Images of representative time points are shown. Tumor burden was measured biweekly by bioluminescent imaging. No treatment indicates mice that were injected with tumor cells but not T cells. Four days later, T cells (4 × 105) described in b were injected intraperitoneally. ( c) NOD-SCID IL2Rγcnull mice were inoculated intraperitoneally with CD19+ Raji human Burkitt lymphoma cell line expressing a green fluorescent protein–firefly luciferase fusion protein (GFP/Luc). Representative plots of two independent experiments. Bottom: black histogram, untransduced cells red histogram, transduced cells. ( b) Flow cytometric analysis of 1928z-T-iPSC-T cells and syngeneic 1928z-transduced γδ (1928z-γδ) and αβ (1928z-αβ) T cells before their injection into tumor-bearing mice. Representative of two independent experiments. ( a) In vitro 51Cr release assay of 7 d-expanded 1928z-T-iPSC-T cells (effectors) and the murine lymphoma cell line EL-4 expressing ovalbumin (EL4-OVA) or human CD19 (EL4-CD19) (targets). Error bars, mean ± s.d.ġ928z-T-iPSC-T cells lyse CD19-positive tumor cells in vitro and in vivo. Graphs represent average of intra-assay technical triplicates. Data were normalized to the values of endogenous GAPDH and pre-expansion expression levels were used as reference. ( f, g) qRT-PCR analysis of the expression of the indicated mRNA transcripts in 1928z-T-iPSC-T cells before and 7 d after expansion on 3T3-CD19 AAPCs. ( e) Flow cytometric analysis of cell surface molecules and cytotoxic receptors in gated CD3 + 1928z-T-iPSC-T cells before and 7 d after expansion on 3T3-CD19 AAPCs. Arrows indicate restimulations with freshly irradiated 3T3-CD19 AAPCs. ( d) Expansion of 1928z-T-iPSC-T cells after weekly stimulations with 3T3-CD19 cells in the presence of IL-7 (10 ng/ml) and IL-15 (10 ng/ml) for 4 weeks. ( c) Intracellular expression of the transcription factor PLZF (red histogram), compared to isotype control (black histogram), and surface expression of CD161 and CD3 in 1928z-T-iPSC-T cells, as assessed by flow cytometry. Transcripts are classified according to known function and expression patterns. ( b) Heatmap comparing the expression of indicated mRNA transcripts expressed in lymphoid and/or NK cells. ( a) Unsupervised hierarchical clustering of 35 total transcriptomes, generated by an mRNA gene expression microarray, from 1928z-TiPCS-T cells at days 30–35 of differentiation (1928z-T-iPSC-T) and other human lymphoid cell subsets isolated for this study or downloaded from the NCBI repository GEO database (naive B cells, TCRVγ9 γδ T-cells before activation (γδ-T GEO) and after activation with BrHPP/IL-2 (bromohydrin pyrophosphate/interleukin-2) for 6 h (γδ-T 6h activ) or 7 days (γδ-T 7d activ) and resting NK cells). Phenotypic profiling of 1928z-T-iPSC-T cells before and after CD19-induced expansion. Data are presented as mean of two independent experiments ± s.d. ( f) Luminex multiplex cytokine analysis of culture supernatant 24 h after seeding of 1928z-T-iPSC-T cells on 3T3 or 3T3-CD19 cells. ( e) Flow cytometric analysis of CD25 and CD69 expression on the surface of 1928z-T-iPSC-T cells 48 h after exposure to 3T3 or 3T3-CD19 cells. Co-cultures shown 24 h after T-cell seeding formation of T-cell clusters and elimination of the 3T3-CD19 monolayer are visible. ( d) 1928z-T-iPSC-T cells were seeded into cultures of 3T3 cells or 3T3 cells expressing CD19 (3T3-CD19). Representative plots are of at least five independent differentiations. ( c) Flow cytometric analysis of 1928z-T-iPSC–derived cells at day 30 of differentiation. Fluorescence microscopy images (below) show mCherry expression was maintained throughout the differentiation process. mCherry + CAR + T-iPSCs are differentiated in three steps: (i) mesoderm formation (days 1–4), (ii) hematopoietic specification and expansion (days 5–10) and (iii) T-lymphoid commitment (days 10–30). T-iPSCs were stably transduced with a bicistronic lentiviral vector encoding the 19–28z CAR and the fluorescent marker mCherry. ( b) In vitro T-lymphoid differentiation protocol. The resulting T-iPSCs are genetically engineered to express a CAR and are then differentiated into T cells that express both the CAR and an endogenous TCR. Peripheral blood lymphocytes are reprogrammed to pluripotency by transduction with retroviruses encoding c-MYC, SOX2, KLF4 and OCT-4 (ref. Differentiation of 1928z CAR–engineered T-iPSCs into CD19-specific functional T lymphocytes.
0 Comments
Leave a Reply. |
AuthorWrite something about yourself. No need to be fancy, just an overview. ArchivesCategories |