Human subjects
We included samples from two prospective observational human cohorts. Demographics and HLA-typing of all included subjects are reported in Supplemental Tables 1 and 2. In the first cohort, human subjects who received the primary two-dose BNT162b2 mRNA vaccine series were prospectively enrolled into an observational study, WU-368. The WU-368 study was approved by the Washington University in St. Louis Institutional Review Board (approval # 2020-12-081). Complete details of the study cohort have been previously published3,26,27. Informed consent was obtained from each subject. Draining dominant lateral axillary lymph nodes ipsilateral to the deltoid muscle mRNA vaccination site were localized with ultrasound and sampled at the indicated time points with multiple passes of 6 separate 25-gauge needles using real-time ultrasound guidance. Each needle was flushed with 3 mL of R10 (RPMI 1640 media containing L-glutamine supplemented with 10% FBS, 100 U/mL penicillin-streptomycin) followed by three 1-mL rinses with R10. Any contaminating RBC were lysed with ACK hypotonic lysis buffer, dLN FNA cells were washed twice with P2 (1x PBS supplemented with 2% FBS and 2 mM EDTA) and cells were then counted and cyropreserved in 90% FBS with 10% DMSO before storage in liquid nitrogen until analysis. Matched blood samples at the indicated time points were obtained into EDTA-anticoagulated tubes and prepared to PBMC using Ficoll density gradient centrifugation. Contaminating RBC were removed from PBMC via hypotonic lysis, PBMC were washed, counted and cryopreserved in 90% FBS / 10% DMSO and kept in liquid nitrogen until analysis.
Human subjects in the second study, WU-350, experienced their first exposure to SARS-CoV-2 spike via acute infection in the first wave of the COVID-19 pandemic during the spring and summer of 2020. Subjects with acute symptomatic viral respiratory illness evaluated at Barnes Jewish Hospital, Saint Louis Children’s Hospital, Christian Hospital or affiliated Barnes Jewish Hospital testing sites, all located in Saint Louis, Missouri, USA were enrolled into a prospective observational cohort study. All subjects included in the present manuscript tested positive for SARS-CoV-2 with a clinical PCR test. Full details of the cohort and inclusion criteria have been previously published41. The WU-350 study was approved by the Washington University in St. Louis Institutional Review Board (approval # 2020-03-085). Informed consent was obtained from each subject or their legally authorized representative. Blood at the indicated time-points post onset of viral respiratory illness symptoms was collected into EDTA anticoagulated tubes and prepared to PBMC using Ficoll density gradient centrifugation. Contaminating RBC were removed by hypotonic lysis, PBMC were washed, counted and cryopreserved in 90% FBS / 10% DMSO and kept in liquid nitrogen until analysis.
HLA-typing
Vaccinated individuals were HLA-typed by nanopore sequencing59. Genomic DNA was purified using the AllPrep DNA/RNA kit (Qiagen). Target HLA genes were amplified by long-range PCR (NGS LR kit, One Lambda) and sequenced following the SQK-LSK109 protocol on the R10.3 MinION flow cells (Oxford Nanopore Technologies). High-resolution HLA typing was assigned using the Athlon2 program.
For HLA-typing of infected individuals we extracted DNA from PBMCs using Zymo Quick-DNA Plus kits for use in the AllTYpe NGS 11-Loci Amplification Kit (One Lambda, Lot 014). HLA libraries were sequenced at 150x150 bp (MiSeq, Illumina), and the data were analyzed with TypeStream Visual (v3.0; One Lambda).
dLN single-cell RNA-seq library preparation and sequencing
dLN FNA samples were thawed, washed with P2 and resuspended in P2. Chromium Single Cell 5’ Gene Expression Dual Index libraries and Chromium Single Cell V(D)J Dual Index libraries (10x Genomics) were prepared according to the manufacturer’s instructions without modifications. Both gene expression and V(D)J libraries were sequenced on a Novaseq S4 (Illumina) instrument, targeting sequencing depth of 50,000 and 5,000 read pairs per cell, respectively.
