An optimized protocol for the retroviral transduction of mouse CD4 T cells

Abstract

Summary Transduction of primary T cells has become prominent with the introduction of chimeric antigen receptor T-cell therapy. Although there are many protocols for the transduction of human T cells, it remains a challenge to transduce murine T cells. We present an optimized protocol for the retroviral transduction of murine CD4 T cells, which overcomes major challenges including large-scale production and long-term culturing of transduced cells. The optimized protocol combines high transduction efficiency with a low rate of cell death. For complete details on the use and execution of this protocol, please refer to Eremenko et al., 2019 .

Protocol to assess cell-intrinsic regulatory mechanisms using an ex vivo murine T cell polarization and co-culture system

“…Retrovirus transduction can substantially negatively impact T-cell viability. Providing transduced CD4 + T cells with recombinant IL-2 and IL-7 may increase their viability and proliferative capacity as previously described ( Eremenko et al., 2021 ), but may negatively impact polarization towards the desired T-helper subsets. In addition, retrovirus transduction efficiency is relatively low (combined NGFR and Thy1.1 transduction efficiencies are ∼40%–60%) in our system.…”

“… Spin inoculates by centrifuging the plate at 900 × g for 90 min at 32°C with an acceleration rate of 3 and a deceleration rate of 1. CRITICAL: Centrifugation at 32°C is crucial for transduction as previously described ( Eremenko et al., 2021 ; Pampusch et al., 2020 ). CRITICAL: The retrovirus-containing plate should be sealed with plastic wrap before centrifugation to avoid the release of retroviral particles into the centrifuge.…”

“… CRITICAL: Centrifugation at 32°C is crucial for transduction as previously described ( Eremenko et al., 2021 ; Pampusch et al., 2020 ).…”

“…The workflow of this protocol is outlined in Figure 2 . Some steps are modified from previous reports ( Eremenko et al., 2021 ; Flaherty and Reynolds, 2015 ). In this section, we describe the steps for preparing the retroviral constructs, culturing the Plat-E retrovirus packaging cell line, and obtaining the appropriate mice before the start of T cell isolation and culture.…”

“…Retrovirus is not suitable for the transduction of non-proliferating cells. Caution must be exercised for retrovirus transduction of mutant T-cells that have diminished or loss of proliferative capacity ( Cepko and Pear, 2001 ; Eremenko et al., 2021 ). Retrovirus transduction can substantially negatively impact T-cell viability.…”