Blood collection and PBMC isolation
The samples from both COVID–19 patients and healthy donors were collected in accordance with the Declaration of Helsinki after approval by the institutional review boards. Each participant provided informed consent. Peripheral blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes, and isolation of peripheral blood mononuclear cells (PBMCs) was immediately performed using Ficoll-Paque density centrifugation according to standard protocol. PBMCs were suspended in fetal bovine serum (FCS) with 10% dimethyl sulfoxide (DMSO) and stored in liquid nitrogen until analysis.
HLA typing of study participants
PBMCs isolated from COVID–19 patients were thawed and washed with RPMI 1640 supplemented with 10% FCS, 1% Penicillin-Streptomycin solution and Benzonase nuclease (Merck-Millipore, 2500 U/mL), resuspended and incubated at 37C for 30 minutes. PBMCs were counted and up to 1,000,000 cells were aliquoted for subsequent DNA isolation. DNA was isolated using the DNeasy Blood & Tissue Kit (Qiagen, cat. #69506) according to manufacturer’s protocol. HLA typing was done using next-generation sequencing according to the manufacturer’s protocol (GenDx). For the healthy donor samples, DNA was isolated directly from whole blood and HLA typing was performed in the same way as for the COVID- 19 patients.
SARS-CoV–2 peptide selection and synthesis
Fifty SARS-CoV–2 peptides were selected for each of the top ten most prevalent HLA alleles in Italy. The selection was primarily based on SARS-CoV–2 epitopes that had the highest predicted binding affinity to the MHC according to NetMHCpan–4.016, as well as receiving a prediction score higher than 0.5 using NetChop–3.117. The SARS-CoV–2 proteome was obtained from UniProt (Proteome ID: UP000464024). Thirteen proteins (pp1ab (ORF1ab), protein 3a, non-structural protein 6, protein 7a, non-structural protein 7b, non-structural protein 8, protein 9b, ORF10 protein, uncharacterized protein 14, envelope small membrane protein (E), membrane protein (M), nucleoprotein (N) and spike glycoprotein (S)) were considered as sources of potential epitopes. All possible 9–11mer peptide sequences were derived from the thirteen SARS-CoV–2 proteins, and epitopes were ranked and selected as described above. In addition, SARS-CoV–2 epitopes that were predicted to be most immunogenic by the science community9–11,37 were included for analysis (Table S. 1). Selected peptides were synthesized by the Chemical Biology group, Leiden University Medical Centre.
Generation of UV-cleavable pHLA monomers
The UV-cleavable peptides were synthesized in-house as described previously (REF). Recombinant HLA-A*01:01, A*02:01, A*03:01, A*11:01, A*24:02, B*07:02, B*08:01, B*15:01, B*18:01 and B*51:01 heavy chains and human beta–2 microglobulin (B2M) were produced in Escherichia coli and isolated from resulting inclusion bodies30. MHC class I refolding reactions and puriﬁcation by gel ﬁltration HPLC were performed, and HLA-A and B heavy chains and B2M were refolded in the presence of UV-cleavable peptides (Table S. 3) following subsequent biotinylation as described previously31.
Generation of fluorescent pHLA multimers
MHC complexes were loaded with the selected SARS-CoV–2 peptides via UV-induced ligand exchange32,33. In brief, pHLA complexes with UV-sensitive peptide were subjected to 254/366 nM UV light for 1 h at 4˚C in the presence of a rescue peptide. The following amounts of 14 different fluorescent streptavidin conjugates were added to 10 μl of pHLA monomer (100 μg/ml): 1 μl of SA-BB790 (BD, custom), 1 μl of SA-BB630 (BD, custom), 1 μl of SA-APC- R700 (BD, 565144), 0.6 μl of SA-APC (Invitrogen, S868), 1 μl of SA-BV750 (BD, custom), 2 μl of SA-BV650 (BD, 563855), 2 μl of SA-BV605 (BD, 563260), 2 μl of SA-BV480 (BD,564876), 2 μl of SA-BV421 (BD, 563259), 1 μl of SA-BUV615 (BD, 613013), 1.5 μl of SA- BUV563 (BD, 565765), 2 μl of SA-BUV395 (BD, 564176), 1.25 μl of SA-BV711 (BD,563262) and 0.9 μl of SA-PE (Invitrogen, S866). For each pHLA monomer, conjugation was performed with two of these fluorochromes resulting in up to 75 dual fluorescent colour codes. Subsequently, milk (1% w/v, Sigma) was added to block and capture unspecific peptide binding residues, and fluorescently labelled pHLA multimers were incubated for 30 min on ice. Finally, D-biotin (26.3 mM, Sigma) in PBS and NaN3 (0.02% w/v) was added to block residual binding sites.
