Journal: Frontiers in Immunology

Article Title: Gene Expression-Based Identification of Antigen-Responsive CD8 + T Cells on a Single-Cell Level

doi: 10.3389/fimmu.2019.02568

Figure Lengend Snippet: Single-cell gene expression analysis of sorted Flu MP 58−66 -responsive CD8 + T cells. (A) Representative flow cytometry dot plots of PBMCs stained with HLA-A2 multimers loaded with Flu MP 58−66 after incubating PBMCs for 20 h in the presence of mock (IGRP 265−273 ) or cognate (Flu MP 58−66 ) peptide. Plots show 5 × 10 4 cells in the CD8 gate. (B) Schematic work-flow of the dye-based activation assay. (C) Top : representative dot plots (donor #1) of multimer and cellular dye-stained PBMCs (left). Cells in the CD8 gate are shown. CD8 cells staining positive for the multimer and cell dye were sorted (red arrow) for use in assays using the K562/A*0201 cell line or autologous PBMCs for antigen presentation. After incubation with control stimuli (peptide solvent and mock peptide) or the cognate peptide for 20 h, CD8 + T cells staining positive for the cell dye were sorted for single-cell targeted gene expression analysis. Lower left : t-SNE analysis for donors #1–3. Gene expression was analyzed in single cells following incubation with an antigen-presenting cell line (open circles) or PBMCs (filled circles) in the presence of solvent (black) or mock peptide (blue) as control stimuli or with the cognate peptide (red). Lower right : Heatmaps of the top 20 ranked differentially expressed genes in cognate peptide-stimulated cells relative to control-stimulated cells for donors #1–3. The numbers of analyzed cells are shown below the individual heatmaps. Genes marked with an asterisk encode proteins expressed on the cell surface. (D) t-SNE of scRNAseq gene expression data for Flu MP 58−66 -directed CD8 + T cells derived from donor #1 and incubated with K562/A*0201 (open) or autologous PBMCs (filled) in the presence of solvent (black) or mock peptide (blue) as control stimuli or with cognate peptide (red). (E) Heatmaps for the top 50 ranked differentially expressed genes in antigen-directed CD8 + T cells incubated with cognate peptide relative to control stimuli (purple: upregulated genes; blue: downregulated genes; CD8B and CD3E are also shown). Data are shown for donor #1 following incubation with K562/A*0201 or autologous PBMCs. Genes were combined for the ranking. The numbers of analyzed cells are shown below the heatmaps.

Article Snippet: Phycoerythrin-labeled HLA-A * 0201 multimers loaded with Flu MP 58−66 (GILGFVFTL), hCMV pp65 495−503 (NLVPMVATV), or IGRP 265−273 (VLFGLGFAI) were purchased from Immudex.

Techniques: Gene Expression, Flow Cytometry, Staining, Activation Assay, Immunopeptidomics, Incubation, Control, Solvent, Targeted Gene Expression, Derivative Assay

Journal: Frontiers in Immunology

Article Title: Gene Expression-Based Identification of Antigen-Responsive CD8 + T Cells on a Single-Cell Level

doi: 10.3389/fimmu.2019.02568

Figure Lengend Snippet: Combined gene and pathway analysis of Flu MP 58−66 - and CMVpp65 495−503 -responsive CD8 + T cells. (A) Venn diagram of overlapping genes upregulated (purple) or downregulated (blue) in Flu MP 58−66 -directed (donor #1) or CMVpp65 495−503 -directed CD8 + T cells (donors #4–6) following stimulation with the cognate peptide relative to control stimuli. The number of genes belonging to the top 100 differentially expressed genes in each individual is shown in parenthesis. Sixteen genes were shared between all individuals, as shown in the central yellow circle. (B) Top 20 KEGG pathways identified based on the differentially expressed genes in individual donors following stimulation with cognate peptide relative to control stimuli. The pathways showing significant enrichment for these genes in at least one donor were included in the analysis and were ranked based on the median pathway coverage (large blue dots) for all donors. (C) Scatter/violin plots showing normalized counts of the five genes that best separated the cognate peptide-stimulated and control-stimulated cells in antigen-directed CD8 + T cells incubated with cognate peptide (red violins) relative to solvent (black) or mock peptide (blue) in the presence of autologous PBMCs (black symbols) or K562/A*0201 (orange symbols). The y -axis represents the normalized read counts for individual Flu MP 58−66 -specific cells from donor #1 (triangles) or CMVpp65 495−503 -specific cells from donors #4 (circles), #5 (squares), and #6 (diamonds). The median values for individual donors and conditions are shown as yellow lines. Similar cell numbers were analyzed per condition for each donor, and data comprise counts for 148 cells incubated with DMSO, 152 cells incubated with mock peptide, and 143 cells stimulated with the cognate peptide.