T cell enrichment of PBMC populations for single-cell RNA-seq
Frozen PBMC samples were thawed, washed once with R10, and then washed with P2. PBMC were counted on a Cellometer Auto 2000 (Nexcelom) and resuspended to a final concentration of approximately 108 cells/mL in P2. Total untouched CD3+ or positively selected CD4+ T cells were enriched using either the EasySep Human T Cell Isolation Kit or the EasySep Human CD4 positive selection kit II, respectively, with the EasyEights magnet (STEMCELL Technologies) all per the manufacturer’s instructions. Following enrichment, T cell populations were washed with P2, re-counted and resuspended in PBS supplemented with 0.05% BSA. Chromium single cell 5’ v2 gene expression and Chromium single cell V(D)J libraries (10x Genomics) were prepared according to the manufacturer’s instructions without modifications. Gene expression and V(D)J libraries were sequenced on a Novaseq S4 (Illumina) instrument.
Remaining T cells were stained for flow cytometry to verify the T cell enrichment. Of the remaining cells, 106 enriched T cells were added to a round-bottom 96-well plate and washed twice in P2. A master mix was added to the cells with the following reagents for 20 minutes at 4oC: CD3 APC Fire 810 (HIT3a, Biolegend); CD4 PerCP (OKT4, Biolegend, to avoid blocking from positive selection); CD8 BV421 (RPA-T8, Biolegend); CD16 BV570 (3G8, Biolegend; CD14 APC (M5E2, Biolegend); CD19 BV750 (HIB19, Biolegend); Zombie NIR (Biolegend) diluted in Brilliant Staining buffer (50µL per test, BD Horizon) and P2. Following staining, cells were washed three times in P2 and then fixed with 1% paraformaldehyde (Electron Microscopy Sciences) for 20 minutes at 4oC. Cells were washed once in P2, then resuspended in P2, and stored at 4oC until analysis within 24-48 hours. Flow cytometry samples were run on an Aurora spectral flow cytometer using SpectroFlo v.2.2 software (Cytek). Flow cytometry data were analyzed using FlowJo v.10 (Treestar).
Single-cell RNAseq processing and analysis
Filtered outputs of 10x Cell Ranger count and V(D)J pipelines were imported into R (v4.1) using the Seurat (v4.1.0 ) R package60. Filtering was applied on a sequencing run basis to remove cells with less than 100 features, more than 2.5-fold the standard deviation of feature numbers, and greater than 15% mitochondrial gene percentage. Doublets were estimated using the scDblFinder (v1.6.0) R package61. Individual cells were annotated using ProjecTILs (v2.0.3) R package62,63 and SingleR (v1.6.1) R package64 using the DICE annotation data set65. Clonotypes were added to the integrated Seurat object using the scRepertoire (v1.7.0) R package66. T cells were isolated based on the assignment of CD4/CD8 T cell annotation from ProjecTIL and the presence of a productive clonotype. Overall T cell dimensional reduction utilized 2,000 variable genes with the TCR genes removed to prevent bias in the manifold by clonality. The harmony (v0.1.0) R package67 was used in integrating multiple sequencing runs and generate the UMAP (dimensions = 1:15, epochs = 500) and clusters (resolution = 0.8, dimensions = 1:15, algorithm = 3). T follicular UMAP embedding and clustering utilized dimensions = 1:20 and a resolution of 0.5. CD8+ T cell designations were based on the examination of the distribution of CD8 expression, and a cut-off was set for CD8A ≥ 0.4. Spike-specific cells from vaccinated and infected donor peripheral blood were integrated using the Harmony R package using the individual sequencing run as the variable and dimensions = 1:30 with calculating UMAP (dimensions = 1:25, epochs = 500) and cluster (resolution = 0.5 and algorithm = 3). Gene expression UMAP overlays utilized the Nebulosa (v1.6.0) R package68. Gene set enrichment analysis was performed using the escape (v1.4.2) R package69 with the UCell approach70 and the Hallmark, Kegg, and BioCarta gene set libraries from GSEA71. TCR rarefication and extrapolation was performed using the iNEXT (v3.0.0) R package72 using the abundance of combined TRA and TRB clonotypes by patient and tissue and default settings in terms of bootstraps, knots and Hill numbers. TCR clustering was performed using the scRepertoire package and the clusterTCR function with the normalized edit distance threshold set to 0.85.
