The methodology for circulating immune profiling has been already extensively described in a previous publication.11 (link) Absolute cell counts of 36 immune subsets in peripheral blood were obtained using flow cytometry. Blood was collected at baseline and at day 63±3 days (first radiological evaluation) and stained with fluorescently labeled antibodies in Trucount Absolute Counting Tubes (Becton Dickinson). Samples were analyzed using a 10-color cytometer and data were analyzed with FlowJo software. Beads were gated out and peripheral blood mononuclear cells (PBMCs) were gated using side scatter (SSC) versus CD45 dot plots. Absolute cell count was calculated using the formula A=X/Y×N/V, where X=number of positive cells events, Y=number of bead events, N=number of beads in the test tube and V=test vol. Validation of flow cytometry cell counts data was obtained through correlation analysis with counts of lymphocytes, granulocytes and monocytes generated on the same samples by an automated hematology cell counter. Thirty-six distinct immune cell subsets were identified and counted using the gating strategy described in Lo Russo et al.11 (link)
Trucount absolute counting tubes
Manufactured by BD
Sourced in United States, Germany
The BD Trucount Absolute Counting Tubes are designed for use with flow cytometry to perform absolute cell counting. The tubes contain a known number of fluorescent beads that serve as an internal standard for quantifying the number of target cells in a sample.
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Lab products found in correlation
Facscanto 2, by BD (7 mentions)
Facs lysing solution, by BD (5 mentions)
7 aad, by BD (2 mentions)
Lymphoprep, by Axis-Shield (2 mentions)
Sdf 1α, by Thermo Fisher Scientific (2 mentions)
Lsrfortessa, by BD (2 mentions)
Facsdiva software, by BD (2 mentions)
Cd31 pe, by BD (1 mentions)
Cd42b fitc, by BD (1 mentions)
Cd11a apc, by BD (1 mentions)
Cd45 apc cy7, by BioLegend (1 mentions)
Rantes, by Thermo Fisher Scientific (1 mentions)
Pore polycarbonate membrane insert, by Corning (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Facsverse flow cytometer, by BD (1 mentions)
Cytofix cytoperm kit, by BD (1 mentions)
Anti ifn γ pe, by BD (1 mentions)
Anti cd19 bv510, by BD (1 mentions)
Facscanto 10c, by BD (1 mentions)
Lysing solution, by Beckman Coulter (1 mentions)
25 protocols using trucount absolute counting tubes
1
Comprehensive Immune Profiling Protocol
2
Absolute Cell Counting in Blood
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Blood was collected in EDTA-containing tubes and then transferred to Trucount Absolute Counting Tubes (Becton Dickinson). Red blood cells were lysed with BD FACS Lysing Solution (Becton Dickinson). The absolute number of cells present in a given volume was determined by using the bead-to-cell ratio.
3
Quantification of Absolute CD57+/CD3- Lymphocytes
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Absolute CD57+/CD3− lymphocytes counts were evaluated with a BD FACS Canto II™ flow cytometer on anticoagulated (K2EDTA) whole blood using a clinically validated lyse no wash protocol. Briefly, 50 µL of whole blood was added to BD Trucount™ absolute counting tubes, with 10 µL of PerCP-anti-CD45 (clone 2D1), 10 µL of PE-anti-CD3e (clone UCHT1) and 10 µL of FITC-anti-CD57 (clone HNK-1) antibody and incubated for 10 min in the dark. The samples where then and lysed with 0.5 mL of BD FACS™ lysing solution and analyzed promptly. Lymphocytes were gated based on forward and side scatter, collecting a minimum of 1000 counting beads, and the cells of interested were identified as the CD57+/CD3− population. Counting beads in the Trucount™ tubes were used to calculate the absolute number of CD57+/CD3− lymphocytes per ml of blood.
