CD8+ T cells specific for conserved coronavirus epitopes correlate with milder disease in COVID-19 patients

Study design

The objective of this study was to measure cross-reactive CD8⁺ T cell immunity between seasonal coronaviruses that cause the common cold and SARS-CoV-2. We measured the frequency of antigen-specific T cells in unexposed pre-pandemic donors and COVID-19 patients presenting mild or severe symptoms to evaluate the contribution of pre-existing immunity to seasonal coronaviruses in disease resolution. For direct, ex vivo detection of antigen-specific T cells at single epitope resolution, we developed an improved multimeric αβ T cell staining “spheromer” reagent.

Design, expression and characterization of multimeric protein scaffolds

In order to develop an optimized self-assembling protein scaffold for the multivalent presentation of peptide-MHC (pMHC) molecules, we designed and tested several (n > 30) protein constructs. All constructs were codon-optimized for expression in mammalian cells. Gene blocks (Integrated DNA Technologies) corresponding to individual constructs were cloned into a vector with a CMV/R promoter by Gibson assembly (New England Biolabs) and sequence confirmed (Elim Biopharm).

We first evaluated the heterologous recombinant expression of self-assembling proteins with different oligomeric states (n = 12, 24 and 60). The sequences corresponding to mini-ferritin (12-mer, UniProt accession ID: P0ABT2), maxi-ferritin (24-mer, UniProt accession ID: Q8U2T8) and lumazine synthase (60-mer, UniProt accession ID: E6PLJ8) were cloned and expressed in Expi293F cells (Thermo Fisher Scientific) as per the manufacturer recommendations. Briefly, 100ml of Expi293F cells sub-cultured at a density of 3×10⁶ viable cells/ml in Expi293 expression media (Thermo Fisher Scientific) were transfected with the expression plasmids complexed with ExpiFectamine 293 transfection reagent. Next day (∼18-22h post-transfection), the cells were supplemented with a cocktail of enhancers. The cell cultures were further incubated for 4 days. Subsequently, the culture supernatants were harvested by centrifugation (2000×g, 30 min, 4°C) for protein purification. The supernatants were filtered (0.45mm PES membrane filters, Thermo Fisher Scientific) and diluted with 20 mM Tris-HCl, pH 8. The proteins were bound to a HiTrap Q FF anion exchange column (Cytiva) using an AKTA pure 25 L1 system (Cytiva). An NaCl gradient (in 20 mM Tris-HCl, pH 8) was used to elute the bound proteins. The yield and purity of the multimeric protein scaffolds was estimated using a NuPAGE Bis-Tris 4-12% gradient gel system (Thermo Fisher Scientific). The homogeneity of the purified proteins was assessed using size-exclusion columns (Superdex 200 Increase 10/300 GL, Superose 6 Increase 10/300 GL (Cytiva)) that were calibrated using a wide range of molecular weight standards (Bio-Rad).

On the basis of protein yield and homogeneity, we further optimized the maxi-ferritin scaffold for pMHC display by testing multiple linkers varying in length and rigidity. A list of all the evaluated linkers is given in Fig. S2A. Each construct was expressed in mammalian cells and purified as described above. The protein construct with linker (SG₂P)₂SG₂ (L6) was chosen for “spheromer” assembly based on yield and optimal radial projection from the scaffold. The sequence of the optimized maxi-ferritin scaffold is given in Fig. S2B. Site-directed functionalization (biotinylation) of the scaffold was performed using BirA biotin-protein ligase. The purified scaffold was incubated with components of the biotinylation reaction as per the manufacturer’s recommendation (Avidity). The functionalized scaffold was subsequently separated from free biotin using a Superdex 200 Increase 10/300 GL (Cytiva) size-exclusion column. Next, the efficiency of protein biotinylation was assessed using a streptavidin gel-shift assay. Briefly, the protein was boiled at 90°C for 7 min before incubation on ice for 10 min. Subsequently, a 2-fold molar excess of streptavidin (SAv, Agilent) was added to the protein and incubated further for an additional 10 min on ice. The shift in mobility of the scaffold resulting from SAv binding was evaluated using the NuPAGE Bis-Tris 4-12% gradient gel system (Thermo Fisher Scientific).

