Journal: bioRxiv

Article Title: Non-invasive imaging of cell-based therapies using acoustic reporter genes

doi: 10.1101/2024.11.01.621111

Figure Lengend Snippet: a , Schematic of lentiviral vectors designed to report T cell activity state by expressing GVs downstream of the NFAT promoter. T cell activation is chemically induced by incubating cells with PMA and ionomycin, which trigger the NFAT promoter to express rtTA, and in the presence of doxycycline, activate transcription of GV genes. b , Representative BURST images (top) and signal quantification (bottom) of Jurkat cells transduced with the vectors in panel ( a ) with or without chemical activation, normalized to wild-type (WT) cells (not shown). c , Percentage of Jurkat cells in panel ( b ) expressing both fluorescent reporters (GFP and BFP), with and without activation. d , Imaging T cell activation in response to CD19-CD3 BTE-mediated engagement with CD19+ Raji cells, using cells engineered with the vectors shown in ( a ). e , Representative BURST images (top) and signal quantification (bottom) of Jurkat cells engineered to express GVs upon receptor-mediated activation in the presence of Dox. f , Percentage of cells from panel ( e ) expressing both fluorescent reporters (GFP and BFP), with and without receptor engagement. Statistical comparisons were performed using Fisher’s LSD test, with each condition compared to resting state T cells (without PMA/ionomycin or BTE stimulation). P-value for ( e ): 0.0173. All other p-values < 0.0001. Significance levels: *p<0 . 05, **p<0 . 01, ***p<0 . 001, ****p<0 . 0001 . Error bars represent mean ± s.e.m. of N = 3 biological replicates. Each data point represents the arithmetic mean of N = 2 technical replicates. Scale bars for all ultrasound images represent 1 mm.

Article Snippet: To induce receptor-mediated T cell cytotoxicity, CD19-CD3 BTE (1 ng/mL, BPS Bioscience) and IL-2 (100 U/mL) were added.

Techniques: Activity Assay, Expressing, Activation Assay, Transduction, Imaging

Journal: bioRxiv

Article Title: Non-invasive imaging of cell-based therapies using acoustic reporter genes

doi: 10.1101/2024.11.01.621111

Figure Lengend Snippet: a , Workflow for engineering T cells isolated from human PBMCs to express doxycycline-inducible GVs and imaging BTE-mediated homing of cytotoxic T cells into target tumors. Top: T cells are transduced with the 3-vector lentiviral system, and GFP expressing cells are sorted to select for transduction of the assembly factor vectors. The cells are then expanded in vitro for downstream characterization and in vivo injections. Bottom: T cells are systemically administered to immunocompromised mice bearing subcutaneous CD19+ Raji cell tumors, accompanied by intraperitoneal (IP) injections of BTE every 48 hours and IP injections of doxycycline for 72 hours prior to ultrasound imaging. b , BURST SBR (left) and representative images (right) of GV-expressing and WT T cells. Paired one-tailed t test, p = 0.0115. Error bars represent mean ± s.e.m. of N = 5 PBMC donors; each data point is the arithmetic mean of N = 3 technical replicates. c , Percentage of live Raji cells in a cytotoxicity assay, where T cells are co-cultured with CD19+ Raji cells at effector-to-target (E:T) ratios of 1:1 and 4:1. Welch’s two-tailed t-test, N = 6 biological replicates (2 PBMC donors and 3 replicates per donor). d , GFP and BFP fluorescence measurements from ( c ), normalized to the maximum mean fluorescence intensity (MFI) of each reporter. e , BURST images overlaid on anatomical B-mode grayscale images of representative Raji tumors in mice injected with GV-expressing T cells, either Dox-induced or uninduced, and mice injected with WT T cells. Red lines indicate tumor boundary used for signal quantification. f , Quantification of BURST signal inside tumors. Welch’s two-tailed t test, Dox+ vs. Dox-, p = 0.0067; Dox+ vs. WT, p = 0.0063. N = 10 mice for Dox+, N = 6 for Dox-, N = 6 for WT. g , Histology of an example tumor infiltrated by GV-expressing T cells and its corresponding BURST image. White: Raji-Antares cells; Magenta: CD8+ cytotoxic T cells; Green: GFP+ T cells; Blue: BFP+ T cells. h , Schematic of T cell distribution in the Dox-induced tumor from ( g ). i , Correlation of the BURST signal within tumors with the absolute numbers of GFP+ and BFP+ T cells in corresponding histological sections. Pearson correlation: r = 0.89, p = 0.0072, N = 7 mice. One mouse (gray data point) was excluded due to a significant number of T cells located outside the imaging plane whose GV expression was not captured by BURST (see ). All scale bars represent 1 mm.

Article Snippet: To induce receptor-mediated T cell cytotoxicity, CD19-CD3 BTE (1 ng/mL, BPS Bioscience) and IL-2 (100 U/mL) were added.

Techniques: Isolation, Imaging, Transduction, Plasmid Preparation, Expressing, In Vitro, In Vivo, One-tailed Test, Cytotoxicity Assay, Cell Culture, Two Tailed Test, Fluorescence, Injection