4
PBMC-Mediated Cytotoxicity Assay
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Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood of volunteer healthy donors by density gradient centrifugation using lymphoprep (Axis – Shield, cat# AXS-1114544). All donors provided written informed consent in accordance with the Declaration of Helsinki. For cytotoxicity assay, EGFR+ and PD-L1+ MDA-MB231Luc and A549 cells were co-cultured with freshly isolated PBMC at two different effector-to-target (E:T) ratios (5:1 and 10:1) in presence of atz, ctx, IgTT-1E or polyclonal control human IgG (6.67 nM). After 48 hours, cells were stained for 30 minutes at 4 ºC with V450-conjugated anti-CD45 mAb (Becton Dickinson, cat# 560367) and 7-AAD (BD Biosciences, cat# 559925) in 50 µl of PBS 2% FBS using TruCount Absolute Counting Tubes (BD Biosciences, cat# 663028). Finally, the samples were diluted by adding 450 µl of PBS before proceeding to flow cytometry analysis. Cytotoxicity was determined by recording the residual live target cells (7AAD− and CD45−).
5
Quantifying Cell-Derived Extracellular Vesicles
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The platelet-poor plasma (50 μl, reserved in the above-described procedure) was incubated with 50 μl FC-block-staining buffer (BD-564219) for 10 min at room temperature. Then, the above-mentioned mixture was incubated with conjugated monoclonal antibodies CD31-PE (BD-555446), CD42b-FITC (BD-555472), CD11a-APC (BD-550852), and CD45-APC-Cy7 (BioLegend-368516) for 20 min in the same environment. After antibodies labeling, the mixture was diluted in 200 μl phosphate-buffered saline and then added to the Trucount™ Absolute Counting Tubes (BD-340334) to determine the absolute MP number. The MPs detection was performed on a flow cytometer (FACSCanto II, BD), and protocol standardization was based on a blend of fluorescent size-calibrated beads 1 μm (Sigma-L2778). Leukocyte-derived MPs (LMPs) were defined as CD45+, CD11a+, and CD11a+/CD45+ (Anne, 2012 (link); Schwarz et al., 2018 (link)) while platelet-derived MPs (PMPs) and endothelial cell-derived MPs (EMPs) were defined as CD31+/CD42b+ and CD31+/CD42b− (Amabile et al., 2012 (link); Camaioni et al., 2013 (link)), respectively. The results were expressed as the number of MP/μl of plasma.
6
Chemokine-mediated T cell migration
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CD4 T cells were used as recipient cells one week after nucleofection and stained with CellTrace as described above. As donor cells, HeLa cells were transfected with CD32B-GFP or H2B-GFP, together with pHR-CCR5 or CXCR4-HA, with Lipofectamine 3000 (Invitrogen) following the manufacturer’s protocol. The recipient cells and donor cells were co-cultured for 24 h in a 96-well plate. After removing the membrane from the Transwell system, 500 μL of RPMI medium supplemented with 0.2% FBS and the chemokine SDF-1α (1,000 ng/mL; Peprotech) or RANTES (800 ng/mL; Peprotech) were added at the bottom of the Transwell-24 well plate with 3.0 μm pore polycarbonate membrane insert (Corning). The membrane was added into the corresponding wells, and for each condition 200 μL of medium containing 2.5 x 105 cells were transferred on top of the membrane. The 24-well plate was incubated at 37°C for 3 h. Subsequently, the membrane was removed and the total number of cells in the 500 μL medium on the bottom of the Transwell was quantified by flow cytometry. For quantification BD Trucount Absolute Counting Tubes were used.
7
Comprehensive Flow Cytometric Analysis of Immune Cell Subsets
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The absolute number of CD4+ T, CD8+ T and B cells in whole blood was measured using BD Trucount absolute counting tubes according to standard flow cytometric procedures, as described previously23 (link). Leukocytes were surface−labeled with fluorescence−conjugated antibodies after treatment with red blood cell lysis buffer to delineate the naïve (CD95dimCD28−), central memory (CD95highCD28+) and effector memory (CD95+CD28−) CD4+ and CD8+ T−cell subsets, as well as the naïve (CD27−IgD+), un−switched memory (CD27+IgD+), switched memory (CD27+IgD−) and double negative (CD27−IgD−) B−cell subsets. To evaluate the immune activation of each subset, anti−CD38, anti−HLA−DR, anti−PD−1, anti−CD80 and anti−CD86 antibodies were also added for surface staining. Surface−labeled cells were fixed and permeabilized using a Cytofix/Cytoperm kit (BD Biosciences) for Ki67 staining to detect proliferation. The acquisition of at least 1,000,000 events was performed using a FACSVerse flow cytometer (BD Biosciences), and data analysis was performed using FlowJo software (Tree Star) (Supplementary Fig. S1 ).
