Journal: medRxiv

Article Title: Human SARS-CoV-2 challenge resolves local and systemic response dynamics

doi: 10.1101/2023.04.13.23288227

Figure Lengend Snippet: ( a ) Fold changes in abundance of cell states in PBMCs. Detailed annotation of interferon stimulated subsets and immunoglobulin class specific cell states are not shown for clarity. Immune cell abundances were scaled to the total amount of detected PBMCs in every sample prior to calculating the fold changes over days since inoculation compared to pre-infection (day -1) by fitting a GLMM on scaled abundances. The mean cell type proportions over all cells and samples is shown in the green heatmap right of the dotplot to aid the interpretation of changes in cell type abundances. ( b ) Dotplot as in (a), but showing nasopharyngeal immune cells. Immune cell abundances were scaled to the total amount of detected epithelial cells in every sample. ( c ) Dotplot as in (a), but showing nasopharyngeal epithelial cells. (d) UMAP of all CD8+ T cells from the Dextramer assay, with cell types predicted by Celltypist model trained on previous PBMC data. Activated T cells form a distinct cluster. (e) Cell counts for CD8+ T cell types by HLA compatibility of donor with the highest-bound Dextramer. Only Activated T cells have positive log2 fold change for HLA-matched Dextramers. (f) UMAP as in (d), colored by HLA compatibility.

Article Snippet: To help avoid aggregates, each individual dextramer reagent was first microcentrifuge at full speed for 5 mins before adding 2 μL from each dCODETM Dextramer® specificity to a low-bind nucleus free 1.5 eppendorf tube (Eppendorf, 30108051) containing 8.8 μl 100μM d-Biotin (Avidity science, BIO200) (0.2 ul d-Biotin per number of dCODETM Dextramer® specificity i.e. 44).The dCODETM Dextramer® master mix was mixed by gently pipetting before the total volume (96.8 μl) was added to the resuspended cells.

Techniques: Infection

Journal: medRxiv

Article Title: Human SARS-CoV-2 challenge resolves local and systemic response dynamics

doi: 10.1101/2023.04.13.23288227

Figure Lengend Snippet: ( a ) Gating strategy used to enrich SARS-CoV-2 antigen specific T cells via MACSQuant Tyto cell sorting. Cells were sequentially stained with a multi-allele panel of dCODE dextramer-PE complexes, with the addition of anti-human CD3-APC and CD14-FITC FACS antibodies as references to help us identify T cell specific binding. Debris and cell aggregates were gated out first using BSB-H (backscatter blue laser-height) SSC-H (side scatter-height). From the cells, DAPI+ dead cells were excluded. T cells (CD3+) and monocytes (CD14+) were then gated for (CD3+ and\or CD14+ population) and the sort gate defined from this population as all PE-dCODE Dextramer® positive cells. This lenient sorting strategy was decided upon in order to collect enough cells for 10X 5’ single cell analysis downstream and to ensure we were capturing all SARS-CoV-2 antigen specific cells. Any non-specific binding (e.g. to monocytes) and background noise could then be removed computationally. ( b ) Calculated quality control metrics for PE-dCODE Dextramer® positive sequenced cells showing gene reads per cell per sample. ( c ) Proportions of activated T cells bound to Dextramers loaded with selected SARS-CoV-2 antigens. The total amount of bound cells to each Dextramer is shown, color-coded by predicted cell state. If barcodes from several Dextramers were detected to be bound to the same cell, we only selected the Dextramer with the highest signal as bound. As a control to separate background and real binding, cells are separated based on the HLA haplotype compatibility with the tested Dextramer. Only Dextramers with at least 10 HLA matched bound cells are shown. FDR corrected p values were determined by a Fisher-exact test comparing the proportion of HLA matched activated T cells in the Dextramer bound cells to the proportion of unbound HLA matched activated T cells. N represents the number of cells in each bar. The right-most bar represents the overall distribution of cell types across all Dextramer experiments.

Article Snippet: To help avoid aggregates, each individual dextramer reagent was first microcentrifuge at full speed for 5 mins before adding 2 μL from each dCODETM Dextramer® specificity to a low-bind nucleus free 1.5 eppendorf tube (Eppendorf, 30108051) containing 8.8 μl 100μM d-Biotin (Avidity science, BIO200) (0.2 ul d-Biotin per number of dCODETM Dextramer® specificity i.e. 44).The dCODETM Dextramer® master mix was mixed by gently pipetting before the total volume (96.8 μl) was added to the resuspended cells.

Techniques: FACS, Staining, Binding Assay, Single-cell Analysis, Control