Tumor samples from 136 patients with relapsed AML from the St. Jude Children's Research Hospital tissue resource core facility were obtained with written informed consent using a protocol approved by the St. Jude Children's Research Hospital institutional review board (IRB). Studies were conducted in accordance with the International Ethical Guidelines for Biomedical Research Involving Human Subjects. These relapsed AML cases were part of multiple clinical studies, and detailed information of these patients and clinical trials are provided in Supplementary Table S1. CD3+ T cells were first depleted from all AML samples by magnetic beads (EasySep Human CD3 Positive Selection Kit II, 17851, StemCell Technologies). For samples with low-tumor purity (<60%), additional enrichment was performed by flow cytometry using a combination of mouse anti-human CD45 PerCP-Cy5.5 (eBioscience, #8045–9459–120, clone:2D1, RRID:AB_1907397) and mouse anti-human CD33 APC (eBioscience, #17–0338–42, clone: WM-53, RRID:AB_10667747), along with negative selection of T cells with mouse anti-human CD3 APC-Cy7 (BD Biosciences, #557832, clone:SK7, RRID:AB_396890). Matched germline DNA was obtained from purified T cells (n = 18), skin or bone marrow fibroblasts (n = 70), or remission samples (n = 3). Twenty-eight samples were sequenced through our clinical pipeline (n = 28; refs. 23, 25 (link)).
Easysep human cd3 positive selection kit 2
Manufactured by STEMCELL
Sourced in Canada
The EasySep Human CD3 Positive Selection Kit II is a laboratory equipment product designed to isolate and enrich CD3-positive cells from human peripheral blood mononuclear cells (PBMCs) or other single-cell suspensions. The kit utilizes magnetic particles coated with antibodies targeting the CD3 surface antigen, allowing for the isolation of CD3-positive cells through a simple, column-free procedure.
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Lab products found in correlation
Easysep human t cell isolation kit, by STEMCELL (2 mentions)
Ficoll paque plus, by Cytiva (2 mentions)
Facsaria 2, by BD (2 mentions)
Human il 2, by Thermo Fisher Scientific (2 mentions)
Anti cd3 okt3, by BioLegend (1 mentions)
Anti cd28 cd28, by BioLegend (1 mentions)
Carboxyfluorescein succinimidyl ester (cfse), by Thermo Fisher Scientific (1 mentions)
Attune nxt focus cytometer, by Thermo Fisher Scientific (1 mentions)
Dasatinib, by Merck Group (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
Easysep magnet, by STEMCELL (1 mentions)
Lymphocyte separation solution, by Nacalai Tesque (1 mentions)
Cts optmizer, by Thermo Fisher Scientific (1 mentions)
Lymphosep lymphocyte separation medium, by MP Biomedicals (1 mentions)
L glutamine, by Thermo Fisher Scientific (1 mentions)
Gentra puregene blood kit, by Qiagen (1 mentions)
Easysep human cd14 positive selection kit 2, by STEMCELL (1 mentions)
Easyseptm human b cell enrichment kit, by STEMCELL (1 mentions)
Facscalibur cytometer, by BD (1 mentions)
Acd a, by BD (1 mentions)
19 protocols using easysep human cd3 positive selection kit 2
1
Relapsed AML Sample Preparation
2
AdCAR T cells for AML treatment
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Healthy donor (HD)-derived AdCAR T cells were co-cultured with irradiated (2.5 Gy) OCI-AML-3 cells in R10 (E:T = 1:4; 1 × 106 cells/ml) in the presence of 10 ng/ml αCD33-AMFab. After 3 days, half of the medium was exchanged with fresh medium containing αCD33-AMFab and irradiated target cells (E:T = 1:2). On day 7, human T cells were isolated using the EasySep Human CD3 Positive Selection Kit II (Stemcell, Vancouver, Canada) according to manufacturer’s instructions. A fraction of isolated T cells was used for functional cytotoxicity and proliferation assays and immunophenotyping [22 (link)]. The remaining T cells were re-cultured with irradiated target cells (E:T = 1:4). To implement TFIs, the cultures were split and treated with or without αCD33-AMFab for a further 7 days. A third CONT round of αCD33-AMFab stimulation was performed until day 21. Co-culture supernatants were harvested to quantify human cytokine secretion.
3
Evaluating CD8+ T-cell Proliferation in HNSCC
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CD3+ T cells were purified from healthy donor human PBMCs using the EasySep Human CD3 Positive Selection Kit II (StemCell Technologies, catalog no. 17851) according to the manufacturer's protocol. Purified CD3+ T cells were stained with 5 µmol/L CFSE (Invitrogen, catalog no. C34554) and resuspended in complete RPMI media. CD3+ T cells (1 × 105) were cocultured with varying ratios of HNSCC cells and 5 µg/mL anti-CD3 (OKT3, BioLegend) and 10 µg/mL anti-CD28 (CD28.2, BioLegend) stimulation with/without 10 µg/mL CDA in 100 µL in round-bottom 96-well plates. Following a 4-day incubation at 37°C and 5% CO2, T cells were stained with APC-conjugated CD8 (SK1, BioLegend) and CD8+ T-cell proliferation was assessed by flow cytometry using the Attune NxT Focus cytometer (Thermo Fisher Scientific) and data were analyzed with FlowJo software.
4
Isolation and Characterization of Allergen-Reactive T Cells
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CD3-positive T cells were purified using the human T cell negative selection kit (EasySep™ Human T cell Isolation Kit, Stemcell Technologies, Vancouver, BC, Canada). APCs were separated from PBMCs in the negative flow-through using the human CD3 positive selection kit (EasySep™ Human CD3 Positive Selection Kit II, Stemcell Technologies, Vancouver, BC, Canada). Allergen-reactive T cell responses were detected from purified cells in the presence or absence of APCs using AIM assay. TCR blocking assessment experiments were performed by incubating human PBMCs with the TCR blocker dasatinib (Sigma-Aldrich, St. Louis, MO, USA) for 24 h in the presence of allergen extracts or LPS (Sigma). Allergen-specific αβ or γδ T cell responses were assessed using AIM assay.