8
Comprehensive Characterization of Leukocyte Populations
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Quality control of LP, preDS, and DS was performed as previously described (Tischer et al., 2014b (link)). Total CD45+ leukocytes, viability, and frequencies as well as total cell numbers were determined using Trucount Absolute Counting Tubes (BD Biosciences) and the following staining reagents: anti-CD45 APC-H7 and anti-CD3 FITC mAbs and 7-AAD (all BD Biosciences). After staining, erythrocytes were lysed using Lysing Solution (Beckman Coulter) and samples were acquired on a BD FACSCanto 10c with at least 10,000–50,000 events in the Trucount beads gate. Purity, memory phenotype and cellular composition were analyzed using combinations of the following staining reagents: anti-CD45 APC-H7, anti-CD3 FITC, anti-CD4 AF700, anti-CD8 APC, anti-CD14 BV510, anti-CD19 BV510, anti-IFN-γ PE, anti-CD45RA BV605, anti-CD62L BV421, anti-CD56 AF700, anti-CD33 APC, anti-CD19 PE-Cy7 mAbs, and 7-AAD (all BD Biosciences). After staining and lysis of erythrocytes, samples were acquired on a BD FACSCanto 10c with at least 10,000–50,000 events in the CD45+ leukocyte gate.
9
Isolation and Cryopreservation of PBMCs
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Peripheral blood mononuclear cells (PBMCs) were isolated from whole blood using either standard Pancoll density gradient centrifugation (anticoagulant: EDTA, Pancoll 1.077 g/mL; PanBiotech, Aidenbach, Germany; AIGI and AICOVI) or using Vacutainer® cell processing tubes (CPT™ Cell Preparation Tubes with Sodium Heparin; BD, Heidelberg, Germany; AICOVI) according to the manufacturer’s instructions. Isolated PBMCs were resuspended in leukocyte medium (RPMI 1640; PanBiotech) and counted cytometrically using Trucount beads (Trucount Absolute Counting Tubes; BD). PBMCs were diluted to 5 × 106 cells/mL in freezing medium (RPMI medium supplemented with 40% fetal calf serum (FCS; Sigma-Aldrich, Taufkirchen, Germany) and 10% dimethyl sulfoxide (AppliChem, Darmstadt, Germany), aliquoted in cryovials and stored at -156°C for at least six days.
10
Cytotoxicity of IL-1RAP CAR T-cells against AML
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IL-1RAP CAR T-cells were cocultured with target tumor cells (AML cell lines or primary AML blasts) overnight at an effector:target (E:T) ratio of 1:5 for the detection of interferon (IFN)-γ intracellular expression. An IL-1RAP CAR T-cell proliferation assay was performed after 3 days of coculture (E:T ratio=1:3) and assessed by FCM after V450 e-Fluor staining. The cytotoxicity of IL-1RAP CAR T-cells against AML leukemic cells was assessed after incubation for 24 hours at different E:T ratios by FCM using trucount Absolute Counting tubes (BD Biosciences). Cell death was evaluated by 7-amino actinomycin D (7-AAD+) labeling. Effector cells were distinguished from target cells with a previously described V450 e-Fluor labeling method. Gating on CD3+/CD19+ cells and on IL-1RAP+ cells allowed discrimination of CAR T-cells from IL-1RAP+ tumor AML cells. In the case of allogeneic mismatch, alloreactivity was taken into account by subtracting the cytotoxicity of untransduced T-cells (C0) or MockT-cells at the respective E:T ratio. C0 and MockT-cells were used as controls.
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