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: 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

Journal: bioRxiv

Article Title: Ly6C defines a subset of memory-like CD27 + γδ T cells with inducible cancer-killing function

doi: 10.1101/2020.09.08.287854

Figure Lengend Snippet: Single-cell suspensions of spleen, lymph node (LN) and lung from WT mice were analyzed by flow cytometry. (A) Representative dot plots of Ly6C and CCR7 staining. Viable single cells were gated on CD3 + and TCRδ + cells, followed by CD27 + cells. (B) Bar graphs showing the proportion of Ly6C + cells among CD27 + γδ T cells in spleen, LN and lung (n = 7/group). Each dot represents one mouse. Data are represented as mean ± SD. * p < 0.05 as determined by one-way ANOVA followed by Dunn’s posthoc test. (C) Representative dot plots of CD160, NKG2A, NKp46 and IFNγ staining, after gating on CD3 + TCRδ + cells followed CD27 + Ly6C + cells. Fluorescence minus one (FMO) controls were used to draw gates. SSC = side scatter. (D) Bar graphs showing the proportion of CD160, NKG2A, NKp46 or IFNγ positive cells for CD27 + Ly6C — and CD27 + Ly6C + γδ T cell populations (n = 4/group) in spleen, LN and lung. Each dot represents one mouse. Data are represented as mean ± SD. * p < 0.05 as determined by Mann-Whitney U test.

Article Snippet: To yield a purer γδ T cell population, positive selection was performed twice. γδ T cells were cultured in IMDM medium supplemented with 10% FCS, 100U/mL penicillin/streptomycin, 2mM glutamine, 50μM 2-Mercaptoethanol (β-ME), 10ng/mL murine interleukin-15 (IL-15) (PeproTech) and CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) at a 1:1 ratio.

Techniques: Flow Cytometry, Staining, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Ly6C defines a subset of memory-like CD27 + γδ T cells with inducible cancer-killing function

doi: 10.1101/2020.09.08.287854

Figure Lengend Snippet: Single-cell suspensions of spleen, lymph node (LN) and lung from WT mice were analyzed by flow cytometry. (A) Representative dot plots of CD44 and CD62L staining, after gating on CD3 + TCRδ + cells followed CD27 + cells. Fluorescence minus one (FMO) controls were used to draw gates. Blue box indicates effector memory cells; dark grey indicates memory cells; and light grey indicates naïve T cells. (B) Bar graphs showing the proportion of naïve T cells (CD62L + CD44 — ), memory (CD62L + CD44 + ) and effector memory (CD62L — CD44 + ) among CD27 + γδ T cells in spleen, LN and lung (n = 4/group). Each dot represents one mouse. Data are represented as mean ± SD. * p < 0.05, ** p < 0.01, and *** p < 0.001 as determined by repeated measures ANOVA followed by Tukey’s posthoc test. (C) Representative dot plots of CD3 + TCRδ + CD27 + cells segregated into Ly6C — and Ly6C + subsets, followed by CD44 and CD62L staining. Blue boxes indicate effector memory cells; dark grey indicates memory cells; and light grey indicates naïve T cells. (D) Bar graphs showing the proportion of effector memory, memory and naïve T cells in CD27 + Ly6C — and CD27 + Ly6C + γδ T cells from spleen, LN and lung (n = 4/group). Data are represented as mean ± SD. * p < 0.05 as determined by Mann-Whitney U test. (E) Bar graphs showing the proportion of Ki67 + cells among CD27 + Ly6C — and CD27 + Ly6C + γδ T cells from spleen, LN and lung (n = 4/group). Each dot represents one mouse. Data are represented as mean ± SD. * p < 0.05 and *** p < 0.001 as determined by Mann-Whitney U test or repeated measures ANOVA followed by Tukey’s posthoc test.

Article Snippet: To yield a purer γδ T cell population, positive selection was performed twice. γδ T cells were cultured in IMDM medium supplemented with 10% FCS, 100U/mL penicillin/streptomycin, 2mM glutamine, 50μM 2-Mercaptoethanol (β-ME), 10ng/mL murine interleukin-15 (IL-15) (PeproTech) and CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) at a 1:1 ratio.

