Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Schematic showing the domain organization of the reference HER2-specific CAR constructs and modifications made to introduce programmed membrane protein (proMP) transmembrane domains (TMDs). Bold, boxed sequence indicates the human CD28 TMD in the reference CD28TM and no cys CARs and designed proMP sequences in the monomeric (proCAR-1), dimeric (proCAR-2), and trimeric (proCAR-3) receptors. ( b ) BW5147 murine thymoma cells stably expressing proCARs and a destabilized GFP NF-κB reporter were surface labeled with anti-Myc antibody and analyzed by flow cytometry to assess surface expression levels. ( c ) Live cells from ( b ) were coated with polyclonal anti-IgG to bind CARs through the scFv domain and immunoprecipitated using protein G beads. Products were separated by nonreducing SDS-PAGE and immunoblotted using anti-Myc antibody to visualize surface-expressed CAR proteins. Molecular weight of the unglycosylated CAR polypeptide is 55 kDa. ( d, e ) Cells from ( b ) were co-cultured with HER2+ SKBR3 human breast adenocarcinoma cells for the indicated times and analyzed by flow cytometry for upregulation of activation marker CD69 ( d ) and GFP expression from the NF-κB reporter ( e ). All activation levels are normalized to the 8 hr time point in cells expressing the CD28TM CAR (% CD28TM Max). Bars represent the mean ± SD, and dots show the individual data points for three independent experiments. ( f ) Maximum target killing percentage at 20:1 effector to target ratio from 4 hr 51 Cr release assay. Bars show mean ± SEM with each data point representing an individual experiment (n = 3). p-Values determined from paired t -tests. ( g ) Cytokine production by primary mouse HER2 proCAR T cells following 24 hr co-culture with MC57-HER2 target tumor cells. Bars show mean concentration ± SEM with each data point representing an individual experiment (n = 5). Significance was determined from one-way ANOVA with multiple comparisons. Cytokine production on antigen-negative parental MC57 cells shown separately in .

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Construct, Introduce, Sequencing, Stable Transfection, Expressing, Labeling, Flow Cytometry, Immunoprecipitation, SDS Page, Molecular Weight, Cell Culture, Activation Assay, Marker, Release Assay, Co-Culture Assay, Concentration Assay

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Model of the CD28TM interface generated by mutagenesis of the CD3ζ TMD (PDB: 2HAC). Polar residues of the CD28 dimerization motif (orange) with predicted hydrogen bonds depicted (dotted lines). ( b ) Surface expression and ( c ) SDS-PAGE and immunoblot analysis of HER2 CARs possessing WT CD28TM or CD28TM mutations depicted in ( a ) expressed in the BW5147 cell line. ( d ) Quantitation of target cell killing measured by chromium release assay and cytokine production by primary mouse CD8 + CAR T cells in response to the MC57-HER2 target cell line (n = 4). Experiments performed as in . p-Values determined by paired t -tests. ( e ) Representative immunofluorescent confocal images of CAR-CD28 co-clustering in primary mouse CAR T cells. CAR clustering was induced with anti-Myc primary followed by crosslinking with fluorescent secondary antibody (magenta). Cells were then labeled for CD28 (cyan). Images are Z-projections over 12 m, scale bar represents 3 m. ( f ) Quantitation of CAR-CD28 co-clustering, each dot representing the percentage of CAR clusters in one cell that co-localized with a CD28 cluster. Lines show mean CAR-CD28 co-clustering percentage/per cell ± SEM, n ≥ 30 cells. p-Values determined by unpaired t -tests.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Generated, Mutagenesis, Expressing, SDS Page, Western Blot, Quantitation Assay, Release Assay, Labeling

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: ( a ) Flow cytometry gating strategy to determine the transduction efficiency of primary murine CAR T cells. Lymphocytes selected via morphology, live cells selected as zombie aqua negative, T cells selected as CD3 + CD8 + and mCherry + cells defined as CAR T cells. c-Myc co-expression with mCherry indicates surface CAR expression. ( b ) Example 2D plots showing extracellular c-Myc labeling (y-axis) vs. intracellular mCherry (x-axis) of CD3 + CD8 + T cells on day 5 post-transduction with CD28TM CARs and ProCARs 1–3, demonstrating the percentage of cells expressing the CARs. Empty mCherry vector included as c-Myc-negative control.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Flow Cytometry, Transduction, Expressing, Labeling, Plasmid Preparation, Negative Control

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet: Example 2D plots showing extracellular c-Myc labeling (y-axis) vs. intracellular mCherry (x-axis) of CD3 + CD8 + T cells on day 5 post-transduction with CD28TM chimeric antigen receptors (CARs) and proCAR-4, demonstrating the percentage of cells expressing the CARs. Empty mCherry vector included as c-Myc-negative control.

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Labeling, Transduction, Expressing, Plasmid Preparation, Negative Control

Journal: eLife

Article Title: De novo-designed transmembrane domains tune engineered receptor functions

doi: 10.7554/eLife.75660

Figure Lengend Snippet:

Article Snippet: Commercial assay or kit , Mouse T-activator CD3/CD28 Dynabeads , Gibco , Cat# 11456D , .

Techniques: Flow Cytometry, Fluorescence, Microscopy, Recombinant, Sequencing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Modification, Software