TCR sequencing analysis and visualization
Spike-specific clonotype annotations were assigned for both TRA and TRB and derived from previously published data3,28 and the VDJdb database29. TCR sequencing motifs were created with the msa (v1.28.0) R package set to protein alignment with the ClustalW algorithm and max iteration = 30. The resulting aligned sequences were converted into seginer format and plotted with ggseqlogo (v0.1) R package. Single clonotype representation for single-cell analysis was performed similarly to the previously described CoNGA36. For a given combined TRA and TRB, a single transcriptome was selected based on the minimal Euclidean distance across all cells in the individual clonotype. Vectors for the TRA and TRB were calculated using the TCR autoencoder Trex (v0.99.7) R package translating the CDR3 amino acid sequence into a matrix based on the Kidera factors73. For the resulting RNA principal components and embedded TCR values, the first 15 dimensions were selected and rescaled using the mutual nearest neighbor approach with k=100 with the mumosa (v1.4.0) R package. The resulting values were then subjected to the phate algorithm with default settings with the PhateR (v1.0.7) R package74. Clustering was performed by generating a k-nearest neighbor igraph with the bluster (v1.6.0) R package and clusters were calculated using the Leiden algorithm from the leidenAlg (v1.0.3) R package with a resolution = 0.7 and number of iterations = 5. Putative spike-specific TCRs were derived from clusters where previously identified spike-specific TCRs were present. In addition, the putative TCRs were selected for the presence of either an alpha or beta chain that appeared in 2 or more donors and had not been previously shown to bind the spike epitope. Related putative-spike specific clones were called by identifying TRA or TRB CDR3 sequences within Levenshtein distance of two and shared V genes.
Development of Trex autoencoding models
TCR embedding utilized training variational autoencoders on TRA and TRB CDR3 amino acid sequences, taking the AF, KF, or both converted numeric matrices with 0 padding to set CDR3 length of 60. The matrices were transformed into a 1-dimensional array, and values normalized across all sequences. Values with no variation were transformed into 0s. Alternatively, a one-hot autoencoding approach was also trained by converting the amino acid sequence to a matrix based on the individual amino acid along the sequence. A stacked autoencoder approach was utilized, similar to the previously described method50 with a 128-64-30-64-128 neuron structure. The bottleneck layer consists of a 30 neuron/vector embedding. Each autoencoder model was trained using the keras (v2.4.0) R package across 288,043 unique CDR3 AA TRA and 453,111 unique CDR3 AA TRB sequences across 15 single-cell data sets and 4 curated TCR databases – McPAS-TCR75, VDJdb76, IEDB77, and PIRD78 resulting in 8 models: TRA-AF, TRA-KF, TRA-both, TRA-OHE, TRB-AF, TRB-KF, TRB-both, TRB-OHE. The models were trained using 80:20 data split and hyperparameters were selected based on minimal Kullback-Leibler divergence value with a batch size of 128, learning rate of 0.001, and optimization using root mean square propagation. The TCR models and corresponding R package to run the embeddings with a Seurat or Single-cell Experiment object is available at https://github.com/ncborcherding/Trex.
Putative spike-specific TCR transductants
Putative spike-specific TRA and TRB variable regions were combined in silico with murine constant regions (murine TRAC and murine TRBC2) modified to include additional cysteine residues in place of serine at position 57 in murine TRBC2 and threonine at position 47 in murine TRAC. Use of murine constant regions prevents pairing with endogenous human TCR following retroviral transduction of primary human T cells and the additional cysteine residues enhance alpha/beta constant region binding affinity increasing chimeric human variable/mouse constant TCR surface expression. Constructs containing the modified TRA and TRB were separated by a T2A sequence and synthesized to include a NotI and EcoRI restriction site at the 5’ and 3’ ends of the region of interest, respectively (GenScript). Synthesized constructs from GenScript were double-digested with NotI and EcoRI and cloned into the pMP71 retroviral vector37 and ligation was confirmed via sequencing of the recombinant plasmid. Recombinant pMP71 was used to transfect the 293Vec-RD114 retroviral packaging cell line (provided by BioVec Pharma) with the TransIT-LT1 (Mirus Bio) transfection reagent using the manufacturer’s protocol and recommended conditions. Transfection media was removed after 24 hours, replaced with fresh media, and retrovirus containing supernatents were harvested 24 hours later. Retroviral supernatants were stored at -80℃ until used.