Spheromer assembly and characterization

The spheromer assembly is a two-step process: i) Generation of a semi-saturated SAv-pMHC₂ complex, and ii) Conjugation of SAv-pMHC₂ to the functionalized maxi-ferritin scaffold. We optimized the reaction conditions for getting the maximum yield of SAv-pMHC₂ by varying the reactant concentrations, incubation time, agitation conditions and reaction temperature. We evaluated the formation of SAv-pMHC₂ by size-exclusion chromatography (Cytiva) and NuPAGE Bis-Tris 4-12% gradient gel system (Thermo Fisher Scientific). The maximum yield of SAv-pMHC₂ was obtained by incubating 1 μM of the pMHC (monomer) with 0.45 μM of SAv at 25°C for 30 min without agitation. Subsequently, the spheromer complex was assembled by incubating SAv-pMHC₂ with the functionalized scaffold for 1h at room temperature with mild rotation. The unconjugated and fluorophore-conjugated SAv were sourced from Agilent and Invitrogen, respectively. We determined the stoichiometry of pMHC saturation on the spheromer by incubating the functionalized scaffold with increasing concentrations of SAv-pMHC₂ and analyzing the resulting product on size-exclusion columns calibrated using a broad range of molecular weight standards. The complexes were also assessed by Blue native PAGE (BN-PAGE) as per the manufacturer recommendations (Thermo Fisher Scientific). We further purified the spheromer assembly using a size-exclusion column to mitigate the confounding effects from any unreacted SAv-pMHC₂.

We also validated the conjugation of pMHC onto the functionalized scaffold by negative stain electron microscopy. 5μl of the purified samples (0.005-0.5 mg/ml) was applied on glow discharged carbon-coated grids, blotted and stained with 1% uranyl formate according to standard protocols (61). Negative stained grids were imaged on an FEI Morgagni at 100kV.

The number of pMHC molecules conjugated to the engineered maxi-ferritin scaffold was also quantified by ELISA using standard curves generated for pMHC and SAv. Briefly, test samples were coated on 96-well Nunc plates (Thermo Fisher Scientific) at 2 mg/ml in 50 μl PBS, pH 7.4 at 37°C for 1 hour. Plates were then washed with PBS containing 0.05% Tween-20 (PBST) and blocked with 3% skim milk in PBST for 1h. The plates were washed and incubated at room temperature with 50 μl of HRP-conjugated anti-streptavidin IgG (Abcam) in blocking buffer at a predetermined dilution (1:5000) for 1h for the detection of SAv. Alternatively, MHC-I and MHC-II molecules were detected using HRP-conjugated anti-human HLA-A2 antibody (LSBio) or HRP-conjugated anti-human HLA-DR antibody (LSBio). Plates were washed with PBST and developed with 75 μl/well of the substrate 3,3′,5,5′-tetramethylbenzidine (TMB) solution (MilliporeSigma). The reaction was stopped with 100 μl/well of ELISA stop solution for TMB (Thermo Fisher Scientific). The optical density at 450 nm was measured using the FlexStation 3 Multi-Mode Microplate Reader (Molecular Devices) and corrected for any non-specific background signal from ovalbumin coated wells.

Cloning, expression and purification of soluble TCRs

The soluble TCRs were expressed and purified as described previously (62). Briefly, for each TCR, the extracellular domains corresponding to the TCRα and TCRβ chains were codon-optimized for expression in insect cells and cloned independently into a baculovirus expression vector optimized for TCR expression by Gibson assembly (New England Biolabs). The sequence confirmed (Elim Biopharm) plasmids were amplified in E.coli (New England Biolabs). Each plasmid was co-transfected with BestBac Linearized Baculovirus DNA (Expression Systems) into Sf9 insect cells (Expression Systems) using Cellfectin II for the production of baculoviruses. The P1 stocks of TCRα and TCRβ baculoviruses of a given TCRαβ pair were titrated to ensure a 1:1 TCRαβ hetero-dimer formation and then co-transduced into High Five cells (Thermo Fisher Scientific). After 3 days, the supernatant was collected by centrifugation. A precipitation mix (50 mM Tris-HCl (pH 8), 1 mM NiCl₂, and 5 mM CaCl₂) was added to the supernatant while stirring for 15 min at 25°C. The precipitation was subsequently removed by centrifugation and the supernatant was incubated with buffer-equilibrated Ni-NTA beads (Qiagen) for 4h at 25°C under mild mixing conditions. Then, the Ni-NTA beads were collected and washed with 20 mM imidazole in HBS (pH 7.2). The bound protein was eluted using 200 mM imidazole in HBS (pH 7.2). The TCRαβ heterodimer was further purified by a size-exclusion column (Superdex 200 Increase 10/300 GL (Cytiva)) using an AKTA pure 25 L1 system (Cytiva) equilibrated with HBS (pH 7.2). The eluted fractions were analyzed for purity using SDS-PAGE and subsequently pooled.

