Journal: eLife

Article Title: Single-cell transcriptomics identifies altered neutrophil dynamics and accentuated T-cell cytotoxicity in tobacco-flavored e-cigarette-exposed mouse lungs

doi: 10.7554/eLife.106380

Figure Lengend Snippet: The levels of pro-inflammatory cytokines/chemokines in the lung digests from experimental (PG:VG, PG:VG + Nic, fruit, menthol, and tobacco) and control (air) were assessed using multianalyte assay. The results obtained were plotted as a heatmap with the z -scores represented between the scale of orange (low) to blue (high) ( A ). Heatmap showing the fold changes in the expression of commonly dysregulated genes in the myeloid and lymphoid clusters in mouse lungs exposed to flavored e-cig aerosols as compared to ambient air as determined after DESeq2 analyses ( B ). CNET plot results showing the pathways regulated by the common differentially expressed genes (DEGs) (across all cell types) on acute (5-day) exposure to differently flavored (fruit, menthol, and tobacco) e-cig aerosols in C57BL/6J mouse lungs ( C ). Data is representative of n = 2/sex/group. Figure 9—figure supplement 1—source data 1. Z -score values of the levels of cytokine/chemokine obtained in per mg of protein from mouse lung tissue samples following 5-day nose-only exposure to PG:VG, PG:VG + Nic, fruit, menthol, or tobacco-flavored e-cig aerosols as plotted in . Figure 9—figure supplement 1—source data 2. Z -score values of the gene expression of commonly dysregulated genes in the mouse lung tissue samples following 5-day nose-only exposure to PG:VG, fruit, menthol, or tobacco-flavored e-cig aerosols as compared to air controls as plotted in .

Article Snippet: We used a multiplex assay to determine the levels of cytokine/chemokine in the lung homogenates from control and e-cig aerosol exposed mouse lungs using commercially available Bio-Plex Pro Mouse Chemokine Assay (Cat# 12009159, Bio-Rad, Hercules, CA) per the manufacturer’s instructions.

Techniques: Control, Expressing, Gene Expression

Journal: Molecular Systems Biology

Article Title: Quantifying persistence in the T‐cell signaling network using an optically controllable antigen receptor

doi: 10.15252/msb.202010091

Figure Lengend Snippet: A–C Flow cytometry data of Jurkat T cells either untransduced (gray) or expressing the OptoCAR construct (red) after lentiviral transduction. The distribution of mScarlet fluorophore genetically fused to OptoCAR is shown (A), along with surface staining for the receptor using an anti‐CD86 antibody (B). The bivariate plot of these two distributions shows that they are extremely well‐correlated (C), demonstrating mScarlet intensity is a good marker for OptoCAR cell surface expression. D, E Conjugates formed between Jurkat (J.NFAT; iRFP713 + ) and Raji‐FRB Ex cells (mTagBFP + ) can be directly gated on during acquisition of flow cytometry data. Simply mixing the two cell types together produces essentially no double‐positive events (D), but allowing them to interact at high density at 37°C for 10 min creates a distinct and readily observable population of cell conjugates (E). F Photograph of the custom‐built illumination device that can expose the sample to blue light while maintaining it at 37°C on the flow cytometer. G, H Repeating the Ca 2+ flux assay described in the main text with a variant of the LOV2 domain in the OptoCAR that is unresponsive to blue light illumination (OptoCAR C450G ) drives increased intracellular [Ca 2+ ] independently from the illumination state of the sample (G). Bounded line shows mean ± SEM ( n = 3). Conversely, the complementary mutation of the LOV2 domain that maintains it in the “light” state (OptoCAR I539E ) cannot drive signaling even in the dark (H). Bounded line shows mean ± SEM ( n = 4).

Article Snippet: pHEFI‐OptoCAR CD19 ‐mScarlet , This work , Addgene#: 170465.

Techniques: Flow Cytometry, Expressing, Construct, Transduction, Staining, Marker, Flux Assay, Variant Assay, Mutagenesis

Journal: Molecular Systems Biology

Article Title: Quantifying persistence in the T‐cell signaling network using an optically controllable antigen receptor

doi: 10.15252/msb.202010091

Figure Lengend Snippet: Schematic showing the anti‐CD19 OptoCAR engineered to be light‐responsive. Binding of the OptoCAR CD19 expressed in T cells to CD19‐expressing B cells drives signaling through an equivalent intracellular sequence used in the OptoCAR, where illumination with blue light causes the reversible disruption of receptor signaling. Stimulating primary CD4 + T cells expressing OptoCAR CD19 over 24 h in the dark drives expression of CD137 (4‐1BB), a robust activation marker. Continuous illumination completely abolishes this to CD137 levels equivalent to unstimulated T cells. Plot of geometric mean of CD137 expression when the length of an individual signaling pulse is varied. Single pulse ranged from 0 to 24 h, with sample illumination initiated at the end of the pulse and CD137 expression measured for all samples 24 h after initiation of signaling. Bars show mean ± SEM of biological replicates ( n = 4). The cRQ values from RT‐qPCR of indicated cells. Total RNA was extracted and CD137 mRNA levels measured relative to that from HEK293T cells. Conjugated OptoCAR CD19 ‐T cells were also assayed either before or after 24 h stimulation in the dark state. Individual values from three biological replicates are shown, with mean depicted by bar plot. Series of experiments showing how CD137 expression is modulated by pulsatile trains of signaling in primary CD4 + T cells activated through OptoCAR CD19 . A combined signaling period of 6 h was broken into pulses ranging from 15 min to 2 h and the refractory period between these pulses varied (x‐axis), shown in the schematic above each plot. Geometric mean of CD137 intensity is shown plotted as a function of inter‐pulse interval, scaled to continuous 6‐h output. Bars show mean ± SEM ( n = 5). Representative dataset from (E), demonstrating how pulsatile stimulation of OptoCAR CD19 ‐T cells drives more efficient activation. Dotted and gray histograms show CD137 expression in resting cells and 24‐h activation, respectively. The filled histogram shows either a continuous 6‐h stimulation (left) or cumulative pulsed signals over 24 h driving substantially more potent activation.

Article Snippet: pHEFI‐OptoCAR CD19 ‐mScarlet , This work , Addgene#: 170465.

Techniques: Binding Assay, Expressing, Sequencing, Disruption, Activation Assay, Marker, Quantitative RT-PCR

Journal: Molecular Systems Biology

Article Title: Quantifying persistence in the T‐cell signaling network using an optically controllable antigen receptor

doi: 10.15252/msb.202010091

Figure Lengend Snippet:

Article Snippet: pHEFI‐OptoCAR CD19 ‐mScarlet , This work , Addgene#: 170465.

Techniques: Blocking Assay, Virus, Recombinant, TaqMan Assay, Derivative Assay, Software