Human CD4+ T cells were enriched from cryopreserved PBMC using the EasySep Human CD4 Positive Selection Kit II (STEMCELL Technologies). Isolated T cells were cultured in R10-500 (R10 supplemented with 500 U/mL recombinant human IL-2 [BioLegend]) at 37℃ with 5% CO2 and activated with the Miltenyi Biotec human T Cell Activation/Expansion kit according to the manufacturer's instructions. 2 days after activation/expansion, activated T cells were purified from dead cell debris and activation beads with a Ficoll gradient. Cells were washed in R10, resuspended at 2x106 per mL in R10-500, and plated on 24-well flat-bottom tissue culture plates.
TCR RD114 retroviral supernatants were thawed, layered on top of a 20% sucrose (w/v) gradient, and centrifuged in a microcentrifuge at 20,000 x g at 4℃ for 1 hour. The supernatant was discarded and residual volume including the retroviral pellets were incubated with ViroMag beads (OZ Biosciences) for 15 minutes at room temperature. Retrovirus/beads were then added to the activated T cells in the 24-well plate and the plate was briefly centrifuged at 1600 x g for 1 minute before being placed on a pre-warmed magnet (OZ Biosciences) and incubated at 37℃ with 5% CO2 for 15 minutes. Transduced T cells were cultured for at least 1 week prior to analysis with changes of R10-500 media, as needed.
Intracellular cytokine staining mapping of human TCR transductants
250,000 to 500,000 transduced CD4+ T cells, a portion of which were confirmed to express the recombinant chimeric TCR using a murine TCR beta chain-specific monoclonal antibody (BV510, clone H57-597, BioLegend), were co-cultured with 100,000 EBV-transformed B cells from the experimental subject who expressed the index paired putative spike-specific TCR in the presence of various mapping pools of SARS-CoV-2 spike overlapping 17-mer peptides (NR-52402, BEI Resources). Each peptide was incubated at a final concentration of 1 µg/mL. Separate unstimulated control wells with equivalent concentrations of DMSO to the final concentration of DMSO found in the peptide-stimulated condition were included. Positive control phorbol 12-myristate 13-acetate (PMA, InvivoGen) and Ionomycin (InvivoGen) were added to separate wells. Cells in all conditions were co-cultured in R10 media supplemented with co-stimulatory antibodies against CD28 and CD49d (BD Biosciences). Samples with the appropriate stimulus were incubated for 1.5 hours before the addition of Brefeldin A and monensin (both from BD Biosciences) and then incubated for an additional 12-16 hours. Surface staining was performed followed by fixation in 1% paraformaldehyde, permeabilization with washing buffer supplemented with 0.1% w/v saponin (Sigma) and intracellular staining using fluorescently labeled antibodies directed against cytokine antigens. We used the following antibodies: CD3 PE-Cy7 (clone UCHT1, BioLegend), CD4 APC-Cy7 (clone SK3, BioLegend), murine TCR beta chain BV510 (clone H57-597, BioLegend), CD69 BV711 (clone FN50, BioLegend), IFN-gamma PE (clone B27, BioLegend), TNF-alpha PerCP-Cy5.5 (clone MAb11, BioLegend) and IL-2 APC (clone 5344.111, BD Biosciences). The panel included Zombie NIR viability stain (BioLegend). All antibodies were used at pre-titrated optimal staining concentrations. In a separate experiment performed on unstimulated TCR2 transductants, we performed the surface stain portion of the panel following incubation with an S167-180 HLA-DPB1*04:01 PE-labeled tetramer reagent (Washington University in Saint Louis tetramer core facility) for 15 minutes to confirm ICS results. All samples were acquired on a Cytek Aurora spectral flow cytometer and unmixed files were analyzed using FlowJo software (version 10, BD Biosciences). Final analysis was gated on live CD4+ T cells positive for murine TCR beta chain.
Statistics
Heatmaps of gene sets were derived from the intersection of significant enrichment comparison (Bonferroni-adjusted p-value < 0.05) by ANOVA and Kruskal-Wallis H test for multiple comparisons and T-test and Wilcoxon Rank Sum test for binarized comparisons. Differential gene expression utilized MAST79 using the donor as a latent variable and a pseudocount of 0.1. Cluster proportion comparisons between antigen-specific T cells used the scProportionTest (v0.0.0.9) R package with 1,000 permutations. Code for the entire analysis is available at https:://github.com/ncborcherding/COVID_TCR.