MHC-I protein purification and peptide exchange

In order to generate HLA-A*02:01 (MHC-I) monomers, the corresponding α-chain and β2m protein constructs were overexpressed separately in E.coli. The protein was refolded from the inclusion bodies in the presence of a UV-cleavable peptide and biotinylated for downstream applications as described previously (63). After purification, the protein was concentrated and stored with 20% glycerol at −80°C. For each epitope specificity tested in this study, peptide exchange reactions were set up in a volume of 100 μl containing 0.2 mM peptide and 100 μg/ml HLA-A*02:01 protein in PBS (pH 7.4). The reaction mixture was exposed to 365nm UV-light irradiation for 20 min using a Stratagene UV Stratalinker 2400 in 96-well U-shaped-bottom microplates (Corning). The plate was then transferred to 4°C overnight to complete the exchange. The protein was subsequently buffer exchanged against PBS (pH 7.4) using Microcon centrifugal filters (10 kDa cut-off, MilliporeSigma) to remove the excess free peptide and subsequently spun at 13000×g for 15 min at 4°C to remove aggregates. The protein was filtered and stored at 4°C until further use.

Purification of MHC-II heterodimers and peptide exchange

The ectodomains of HLA-DRA, HLA-DRB1*04:01 and HLA-DRB1*15:01 were cloned into a CMV/R promoter-based vector by Gibson assembly (New England Biolabs). The gene constructs were codon-optimized for mammalian expression. The sequence confirmed (Elim Biopharm) plasmids were amplified in E.coli. Plasmids encoding the MHCα and MHCβ-chains of a given MHCαβ hetero-dimer were co-transfected into Expi293F cells (Thermo Fisher Scientific) following the manufacturer recommendations. The transfected cells were enhanced ∼18-20h post-transfection with the ExpiFectamine 293 transfection enhancers 1 and 2 (Thermo Fisher Scientific). The supernatant was harvested 5 days post-transfection and incubated with buffer-equilibrated Ni-NTA beads (Qiagen) for 5h at 4°C. The Ni-NTA beads were then collected, washed (20 mM imidazole in HBS (pH 7.2)) and the bound protein was eluted under gravity flow with 200 mM imidazole in HBS (pH 7.2). The protein was buffer-exchanged to remove the imidazole and biotinylated using the BirA biotin-protein ligase reaction kit (Avidity) as per the manufacturer recommendations. The MHC-II heterodimer was subsequently purified by via size-exclusion chromatography (Superdex 200 Increase 10/300 GL (Cytiva)) using an AKTA pure 25 L1 system (Cytiva) equilibrated with HBS (pH 7.2). The eluted fractions were analyzed for purity and pooled, and also assessed for biotinylation efficiency using SDS-PAGE. Thrombin (Novagen) was used to cleave the invariant CLIP peptide from the purified MHC-II molecules to enable exchange with the test peptide. After 2h incubation of MHC-II molecules with thrombin at room temperature, the reaction was stopped by the addition of a protease inhibitor cocktail (MilliporeSigma). The cleaved MHC-II protein was incubated at 30°C overnight in an aqueous solution of 1% octyl β-D-glucopyranoside, 0.1 M NaCl, 50 mM citrate (pH 5.2), 1 mM EDTA, and 0.4 mg/mL test peptide for completion of exchange. Next day, the reaction was neutralized with 1M Tris-HCl (pH 8). The excess peptide was removed during buffer exchange against PBS (pH 7.4) using Microcon centrifugal filters (10 kDa cut-off, MilliporeSigma). The protein was further spun at 13000×g for 15 min at 4°C to remove aggregates and filtered before storing at 4°C until further use.

