Samples
The study protocol was approved by the Institutional Review Board of OncoTherapy Science and the written informed consent was obtained from each of peripheral blood mononuclear cells (PBMCs) donors.
PBMCs
For in vitro CTL induction, human leukocyte antigen (HLA)-A*24:02-positive PBMCs were isolated from blood of healthy volunteers by density gradient centrifugation using Ficoll-Paque PLUS (Cytiva) according to an industrial instruction manual. HLA-A*02:01- or HLA-A*02:06-positive PBMCs were purchased from Cellular Technology Limited (Cleveland, OH). For tetramer staining, HLA-A*24:02-positive PBMCs derived from COVID-19-recovered individuals with a prior positive SARS-CoV-2 PCR test were purchased from Precision For Medicine (Bethesda, MD). Healthy individuals-derived PBMCs that were collected before the SARS-CoV-2 pandemic (March 2018–May 2019) were purchased from Cellular Technology Limited.
Cell lines
TISI cells (HLA-A*24:02/-, lymphoblastoid cell) were purchased from the IHWG Cell and Gene Bank (Seattle, WA). T2 (HLA-A*02:01/-, lymphoblast), Jiyoye (HLA-A32, Burkitt’s lymphoma), and EB-3 (HLA-A3/Aw32, Burkitt’s lymphoma) cells were purchased from American Type Culture Collection (Manassas, VA). HEV0011 (HLA-A*02:06/-, B lymphocytes) cells were purchased from RIKEN Bioresource Research Center (Tsukuba, Japan). All cells were cultured in RPMI1640 media (GIBCO) supplemented with 10% fetal bovine serum (GIBCO) and 1% antibiotic solution (Wako).
Peptides
SARS-CoV-2-derived 9-mer and 10-mer peptides were synthesized by Cosmo Bio Co., Ltd. (Otaru, Japan). The purity (>90%) and the sequences of peptides were confirmed by analytical HPLC and a mass spectrometry analysis. Peptides were dissolved in dimethyl sulfoxide at 20 mg/mL and stored at −80 °C until the use for in vitro CTL induction, an IFN-γ enzyme-linked immunospot (ELISPOT) assay and an IFN-γ enzyme-linked immunosorbent assay (ELISA).
in vitro CTL induction
Monocyte-derived dendritic cells (DCs) were generated from PBMCs. Monocytes were isolated from PBMCs by adherence to a plastic tissue culture dish (Becton Dickinson) and cultured in the presence of 1000 IU/mL of granulocyte-macrophage colony-stimulating factor (R&D System) and 1000 IU/mL of interleukin (IL)−4 (R&D System) in AIM-V medium (Invitrogen) supplemented with 2% human AB serum (SIGMA) for 7 days. On day 5, OK-432 (Chugai Pharmaceutical) was added in the culture medium to induce the maturation of DCs (final concentration: 0.1 KE/mL). Autologous CD8+ T cells (3.0 × 105 cells) purified from PBMCs with CD8-Positive Isolation Kit (Dynal) were cultured with DCs (1.5 × 104 cells) with 20 μg/mL of each peptide in AIM-V medium containing 2% human AB serum, 10 ng/mL of IL-7 (R&D System), and 30 ng/mL of IL-21 (Cell Genix). After 3 days of the culture, CD8+ T cells were restimulated with DCs and 20 μg/mL of each peptide. DCs were prepared by the same procedure described above. At day 7 of the culture, CD8+ T cells were further stimulated with 48 IU/mL of IL-2 (Novartis), 5 ng/mL of IL-7 (Novoprotein), and 5 ng/mL of IL-15 (Novoprotein) in AIM-V medium supplemented with 2% human AB serum. At day 11 of the culture, CD8+ T cell responses were examined by an IFN-γ ELISPOT assay.
IFN-γ ELISPOT assay
The human IFN-γ ELISPOT kit and AEC substrate set (BD Biosciences) were used to analyze CD8+ T cell responses to SARS-CoV-2-derived peptides. The ELISPOT assay was performed according to the industrial instruction manual. Briefly, TISI, T2, or HEV0011 cells were used as stimulator cells. These cells were cultured with or without 20 μg/mL of peptide overnight at 37 °C. To analyze CD8+ T cell responses, 500 μL of supernatant was removed from each well of culture plates for in vitro CTL induction. Subsequently 100 μL of cell suspensions and stimulator cells (2 × 104 cells) with or without each peptide were co-cultured in 96-well plates coated with anti-IFN-γ antibody. After 16–18 h incubation, IFN-γ spots were detected and counted using ImmunoSpot S6 analyzer (Cellular Technology Limited).
Limiting dilution
CD8+ T cells were diluted to 0.5 cell or 10 cells per well in 96-well round-bottom plates (Corning) and cultured with feeder cells in AIM-V medium containing 5% human AB serum, 30 ng/mL of anti-CD3 monoclonal antibody (clone UCHT1; BD Biosciences), and 150 IU/mL of IL-2. Jiyoye and EB-3 cells (1 × 104 cells each) were used as feeder cells after treating with 30 μg/mL of Mitomycin C (MEDAC) at 37 °C for 30 min. After 10 days of the culture, IL-2 was added to each well (final concentration: 150 IU/mL). After additional 4 days of the culture, IFN-γ ELISPOT was performed for screening CD8+ T cell responses against each peptide.