Article Snippet: Phycoerythrin-labeled HLA-A * 0201 multimers loaded with Flu MP 58−66 (GILGFVFTL), hCMV pp65 495−503 (NLVPMVATV), or IGRP 265−273 (VLFGLGFAI) were purchased from Immudex.

Techniques: Control, Incubation, Solvent

Journal: Frontiers in Immunology

Article Title: Gene Expression-Based Identification of Antigen-Responsive CD8 + T Cells on a Single-Cell Level

doi: 10.3389/fimmu.2019.02568

Figure Lengend Snippet: CD137 expression marks antigen-responsive CD8 + T cells in the absence of multimer staining. (A) Representative flow cytometry dot plots of PBMCs from donor #4 stained with HLA-A2 multimers loaded with CMVpp65 495−503 peptide after incubating PBMCs for 20 h with mock (Flu MP 58−66 ) or cognate (CMVpp65 495−503 ) peptides (top) and corresponding plots showing CD25 and CD137 expression (bottom). All plots show 5 × 10 4 cells in the CD8 gate. (B) TCR repertoire analysis of CD8 + T cells subjected to single-cell sorting based on positive staining with CMVpp65 495−503 peptide-loaded HLA-A2 multimers from PBMCs stimulated with mock peptide (blue gate in A ; antigen-specific) or based on CD137 expression after incubation with cognate peptide (representing cells in the red gate in A ; antigen-responsive). The frequencies of genes used for the production of TCR α and β chains in each of the two populations of cells are shown. (C) Heatmaps of the top 25 ranked differentially expressed genes in CMV pp65 495−503 -specific relative to CMV pp65 495−503 -responsive CD8 + T cells from donor #4. The numbers of analyzed single cells are shown beneath the heatmaps. (D) t-SNE plot of gene expression data for single-cell-sorted antigen-specific (black outline filled blue) and antigen-responsive CD8 + T cells (black outline filled red). Data for CMV-directed cells incubated with mock (blue) or cognate (red) peptide in the dye-based activation assay are included as a reference. (E) Scatter plots of flow cytometric analyses comparing the median fluorescence intensities for the indicated surface markers between antigen-specific and antigen-responsive CD8 + T cells for donors #1–3 (donors with Flu-directed cells) and #4–6 (donors with CMV-directed cells).

Article Snippet: Phycoerythrin-labeled HLA-A * 0201 multimers loaded with Flu MP 58−66 (GILGFVFTL), hCMV pp65 495−503 (NLVPMVATV), or IGRP 265−273 (VLFGLGFAI) were purchased from Immudex.

Techniques: Expressing, Staining, Flow Cytometry, FACS, Incubation, Gene Expression, Activation Assay, Fluorescence

Journal: Frontiers in Immunology

Article Title: Gene Expression-Based Identification of Antigen-Responsive CD8 + T Cells on a Single-Cell Level

doi: 10.3389/fimmu.2019.02568

Figure Lengend Snippet: Autoantigen-directed CD8 + T cells may differ in their responsiveness to the cognate peptide. (A) Representative flow cytometry dot plots of PBMCs from a donor with type 1 diabetes stained with HLA-A2 multimers loaded with Flu MP 58−66 or IGRP 265−273 peptide. (B) Representative dot plots of bulk (cell dye-negative) and IGRP 265−273 directed (cell dye-positive) CD8 + T cells incubated for 20 h with the indicated stimuli using K562/A*0201 cells for antigen presentation in the dye-based CD8 + T cell activation assay. Cells in the CD8 gate are shown. (C) t-SNE plots of single-cell gene expression data from IGRP 265−273 -directed CD8 + T cells after incubation for 20 h with the indicated antigen-presenting cells in the presence of solvent (black), mock peptide (blue), or cognate peptide (red). (D) Heatmaps of single-cell gene expression data for the previously identified 16 genes of antigen-responsive virus-directed CD8 + T cells derived from IGRP 265−273 -directed CD8 + T cells stimulated with the control or cognate peptide (see ). The reference genes CD3E and CD8B are also shown.

Article Snippet: Phycoerythrin-labeled HLA-A * 0201 multimers loaded with Flu MP 58−66 (GILGFVFTL), hCMV pp65 495−503 (NLVPMVATV), or IGRP 265−273 (VLFGLGFAI) were purchased from Immudex.

Techniques: Flow Cytometry, Staining, Incubation, Immunopeptidomics, Activation Assay, Gene Expression, Solvent, Virus, Derivative Assay, Control