276 control renilla luciferase rluc lentivirus  (BPS Bioscience)

Journal: Science Advances

Article Title: Optimally engineered HLA/peptide-specific CAR-T cells outperform TCR-T cells to eradicate solid tumors

doi: 10.1126/sciadv.adx9371

Figure Lengend Snippet: ( A ) Sorted CD8 + MAGEA4 TCR-T and CAR-T cells were cocultured with A375 cells for the indicated time course. Cell lysates were analyzed for activation of CD3 proximal signaling by phosphorylation of PLCγ1 and LAT. ( B ) Sorted CD8 + MAGEA4 TCR-T and CAR-T cells were cocultured with A375 cells at a 1:1 E:T ratio with additional tumor cells added every 24 hours to maintain antigen stimulation. Absolute numbers of TCR + /CAR + T cell numbers were assessed over time. ( C to E ) Induction of T cell activation/dysfunction markers PD-1, LAG3, and TIM3 was assessed by flow cytometry at the 72-hour time point shown in (B). [(B) to (E)] Significant differences between indicated groups are shown: ** P < 0.01, *** P < 0.001, **** P < 0.0001 by one-way ANOVA. ( F ) A375 cells stably expressing NucLightRed (A375/NLR) were cocultured with MAGEA4 TCR-T and CAR-T cells at a 1:2 E:T ratio, normalized by TCR/CAR + frequency, and imaged on the Incucyte S3 system. Additional tumor cells were added every 3 days as indicated by vertical dashed lines. Values represent mean red image intensity as a measure of tumor cell growth. [(A) to (F)] Results are representative of three to five individual experiments.

Article Snippet: NucLightRed-expressing cell lines were generated by transduction with NucLightRed Lentivirus (Sartorius, 4476) and maintenance in puromycin.

Techniques: Activation Assay, Phospho-proteomics, Flow Cytometry, Stable Transfection, Expressing

Journal: Science Advances

Article Title: Optimally engineered HLA/peptide-specific CAR-T cells outperform TCR-T cells to eradicate solid tumors

doi: 10.1126/sciadv.adx9371

Figure Lengend Snippet: ( A and B ) MAGEA4 TCR-T and CAR-T cells were cocultured with A375 cells in triplicate for 72 hours. (A) Representative CCR7 and CD45RO expression flow cytometry. (B) Quantification of memory populations analyzed in (A). Significant differences between MAGEA4 TCR and MAGEA4 TCR-41BBL: **** P < 0.0001 by one-way ANOVA. ( C ) A375 cells expressing NucLightRed (A375/NLR) were cocultured with MAGEA4 TCR-T and CAR-T cells (1:1 E:T in triplicate, normalized for TCR/CAR + frequency). Additional tumor cells were added every 4 days (dashed lines). Red fluorescence was measured at 4-hour intervals. [(A) to (C)] Results representative of three experiments. ( D to G ) NSG mice ( n = 5 per group) were implanted with A375-pLVX control or A375-41BBL tumor cells subcutaneously and dosed with 1.5 × 10 6 tetramer + TCR-T cells 11 days post-implant. Tumor growth (D), serum IL-2 (E) and IFN-γ (F) concentrations, and TCR-T cells in the blood (G) were measured. Significant differences by two-way ANOVA: ** P < 0.01 and *** P < 0.001 between A375-pLVX + MAGEA4 TCR and A375-41BBL + MAGEA4 TCR. ( H ) Absolute numbers of T cells after 6 days of in vitro chronic antigen stimulation. Significant differences between MAGEA4 TCR + EGFR/41BB CCR and MAGEA4 TCR + Ctrl/41BB CCR groups shown: ** P < 0.01 one-way ANOVA. ( I and J ) NSG mice ( n = 5 per group) were implanted subcutaneously with A375 tumor cells and dosed with 2 × 10 6 TCR + T cells 11 days later. Tumor growth (I) and blood TCR-T cell numbers are shown. Significant differences by two-way ANOVA: * P < 0.05 and *** P < 0.001 between MAGEA4 TCR + EGFR/41BB CCR and MAGEA4 TCR + Ctrl/41BB CCR groups; # P < 0.05 and ### P < 0.001 between MAGEA4 TCR and MAGEA4 TCR + Ctrl/41BB CCR groups. [(D) to (J)] Results are from two experiments.

Article Snippet: NucLightRed-expressing cell lines were generated by transduction with NucLightRed Lentivirus (Sartorius, 4476) and maintenance in puromycin.

Techniques: Expressing, Flow Cytometry, Fluorescence, Control, In Vitro