T Cell Receptor (TCR) Gene Transfer with Lentiviral Vectors Allows Efficient Redirection of Tumor Specificity in Naive and Memory T Cells Without Prior Stimulation of Endogenous TCR

Abstract

We investigated the possibility of introducing exogenous T cell receptor (TCR) genes into T cells by lentiviral transduction, without prior stimulation of endogenous TCR with anti-CD3. TCR transfer is used to impose tumor antigen specificity on recipient T cells, but sustained activation required for retroviral transduction may affect the clinical efficacy of engineered T cells. Cytokine stimulation makes T cells susceptible to lentiviral transduction in the absence of TCR triggering, but this advantage has never been exploited for TCR transfer. Autoimmune diseases are a source of high-affinity TCRs specific for self/tumor antigens. We selected, from a patient with vitiligo, a Mart1-specific TCR based on intrinsic interchain pairing properties and functional avidity. After lentiviral transduction of human peripheral blood mononuclear cells, preferential pairing of exogenous alpha and beta chains was observed, together with effective recognition of Mart1(+) melanoma cells. We tested transduction efficiency on various T cell subsets prestimulated with interleukin (IL)-2, IL-7, IL-15, and IL-21 (alone or in combination). Both naive and unfractionated CD8(+) T cells could be transduced without requiring endogenous TCR triggering. IL-7 plus IL-15 was the most powerful combination, allowing high levels of transgene expression without inducing T cell differentiation (34 +/- 5% Mart1-TCR(+) cells in naive CD8(+) and 16 +/- 6% in unfractionated CD8(+)). Cytokine-prestimulated, Mart1-redirected naive and unfractionated CD8(+) cells expanded better than CD3-CD28-prestimulated counterparts in response to both peptide-pulsed antigen-presenting cells and Mart1(+) melanoma cells. This strategy allows the generation of tumor-specific T cells encompassing truly naive T cells, endowed with an intact proliferative potential and a preserved differentiation stage.

miR-214 Coordinates Melanoma Progression by Upregulating ALCAM through TFAP2 and miR-148b Downmodulation

“…Hynes (Massachusetts Institute of Technology, Cambridge, MA) and maintained as described previously (17,18). WK-Mel, SK-Mel-28, and human umbilical vein endothelial cells (HUVEC)-GFP were provided by P. Circosta (Molecular Biotechnology Center, Torino, Italy), L. Poliseno (Core Research Laboratory -Istituto Toscano Tumori, CRL-ITT, Pisa, Italy), and L. Primo (Institute for Cancer Research and Treatment, Candiolo, Italy), respectively and maintained as described (19)(20)(21). MDA-MB-231 were from American Type Culture Collection.…”

T-cells “à la CAR-T(e)” – Genetically engineering T-cell response against cancer

“…The most common viral platforms of expression are based on either γ-retroviral vectors such as the MSCV (Mouse stem cell virus) -based or MPSV (myeloproliferative sarcoma virus) [13,264] and lentiviral vectors [265][266][267][268]. The latter may represent a better alternative than γretroviral vectors due to their ability to transduce non-dividing cells, to be more resistant to gene silencing [269] and to their safer integration site profile [60,270].…”

Adenoviral Transduction of Naive CD4 T Cells to Study Treg Differentiation

“…For example, electroporation and retroviral transduction are only effective in activated T cells. In contrast to initial expectations, lentiviral transduction, which is typically effective in resting cells, requires pre-activation of naive T cells by cytokines 13 . Furthermore, the transfer of cDNA or mRNA during electroporation involves depolarization of the plasma membrane, which itself confers features of T cell activation and may even mobilize Ca 2+ signaling and activate NFAT proteins (unpublished observation and ref.…”