5
Engrafting AML Cells in NSG Mice
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AML samples were thawed and CD3 depleted using EasySep Human CD3 Positive Selection Kit II (STEMCELL Technologies) and EasySep Magnet (STEMCELL Technologies). Immune-deficient NOD LtSz-scidIL2Rγnull (next generation sequencing, NGS) mice were bred in a barrier facility, and all experimental protocols were approved by the Animal Research Ethics Board of McMaster University. NGS mice 6-10 weeks of age were sublethally irradiated at 315 Rads using a 137Cs γ-irradiator 24-hours prior to transplantation. 5-15 × 106 cells were intravenous (IV) injected, and BM aspirates were performed to identify human chimerism prior to harvesting. BM was harvested from legs and spines 6-12 weeks post-engraftment and cells recovered by mechanical dissociation as previously described35 (link) and analyzed by flow cytometry.
6
Isolation and Analysis of Human T Cells
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Peripheral blood mononuclear cells (PB-MNCs) and granulocytes (PB-Gs) were collected with Lymphosep Lymphocyte Separation Medium (MP Biomedicals) and Lymphocyte Separation Solution (Nacalai Tesque), respectively. CD3-positive cells were collected with the EasySep™ Human CD3 Positive Selection Kit II (STEMCELL Technologies) and expanded in complete medium supplemented with GTS™ OpTmizer™, CTS™ OpTmizer™ (Gibco™), L-glutamine (Gibco™), and human IL-2 (PeproTech). Genomic DNA was purified with the Gentra Puregene Blood Kit (QIAGEN). HUMARA assays, including fragment analysis by capillary electrophoresis using GeneMapper® software, were performed as previously described14 (link),32 (link).
7
Isolation and Purification of Immune Cell Populations
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Peripheral blood mononuclear cells (PBMCs) were separated using Ficoll. We isolated 3–6 × 107 of the PBMCs from each patient and control. We used 1 × 107 of the PBMCs for the isolation of B cells with negative selection by magnetic-activated cell sorting (MACS; B Cell Isolation Kit II human, Miltenyi Biotec; CA, USA) or EasySep (EasySepTM Human B Cell Enrichment Kit, STEMCELL Technologies Inc., Vancouver, Canada). The separated B cells (mean purity: 97%) were used for the total B cell studies, as described below. The remaining PBMCs were divided into two groups: one group (2–5 × 107 cells) was used to separate memory B cells (CD19 + CD27 +), naïve B cells (CD19 + CD27 −), pDCs (CD304 +), and mDCs (CD1c +) by BD FACSAria III, and the other (1 × 106 cells) to separate monocytes and CD3 + T cells by MACS positive selection (EasySep Human CD14 Positive Selection Kit II, STEMCELL Technologies Inc.; Vancouver, Canada.; EasySep Human CD3 Positive Selection Kit II, STEMCELL Technologies Inc.; Vancouver, Canada).
8
Purifying T cells and APCs
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CD3+ T cells were purified using the human T cell negative selection kit (EasySep Human T cell Isolation Kit, Stemcell Technologies). APCs were separated from PBMCs in the negative flow through using the human CD3 positive selection kit (EasySep Human CD3 Positive Selection Kit II, Stemcell Technologies). We conducted experiments by selecting 4 subjects whose responses were observed to be downmodulated in previous assays; their purified T cells and APCs from PBMCs before reexposure and ARE were mixed according to the original ratio, and mouse allergen-reactive T cell responses were detected using AIM assay.
9
Isolation and Characterization of Human T Cells
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Peripheral blood was anticoagulated by sodium heparin. Peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll isolation (Ficoll-Paque PLUS, Cytiva). Human T cells were enriched by the positive CD3 cell selection Kit (EasySep Human CD3 Positive Selection Kit II, STEMCELL Technology) with a purity of 90 to 95%. For flow cytometry, PBMCs were stained with monoclonal antibodies against human CD3 (OKT3) and CD25 (CD25-4E3) (eBioscience, USA) in phosphate-buffered saline with 5% fetal bovine serum and washed twice before acquisition. Forward scatter and side scatter dot plots were used to determine proportion of lymphocytes, monocytes, and granulocytes (fig. S10). Data were obtained by a FACSCalibur cytometer (Becton, Dickinson and Company) and analyzed using the CellQuest Pro software.
10
Isolation and Characterization of Immune Cells
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Peripheral blood (8.5 mL) was collected from ICH patients and healthy controls in BD Vacutainer collection tubes with acid citrate dextran (Fisher Scientific) as an anti-coagulant and shipped overnight at 4 °C as described above. Leukocytes were isolated as described in the methods section titled “Leukocyte Isolation by Filtration and Subsequent Hypotonic Lysis”. Leukocytes were incubated in CellCover for 10 min on ice and then washed with HBSS. T cells were separated from other leukocytes by magnetic selection prior to FACS using an EasySep™ Human CD3 Positive Selection Kit II (Stem Cell) according to the manufacturer’s instructions and subsequently, the CD3+ and CD3− fractions were stained individually. Briefly, cells were washed in ice-cold EasySep buffer (2% FBS, 1 mM EDTA in PBS) and subsequently stained with antibodies detailed in Additional file 1 : Table S1 on ice for 15 min. Cells were incubated on ice prior to sorting on a BD FACS Aria II for CD8+ T cells and CD14+ monocytes.
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