Journal: Frontiers in Immunology

Article Title: Unveiling spatial complexity in solid tumor immune microenvironments through multiplexed imaging

doi: 10.3389/fimmu.2024.1383932

Figure Lengend Snippet: Immunophenotyping panel for multiplexed tissue imaging of cancer.

Article Snippet: CD3 , REA1151 , 50 , 130-120-267 , FITC (APC) , Miltenyi Biotec.

Techniques: Imaging

Journal: bioRxiv

Article Title: Improving the sensitivity of in vivo CRISPR off-target detection with DISCOVER-Seq+

doi: 10.1101/2022.10.29.514376

Figure Lengend Snippet: a , VEGFA site 2 Cas9 target sites detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right) in WTC-11 iPSCs. b-c , Genome browser visualization of MRE11 enrichment at an (b) on-target and (c) representative off-target position with 4 mismatches (‘4 mm’) in WTC-11 iPSCs with Cas9 targeting VEGFA site 2 . DISCOVER-Seq+ data in red (with Ku-60648), DISCOVER-Seq data in blue (with no drug exposure). d , Schematic of the DISCOVER-Seq+ protocol in the knock-in of a cancer neoantigen-specific, transgenic TCR (tgTCR) into the TRA locus of primary human T cells. e , TRA Cas9 target sites in primary T cells detected using DISCOVER-Seq (left) versus DISCOVER-Seq+ (right). f-g , Genome browser visualization of MRE11 enrichment at two representative 4-mismatch (‘4mm’) off-target positions in primary human T cells with Cas9 targeting TRA for knock-in of a tgTCR template. DISCOVER-Seq+ data in red (with Ku-60648), DISCOVER-Seq data in blue (with no drug exposure). h-i , (h) Plots of MRE11 ChIP-seq reads-per-million enrichment within a 1.5 kb window for samples with (y-axis) or without (x-axis) DNA-PKcs inhibition, at all TRA Cas9 off-target sites in primary human T cells from the DNA-PKcs inhibited samples. Each point in the plot (20 total) corresponds to a putative target site. Significant differences (p < 1E-3 or p < 1E-5) between y-axis and x-axis values were determined using two-sided Wilcoxon rank sum test. (i) Same as panel h, for cells delivered with Cas9 but without gRNA (negative control). j , TRA Cas12a (Cpf1) target sites in primary T cells. The results were the same between DISCOVER-Seq and DISCOVER-Seq+; only the on-target site was detected.

Article Snippet: Purified CD3+ T cells were activated with Dynabeads Human T-Activator CD3/CD28 (ThermoFisher) at a 1:2 bead-to-cell ratio in RPMI-1640 (ATCC) supplemented with 10% fetal bovine serum (HyClone Defined), 100 units/mL Penicillin (Gibco), 100 ug/mL Streptomycin (Gibco), 100 IU/mL recombinant human IL-2 (Proleukin, Prometheus Laboratories) and 5 ng/mL recombinant human IL-7 (BioLegend) at 37C, 5% CO 2 .

Techniques: Knock-In, Transgenic Assay, ChIP-sequencing, Inhibition, Negative Control

Journal: Nature Communications

Article Title: Predicting bacterial infection outcomes using single cell RNA-sequencing analysis of human immune cells

doi: 10.1038/s41467-019-11257-y

Figure Lengend Snippet: scRNA-seq analysis of human PBMCs before and after ex vivo Salmonella infection. a Overview of the scRNA-seq experiment: PBMCs were isolated from a blood sample of a healthy individual and were infected ex vivo with Salmonella (exposed), or remained unexposed (naïve). Overall ~7000 cells were sequenced using 10x genomics Chromium. b Visualization of the scRNA-seq data using forced layout on a two-dimensional space by k-nearest neighbor (KNN)-graph ( k = 20; naive cells (gray) and exposed cells (black)). K-means clustering of the cells revealed the seven main cell types: NK cells (red), CD8 T cells (orange), CD4 T cells (yellow), NKT cells (brown), B cells (green), monocytes (purple), and dendritic cells (DC; pink), as inferred using cluster-specific genes and marker genes expression (see Supplementary Data 1 and Supplementary Fig. ). Colored contours represent cells which belong to the same cell type in each sample (see also Supplementary Fig. for complete KNN-graph with edges and clusters). c Expression levels of representative genes from the infection signature (see methods and Supplementary Fig. ). Top: general infection genes which are upregulated following Salmonella infection in all exposed cells, and bottom: cell-type specific infection genes. Gene expression is shown using the same layout as in b , with the nodes colored by the indicated gene expression in each cell (see colorbar). d KNN-graph ( k = 20) of the scRNA-seq data after removal of the global infection signature eliminated the separation between naïve and exposed cells for all cell types, except for the monocytes, which contain intracellular bacteria. Colors and contours are the same as in b (see also Supplementary Fig. )

