Journal: Cell Reports Medicine

Article Title: CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance

doi: 10.1016/j.xcrm.2023.100917

Figure Lengend Snippet: CAR-T cells can transduce T cell signaling without LCK (A) Top: western blot detection of LCK or FYN expression in CAR or TCR-Jcam1.6 cell. Bottom: CAR or TCR responsiveness in LCK-deficient Jcam1.6 cells. CHO-L2 is the artificial APC presenting the LMP2A 426-434 (CLGGLLTMV) (L2) target peptide on HLA-A2. CHO-GAG presents the irrelevant GAG (SLYNTVATL) peptide. (B) Phosphorylation of TCR signaling pathway molecules PLCγ1, ERK, and CD3ζ at different time points by LCK-deficient CD28CAR-Jcam cells. LCK-sufficient TCR-Jurkat cells were used as positive control. Phosphorylation of PLCγ1, ERK1/2, or CD3ζ was calculated relative to intensity of total PLCγ1, ERK, or ERK, respectively. (C) Schematic of LCK locus-targeted homologous directed repair (HDR) by CRISPR-Cas9 editing. The anti-CD19-CD28CAR construct has either a Myc or His tag. (D) CAR-His and CAR-Myc expression after LCK locus-targeted HDR. (E) Western blot detection of LCK expression after sorting of CAR-His + , CAR-Myc + , and CAR-DP (Myc + /His + ) CD8 + -T cells. Control represents mock-edited CD8 + -T cells. (F) Cytotoxicity of CAR-His + , CAR-Myc + , and CAR-DP CD8 + -T cells. Control group was the target cells with mock-edited CD8 + -T cells. (G) Cytokine secretion of CAR-His + , CAR-Myc + , and CAR-DP CD8 + -T cells upon activation. CAR-T cells were incubated with CD19-expressing Daudi cells at a 1:1 ratio for 18 h, then stained for intracellular TNF or IFNγ. The left panel is the representative fluorescence-activated cell sorting (FACS) data, and the right panel is the statistical summary and analysis. All data are representative of 3 independent experiments, plotted as mean ± SD of technical triplicates. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001; not significant (NS) p > 0.05. (A)–(G) were analyzed by Student’s t test. See Figure S1 for additional data.

Article Snippet: Cas9 lentivector was obtained from Addgene (#52961).

Techniques: Western Blot, Expressing, Phospho-proteomics, Positive Control, CRISPR, Construct, Control, Activation Assay, Incubation, Staining, Fluorescence, FACS

Journal: Cell Reports Medicine

Article Title: CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance

doi: 10.1016/j.xcrm.2023.100917

Figure Lengend Snippet: FYN is suggested to mediate CD28-CAR-T cell activation in the absence of LCK (A) The cytotoxicity of primary CAR-T and TCR-T cells with or without PP2 inhibitor (10 μM). CAR-T targets CD19, and TCR-T targets HLA/A2-L2. Daudi cells, which express CD19 and were transduced with HLA/A2-L2, were used as target cells. The CAR-T cells were mixed with target cells at an E:T ratio of 4:1. (B) Phosphorylation (pY420) of FYN in LCK-deficient CAR- or TCR-Jcam cells at different time points. APC stands for artificial antigen-presenting cell CHO-L2, to which the CAR and TCR responded. The number shown below indicates band intensity of pY420 relative to total FYN (FYN pY420 was stained by anti-pSRC family pY416). (C) LCK and FYN expression in LCK or FYN-KO Jurkat clone after CRISPR-Cas9 editing. C-CBL was used as loading control. (D) IL-2 production of TCR and CAR in LCK or FYN-KO Jurkat upon activation by CHO-L2. (E) Association of FYN with CD28CAR variants. Jcam cells with different CD28CAR variants were activated by CHO-L2 for 5 min, and anti-Myc antibody was used to immunoprecipitate CAR. The association strength (intensity ratio) was calculated by the relative intensity of FYN to that of immunoprecipitated CAR. (F) Correlation of FYN association strength with IL-2 production by Jcam cells with different CD28-CAR variants. The intensity ratio in abscissa is from (E), and the statistical significance is calculated by compare IL-2 production by CD28CAR variants with that of wild type (WT). The CAR and TCR mentioned above (B–E) both specifically target HLA/A2-L2, and CD28CAR was used. Data are representative of 3 independent experiments (except 2 for E), plotted as mean ± SD of technical triplicates. p values denoted as in the Figure 1 legend by Student’s t test. See Figure S3 for additional data.

Article Snippet: Cas9 lentivector was obtained from Addgene (#52961).

Techniques: Activation Assay, Transduction, Phospho-proteomics, Staining, Expressing, CRISPR, Control, Immunoprecipitation

Journal: Cell Reports Medicine

Article Title: CD28-CAR-T cell activation through FYN kinase signaling rather than LCK enhances therapeutic performance

doi: 10.1016/j.xcrm.2023.100917

Figure Lengend Snippet:

Article Snippet: Cas9 lentivector was obtained from Addgene (#52961).

