Overcoming the challenge of T cells gene transfer with lentiviral vectors
Vectalys extensive in vitro transduction data show that the use of highly concentrated (1E9 TU/mL) and pure (1E8 TU/mg of proteins) lentiviral vectors increases significantly the transduction level of naive and regulatory CD4+ T cells.
Naive and activated human total T lymphocytes
Human naive or activated (CD3/CD28) T lymphocytes are transducted by a ZsGreen-expressing lentiviral vector using a range of MOI (Multiplicity of Infection) from 0 to 50 in presence of polybrene 4 µg/mL for 5h at 37°C, 5% CO2.
Five days post-tranduction, the % of ZsGreen positive cells and Fluorescence Intensity are analyzed by flow cytometry.
Naive and activated murine CD4+ T lymphocytes
Naive CD4+ lymphocytes are obtained from lymph nodes by negative selection (CD8b, CD45R (B220), CD11b (Mac1), Ter-119 and CD16/CD32). Purified CD4 cells are then labelled with antibodies against Lineage markers (CD8a, NK1.1, gdTCR and CD25) and with an anti-CD44 antibody.
Naive CD4+ T lymphocytes are FACS-sorted as CD44lo Lin-cells and transducted by a ZsGreen-expressing lentiviral vector using a range of MOI from 0 to 30 in presence of polybrene 4µg/mL for 5h at 37°C, 5% CO2.
Four days later, the % of ZsGreen+ cells and Fluoresence Intensity are analyzed by flow cytometry. Cell activation (anti-CD3/CD28 coupled beads) is performed 16h after transduction as a control.
T cells keep their phenotype and specific markers expression
Furthermore, transduced T cells exhibit an original cell phenotype without any changes of T cells specific markers expression (CD44, CD69, CD25, Ly-6C) attesting that the composition of this pure batch of lentiviral vectors does not have any impact on T cells phenotype.
Naive phenotype of murine CD4+ T lymphocytes is maintained
Naive CD4+ T lymphocyte are transduced by a ZsGreen-expressing lentiviral vector at MOI 10 in presence of polybrene 4µu/mL for 5h at 37°C, 5% CO2.
Four days later, the expression pattern of naive markers (CD44, CD25, CD69 and Ly-6C) and Fluorescence Intensity are analyzed by flow cytometry. Cell activation (anti-CD3/CD28 coupled beads) is performed 16h after transduction as a control. The naive phenotype of T cells is characterized by a low expression of CD44, which is an indicative marker for effector-memory T-cells, no expression of CD25 which is expressed specifically on stimulated T cells, nor CD69 which is an early marker of activation. Ly6 is down regulated on activated cells and it is thus found expressed on these naive T cells.
Regulatory murine CD4+ T lymphocytes
GFP-expressing CD4+ regulatory T lymphocyte (Tregs) are obtained from FoxP3-GFP mice. Tregs are FACS-sorted as GFP positive cells, then transduced by a tdTomato-expressing lentiviral vector at MOI 10 in presence of ploybrene 4µu/mL for 5h at 37°C, 5% CO2. Five days post-transduction, the % of tdTomato positive cells and Fluorescence Intensity are analyzed by cytometry.
Lentiviral vectors: a major technological leap
Our data bring out that the composition of lentiviral vectors in terms of titer, specific activities and purity are the success keys to ensure transduction of murine T cells. This major technological leap will allow to tightly control expression of various modifiers of these cells and thus paves the way to design original tools and develops cell-based cancer models.
Gene transfer using concentrated and highly purified lentiviral vectors is the best way to obtained a stable expression of the sequence of interest (cDNA, shRNA, miRNA, CRISPR/Cas9). Compared to transfection or standard lentiviral vectors, they allow time, money, and energy saving, providing a single tool from in vitro to in vivo applications.
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