Article Snippet: For NKT enrichment, CD3 + CD56 + NKT Cell Isolation Kit (Miltenyi Biotec, 130-093-064) was used according to the manufacturer’s manual.

Techniques: Ex Vivo, Infection, Isolation, Marker, Expressing, Gene Expression, Bacteria

Journal: Nature Communications

Article Title: Predicting bacterial infection outcomes using single cell RNA-sequencing analysis of human immune cells

doi: 10.1038/s41467-019-11257-y

Figure Lengend Snippet: scRNA-seq based dynamic deconvolution to infer cell-type composition and infection-induced states. a Illustration of the dynamic deconvolution approach: transformation of the scRNA-seq data into two properties that can be inferred from bulk measurements - immune cell-type composition and infection-induced cell state. Cell-type composition is represented as a one-dimensional vector, where k j is the number of cells from a specific cell type j . The infection-induced cell state ( S j ) is represented as the induction of cell-type specific genes following infection. Using our deconvolution algorithm (equations at the bottom, see methods) we infer robust estimators for the relative abundance ( K j ) and infection-induced state ( S j ) of each cell type across individuals from bulk RNA-seq measurements, as illustrated on the right. b and c Reduction of the scRNA-seq data into two sets of genes which represent intrinsic cell-type properties ( b ) and cell-type specific infection-induced states ( c ). Cells are ordered by their cell type (color-coded at the bottom) and cell origin (white for naïve and black for exposed cells); see colorbar for expression levels. d Validation of our deconvolution algorithm using FACS experiment. Comparison between the percentages of each cell type as measured by FACS ( x -axis) to the relative abundance by our deconvolution ( y -axis). There is a high concordance between the deconvolution prediction and the cellular composition as determined by FACS. Each dot is the mean of 3–4 replicates for the FACS and bulk RNA-seq. Presented also are the standard error (SEM) for the replicates. e Validation of the infection-induced signatures in sorted populations. Presented are the expression levels of the intrinsic cell types (from b ) and infection-induced marker genes (from c ) in bulk measurements of sorted naïve and exposed NKT cells and monocytes. The NKT infection-induced state is upregulated following infection solely in the exposed NKT cells (left). Similarly, the monocytes cell-type signature is expressed exclusively in naïve and exposed monocytes, and the monocytes infection-induced signature is upregulated following infection exclusively in the exposed monocytes (right). Each sample is the mean of 2–4 technical replicates; cell-type signatures are color-coded ( n denotes the number of genes in each signature)

Article Snippet: For NKT enrichment, CD3 + CD56 + NKT Cell Isolation Kit (Miltenyi Biotec, 130-093-064) was used according to the manufacturer’s manual.

Techniques: Infection, Transformation Assay, Plasmid Preparation, RNA Sequencing, Expressing, Biomarker Discovery, Comparison, Marker

Journal: Nature Communications

Article Title: Predicting bacterial infection outcomes using single cell RNA-sequencing analysis of human immune cells