Generation of pMHC multimer reagents

Here, we generated different multivalent formulations of a given pMHC specificity to enable comparative analysis. In order to ascribe the observed differences to the multimerization scaffold, all the multivalent pMHC formulations (tetramer, dextramer and spheromer) were made using the same stock of purified MHC molecules. The pMHC-tetramers were generated as described previously (63). Briefly, fluorophore-conjugated streptavidin (Invitrogen) was added to each pMHC monomer incrementally to achieve a 4:1 (pMHC:SAv) molar ratio. Next, streptavidin agarose was added to each tetramer for quenching any unbound, biotinylated pMHC. After filtration, biotinylated agarose beads were added to remove any unsaturated streptavidin molecules. The protein was filtered and stored at 4°C until further use. We also used a previously described protocol for generating the pMHC-dextramers (19). The biotinylated pMHC molecules were incubated with fluorophore-conjugated streptavidin (Invitrogen) at a molar ratio of ∼3.5:1 (pMHC:SAv) for 30 min at room temperature. To this mixture, biotin-dextran (MW = 70 kDa, Thermo Fisher Scientific) was added at a molar ratio of ∼30:1 (pMHC:Dextran) and incubated further for another 30 min at room temperature. The spheromer assembly has already been described above.

Binding affinity measurements using biolayer interferometry (BLI)

Binding affinity for the cognate TCR-pMHC pairs was determined by BLI using an Octet QK instrument (ForteBio). The purified, soluble TCRs were captured onto amine reactive second-generation (AR2G) biosensors using the amine reactive second-generation reagent kit. The ligand-bound biosensors were then dipped into a decreasing concentration series (50 μM followed by 2-fold dilutions) of the indicated analytes in PBST (PBS with 0.05% Tween-20) to determine the binding kinetics. A series of unliganded biosensors dipped into the analytes served as controls for referencing. In addition, signals from analyte binding to an irrelevant TCR was used for non-specific binding correction. The traces were processed using ForteBio Data Analysis Software.

Lentiviral transduction for generating T cell lines

The T cell lines were generated as described previously (34). Briefly, gene blocks (Integrated DNA Technologies) corresponding to the TCRα and TCRβ chains of a given TCRαβ pair were cloned into the EF1a-MCS-GFP-PGK-puro lentiviral vector. Each sequence confirmed (Elim Biopharm) lentiviral plasmid was separately co-transfected with the gag-pol and VSV-G envelope plasmids into Lenti-X 293T cells (Takara Bio) cultured in DMEM media (Thermo Fisher Scientific) supplemented with 10% FBS (R&D Systems) and 100U/ml of penicillin-streptomycin using FuGENE (Promega) transfection reagent. After 72h, lentiviruses for both TCRα and TCRβ constructs were harvested by collecting the culture supernatant. TCR-deficient Jurkat cells (α⁻β⁻) (ATCC) were transduced with the viral supernatant. TCR and CD3 expression was assessed by flow cytometry after staining the cells with anti-TCR α/β (PE, clone 3C10, BioLegend) and anti-CD3 (BV421, clone OKT3, BioLegend) antibodies for 30 min on ice. The cells were washed, resuspended in FACS buffer (PBS with 1% BSA and 2 mM EDTA) and acquired on a BD LSRII flow cytometer. The data was analyzed using FlowJo (v10) software. If TCR expression after lentiviral transduction was <80%, enrichment for TCR expression was performed using anti-TCR α/β (APC, clone 3C10, BioLegend) antibody in conjunction with anti-APC microbeads (Miltenyi Biotec).

