Author: Han, Lu ; Liu, Xue ; Yuan, Long ; Wang, Yao ; Zhang, Kuang ; Li, Tiepeng ; Li, Linlin ; Gao, Quanli ; Qin, Peng ; Guo, Jindong ; Zhang, Yong ; Xu, Benling ; Chen, Guangyu ; Zhang, Mengyu ; Yuan, Peng ; You, Hongqin

Adoptive cell therapy (ACT) has revolutionized the treatment of patients with cancer. The success of ACT depends largely on transferred T cell status, particularly their less-differentiated state with stem cell-like properties, which enhances ACT effectiveness. Stem cell-like memory T (T_SCM) cells exhibit continuous self-renewal and multilineage differentiation similar to pluripotent stem cells. T_SCM cells are promising candidates for cancer immunotherapies, whereas maintenance of a more stem-cell-like state before transfer is challenging. Here, we established a highly efficient protocol for generating CD8⁺ T_SCM cells from peripheral blood mononuclear cells (PBMCs). The process involved activating PBMCs using anti-CD3 monoclonal antibody and RetroNectin, followed by a transient-resting culture period (24 h) and subsequent long-term expansion in vitro with interlukien-2. We report that this transient-resting culture after activation preserves CD8⁺ T cells in a stem memory phenotype (CD95⁺ CD45RA⁺ CCR7⁺) compared to the conventional culture method. Further, this approach reduces the expression of T cell immunoglobulin mucin-3, an exhaustion marker, and increases the expression of T cell factor-1, a master regulator of stemness even after long-term culture compared to the conventional culture method. In conclusion, our study presents a simplified and cost-effective method for generating and expanding CD8⁺ T_SCM cells ex vivo. This approach streamlines the optimization of cancer immunotherapy using ACT.

01 Jan 2024·Methods in Molecular Biology

Expansion and Retroviral Transduction of Primary Murine T Cells for CAR T-Cell Therapy

Article

Author: Short, Lauralie ; Evgin, Laura ; Savage, Gillian ; Loos, Pauline

The development of chimeric antigen receptor (CAR) T cells has been a revolutionary technology for the treatment of relapsed and refractory leukemias and lymphomas. The synthetic CAR molecule redirects T cell function toward tumor surface-expressed antigens through a single-chain variable fragment (scFv) fused to CD3z and intracellular costimulatory domains. Here, we describe a protocol for the generation of CAR T cells using primary mouse T cells and a gammaretroviral vector encoding a CAR transgene. This protocol outlines several transduction and expansion methods based on the use of two transduction enhancers, RetroNectin^® and Vectofusin^®-1, and cell culture systems such as conventional plates or G-Rex^® devices.

01 Jan 2022·BIO-PROTOCOL

High-Efficiency Retroviral Transduction for Type 1 Regulatory T Cell Differentiation

Article

Author: August, Avery ; McGee, Michael C ; Huang, Weishan

Type 1 regulatory T (Tr1) cells are an immunoregulatory CD4 ⁺ Foxp3- IL-10 ^high T cell subset with therapeutic potential for various inflammatory diseases. Retroviral (RV) transduction has been a valuable tool in defining the signaling pathways and transcription factors that regulate Tr1 differentiation and suppressive function. This protocol describes a method for RV transduction of naïve CD4 ⁺ T cells differentiating under Tr1 conditions, without the use of reagents such as polybrene or RetroNectin. A major advantage of this protocol over others is that it allows for the role of genes of interest on both differentiation and function of Tr1 cells to be interrogated. This is due to the high efficiency of RV transduction combined with the use of an IL10 ^GFP /Foxp3 ^RFP dual reporter mouse model, which enables successfully transduced Tr1 cells to be identified and sorted for functional assays. In addition, this protocol may be utilized for dual/multiple transduction approaches and transduction of other lymphocyte populations, such as CD8 ⁺ T cells.

100 Deals associated with Retronectin-induced T cell therapy (Takara Bio)

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Indication	Highest Phase	Country/Location	Organization	Date
Gastrointestinal Neoplasms	Phase 2	Japan	Kyoto Prefectural University of Medicine	-
Gastrointestinal Neoplasms	Phase 2	Japan	Takara Bio, Inc.	-
Liver Cancer	Phase 2	Japan	Takara Bio, Inc.	-
Liver Cancer	Phase 2	Japan	Kyoto Prefectural University of Medicine	-
Lung Cancer	Phase 2	Japan	Takara Bio, Inc.	-
Lung Cancer	Phase 2	Japan	Kyoto Prefectural University of Medicine	-
Neoplasms	Phase 2	China	Tianjin Cancer Institute	-
Neoplasms	Phase 2	China	Takara Bio, Inc.	-
Pancreatic Cancer	Phase 2	Japan	Kyoto Prefectural University of Medicine	-
Pancreatic Cancer	Phase 2	Japan	Takara Bio, Inc.	-

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