doi: 10.1038/s41467-019-11257-y

Figure Lengend Snippet: Dynamic deconvolution of immune cell states reveals differences between WT and TLR10 individuals. a Overview of the bulk RNA-seq experiment: isolated PBMCs from blood of eight healthy individuals: WT (green) and TLR10 (purple), were infected ex vivo with Salmonella and bulk RNA-seq was measured before infection ( t = 0), 4 ( t = 4), and 8 ( t = 8) hours post-infection in triplicates. b Box-plots of the relative abundance or infection-induced state of each cell type before and 4 or 8 h post-infection in WT vs. TLR10 uncover significant difference in NKT infection-induced states following infection. The box represents the median and 25–75th percentile, whiskers encompass all data points. * p -value < 0.05, two sample t -test. Values are inferred from bulk measurements using our deconvolution algorithm; estimators of cell-type index are in arbitrary units (au). c Unique molecular identifier (UMI) counts of IFNγ from each cell by scRNA-seq data revealed production of IFNγ exclusively from NKT cells 4 h post-infection; color-coded cell types are indicated at the bottom. d Gene Set Enrichment Analysis (GSEA) of the ‘monocytes infection-induced genes’ in the genes that are higher in WT relative to TLR10 individuals 8 h post-infection (see methods) reveals partition of the gene signature into two sets which imply differences in sub-types activation following infection. Red to blue bar at the bottom represents the gene expression fold change between WT and TLR10 individuals (see also colorbar to the right); the black bars below indicate positions of the ‘monocytes infection-induced genes’ in the ordered list of genes. p -value is calculated by the maximal Enrichment Score (ES), which also defines the group of enriched genes (all genes to the left of the maximal ES position, i.e. the dashed line). e Expression matrix (scRNA-seq data) of the set of ‘monocytes infection-induced genes’ that were enriched in the genes that are higher in WT relative to TLR10 (genes to the left of the dashed line in d ). Presented is the mean expression of these genes from each cell sub-type of the naive and exposed monocytes. The left bar represents the mean expression of these genes in each sub-types; monocytes sub-types color-coded as in Fig.

Article Snippet: For NKT enrichment, CD3 + CD56 + NKT Cell Isolation Kit (Miltenyi Biotec, 130-093-064) was used according to the manufacturer’s manual.

Techniques: RNA Sequencing, Isolation, Infection, Ex Vivo, Activation Assay, Gene Expression, Expressing

Journal: Nature Communications

Article Title: Predicting bacterial infection outcomes using single cell RNA-sequencing analysis of human immune cells

doi: 10.1038/s41467-019-11257-y

Figure Lengend Snippet: Differences in cell–cell signaling is associated with different bacterial control in WT and TLR10 individuals. a Isolated PBMCs from a WT individual were infected ex vivo with Salmonella in the presence of isotype control or anti-IFNγ neutralizing antibodies. Intracellular bacterial growth was determined by CFU 8 h post-infection. Data are presented as bar chart with the average of three independent experiments with four replicates and SEM, all data points are presented by dots. Blocking IFNγ in WT individual increases bacterial load; statistical significance was determined using Friedman’s test, p -value is indicated. b Intracellular bacteria number was determined by CFU 8 h after ex vivo Salmonella infection of monocytes alone or co-culture of monocytes and NKT cells. Data are presented as bar chart with mean and SEM of eight replicates; data points are presented by dots. Co-culture of monocytes with NKT cells provided better control of intracellular bacterial infection relative to monocytes alone; statistical significance was determined using the unpaired Mann–Whitney U test, p -value is indicated in the figure. c Secreted IFNγ levels from monocytes alone or co-culture of monocytes and NKT cells were measured before and after ex vivo Salmonella infection. Data are presented as bar chart with mean and SEM of eight replicates; data points are presented by dots. Co-culture of monocytes and NKT cells secreted significantly higher levels of IFNγ relative to monocytes alone; statistical significance was determined using the unpaired Mann–Whitney U test, p -value is indicated. d Isolated PBMCs from eight individuals (WT in green and TLR10 in purple) were infected ex vivo with Salmonella . Intracellular bacterial growth was determined by CFU 8 h post-infection. Data are presented as mean and SEM of three replicates. TLR10 individuals exhibit higher bacterial load than WT individuals; statistical significance was determined using the unpaired Mann–Whitney U test, p -value is indicated

Article Snippet: For NKT enrichment, CD3 + CD56 + NKT Cell Isolation Kit (Miltenyi Biotec, 130-093-064) was used according to the manufacturer’s manual.

Techniques: Control, Isolation, Infection, Ex Vivo, Blocking Assay, Bacteria, Co-Culture Assay, MANN-WHITNEY