Binding of T cell lines with pMHC multimers

The binding of pMHC to T cell lines was monitored by flow cytometry. pMHC multimers with Alexa 647 conjugated streptavidin (Invitrogen) were generated as described above. Binding curves (MFI) were determined using a concentration series of the pMHC multimer reagents. The cells were stained with pMHC multimers (tetramer, dextramer and spheromer) for 1h in FACS buffer. The pMHC multimer staining was done at 4°C or 25°C for MHC-I- and MHC-II-restricted T cell specificities, respectively. The cells were washed and subsequently stained with anti-CD3 (BV421, clone OKT3, BioLegend) antibody for 20 min on ice. The cells were then washed twice, resuspended in FACS buffer and acquired on a Attune NxT Flow Cytometer (Thermo Fisher Scientific). The data was analyzed using FlowJo (v10) software.

Human biological sample collection

Peripheral blood mononuclear cells (PBMCs) from healthy donors were obtained from the Stanford Blood Center according to our IRB approved protocol. All healthy donor samples used in the current study were confirmed to be HLA-A*02:01⁺ and were collected between April 2018 – Feb 2019 before the SARS-CoV-2 pandemic. The EBV and HCMV infection status for these donors was also determined by the Stanford Blood Center.

The COVID-19 patient sample collection for this study was conducted at the Stanford Occupational Health under an IRB approved protocol (Protocol Director, Nadeau). We obtained samples from all COVID-19+ adults who had a positive-test result for the SARS-CoV-2 virus from analysis of nasopharyngeal swab specimens obtained at any point from March 2020 – June 2020. Stanford Health Care clinical laboratory developed internal testing capability with a reverse-transcriptase based polymerase-chain-reaction assay. All participants consented prior to enrolling in the study. We obtained clinical data from Stanford clinical data electronic medical record system as per consented participant permission. This database contains all the clinical data available on all inpatient and outpatient visits to Stanford facilities. The data obtained included patients’ demographic details, vital signs, laboratory test results, medication administration data, historical and current medication lists, historical and current diagnoses, clinical notes, and radiological results. Participants were excluded if they were taking any experimental medications (i.e., those medications not approved by a regulatory agency for use in COVID-19). The severity of COVID-19 illness was defined based on the symptom score described by Chen et. al. (64).

PBMC staining and flow cytometry

PBMCs were thawed in a water bath set at 37°C and the cells were immediately transferred to warm RPMI media (Thermo Fisher Scientific) supplemented with 10% FBS (R&D Systems) and 100U/ml of penicillin-streptomycin. After washing, the cells were filtered (70 μm cell strainer) and rested for 1h at 37°C. CD8⁺ T cells were enriched from PBMCs by negative selection using a FITC-conjugated antibody cocktail against non-CD8⁺ T cells (anti-CD14 (Clone HCD14, BioLegend), anti-CD19 (Clone HIB19, BioLegend), anti-CD33 (Clone HIM3-4, BioLegend) and anti-γδ TCR (Clone 5A6.E9, ThermoFisher Scientific)) followed by magnetic bead depletion using anti-FITC microbeads (Miltenyi Biotec). The enriched CD8⁺ T cells were washed and resuspended in FACS buffer for staining. All pMHC-multimer staining was done for 1h at 4°C after incubating the cells with Human TruStain FcX (BioLegend) for 15 min. In order to compare the frequency of viral (influenza and HCMV) antigen-specific T cells detected using tetramer or spheromer, each sample was divided equally after CD8⁺ T cell enrichment and stained with M1-A*02:01 (Alexa 647) and pp65-A*02:01 (PE) formulated as tetramer or spheromer. The pMHC-multimer formulations were used at a monomeric concentration of 100nM. The gag-A*02:01 (Alexa 488) pMHC-multimer (200nM) was used as an irrelevant specificity control. The cells were subsequently stained with anti-CD19 (BV510, clone HIB19), anti-γδ TCR (BV510, clone B1), anti-CD33 (BV510, clone HIM3-4), anti-CD3 (PE/Cyanine7, clone OKT3), anti-CD8 (BUV396, clone RPA-T8, BD Biosciences), anti-CD4 (BV785, clone RPA-T4), anti-CCR7 (PE/Dazzle 594, clone G043H7), anti-CD45RA (BV711, clone HI100) and an amine-reactive viability stain (Live/dead fixable aqua dead cell stain kit; Invitrogen) for 30 min on ice, washed, resuspended in FACS buffer and acquired on a BD LSRII flow cytometer. All the antibodies for flow cytometry were purchased from BioLegend unless mentioned otherwise. The data was analyzed using FlowJo (v10) software.