Combinatorial encoding of pHLA multimers and surface marker staining
PBMCs isolated from COVID–19 patients were thawed and washed with RPMI 1640 supplemented with 10% FCS, 1% Penicillin-Streptomycin solution and Benzonase nuclease (Merck-Millipore, 2500 U/mL), resuspended and incubated at 37C for 30 minutes. The following amounts of fluorescently labelled pHLA multimers were used to stain T cells: 1 μl of SA-BB790-pHLA, SA-BB630-pHLA, SA-APC-R700-pHLA, SA-BV750-pHLA, SA- BV650-pHLA, SA-BV605-pHLA, SA-BV480-pHLA, SA-BV421-pHLA, SA-BUV615- pHLA, SA-BUV563-pHLA, SA-BUV395-pHLA, SA-BV711-pHLA, SA-PE-pHLA and 2 μl of SA-APC-pHLA. The cells were stained in Brilliant Staining Buffer Plus (BD, 563794) according to manufacturer’s protocol. Final staining volume was 100 up to 194 μl depending on the amount of fluorescent pHLA multimers for each individual sample. Cells were incubated for 15 min at 37 °C. Subsequently cells were stained with 2 μl of anti-CD8-BUV805 (BD, 564912), 1 μl of anti-CD4-APC-H7 (BD, 641398), 1 μl of anti-CD14-APC-H7 (BD, 560180) 1 μl of CD16-APC-H7 (BD, 560195), 1 μl of anti-CD19-APC-H7 (BD, 560252), 1 ul of 2B4- FITC (BD, 550815), Tim–3-BV786 (BD, 742857), PD1-BUV737 (BD, 612791), NKG2A-PE-Cy7 (Beckman, B10246) and 0.5 μl of LIVE/DEAD Fixable IR Dead Cell Stain Kit (Invitrogen, L10119) and incubated on ice for 20 min. Samples were analysed on the BD FACSymphony A5.
Identification of antigen-specific CD8 T cell responses
Analysis of antigen-specific CD8 T cell responses was carried out without prior knowledge about clinical patient characteristics to avoid experimental bias. The following gating strategy was applied to identify CD8+ T cells: (i) selection of live (IRDye low-dim) single-cell lymphocytes [forward scatter (FSC)-W/H low, side scatter (SSC)-W/H low, FSC/SSC-A], (ii) selection of anti-CD8+ and ‘dump’ (anti-CD4, anti-CD14, anti-CD16, anti-CD19) negative cells. Antigen-specific CD8 T cell responses that were positive for two none of the other pHLA multimer channels were identified using Boolean gating. The full gating strategy used on the BD FACSymphony A5 is shown in Error! Reference source not found.. Cut-off values for the definition of positive responses were ≥ 0.005% of total CD8 T cells and ≥ 5 events. A minimum of 1,000 CD8 T cells were acquired per sample. To reduce researcher-bias caused by manual gating, only positive responses that were confirmed by three independent people were defined as real. Data was analysed using either the BD FACSDiva v.8.0.1 or the FlowJo 10.6.2 software. To monitor the reproducibility of the assay system, reference samples with up to 10 CD8 T cell responses present at varying frequencies were included in each analysis.
Flow cytometer settings
The following 21-color instrument settings were used on the BD FACSymphony A5: blue laser (488 nm at 200 mW): FITC, 530/30BP, 505LP; BB630, 600LP, 610/20BP; BB790, 750LP, 780/60BP. Red laser (637 nm at 140 mW): APC, 670/30BP, APC-R700, 690LP, 630/45BP, IRDye and APC-H7, 750LP, 780/60BP. Violet laser (405 nm at 100 mW): BV421, 420LP, 431/28BP; BV480, 455LP, 470/20BP; BV605, 565LP, 605/40BP; BV650, 635LP, 661/11BP; BV711, 711/85, 685; BV750, 735LP, 750/30BP, BV786, 780/60BP, 750LP. UV laser (355 nm at 75 mW): BUV395, 379/28BP, BUV563, 550LP, 580/20BP; BUV615, 600LP, 615/20BP; BUV737, 735/44BP, 770LP; BUV805, 770LP, 819/44BP. Yellow-green laser (561 nm at 150mW): PE, 586/15BP; PE-Cy7, 750LP, 780/60BP. Appropriate compensation controls were included in each analysis.
Differences between two or multiple patient groups were assessed using the non-parametric Mann-Whitney U-test or the ordinary one-way ANOVA test, respectively. The data cut-off for all analyses was 21 May 2020. Statistical analysis was performed using Excel 16.36 and PRISM 8 (Version 8.4.0).