The authors acknowledge the concept of the minimal information about T cell assays (MIATA) framework which was recently published [9, 10]. Detailed information is structured in the 5 modules proposed by MIATA: the sample, the assay, the data acquisition, the data analysis and the laboratory environment in which the T cell assays were performed.
Media and reagents
IMDM (Lonza, Verviers, Belgium), supplemented with 100 U/mL penicillin/100 μg/mL streptomycin (Invitrogen, Grand Island, NY, USA), 2 mM l-glutamine (Cambrex, East Rutherford, NJ, USA) and 10 % fetal calf serum (FCS; PAA Laboratories, Pasching, Austria), assigned as complete IMDM, or X-Vivo 15 medium (Lonza) were used as indicated. Retronectin used was from Takara (Otsu, Japan). The peptidic epitope of NY-ESO-1157–165 (LLMWITQV) was used in this study and was synthesized with >95 % purity , dissolved in DMSO at the concentration of 50 mg/mL, further diluted to a concentration of 1 mg/mL in phosphate-buffered saline (PBS) and stored in aliquots at −20 °C. The cytokines used in this study were GM–CSF (800 IU/mL; Immunotools, Friesoythe, Germany), IL-2 (150 IU/mL; (Aldesleukin, Novartis, Arnhem, the Netherlands)) and IL-15 (5 ng/mL Pepro tech, Rocky Hill, NJ, USA). The APC-conjugated NY-ESO-1157–165 peptide tetramer was made in the LUMC in-house facility. The antibodies used were CD45-FITC (clone 2D1), CD3-PE (clone SK-7), CD8-FITC (clone SK-1) and CD8-PerCP-Cy5.5 (clone SK1), all purchased from BD biosciences.
PBMC used in this study were isolated from HLA-A*0201 anonymous healthy blood bank donors (Sanquin, The Netherlands) after informed consent. PBMC were isolated within 24 h after blood draw by Ficoll density gradient centrifugation, resuspended in FCS, put on ice for 15 min and after dropwise addition of an equal volume of 80 % FCS and 20 % DMSO (Sigma, St. Louis, MO, USA) cryopreserved using a freezing container (Mr. Frosty, Thermo Fisher Scientific, Langenselbold, Germany). Equal aliquots of cells (107/vial) were stored in the vapor phase of the liquid nitrogen vessel until further use. In addition, PBMC from 7 stage IV melanoma patients were isolated from heparinized venous blood and stored in liquid nitrogen until use. All patients gave written informed consent. The treatment protocol was approved by the Medical Ethics Committee of the Leiden University Medical Center. All patients were HLA-A*0201 positive as determined by HLA genotyping. The expression of NY-ESO-1 on the corresponding tumor cells of the melanoma patients was determined by specific RT-PCR . The handling and storage of the blood samples were performed according to the standard operating procedure (SOP) of the Department of Clinical Oncology, Section Experimental Cancer Immunology and Therapy at the Leiden University Medical Center by well-trained personnel.
The packaging cell line FLY-RD18 was used to produce retroviruses encoding NY-ESO-1-specific TCR. Briefly, the HLA-A*0201 restricted NY-ESO-1157–165-specific TCR construct (the full-length codon-optimized TCR AV23.1 and TCR BV13.1 ) chain sequences of a dominant TCC of patient LAU 155  was used. At day 0, FLY-RD18 were seeded at the density of 1.5 × 105 cells per well in 6-well plate in 2 mL of complete IMDM. At day 1, cells were transfected with 2.5 μg retroviral vector DNA-encoding NY-ESO-1 TCR using transfection reagent Fugene6 (Roche) according to manufacturer’s protocol. After 48 h, the virus-containing supernatant was collected, centrifuged at 2,000 rpm for 8 min, aliquoted and snap-frozen. Viruses were stored at −80 °C until further use.
The transfected FLY-RD18 were trypsinized, washed with cold PBS/0.5 % bovine serum albumin (BSA) and fixed with 4 % paraformaldehyde (PFA) on ice for 4 min. Cells were intensively washed and then permeabilized with PBS containing 0.5 % BSA, saponin and 10 % FCS for 10 min on ice. Cells were then washed and stained with PE-conjugated anti-TCR Vβ13.1 (NY-ESO-1-specific TCR Vβ chain) in dark on ice for 30 min. After 30 min, cells were washed twice, fixed with 1 % PFA and acquired by FACS Calibur (BD biosciences, USA, CellQuest Pro). The analysis of the flow cytometry standard (FCS)-format data files was performed by using FlowJo (Tree Star Inc., OR, USA).