CTL expansion culture
CTLs that showed positive reactivity in the IFN-γ ELISPOT assay after limiting dilution were expanded. They were cultured with feeder cells in AIM-V medium containing 5% human AB serum and 40 ng/mL of anti-CD3 monoclonal antibody. Jiyoye and EB-3 cells (5 × 106 cells each) were used as feeder cells after treating with 30 μg/mL of Mitomycin C at 37 °C for 30 min. The half volume of culture medium was exchanged with fresh AIM-V containing 5% human AB serum and 72 IU/mL of IL-2 every 3 or 4 days. After 14 days of the culture, IFN-γ secretion from CTLs was measured by ELISA.
IFN-γ ELISA
ELISA were performed using IFN-γ ELISA kit (BD Biosciences) according to the industrial instruction manual. Briefly, CTLs (Responders) and TISI, T2, or HEV0011 cells (Stimulators) with or without each peptide were co-cultured in 200 μL of AIM-V medium supplemented with 5% human AB serum at several ratio of Responders to Stimulators (R/S ratio). Responders at 5 × 104, 2.5 × 104, 1.25 × 104, 0.625 × 104, or 0.3125 × 104 cells/well concentration were plated in 96-well round-bottom plates together with 1 × 104 Stimulators. After 16–18 h of the incubation, supernatants were collected for measurement of IFN-γ. IFN-γ production was calculated as pg/mL using a standard curve.
TCR sequencing
Total RNAs were extracted from peptide-specific CTL clones using RNeasy mini kit (QIAGEN). cDNAs were synthesized using SMARTScribe Reverse Transcriptase (Clontech). SARS-CoV-2-derived peptides-reactive TCR sequences were identified by Sanger sequencing. TCRα cDNA was amplified by PCR using a common forward primer for adaptor (5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGTATCAACGCAGAGTGGCCAT-3′) and a reverse primer specific for the constant region (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDBDHHCAGGGTCAGGGTTCTGGATA-3′). Sanger sequencing was performed using M13 forward primer (5′-TGTAAAACGACGGCCAGTG-3′) and M13 reverse primer (5′-CAGGAAACAGCTATGACCAT-3′) after TA cloning. TCRβ cDNA was amplified using a common forward primer for adaptor and a reverse primer specific for the constant region (5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGDVHDVTCTGATGGCTCAAACACAGC-3′) [21].
in vitro stimulation
PBMCs (5 × 105 cells/mL) obtained from COVID-19-recovered individuals or healthy individuals without the history of SARS-CoV-2 infection were stimulated with 10 μg/mL of each peptide in mixture of 50% AIM-V and 50% RPMI1640 medium supplemented with 10% fetal bovine serum. After 4 days of the stimulation, PBMCs were restimulated with 10 μg/mL of each peptide. PBMCs were cultured for 12 days adding 120 IU/mL of IL-2 on days 5, 7, and 10.
Preparation of tetramers
SARS-CoV-2-derived peptides-loaded MHC class I (pMHCI) tetramers were generated by QuickSwitchTM Quant HLA-A*24:02 Tetramer Kit-PE (MBL International Corporation) according to an industrial instruction manual [22]. Briefly, 50 μL of tetramer folded with an irrelevant exchangeable peptide at 50 μg/mL was mixed with 1 μL of peptide at 1 mg/mL and 1 μL of Peptide Exchange Factor for 4 h at room temperature. Rate of peptide exchange was quantitated by flow cytometry. When irrelevant exchangeable peptides were exchanged for SARS-CoV-2-derived peptides at a rate of more than 75%, tetramers were used for staining PBMCs.
Tetramer staining
After in vitro stimulation, PBMCs were incubated with phycoerythrin (PE)-conjugated tetramer on ice for 30 min. Following wash in Dulbecco’s phosphate-buffered saline (DPBS, GIBCO) with 0.5% bovine serum albumin (BSA, Iwai Chemicals), the cells were stained with fluorescein isothiocyanate-conjugated anti-human CD8 antibody (clone RPA-T8, BD Biosciences), allophycocyanin-conjugated anti-human CD3 antibody (clone UCHT1, BD Biosciences), and phycoerythrin-Cy7 (PE-Cy7)-conjugated anti-human CD4 antibody (clone RPA-T4, BD Biosciences) on ice for 20 min. The cells were washed in DPBS with 0.5% BSA and then stained with DPBS containing 0.1 μg/mL 4′,6-diamidino-2-phenylindole (DAPI, BD Biosciences). CD8+ T cells that bound to pMHCI tetramer were identified using SH800 (Sony Biotechnology). PBMCs were treated with 450 nM dasatinib (Cayman Chemical) for inhibiting TCR downregulation from surface of T cells before tetramer staining [23].
