Journal: Nature Immunology

Article Title: Trimodal single-cell profiling reveals a novel pediatric CD8αα + T cell subset and broad age-related molecular reprogramming across the T cell compartment

doi: 10.1038/s41590-023-01641-8

Figure Lengend Snippet: (a) Heatmap of all differentially expression genes (DEGs) using Seurat’s FindAllMarkers function (parameters: logfc.threshold = 0.25, P < 0.05 determined by two-tailed Wilcoxon’s rank sum test) between CD4 T cell subsets in TEA-seq dataset. (b) Heatmap of all differentially accessible peaks using ArchR’s getMarkerFeatures (parameters: FDR< = 0.1, Log2FC ≥ 0.5) between CD4 T cell subsets in TEA-seq dataset. (c) Pseudo-bulk expression values from our confirmatory scRNA-seq dataset from pediatric (Ped), young adult (YA), and older adult naive CD4 T cells of select age-specific genes identified in TEA-seq. (d) Gene expression of SOX4, TOX, CPQ, and STAT4 in bulk-sorted naive CD4 T cells (CD3 + CD4 + CD8 neg CD45RA + CCR7 + CD27 + CD95 neg ) from newborn cord blood (n = 7 donors) and older adult peripheral blood (n = 6 donors) using qRT-PCR. Two-tailed Mann-Whitney test. * P < 0.05, **** P < 0.0001. In panels a–c, values have been scaled (z-score) per gene or peak.

Article Snippet: Hashed 10x Genomics scRNA-seq data processing was carried out using CellRanger (10x Genomics) and BarWare to generate sample-specific output files.

Techniques: Expressing, Two Tailed Test, Quantitative RT-PCR, MANN-WHITNEY

Journal: Nature Immunology

Article Title: Trimodal single-cell profiling reveals a novel pediatric CD8αα + T cell subset and broad age-related molecular reprogramming across the T cell compartment

doi: 10.1038/s41590-023-01641-8

Figure Lengend Snippet: a , Heat map of the top 20 DEGs for each age group in individual true naive CD4 + T cells. For visualization, values are scaled ( z score) per gene. Exp, scaled expression. b , Dot plots of average pseudobulk gene expression for select transcripts in true naive CD4 + T cells separated by age ( n = 8 per group; P, pediatric; OA, older adult). The line indicates the median value. P values were determined by a two-tailed Mann–Whitney test. ** P = 0.0006, *** P = 0.0002. c , TF binding motif enrichment comparison between age groups in true naive CD4 + T cells. The P adj of enrichment was determined by hypergeometric testing. ETV1/ETV2, ETS translocation variant 1/2; NFATC3, nuclear factor of activated T cells, cytoplasmic 3; ATF3/ATF7, activating TF 3/7; TCFL5, TF-like 5 protein; CREM, cAMP-responsive element modulator; SPIB, Spi-B TF; SOX4/SOX10, SRY-box TF 4/10. d , ChromVar motif enrichment UMAP plots. Areas enriched for true naive CD4 + T cells in older adults (orange) and children (green) are outlined. dev, deviation. e , Overview of the scRNA-seq confirmatory cohort ( n = 16 per age group). f , RNA-based UMAP plot of naive CD4 + T cells from the confirmatory cohort. g , Average pseudobulk expression of select signature genes in the naive CD4 + T cell subset for each donor across all age groups, including an external cord blood ( n = 3) dataset. Best-fit lines with 95% confidence intervals are shown. AvgExp, average expression.

Article Snippet: Hashed 10x Genomics scRNA-seq data processing was carried out using CellRanger (10x Genomics) and BarWare to generate sample-specific output files.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Binding Assay, Comparison, Translocation Assay, Variant Assay

Journal: Nature Immunology

Article Title: Trimodal single-cell profiling reveals a novel pediatric CD8αα + T cell subset and broad age-related molecular reprogramming across the T cell compartment

doi: 10.1038/s41590-023-01641-8

Figure Lengend Snippet: a , Identification of subsets within CD8 + CD27 + CD197 + CD45RA + T cells through a trimodal analysis, shown in a 3WNN UMAP plot with the true naive, SCM, MNP-1, MNP-2 and MAIT subsets colored. b , Expression of select RNA and ADT cell type markers, shown in 3WNN UMAP plots. The modality of detection is indicated in square brackets. Density, gene-weighted 2D kernel density. c , Chromatin accessibility tracks of the IFNG gene region in naive CD8 + T cell subsets, showing normalized read coverage. d , Bar plot (median value shown) of the frequencies of naive CD8 + T cell subsets within the overall naive CD8 + compartment by age group ( n = 8 per group). P values were determined by a two-tailed Mann–Whitney test with the Holm–Sidak multiple-comparison method. * P < 0.05 ( P = 0.02), ** P < 0.01 ( P = 0.003), *** P < 0.001 ( P = 0.0008). e , Age-specific composition of the non-naive compartment found within naive CD8 + T cells. f , 3WNN UMAP plot of all T cells overlaid with naive CD8 + T cell subsets and separated by age. Only cells from the naive CD8 + T cell compartment of children (left) or adults (right) are colored; all other cells are gray. g , Comparison of differential chromatin accessibility across all CD8 + T cell subsets (24,874 features). For visualization, all values are scaled ( z score) per differential region. h , Dot plot of select DEGs across naive CD8 + T cell subsets. The size of points corresponds to the fraction of cells expressing each gene; color corresponds to average expression. AvgExp, scaled average expression. i , Identification of the MNP-2 subset through gene expression profiling in the scRNA-seq confirmatory cohort. Density, gene-weighted 2D kernel density. j , MNP-2 subset frequencies within the total T cells across all age groups including an external cord blood ( n = 3) dataset.

Article Snippet: Hashed 10x Genomics scRNA-seq data processing was carried out using CellRanger (10x Genomics) and BarWare to generate sample-specific output files.

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Comparison

Journal: Nature Immunology

Article Title: Trimodal single-cell profiling reveals a novel pediatric CD8αα + T cell subset and broad age-related molecular reprogramming across the T cell compartment

doi: 10.1038/s41590-023-01641-8

Figure Lengend Snippet: a , Overview of a fixed CITE-seq experiment ( n = 4 pediatric donors) for TCR stimulation. b , RNA-based UMAP plot of unstimulated, anti-CD3/anti-CD28 (TCR; 0.5:1 beads per cell)-stimulated and PMA/iono (PMA 50 ng ml −1 , iono 1 μg ml −1 )-stimulated pediatric CD8 + T cells. Stimulated subsets are indicated with the stimulation condition. c , Comparison of DEGs across each subset of unstimulated, TCR-stimulated and PMA/iono-stimulated CD8 + T cells. d , Violin plots of the single-cell expression of select effector genes for naive, MNP-2 and memory CD8 + T cells before and after stimulation with TCR and PMA/iono. e , Expression density of select RNA and ADT cell type markers, shown in UMAP plots of PMA/iono-stimulated and unstimulated cells. The modality of detection is indicated in square brackets. Density, gene-weighted 2D kernel density; exp, average expression level. f , Overview of an external pediatric MIS-C scRNA-seq dataset used for MNP-2 cell identification and frequency comparison. g , Frequency of MNP-2 cells in the total peripheral T cells of healthy children ( n = 6), children with active MIS-C ( n = 7) and children who had recovered from MIS-C ( n = 2).

Article Snippet: Hashed 10x Genomics scRNA-seq data processing was carried out using CellRanger (10x Genomics) and BarWare to generate sample-specific output files.

Techniques: Comparison, Expressing