Fig 2: Comparative analysis between the standard method and PeptiCHIP technology. (A) Peptide length distribution is reported for the standard method (blue bars) and the microchip (green bars) for each number of cells (left, 1 × 106 cells; right, 10 × 106 cells). (B–E) The 9mers consensus binding motifs were deconvoluted by unsupervised Gibbs clustering analysis for both the standard method and the microchip and for each number of cells. The reference motif is shown in the upper right corner. The clusters with higher KLD values were chosen (orange star), and the sequence logo is reported. (F–I) The binding affinities to HLA-A*02:01 and HLA-B*07:02 were predicted for the eluted 9mers, and the percentages of binders and not binders are depicted as a part to the whole for both the standard method and the microchip and for both numbers of cells.

Fig 3: Magnitude, dynamics, and cross-reactivity of CD8+ epitope-specific responses after diverse SARS-CoV-2 exposures.a. Antigen specificity of each T cell inferred from dextramer-barcode UMI counts. Representative distribution of the number of UMIs in cells called dextramer-positive (pink) and dextramer-negative (yellow). b. T cells within a clone have largely consistent specificity assignments, except T cells that cross-react with common cold coronavirus epitopes (B15_NQK_A/B15_NQK_Q pair). Each bar shows a fraction of cells of a given clonotype attributed to different dextramers. The 43 most abundant clones (more than 20 cells) are shown. c. The correlation between the number of UMIs for B15_NQK_Q (SARS-CoV-2) and B15_NQK_A (OC43 and HKU1) dextramers (Spearman ?=0.8, p<0.001). d. Cross-reactivity between HLA-B*15:01-NQK epitope variants confirmed in vitro. Jurkat cell line expressing aßTCR identified from scTCRseq data binds pMHC multimers loaded with both SARS-CoV-2 and CCCoV variants of the epitope. e. The magnitude of epitope-specific CD8+ T cell responses. Each point depicts an estimated frequency of epitope-specific T cells in a sample. Estimated frequency was calculated as a fraction of dextramer-specific T cells in scRNAseq results multiplied by bulk frequency of dextramer-stained CD8+ cells of all CD8+ cells measured by flow cytometry. Central line on boxplot shows the median. Epitopes from spike protein are in bold font. f. Composition of HLA-A*01-restricted T cell response in HLA-A*01 positive donors. Increasing proportion of spike-targeting T cells (pink) is observed after vaccination of infected individuals. g. Boosting of spike-specific epitope fraction after vaccination (donor R6). h. Previously infected individuals have a higher proportion of spike-specific T cells after vaccination than before vaccination (p=0.025, one-sided Wilcoxon signed-rank test). Spike T cell proportion (shown on a log10-scale) was calculated as a fraction of spike-specific T cells out of all CD8+ epitope-specific T cells of a donor in scRNAseq data. Central line on the violin shows the median.

Fig 4: Counts for mRNA expression in tumor tissue through NanoString analysis for STAT1(A), HLA-A (B), HLA-B (C), HLA-C (D), HLA-E (E), HLA-G (F), and IFN-? (G). Blue bar indicates the results of the high-STAT1 group in the Table 1. Red bar indicates the results of the low-STAT1 group in Table 1. *p < 0.05

Fig 5: Transcriptional repression of multiple class I pathway genes and NLRC5 alterations underlie the loss of HLA-I surface expression in the panel of MCC lines.(A) RNA-Seq heatmaps of class I antigen presentation gene expression. Middle heatmap: Unsupervised clustering by Euclidean distance of the MCC cell line panel, with and without IFN-? treatment. Left: Reference heatmap of MCC lines MKL-1 and WaGa. Right: Reference heatmap of epidermal keratinocytes and dermal fibroblasts. (B) Unsupervised clustering by Euclidian distance of protein expression values for class I genes, with and without IFN-? treatment. (C) scRNA-Seq data from MCC-336 (MCPyV+) and MCC-350 (MCPyV–) fresh tumor samples. Right panel: UMAP (uniform manifold approximation and projection) visualization of all cells is displayed, colored by cluster (left) and by sample (right). Left panel: Expression levels of HLA-A, -B, and -C and B2M across all clusters (clusters 0–5, MCC cells; cluster 6, immune cells). (D) log2 copy number ratios for class I genes (left) and for chromosome 16 (right), where NLRC5 is located.