Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of male and female blood donors (Transfusion Medicine, University Medicine Greifswald, Germany) using standard Ficoll gradient centrifugation. CD4+ cells and CD8+ cells were positively selected using Dynal CD4 Positive Isolation Kit and Dynal CD8 Positive Isolation Kit (Invitrogen GmbH, Karlsruhe, Germany) according to the manufacturer’s instructions. All cells were freshly isolated directly before their use. No cryopreserved cells were used in any experiment.
Dynal cd4 positive isolation kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Dynal CD4 Positive Isolation Kit is a laboratory equipment used for the isolation and enrichment of CD4-positive cells from various biological samples. It employs magnetic beads coated with anti-CD4 antibodies to capture and isolate CD4-positive cells from the sample.
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Lab products found in correlation
Human ifn γ elispot plus kit, by Mabtech (3 mentions)
Interleukin 2 (il 2), by Novartis (3 mentions)
Ficoll paque plus gradient, by GE Healthcare (2 mentions)
Anti cd3 and anti cd28 coated beads, by Thermo Fisher Scientific (2 mentions)
X vivo 20 serum free medium, by Lonza (2 mentions)
Ficoll paque plus, by GE Healthcare (2 mentions)
Penicillin, by Merck Group (2 mentions)
Streptomycin, by Merck Group (2 mentions)
L glutamine, by Merck Group (2 mentions)
Interleukin 6 (il 6), by Roche (2 mentions)
Dynal cd8 positive isolation kit, by Thermo Fisher Scientific (1 mentions)
Profection, by Promega (1 mentions)
Golgistop protein transport inhibitor, by BD (1 mentions)
Anti rorα, by Abcam (1 mentions)
Ionomycin, by Merck Group (1 mentions)
Aec substrate set, by BD (1 mentions)
L glutamine, by Corning (1 mentions)
Penicillin streptomycin, by Corning (1 mentions)
Mem vitamins, by Corning (1 mentions)
Sodium pyruvate, by Corning (1 mentions)
12 protocols using dynal cd4 positive isolation kit
1
PBMC Isolation and CD4/CD8 Cell Separation
2
Isolation and Activation of CD4+ T Cells
3
CTL Response Measurement using IFN-γ ELISPOT
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IFN-γ enzyme-linked immunospot (ELISPOT) kit and AEC substrate set (BD Pharmingen, San Diego, CA) were used to measure CTL responses in the clinical study, as reported previously [14 (link)]. Peripheral blood mononuclear cells (PBMCs) were obtained from patients and were frozen before vaccination and at the end of each course. Frozen PBMCs were thawed and used for in vitro sensitization. Briefly, PBMCs were cultured in 1 mL of complete media (prepared with a mix of AIM-V and RPMI, 50 % of each) containing 10 % fetal bovine serum in a 48-well plate with 10 μg/mL of HIG2-9-4 peptide and 20 IU/mL of IL-2 at 37 °C with 5 % CO2, for 2 weeks. On day 7, half of the medium was removed from each well and 500 μL of fresh medium containing epitope peptide, as described above, was added to sensitize. After a 2-week incubation, CD4-positive cells were removed by using a Dynal CD4 positive isolation kit (Invitrogen, Carlsbad, CA, USA), and harvested cells were co-cultured with peptide-pulsed T2 cells (1 × 105 cells per well) at 37 °C for 20 h. HLA-A*0201-restricted HIV-derived epitope peptide (ILKEPVHGV) was used as the control peptide. ELISPOT assay was performed in triplicate. HIG2-9-4-specific CTL response was defined according to an evaluation tree algorithm [15 (link)].
4
Quantifying Antigen-Specific T-Cell Responses via ELISPOT
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An enzyme-linked immunospot (ELISPOT) assay was performed to measure the peptide specific CTL response, as described previously [23 (link),25 (link)]. For the evaluation of CTL and Flow cytometry, blood samples were obtained from the patients at the pre-vaccination period and after the 4th, 8th, 12th and 16th vaccinations. Briefly, peripheral blood mononuclear cells (PBMCs) of blood samples were cultured with respective peptide and IL-2 (Novartis, Emeryville, CA) at 37°C for two weeks. Peptide was added into the culture at day 0 and day 7. Following CD4+ cell depletion by Dynal CD4 positive isolation kit (Invitrogen, Carlsbad, CA), IFN-γ ELISPOT assay was performed using Human IFN-γ ELISPOT PLUS kit (MabTech, Nacka Strand, Sweden) according to the manufacturer’s instructions. The number of peptide specific spots was calculated by subtracting the spot number in the control well from the spot number of wells with peptide-pulsed TISI cells. The positivity of the antigen-specific T cell responses were classified into four grades (-, +, ++ and +++), depending on the peptide-specific spots at different responder/stimulator ratios. When the algorithm indicated +, ++ or +++, we judged it to be a positive case.