For the simultaneous detection of multiple SARS-CoV-2 epitopes (described below) using the spheromer technology, we adapted a combinatorial staining approach developed previously (49). Briefly, each peptide was assigned a unique fluorophore-barcode that allows the simultaneous detection of 2ⁿ-1 specificities in a sample, where n is the number of distinct fluorophore labels. The relative concentrations for pMHC monomers associated with each fluorophore label (Alexa 647, eFluor 450, PE and PE/Cyanine7) was experimentally determined. Four T cell lines with distinct antigen specificities (M1-A*02:01, pp65-A*02:01, BMLF1-A*02:01 and BHW58-A*02:01) were mixed at a pre-determined ratio with TCR-deficient Jurkat cells (α⁻β⁻) and stained with a pool of spheromers, wherein each cognate pMHC was associated with a unique fluorescent tag. The cells were further labeled with anti-CD3 (FITC, clone OKT3, BioLegend) for 30 min, washed, resuspended in flow cytometry buffer and acquired on a BD LSRII flow cytometer. The data was analyzed to determine the optimal concentration for pMHC monomers associated with each fluorophore label (Alexa 647; 100 nM, eFluor 450; 125 nM, PE; 75 nM and PE/Cyanine7; 50 nM) that provided the maximum separation between the distinct T cell lines. The gag-A*02:01 pMHC-spheromer defined by the fluorophore-barcode (Alexa 647 + eFluor 450 + PE + PE/Cyanine7) was used as irrelevant specificity control. After staining the PBMC samples with spheromer pools displaying SARS-CoV-2 epitopes, magnetic enrichment of spheromer-positive population was performed using super-paramagnetic beads conjugated to an anti-c-myc monoclonal antibody (Miltenyi Biotec). The α-chain of HLA-A*02:01 is engineered to contain an exposed, C-terminal c-myc tag. The cells were subsequently stained with anti-CD19 (BV510, clone HIB19), anti-γδ TCR (BV510, clone B1), anti-CD33 (BV510, clone HIM3-4), anti-CD3 (FITC, clone OKT3), anti-CD8 (BUV396, clone RPA-T8, BD Biosciences), anti-CD4 (BV785, clone RPA-T4), anti-CCR7 (PE/Dazzle 594, clone G043H7), anti-CD45RA (BV711, clone HI100) and an amine-reactive viability stain (Live/dead fixable aqua dead cell stain kit; Invitrogen) for 30 min. The antigen-specific T cell enumerated as described previously (29, 39). Briefly, the frequency was calculated based on the total number of pMHC multimer⁺ cells divided by the total CD8⁺ T cells. The absolute counts of the desired cell populations were determined using BD Trucount beads as per the manufacturer’s recommendation (BD Biosciences) by measuring the number of bead events in 1/10^th of the initial staining reaction (pre-enriched) and the eluted fraction after magnetic enrichment. The % recovery after enrichment is estimated by bead count in the eluted fraction. In experiments wherein magnetic enrichment of the pMHC multimer stained cells was not performed, the entire sample was recorded, and the total cell count of the desired populations determined using BD Trucount beads (BD Biosciences) was used for calculating the frequency of antigen-specific T cells. The sensitivity of pMHC multimer staining after magnetic enrichment was determined by comparing the expected versus the actual numbers of TCR1 cells (BHW58-A*02:01 specificity) recovered from a serial dilution of TCR1 cells into TCR-deficient Jurkat cells (α⁻β⁻). The sensitivity of multimer staining was also determined independently by calculating the recovery of TCR1 cells spiked into PBMCs from a healthy HLA-A*02:01 donor. The TCR1 cells were labeled with a viability dye before spiking them into a PBMC sample. The limit of detection after magnetic enrichment was determined to be ∼2´10⁻⁷ (i.e., one antigen-specific T cell in several million total CD8⁺ T cells). ∼0.1´10⁶ cells from each COVID-19 patient sample was also separately stained (without spheromer pools) with anti-CD19 (BV510, clone HIB19), anti-γδ TCR (BV510, clone B1), anti-CD33 (BV510, clone HIM3-4), anti-CD3 (FITC, clone OKT3), anti-CD8 (BUV396, clone RPA-T8, BD Biosciences), anti-CD4 (BV785, clone RPA-T4), anti-CCR7 (PE/Dazzle 594, clone G043H7), anti-CD45RA (BV711, clone HI100), anti-HLA-A2 (Alexa 700, clone BB7.2) antibody and an amine-reactive viability stain (Live/dead fixable aqua dead cell stain kit; Invitrogen) for 30 min on ice. All the antibodies for flow cytometry were purchased from BioLegend unless mentioned otherwise. The cells were washed, resuspended in FACS buffer and processed using a BD LSRII flow cytometer. The data was analyzed using FlowJo (v10) software.