Techniques: Flow Cytometry, Staining, Fluorescence, MANN-WHITNEY

Journal: bioRxiv

Article Title: Ly6C defines a subset of memory-like CD27 + γδ T cells with inducible cancer-killing function

doi: 10.1101/2020.09.08.287854

Figure Lengend Snippet: T cell receptor (TCR) usage was analyzed on γδ T cells from WT mice by flow cytometry. To assess the function of Vγ1 cells in tumour-bearing mice, mice were transplanted with KB1P tumour fragments; 14 days after tumours were palpable, mice received isotype control antibodies or anti-Vγ1 depleting antibodies daily until tumours reached 15mm. (A) Gating strategy for Vγ1 + (blue), Vγ4 + (brown) and Vγ1 — Vγ4 — (grey) cells after selection of CD3 + TCRδ + CD27 + cells. (B) Bar graphs showing the proportion of CD27 + γδ T cells expressing Vγ1, Vγ4 or Vγ1 — Vγδ4 — TCRs in spleen, LN and lung (n = 4/group). Data are represented as mean ± SD. * p < 0.05 and ** p < 0.01 as determined by repeated measures ANOVA followed by Tukey’s posthoc test. (C) Bar graphs showing the ratio of CD27 + Ly6C — and CD27 + Ly6C + γδ T cell populations among Vγ1 + , Vγ4 + and Vγ1 — Vγ4 — cells from spleen, LN and lung (n = 4/group). Data are represented as mean ± SD. ** p < 0.01 as determined by repeated measures ANOVA followed by Tukey’s posthoc test. (D) Bar graphs showing the ratio of Vγ1 — Vγ4 — , Vγ4 + and Vγ1 + cells among CD27 + Ly6C — and CD27 + Ly6C + γδ T cell populations (n = 4/group) from spleen, LN and lung (n = 4/group). Data are represented as mean ± SD. * p < 0.05 as determined by Mann-Whitney U test. (E) Bar graph showing the proportion of CD27 + γδ T cells among CD3 + T cells in lungs from mice treated with anti-Vγ1 antibodies (n = 6) or isotype control antibodies (n = 5). Each dot represents one mouse. Data are represented as mean ± SD. ** p < 0.01 as determined by Mann-Whitney U test. (F) Tumour growth curve of KB1P mammary tumour-bearing mice treated with anti-Vγ1 antibodies (n = 6 mice) or isotype control antibodies (n = 5 mice). Each dot represents mean ± SD. (G) Kaplan-Meier survival analysis of KB1P mammary tumour-bearing mice treated with anti-Vγ1 antibodies (n = 6 mice) or isotype control antibodies (n = 5 mice).

Article Snippet: To yield a purer γδ T cell population, positive selection was performed twice. γδ T cells were cultured in IMDM medium supplemented with 10% FCS, 100U/mL penicillin/streptomycin, 2mM glutamine, 50μM 2-Mercaptoethanol (β-ME), 10ng/mL murine interleukin-15 (IL-15) (PeproTech) and CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) at a 1:1 ratio.

Techniques: Flow Cytometry, Selection, Expressing, MANN-WHITNEY

Journal: bioRxiv

Article Title: Ly6C defines a subset of memory-like CD27 + γδ T cells with inducible cancer-killing function

doi: 10.1101/2020.09.08.287854

Figure Lengend Snippet: CD27 + γδ T cells were isolated from LN and spleen of WT mice. The cells were cultured and expanded with CD3/CD28 beads and IL-15. Flow cytometry was used to measure expression of cytotoxic markers before and expansion. (A) Line graph depicting the number of CD27 + γδ T cells in vitro over 4 days (n = 4). Each dot represents mean ± SD. (B) Line graphs of the proportion of cytotoxic markers identified by scRNAseq on CD27 + γδ T cells before and after expansion (n = 4). Each dot and line represent cells from two pooled mouse samples. * p < 0.05, ** p < 0.01 and *** p < 0.001 as determined by paired t test. (C) Bar graphs of cytotoxic markers expressed by CD27 + Ly6C — and CD27 + Ly6C + γδ T cells after expansion in vitro (n = 4). Each dot represents cells from one two pooled mice. Data are represented as mean ± SD. * p < 0.05 as determined by Mann-Whitney U test.

Article Snippet: To yield a purer γδ T cell population, positive selection was performed twice. γδ T cells were cultured in IMDM medium supplemented with 10% FCS, 100U/mL penicillin/streptomycin, 2mM glutamine, 50μM 2-Mercaptoethanol (β-ME), 10ng/mL murine interleukin-15 (IL-15) (PeproTech) and CD3/CD28 coated Dynabeads (Thermo Fisher Scientific) at a 1:1 ratio.

Techniques: Isolation, Cell Culture, Flow Cytometry, Expressing, In Vitro, MANN-WHITNEY