5
Isolation and Activation of CD4+ T Cells
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Peripheral blood and BAL fluid were processed as previously described [5 (link), 16 (link)]. CD4+ T cells were purified from fresh or cryopreserved PBMC by magnetic separation (Dynal CD4 Positive Isolation Kit; Invitrogen, Carlsbad, CA). Resting CD4+ T cells were activated by cross-linking with plate-bound anti-CD3 (OKT-3; American Type Culture Collection) and soluble anti-CD28 (1 μg/mL; BD Biosciences, San Jose, CA) as previously described [17 (link)]. CD4+ T cells were cultured and maintained in RPMI 1640 supplemented with 10% fetal bovine serum and 1.2% each of Penicillin Streptomycin and L-Glutamine, along with MEM vitamins, amino acids, and sodium pyruvate (all from Mediatech, INC, Manassas, VA). PBMC viability of >95% was confirmed by trypan blue in all analyses.
6
Purification and Th17 Polarization of CD4+ T Cells
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Human mononuclear cells were isolated from the umbilical cord blood of healthy neonates (Turku University Central Hospital, Turku, Finland) using Ficoll-Paque isolation (Ficoll-Paque PLUS; GE Healthcare). CD4+ cells were further purified (Dynal CD4 Positive Isolation Kit; Invitrogen) and after the isolation cells from several individuals were pooled. Cells were activated with plate-bound αCD3 (750 ng/24-well culture plate well; Immunotech) and soluble αCD28 (1 μg/mL; Immunotech) in a density of 0.5 × 106 cells/mL of X-vivo 20 serum-free medium (Lonza). The media was supplemented with 2 mM L-glutamine (Sigma-Aldrich), and 50 U/mL penicillin and 50 μg/mL streptomycin (Sigma-Aldrich). Cells were polarized toward Th17 direction with IL6 (20 ng/mL; Roche), IL1β (10 ng/mL) and TGFβ (10 ng/mL) in the presence of neutralizing anti-IFNγ (1 μg/mL) and anti-IL4 (1 μg/mL). Cells activated without differentiating cytokines but with only neutralizing antibodies were also cultured as controls (Th0). All cytokines and neutralizing antibodies were from R&D Systems unless otherwise stated.
7
Quantifying Antigen-Specific CTL Responses
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Specific CTL response was estimated by enzyme‐linked immunoSpot (ELISPOT) assay following in vitro sensitization.26 Frozen peripheral blood mononuclear cells (PBMCs) derived from the patient were thawed at the same time, and viability was confirmed as >90%. PBMCs (5 × 105/mL) were cultured with 10 μg/mL of the candidate peptide and 100 IU/mL of interleukin (IL)‐2 (Novartis, Emeryville, CA, USA) at 37°C for 2 weeks. Peptide was added into the culture on days 0 and 7. Following CD4+ cell depletion using a Dynal CD4‐positive isolation kit (Invitrogen, Carlsbad, CA, USA), an IFN‐γ ELISPOT assay was performed using a Human IFN‐γ ELISpot PLUS kit (MabTech, Nacka Strand, Sweden) according to instructions from the manufacturer. The number of peptide‐specific spots was calculated by subtracting the spot number in the control well from the spot number of a well with peptide‐pulsed stimulator cells. Peptide‐specific T‐cell response was classified into four grades (−, +, ++, or +++) according to the algorithm flow chart described in the previous report (Fig. S1).27 Sensitivity of this ELISPOT assay was estimated as being at an approximately average level by the ELISPOT panel of the Cancer Immunotherapy Consortium.28
8
Isolation and Activation of Immune Cells
9
Isolation of Mouse CD4+ T-cells
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Human CD4+ T-cells were isolated from mouse spleen cells by positive immunoselection with the Dynal® CD4-positive isolation kit (Invitrogen), according to the manufacturer’s protocol. In brief, mouse spleen cells were incubated with anti-CD4-coated beads for 30 min at 4 °C under gentle tilt rotation. Captured CD4+ T-cells were collected with a magnet (Dynal MPC-S) and detached from beads with DETACHaBEAD CD4/CD8® (Invitrogen). Purity was >99% CD4+ T-cells as determined by flow cytometry.
10
Quantifying Peptide-Specific CTL Responses
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Peptide-specific CTL responses were estimated by the in vitro ELISPOT assay as previously described (20 (link)). Briefly, frozen peripheral blood mononuclear cells (PBMCs) from each patient were thawed simultaneously. PBMCs (5×105/ml) were then cultured with 10 microgram/ml of each peptide and 100 IU/ml of interleukin-2 (Novartis, Emeryville, CA, USA) at 37°C for 2 weeks. Peptides were added to the cultures on day 0 and day 7. Following CD4+ cell depletion by the Dynal CD4 positive isolation kit (Invitrogen, Carlsbad, CA, USA), interferon (IFN)-γ ELISPOT assays were performed using the Human IFN-γ ELISpot PLUS kit (MabTech, Nacka Strand, Sweden), according to the manufacturer's instructions. The positivity of antigen-specific T cell response was quantitatively defined according to the evaluation tree algorithm described by Kono et al (21 (link)). Briefly, the peptide-specific T cell responses were classified into four grades (−, +, ++, and +++) depending on the peptide-specific spots at different responder/stimulator ratios. We judged to be positive case, when the algorithm indicated +, ++, or +++.
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