Selection of SARS-CoV-2 peptides and sequence conservation analysis

The complete genome sequence for SARS-CoV-2 isolate SARS-CoV-2/USA/WA-CDC-WA1/2020 (GenBank accession ID: MN985325) was obtained from the NCBI database. The binding of all possible 9-mers from SARS-CoV-2 ORF1ab, S, M and N proteins to HLA-A*02:01 was predicted following the immune epitope database and analysis resource (IEDB) recommendations (http://tools.iedb.org/mhci/) (44). The peptide binding predictions were cross validated using the SYFPEITHI algorithms (45). We further prioritized peptides based on the biochemical properties of amino acids at positions P2, P5 and P9 (40, 46). The binding of selected peptides to HLA-A*02:01 was further experimentally validated by an MHC stabilization assay using the transporter associated with antigen processing (TAP) deficient T2 cell line (ATCC) expressing HLA-A*02:01. Briefly, T2 cells were incubated with a concentration series of the test peptide (GenScript) in AIM V serum free media (Thermo Fisher Scientific) for 1h at 37°C. The cells were then transferred to a lower temperature (26°C) for another 14h, before returning them to 37°C for 3h prior to antibody staining. The cells were washed free of any unbound peptide and incubated with anti-HLA-A2 (PE, clone BB7.2) antibody and an amine-reactive viability stain (Live/dead fixable aqua dead cell stain kit; Invitrogen) for 30 min on ice. Subsequently, cells were washed, resuspended in FACS buffer and acquired on a BD LSRII flow cytometer. T2 cells incubated in AIM V serum free media alone (no peptide) served as a negative control. The list of SARS-CoV-2 peptides evaluated using the spheromer technology in this study are listed in Table S1.

To perform a sequence conservation analysis of the peptides selected from SARS-CoV-2 across other seasonal hCoVs, we obtained representative whole genome sequences for 229E (HCoV_229E/Seattle/USA/SC0865/2019, GenBank accession ID: MN306046) , HKU1 (HCoV_HKU1/SC2628/2017, GenBank accession ID: KY983584) , NL63 (HCoV_NL63/UF-2/2015, GenBank accession ID: KX179500) and OC43 (HCoV_OC43/Seattle/USA/SC9430/2018, GenBank accession ID: MN306053) from the NCBI database. The binding of all possible 9-mers from ORF1ab, S, M and N proteins to HLA-A*02:01 for each of the seasonal hCoV reference strains listed above was predicted following the immune epitope database and analysis resource (IEDB) recommendations (http://tools.iedb.org/mhci/). We then filtered the peptides based on percentile rank (<5.0). A lower percentile rank indicates higher affinity. This was done to restrict the search for cross-reactive peptides in hCoVs that are potentially functional owing to their ability to bind HLA-A*02:01, a pre-requisite to activate T cells. We then calculated the pairwise sequence similarity score for each of the selected SARS-CoV-2 peptides against all filtered seasonal hCoV peptides using the sequence manipulation suite (65). The sequence similarity score was calculated allowing for amino acid substitutions (GA, VLI, FYW, ST, KR, DE and NQ) with similar biochemical properties (47, 48). The list of seasonal hCoV peptides identified based on the similarity score is given in Table S1. The sequence similarity (%) and the percentile rank are also mentioned. The sequences of the SARS-CoV-2 variants of concern for conservation analysis were obtained from the GISAID database.

