(F) Cytotoxicity assay of TCR 1-CD8+ T cells against HLA-A?11:01+ lung organoids endogenously expressing the N protein (left). these different types of antigen presenting cells, to elicit successful activation and effective cytotoxicity of CD8+ T cells to separate the cellular fraction and plasma. The plasma was then carefully separated from the cell pellets and stored at ?20?C. The PBMCs were isolated via density gradient centrifugation (Lymphoprep, STEMCELL Technologies, Canada). Isolated PBMC were cryopreserved and stored in liquid nitrogen until used in the JNJ-47117096 hydrochloride assays. Preparation and maturation of DCs Monocytes were enriched from the PBMCs of healthy individuals by using the EasySep CD14 positive selection kit according to the manufacturer’s instructions (STEMCELL Technologies, Canada). Monocytes were cultured in the DC medium for 6 days, which was made with RPMI-1640 medium supplemented with 10% FBS, 100 JNJ-47117096 hydrochloride IU/ml penicillin, 100?g/ml streptomycin, 2?mM l-glutamine (Gibco, USA), 800 IU/ml GM-CSF (PeproTech, USA) and 800 IU/ml IL-4 (PeproTech, USA). On day 3, fresh DC media were added to the cultures. On day 6, cells RaLP were cryopreserved and stored in liquid nitrogen until used in the assays. Lung organoid culture All lung tissues used in this study were obtained after surgical resections from volunteers, and processed for organoid culture within 4?h. Normal lung tissue organoids were established as reported.39 Briefly, a piece of lung tissue was minced, washed with 10?ml Advanced DMEM/F12 (Thermo Fisher Scientific, USA) containing 1??Glutamax (Thermo Fisher Scientific, USA), 10?mM HEPES (Thermo Fisher Scientific, USA) and antibiotics, termed as AdDF+++, and digested in 10?ml AO medium (Table S3) containing 1?g/ml collagenase (SigmaCAldrich, USA) on a shaker at 37?C for 1?h. The digested lung tissue suspension was sheared using 5?ml plastic Pasteur pipettes before straining over a 100?m filter. The retained tissue pieces were resuspended in 10?ml AdDF+++ following another shearing step. Strained suspensions were combined and centrifuged, and the pellet was resuspended in 10?ml AdDF+++ and washed once. Lung cell pellets were resuspended in 10?mg/ml cold Cultrex growth factor reduced basement membrane extract (BME), type 2 (Trevigen, USA). Drops of 40?l BME-cell suspension were added into 24-well plates and solidified at 37?C for 10C20?min. Then, JNJ-47117096 hydrochloride 400?l of AO medium was added to each well and culture in the 37?C incubator with 5% CO2. The medium was changed every 4 days and the organoids were passaged every 14C18 days. Peptide library To define the SARS-CoV-2-specific CD8+ T cell epitopes of the S and N proteins derived from SARS-CoV-2 reference sequences (GenBank: “type”:”entrez-nucleotide”,”attrs”:”text”:”MN908947″,”term_id”:”1798172431″,”term_text”:”MN908947″MN908947). The NetMHCpan 4.0 algorithm40 was used to analyze the binding of 9-mer peptides derived from the full lengths of SARS-CoV-2 S and N proteins, with HLA-A?02:01, HLA-A?24:02 or HLA-A?11:01. For each allele, binding peptides with high strength (Rank Threshold? ?0.5) were selected, and a total of 72 predicted epitopes were generated. Additional 6 SARS-CoV epitopes were selected (Table S4).41,42 Individual peptide’s HLA-A specificity was listed in Table S4. All 78 epitopes were synthesized (GenScript) and dissolved in the corresponding solvents for the subsequent experiments. Detailed information for peptide grouping was summarized in Table S5. HLA-A typing for COVID-19 convalescent patients and healthy individuals To screen the HLA-A?11:01 phenotypes of the COVID-19 convalescent patients and health individuals, we used Dynabeads? mRNA DIRECT? Purification kit (Thermo Fisher Scientific, USA) to extract RNAs from the PBMC samples. Then, reverse transcription and PCR were performed to amplify HLA-A molecules, with available PCR primer information.43 The PCR products were ligated into the T vector (TransGen Biotech, China) and then transformed into the Trans5 competent cells (TransGen Biotech, China). Single bacterial clones of each sample were selected for PCR verification and sequencing. The sequencing results were analyzed in the IMGT/HLA (https://www.ebi.ac.uk/ipd/imgt/hla/) website. The HLA-A2 and HLA-A24 typing of the COVID-19 convalescent patients and health individuals were analyzed using flow cytometry. For staining, 5??105 PBMCs were resuspended in PBS with.