Transduction of PBMC
At d0, PBMC were thawed, counted and resuspended in X-Vivo 15 medium containing 0.5 μg/mL mitogen Phytohaemagglutinin HA-16 (PHA; Remel Europe) and IL-2 (20 U/mL) at a concentration of 1 × 106 cells/mL and cultured in 24-well plate for 36 h at 37 °C, 5 % CO2 and 92 % humidity. At day 1, a 24-well culture plate (Falcon) was coated with 0.5 mL/well of 50 μg/mL retronectin in PBS (Takara, Otsu, Japan) overnight at 4 °C. On day 2, retronectin was removed and wells were blocked for 30 min at room temperature (RT) with PBS/2 % BSA. After 30-min incubation, wells were washed with PBS and incubated with 0.5 mL supernatant containing virus particles/well at 37 °C for 2–3 h. Then, the PHA-activated PBMC were harvested and washed with PBS + 0.5 % BSA + 2 mM EDTA (EDTA buffer) and CD4+ T cells were depleted using Dynal beads according to manufacturer’s protocol (Invitrogen). The enriched CD8+ T cell fraction was counted, washed and resuspended in warm X-Vivo 15 medium at a concentration of 1 × 106 cells/mL. The enriched CD8+ T cells (0.5 × 106 cells/well) were added to the retronectin-coated plate containing virus, and the plate was centrifuged at 2,000 rpm (acceleration 3, deceleration 0) for 90 min. Cells were then cultured at 37 °C for 2 days. On day 5, transduced cells were harvested, counted and washed with EDTA buffer. Dead cells were removed by spinning cells on 4–5 mL Ficoll at 2,000 rpm for 15 min. Cells obtained from the interphase were washed twice with EDTA buffer and incubated with dynal beads to remove CD4+ T cells according to manufacturer’s protocol. After purification, the cells were washed and stained with APC-conjugated NY-ESO-1157–165 peptide tetramer at RT, 30 min in the dark, washed, then resuspended in EDTA buffer plus 20 μL anti-APC microbeads (Miltenyi Biotec, Germany) and incubated in the dark at 5–8 °C for 15 min. Subsequently, the cells were washed with EDTA buffer and resuspended in 0.5 mL cold EDTA buffer, after which the cells were purified on two successive MS column as per manufacturer’s protocol. Purified cells were stained with anti-CD8-FITC and CD3-PE antibodies to assess purity of NY-ESO-1-specific TCR-expressing CD3+CD8+ T cells, cells were analyzed using FACS Calibur (CellQuest Pro), and the FCS files were analyzed by FlowJo.
Purified cells were clonally expanded by co-culturing them with NY-ESO-1 CTL epitope–loaded GM–CSF-activated autologous plastic adherent monocytes preloaded with 5 μg/mL NY-ESO-1157–165 peptide for 3–4 days in X-Vivo 15 containing 150 U/mL IL-2 and 5 ng/mL IL-15 in flat-bottom plates. After 3–4 days, T cells were harvested, medium was refreshed, and the cell cultures were split if required and cultured in round-bottom plates until the cells started to round off; this was considered the appropriate moment to generate the standard sample by spiking desired percent of NY-ESO-1-specific TCR-expressing CD3+CD8+ T cells into autologous PBMC.
The assay, acquisition and analysis of PBMC and spiked standard samples for antigen-specific T cells
To analyze the number of cells expressing the NY-ESO157–165 peptide–specific TCR, cells were stained first with the HLA–TM (25 μL/sample; 1:100 diluted in EDTA buffer) for 30 min at RT, washed with 100 μL EDTA buffer, divided into two wells and then stained (25 μL/sample) either with antibodies (diluted in EDTA buffer) to CD3 (PE labeled; 1:10) and CD8 (PerCP-Cy5.5 labeled; 1:30) or with antibodies to CD45 (FITC labeled; 1:200) for 20 min at 4 °C. The cells were then washed twice and fixed with 1 % PFA. The stained samples (at least 25,000 cells per sample) were acquired by FACS Calibur flow cytometer (software CellQuest Pro). Analysis of the FCS files was performed using FlowJo.
In order to test the performance of our standard sample in the hands of external investigators, a proficiency panel was run by the CIP, designated as CIP_ID10_2010_MUL/E. The participating laboratories received a dry ice package containing 2 vials of TCR-transduced cells—spiked at 0.25 %—in autologous PBMC (5 × 106) and an aliquot of 5 μL APC-labeled NY-ESO157–165 peptide tetramer. The laboratories were asked to perform their analyses within 7–10 days after the package had arrived. The laboratories were instructed to first stain with the HLA/peptide tetramer (1:100) 30 min at RT, to wash the cells and then to perform CD3–CD8 or CD3–CD45 staining for 20 min at 4 °C. They were required to fix the cells directly after staining for safety reasons as part of the cells express retrovirally transduced TCR. Along with these instructions, they received the flow cytometer plots with the gating strategy as generated by the Laboratory of Clinical Oncology of the LUMC, when analyzing the standard sample freshly after spiking, as a guide for their own analysis. The laboratories were asked for their staining protocol, the antibodies used (clone, company, dilution) and the type of flow cytometer used besides their results (FACS plots).
Mean, standard deviation (SD) and coefficient of variation (CV) are shown for experiments when required. The accuracy of detection was calculated by the following formula: (the percentage of detected HLA–TM+ cells/the percentage of expected HLA–TM+ cells) × 100.
The Laboratory of the Clinical Oncology, Section Experimental Cancer Immunology and Therapy at the Leiden University Medical Center, is a research laboratory where the assays are performed according to SOPs, including the predefined criteria for positive responses, by well-trained personnel. The laboratory—as well as those participating in the small proficiency panel described here—has participated in all proficiency panels on HLA tetramer analysis of the CIMT immunoguiding program (CIP; http://cimt.eu/workgroups/cip/proficiency-panel-program/).