Single-cell paired αβ-TCR sequencing

Multiplexed αβ-TCR sequencing was done following previously established protocols (33). In brief, single spheromer⁺ CD8⁺ T cells (for influenza-M1, HCMV-pp65 and SARS-CoV-2 specificities) were sorted into 96-well plates containing 12 μl OneStep RT-PCR buffer (Qiagen). Reverse transcription was done using the OneStep RT-PCR kit (Qiagen) and the resulting cDNA was used for TCRα and TCRβ amplification using multiplex primers. DNA barcodes were also incorporated within the amplified sequences before processing the samples in a single MiSeq2 ´ 300bp sequencing run. The paired sequencing reads were joined, demultiplexed, and mapped to the human TCR reference dataset available at the international ImMunoGeneTics information system (IMGT) as reported previously (33).

Identification of TCR ‘motifs’ with shared antigen specificity using GLIPH2

We benchmarked the TCR repertoire of antigen-specific (influenza-M1 and HCMV-pp65) CD8⁺ T cells detected using the spheromer by comparing them to tetramer or dextramer derived sequences retrieved from the VDJdb database (28). For each antigen specificity, we implemented the GLIPH2 algorithm to quantify the number of clusters (characterized by a distinct TCR CDR3β motif) that were unique to the spheromer or had an overlap with TCR sequences reported using the tetramer or dextramer. Briefly, the GLIPH2 algorithm compared the antigen-specific TCRs (input dataset) against a reference dataset of 273,920 distinct TCR CDR3β sequences from 12 healthy individuals to generate clusters with unique TCR CDR3β motifs that are significantly enriched (p-value ≤ 0.05, Fisher’s exact test) in the input dataset as previously described (34).

We also analyzed the SARS-CoV-2 epitope-specific TCR sequences identified from unexposed, healthy individuals using the spheromer by implementing the GLIPH2 algorithm. The TCR sequences from COVID-19 patient samples for this analysis were obtained from a published dataset (66). The inclusion of multiple statistical measurements in the GLIPH2 output accounting for Vβ gene usage biases, CDR3β length distribution (relevant only for local motifs), cluster size, HLA allele usage, and clonal expansion facilitates the calling of high-confidence specificity groups.

In vitro stimulation of T cell lines

The stimulation assay was done as previously described (62). The assay was setup in 96-well clear round bottom microplates (Corning) with a volume of 200μl during all incubation steps. T2 cells expressing HLA-A*02:01 were plated at a density of 50,000 cells/well in IMDM media (Thermo Fisher Scientific) supplemented with 10% FBS (R&D Systems) and 100U/ml of penicillin-streptomycin and pulsed with 100 mM of the test peptide for 3h at 37°C. The cells were then washed and co-cultured with Jurkat cells expressing an exogenous TCR of interest (100,000 cells/well) in RPMI media (Thermo Fisher Scientific) supplemented with 10% FBS (R&D Systems) and 100U/ml of penicillin-streptomycin for 16h. Next day, the cells were washed with FACS buffer and stained with anti-CD3 (APC, clone OKT3) and anti-CD69 (PE, clone FN50) antibodies for 20 min at 4°C. Cells were washed, resuspended in FACS buffer and analyzed on an Attune NxT Flow Cytometer (Thermo Fisher Scientific). The data was analyzed using FlowJo (v10) software.

Statistical analysis

R statistical package was used to perform the Fisher’s exact test to compute TRBV gene enrichment across different pMHC formulations using the fisher.test function. Fisher’s exact test was also used to determine the significance levels of the distribution of GLIPH2 TCR motifs at different WHO scores identified using peptides either unique to SARS-CoV-2 or conserved across human coronaviruses. Next, we performed a meta-analysis to combine the p-values from individual hypothesis tests to assess the significance of the overall distribution. Dimensionality reduction analysis were also performed in R. UMAP to visualize multiparametric flow cytometry data was generated using the “umap” package. Additional data and statistical analyses were done in GraphPad Prism. The statistical details for each experiment are provided in the associated figure legends.