Techniques: Purification, Functional Assay, Virus, Recombinant, Staining, Cloning, Mutagenesis, Enzyme-linked Immunosorbent Assay, Cytotoxicity Assay, Sequencing, Software

Journal: mBio

Article Title: Ubiquitination of SARS-CoV-2 NSP6 and ORF7a Facilitates NF-κB Activation

doi: 10.1128/mbio.00971-22

Figure Lengend Snippet: NEMO and TAK1 were necessary for NSP6- and ORF7a-mediated NF-κB activation. (A) Scheme of canonical NF-κB pathway. (B to D) HEK293T-TAK1-KO (B), HEK293T-NEMO-KO (C), and HEK293T-TRAF2-KO, HEK293T-TRAF6-KO, and HEK293T-RIP2-KO (D) cells were transfected with pcDNA3.1-HA, pcDNA3.1-HA-NSP6, or pcDNA3.1-HA-ORF7a, along with pGL4-NF-κB-LucP2 and pNull-RLuc. The level of luciferase activity was determined at 24 h posttransfection. The firefly luciferase activity was normalized to Renilla luciferase activity. An empty plasmid was used as a control and set to 1. Results are shown as the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01 (Student’s t test). (E) HEK293T cells were transfected with pcDNA3.1-HA, pcDNA3.1-HA-NSP6, or pcDNA3.1-HA-ORF7a. At 48 h after transfection, IP was performed by anti-HA antibody. The immunocomplex was analyzed by Western blotting with the indicated antibodies.

Article Snippet: To generate CRISPR-Cas9-mediated knockout cell lines, MAP3K7 sgRNA CRISPR/Cas9 All-in-One lentivector (catalog no. 280341110595), IKBKG single guide RNA (sgRNA) CRISPR/Cas9 All-in-One lentivector (catalog no. 247991110595), and RIPK2 sgRNA CRISPR/Cas9 All-in-One lentivector (catalog no. 396111110595) were purchased from Applied Biological Materials (Richmond, BC, Canada), and TRAF2 CRISPR/Cas9 KO plasmid (catalog no. sc-400361), TRAF5 CRISPR/Cas9 KO plasmid (catalog no. sc-402073), TRAF6 CRISPR/Cas9 KO plasmid (catalog no. sc-400117), and RIP CRISPR/Cas9 KO plasmid (catalog no. sc-400377) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Control, Western Blot

Journal: mBio

Article Title: Ubiquitination of SARS-CoV-2 NSP6 and ORF7a Facilitates NF-κB Activation

doi: 10.1128/mbio.00971-22

Figure Lengend Snippet: NEMO and TAK1 were necessary for NSP6- and ORF7a-mediated NF-κB activation. (A) Scheme of canonical NF-κB pathway. (B to D) HEK293T-TAK1-KO (B), HEK293T-NEMO-KO (C), and HEK293T-TRAF2-KO, HEK293T-TRAF6-KO, and HEK293T-RIP2-KO (D) cells were transfected with pcDNA3.1-HA, pcDNA3.1-HA-NSP6, or pcDNA3.1-HA-ORF7a, along with pGL4-NF-κB-LucP2 and pNull-RLuc. The level of luciferase activity was determined at 24 h posttransfection. The firefly luciferase activity was normalized to Renilla luciferase activity. An empty plasmid was used as a control and set to 1. Results are shown as the mean ± SD of three independent experiments. *, P < 0.05; **, P < 0.01 (Student’s t test). (E) HEK293T cells were transfected with pcDNA3.1-HA, pcDNA3.1-HA-NSP6, or pcDNA3.1-HA-ORF7a. At 48 h after transfection, IP was performed by anti-HA antibody. The immunocomplex was analyzed by Western blotting with the indicated antibodies.

Article Snippet: To generate CRISPR-Cas9-mediated knockout cell lines, MAP3K7 sgRNA CRISPR/Cas9 All-in-One lentivector (catalog no. 280341110595), IKBKG single guide RNA (sgRNA) CRISPR/Cas9 All-in-One lentivector (catalog no. 247991110595), and RIPK2 sgRNA CRISPR/Cas9 All-in-One lentivector (catalog no. 396111110595) were purchased from Applied Biological Materials (Richmond, BC, Canada), and TRAF2 CRISPR/Cas9 KO plasmid (catalog no. sc-400361), TRAF5 CRISPR/Cas9 KO plasmid (catalog no. sc-402073), TRAF6 CRISPR/Cas9 KO plasmid (catalog no. sc-400117), and RIP CRISPR/Cas9 KO plasmid (catalog no. sc-400377) were purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Activation Assay, Transfection, Luciferase, Activity Assay, Plasmid Preparation, Control, Western Blot