Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function

Abstract

Retroviral (RV) expression of genes of interest (GOIs) is an invaluable tool and has formed the foundation of cellular engineering for adoptive cell therapy in cancer and other diseases. However, monitoring of transduced T cells long term (weeks to months) in vivo remains challenging because of the low frequency and often poor durability of transduced T cells over time when transferred without enrichment. Traditional methods often require additional overnight in vitro culture after transduction. Moreover, in vitro-generated effector CD8+ T cells enriched by sorting often have reduced viability, making it difficult to monitor the fate of transferred cells in vivo. Here, we describe an optimized mouse CD8+ T-cell RV transduction protocol that uses simple and rapid Percoll density centrifugation to enrich RV-susceptible activated CD8+ T cells. Percoll density centrifugation is simple, can be done on the day of transduction, requires minimal time, has low reagent costs and improves cell recovery (up to 60%), as well as the frequency of RV-transduced cells (~sixfold over several weeks in vivo as compared with traditional methods). We have used this protocol to assess the long-term stability of CD8+ T cells after RV transduction by comparing the durability of T cells transduced with retroviruses expressing each of six commonly used RV reporter genes. Thus, we provide an optimized enrichment and transduction approach that allows long-term in vivo assessment of RV-transduced T cells. The overall procedure from T-cell isolation to RV transduction takes 2 d, and enrichment of activated T cells can be done in 1 h.

Optimized gene engineering of murine CAR-T cells reveals the beneficial effects of IL-15 coexpression

“…Lentivirus, however, has demonstrated poor gene transfer in murine T cells, likely due to impaired completion of reverse transcription and of nuclear import of the viral preintegration complex (Baumann et al, 2004;Tsurutani et al, 2007). Most examples of efficient murine T cell retroviral transduction are for small and easily expressed reporter genes like GFP (Kurachi et al, 2017;Zhang et al, 2003) or 1G-CARs comprising the CD3ζ endodomain only (Lee et al, 2009). Retrovirus-mediated expression of 2G-CARs has proven less robust both in terms of percentage transduction and expression level per T cell (Kochenderfer et al, 2010;Davila et al, 2013;Fu et al, 2013).…”

TOX transcriptionally and epigenetically programs CD8+ T cell exhaustion

“…For retroviral (RV) transduction, CD8 + T cells were enriched from spleens of donor mice using an EasySep magnetic negative selection kit (Stem Cell Technologies) and transduced as described previously 55 . In brief, cells were resuspended at 10 6 /ml in “complete RPMI (cRPMI)”: RPMI 1640 supplemented 10% FBS, 50μM β-mercaptoethanol, 100U/ml penicillin, 100U/ml streptomycin, non-essential amino acids (Invitrogen), sodium pyruvate (Invitrogen), and HEPES buffer (Invitrogen).…”

Donor-specific chimeric antigen receptor Tregs limit rejection in naive but not sensitized allograft recipients

“…The resulting A2‐ and HER2‐CARs were cloned into a murine stem cell virus (MSCV)–based retroviral vector that encoded a transduction marker downstream of an internal ribosomal re‐entry sequence. Initial experiments were done with vectors encoding surface‐expressed truncated human nerve‐growth‐factor receptor (ΔNGFR) and then switched to vectors encoding the fluorescent intracellular monomeric Kusabira‐Orange2 (mKO2) protein as the transduction marker as described by Kurachi et al Viral particles were produced by using the Platinum‐E (Plat‐E) Retroviral Packaging Cell Line according to the manufacturer recommendations (Cell Biolabs).…”