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SARS-CoV-2 mutations in MHC-I-restricted epitopes evade CD8+ T cell responses

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Science Immunology  04 Mar 2021:
Vol. 6, Issue 57, eabg6461
DOI: 10.1126/sciimmunol.abg6461
  • Fig. 1

    Nonsynonymous mutations are detected in SARS-CoV-2 CTL epitopes. A) Allele frequency of low-frequency mutations detected in 27 CTL epitopes. Epitopes are indicated on the right. The heatmap to the left indicates change in % ranks predicted by netMHCpan 4.1 (32). Bar plots below the large heatmap indicate viral loads as Ct values. B) Allele frequency of mutations in specified epitopes. Regions present in two epitopes are depicted separately. C) Frequency of global fixed mutations in CTL epitopes. D) Venn diagram depicting overlap between global fixed mutations and low-frequency variants. E) Mutations in CTL epitopes arise late in infection. Mutation frequency over time of two patients which were longitudinally sampled. Shown are variants that lead to nonsynonymous mutations in CTL epitopes. Patient 1 was sampled multiple times on the same day for some time points. Dashed lines indicate the detection limit for calling low-frequency mutations.

  • Fig. 2

    Epitope variants lead to diminished MHC-I binding. A-E) Decreased thermostability of mutant peptide MHC-I complexes. Negative first derivative of relative fluorescence units (rfu) plotted against increasing temperatures. Curves for wild type peptides are black, mutated peptides are colored. The minimum point of the curves represents the melting temperature of peptide-MHC-I complexes. Dashed lines indicate SD. n=2-3 technical replicates. F) Tetramers featuring mutated peptides are unstable at 37°C. FACS plots showing staining of in vitro expanded PBMCs stained with tetramers containing wild type (top) or mutant (bottom) peptides incubated at 4°C (blue) or 37°C (red).

  • Fig. 3

    SARS-CoV-2 epitope mutations are associated with decreased CTL responses. A) Experimental overview. B) CTL responses against wild type epitopes. PBMCs were isolated from HLA-A*02:01 or HLA-B*40:01 positive SARS-CoV-2 patients (black, n=35, 5, 3, or 13 respectively, or pre-pandemic controls with unknown HLA status (white, n=7), expanded 10-12 days with indicated peptides, and stained with wild type tetramers. Boxes show median ± 25th and 75th percentile and whiskers indicate 10th and 90th percentile. C-E) T cells expanded with mutant peptides do not give rise to wild type peptide-specific CTLs. PBMCs were isolated as in B), stimulated with wild type or mutant peptides and stained with tetramers containing the wild type peptide. (n=27, 25, and 2 patients per epitope). F) Representative FACS plots for C-E. G-I) Impact of mutations on CTL response. PBMCs expanded with wild type or mutant peptides as indicated, were analyzed for IFN-γ-production via ICS after restimulation with wild type or mutant peptide (n=14, 8, and 4 patients per epitope). J) Representative FACS plots for G-I. K) Ex vivo IFN-γ ELISpot assays from PBMCs stimulated with the YLQ peptide or the corresponding mutant (n=7, PBMCs obtained 2.7 ± 0.8 weeks after symptom onset) or the MEV peptide (marked in gray) or corresponding mutant (n=1, PBMCs obtained 3 weeks after symptom onset). Two or three wells were evaluated per sample and peptide. Patient ID is as indicated in Table S6. L) CTL killing assay. PBMCs from 4 patients were expanded with wild type or mutant YLQ peptide, mixed with autologous EBV+ B cells that were pulsed with wild type or mutant YLQ peptide and specific killing was assessed (n=2 per patient). Error bars represent mean ± SD. Significance is indicated as *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, tested by Wilcoxon matched-pairs signed rank test (C,D,E,G,H,I,K) or 2-way ANOVA followed by Dunnett’s multiple comparison test (L).

  • Fig. 4

    Single cell transcriptomics and TCR sequencing of CD8+ T cells reveals distinct transcritptional profiles in response to mutant peptide. A) Experimental setup. PBMCs were expanded for 10-12 days in the presence of wild type or mutant YLQ peptide, sorted for YLQ tetramer-positive and tetramer-negative CD8+ cells, labeled with barcoded antibodies (TotalSeq anti-human Hashtag) and subjected to single-cell RNA sequencing (figure generated with BioRender.com). B) Percentages of YLQ tetramer-positive CD8+ T cells in response to wild type or mutant peptide expansion from the two donors analyzed. C-D) UMAP plots displaying an embedding of single-cell transcriptomes in 2-dimensional space. The cells are colored according to their clusters (C), or experimental condition (D). E) Distribution of clonotypes for both patients and the indicated conditions. The top 5 clonotypes are colored. Connecting lines show clonotypes shared between conditions. F) Top 15 TRAV and TRVB genes. G) Volcano plot displaying differentially expressed genes between wild type-positive and mutant-positive cells. P-values of 0 were capped to 10−350 (indicated by gray dotted line). H) Violin plots showing expression levels in tetramer-negative and tetramer-positive cells expanded with mutant or wild type peptide. Expression levels given as log-normalized relative read counts (RC). All plots in C-H show combined data from both patients.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/6/57/eabg6461/DC1

    Figure S1. Supplementary figures for mutation analysis

    Figure S2. Controls and additional DSF assay results

    Figure S3. Supplementary figures for PBMC analysis

    Figure S4. Supplementary figures for scRNA-seq analysis

    Figure S5. Quality control plots for scRNA-seq analysis and outlier clusters

    Table S1. Wild type epitopes investigated in this study

    Table S2. Samples with epitope mutations at allele frequency >0.02 (Excel spreadsheet)

    Table S3. Peptides used in the study and their % rank predicted by netMHCpan v4.1

    Table S4. Tm values for peptides tested in DSF assay

    Table S5. Characteristics of HLA-A*02:01/HLA-B*40:01 positive patients

    Table S6. HLA genotypes

    Table S7. Overview of ELISpot results for wild type peptides

    Table S8. DEGs from scRNA-seq analysis (Excel spreadsheet)

    Table S9. Top clonotypes from TCR sequencing (Excel spreadsheet)

    Table S10. Acknowledgments for sequences downloaded from GISAID (separate PDF file)

    Table S11. Raw data (Excel spreadsheet)

    Reproducibility checklist

  • The PDF file includes:

    • Fig. S1. Supplementary figures for mutation analysis.
    • Fig. S2. Controls and additional DSF assay results.
    • Fig. S3. Supplementary figures for PBMC analysis.
    • Fig. S4. Supplementary figures for scRNA-seq analysis.
    • Fig. S5. Quality control plots for scRNA-seq analysis and outlier clusters.
    • Table S1. Wild type epitopes investigated in this study.
    • Table S3. Peptides used in the study and their % rank predicted by netMHCpan v4.1.
    • Table S4. Tm values for peptides tested in DSF assay.
    • Table S5. Characteristics of HLA-A*02:01/HLA-B*40:01 positive patients.
    • Table S6. HLA genotypes.
    • Table S7. Overview of ELISpot results for wild type peptides.

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2. Samples with epitope mutations at allele frequency >0.02 (Excel spreadsheet).
    • Table S8. DEGs from scRNA-seq analysis (Excel spreadsheet).
    • Table S9. Top clonotypes from TCR sequencing (Excel spreadsheet).
    • Table S10. Acknowledgments for sequences downloaded from GISAID (separate PDF file).
    • Table S11. Raw data (Excel spreadsheet).
    • Reproducibility checklist.

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