blood was mixed with either phosphate buffered saline (negative control), or 50 ng/mL phorbol-12-myristate-13-acetate (PMA) together with 500 ng/mL ionomycin (positive control), or 1.25 µg/mL SARS-CoV-2 peptides, or 0.625 µg/mL HCMV IE1 together with 0.625 µg/mL HCMV pp65 peptides for 24h at 37°C. IFN-γ concentration was measured in supernatants with a chemiluminescence immunoassay (QuantiFERON®) on a LIAISON® XL analyzer (DiaSorin), as recommended by the manufacturer.
8
PBMC ELISPOT for IFN-γ and IL-10 Response
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2 × 105 PBMC were plated onto ELISPOT plates (Millipore, Burlington, MA) pre-coated with anti-IFN-γ (10 μg/ml, Mabtech, clone 1-D1K) or anti-IL-10 (10 μg/ml, Mabtech clone 9D7) capture Abs in X-VIVO15 medium (Lonza, Verviers, Belgium) supplemented with 5% human serum (Lonza) and penicillin/streptomycin (Euroclone) alone or with the gliadin peptide (DQ2.5-glia-α1,2 QLQPFPQPELPYPQPQP, hereafter glia-peptide). After 42–48 h, IFN-γ- or IL-10-producing cells were detected by biotin-conjugated anti-IFN-γ (1 μg/ml, Mabtech clone 7-B6–1) or anti-IL-10 (1 μg/ml, Mabtech clone12G8) Abs, followed by streptavidin-peroxidase (Roche) and reaction with 3-Amino-9-ethylcarbazole substrate (BD Biosciences). Spots were counted by ImmunoSpot-S6-ultraV (Cellular Technology Limited, Shaker Heights, OH, USA). A peptide pool (PepTivator® CMV pp65, Miltenyi Biotec) and phytohemagglutinin (PHA, Sigma-Aldrich) were used as controls. Results were normalized to spot forming units (SFU)/106 PBMC. Patients were considered responders (R) if the absolute SFUs in response to -glia 17mer exceeded the SFUs count of negative control peptide wells.
9
Peptide-based B Cell Activation Assay
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B cells were thawed and plated at 100,000 cells/well in complete RPMI supplemented with 10 ng/mL IL-4 in a 96-well, round-bottomed plate. Long peptides (25 mers, GenScript USA) were added at 5 μg/mL (or the indicated concentrations for titrations), and cells were incubated overnight at 37°C with 5% CO2. The following day, B cells were washed twice prior to coculturing with T cells. For initial screening, long peptides were pooled (9–11 peptides/pool). In subsequent screenings, reactive pools were deconvoluted, and individual peptides were used at 5 μg/mL. For reactivity to common viruses, the following Peptivator peptide pool reagents from Miltenyi Biotec were used: Peptivator CMV pp65, Peptivator Influenza A (H1N1) HA, Peptivator Influenza A (H1N1) NP, Peptivator EBV EBNA-1, and Peptivator EBV BZLF1. In some cases, autologous monocytes were used as APCs. Monocytes were isolated from cryopreserved, autologous PBMCs using CD14 MicroBeads (Miltenyi Biotec), and 10,000–25,000 monocytes/well were pulsed separately with HPV peptide pools for 2 to 4 hours.
10
Dendritic Cell Activation for CMV Immunity
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Freshly isolated PBMC were initially seeded for 2 h to allow the adherence of monocytes. Non-adherent PBMC were then collected and cryopreserved. Monocytes were cultured for 8 days to obtain mature dendritic cells (DC), as described elsewhere (24 (link)). Mature DC were pulsed for 1–2 h at 37°C, 5% CO2, with 10 μg/mL of PepTivator® CMV pp65 (Miltenyi Biotec, Bergisch Gladbach, Germany), referred to here as pp65. PepTivator CMV pp65 is based on peptide pools of mainly 15-mer peptides with 11-amino acid (aa) overlap, covering the complete sequence of the pp65 protein of human cytomegalovirus. Pulsed-DC were used for PBMC coculture.
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