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.

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Figure 1: Schematic overview of retroviral transduction experiments in mouse T cells.
Figure 2: Enrichment of RV-transduced CD8+ T cells before transfer improves the efficiency of retroviral experiments in vivo.
Figure 3: Reduced cell viability after flow-cytometric sorting leads to instability of transferred T cells in vivo.
Figure 4: Optimal number of transferred P14 cells in Percoll density enrichment in LCMV Arm infection.
Figure 5: Optimal timing and facilitator for RV transduction to CD8+ T cells.
Figure 6: Comparing the utility of six different RV reporter genes for the study of CD8+ T-cell stability and memory differentiation in vivo.
Figure 7: Pre-fixation of RV-transduced T cells prevents the loss of RV-derived marker detection.

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Acknowledgements

We thank M.A. Ali for animal care; S. Adamski for technical assistance; K. Lewy for plasmid work; J.E. Wu for protocol verification; T. Yamada and K. Rome for comments on RV transduction of CD4+ T cells; and W.S. Pear (University of Pennsylvania), A. Bhandoola (Center for Cancer Research, National Cancer Institute), S.L. Reiner (Columbia University) and D.P. Beiting (University of Pennsylvania) for plasmids, cell line, template protocols and comments. This work was supported by German Research Foundation fellowship BE5496/1-1 (to B.B.), National Institutes of Health (NIH) grants F30AI129263 (to O.K.) and AI105343, AI112521, AI115712, AI108545, AI117950, AI117718, AI082630, AI095608 (to E.J.W.), U.S. Broad Agency Announcements Grant HHSN272201100018C (to E.J.W.) and funding from the Parker Institute for Cancer Immunotherapy (to E.J.W.).

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Authors

Contributions

M.K. and E.J.W. designed the protocol. M.K. and J.K. made the RV stock. J.K. prepared mouse T cells and analyzed flow cytometry data. M.K. optimized density centrifugation. Z.C., J.J. and O.K. optimized in vitro T-cell stimulation and RV transduction methods. M.K., J.K., Z.C., J.J., O.K., B.B., E.S., J.A. and L.M.M. performed the in vivo experiments. J.A. and L.M.M. developed and verified a new fixation method for RV-transduced cells. M.T. and S.U. contributed to the development of a new Kusabira Orange 2 retrovirus. J.K. prepared all figures. M.K. and E.J.W. wrote the manuscript.

Corresponding author

Correspondence to E John Wherry.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Additional two examples of activated CD8+ T cell enrichment with Percoll centrifugation. (Related to Figure 2b,c)

As shown in Fig.1 and 2b,c, in vitro activated CD8+ T cells were enriched at day 1 using 30% and 60% Percoll centrifugation (a and b: repeat 1 at 18 hrs after stimulation, c and d: repeat 2 at 25hrs after stimulation). (a and c) Flow plots that are gated on the 7AAD negative CD8 positive population show enrichment step from input (left) to shortly after Percoll separation at day 1 (right). (b and d) Graph showing recovery of small (‘resting’) and large (‘blast’) size cells in each interface and bottom after Percoll centrifugation. Note that the recovery of large-sized cells (black bar) was 40% (18 hrs-stimulated; Supplementary Fig. 1b), 60% (22 hrs; Fig. 2c), and 43.4% (25 hrs; Supplementary Fig. 1d).

Supplementary Figure 2 Staining and gating strategy to detect LCMV gp33-specific CD8+ T cells in vivo used in this study.

At the indicated time points in each Figure legend, peripheral blood or spleens were harvested and single cell suspensions were prepared using ACK lysis buffer, followed by staining with anti-CD8, CD44, CD45.1, CD45.2 antibodies, and Dbgp33 tetramer. In some experiments, to detect only transferred P14 cells, TCR Vα2 was used instead of Dbgp33 tetramer. Antigen-specific CD8+ T cells are identified by first gating on singlet cells (FS-A and FS-H), and then on live cells (7AAD negative), and lymphocytes (FS-A and SS-A). Antigen–specific CD8+ T cells were identified by successively gating on CD8, Dbgp33 tetramer (or TCR Vα2) and CD44 positive populations. Transferred P14 cells (and endogenous gp33-specific CD8+ T cells in case of Dbgp33 tetramer staining) were further subgated on the basis of CD45.1 and CD45.2 congenic marker expression. RV-reporter expression was analyzed on the final P14 cell gate. In selected experiments, differentiation and function of effector and memory P14 cells were evaluated by additional surface marker staining such as CD127 and KLRG1 and standard intracellular cytokine staining after 5 hrs restimulation with cognate peptide. Representative blood sample used in Figure 2d as no Percoll condition is shown.

Supplementary Figure 3 Small-sized cell frequency at adoptive transfer inversely correlates with RV-transduction efficiency in vivo. (Related to Figure 2a–g)

(a) Experimental design. Donor P14 cells and recipient mice were stimulated in vitro and infected with LCMV Arm at day 0, respectively. At day 1, activated cells were harvested from the flask and analyzed immediately by flow cytometry to determine the ratio of small-sized cells to large-sized cells in ‘Input’ population. An aliquot of activated cells was set aside for a no Percoll control prior to Percoll centrifugation. After Percoll, interface (blasting cells) and bottom (resting cells) cells were separately collected, and a re-mixture of the interface and bottom cells was made. Then the equal numbers of the 4 populations (3x106 cells per well) were transduced with empty-GFP RV. After 4 hrs, aliquots of the 4 populations were checked by flow cytometry (‘transfer’ sample), and 1x105 cells of each population were adoptively transferred to infected mice. RV-transduction efficiency and engraftment of RV-transduced cells were analyzed at day 2 and 8, respectively. (b) Flow plots gated on live P14 cells show the composition of blasting and resting cells in input (left) and 4 populations at transfer (right-left) at day 1, and RV transduction efficiency at day 2 (right-middle), and frequency of RV-transduced cells at day 8 (right-right). (c) Graph showing ‘Retention rate’ of RV-transduced cells among total P14 cells from day 2 (in vitro) to day 8 (blood). Bars show mean ± s.e.m. *p<0.005 (two-tailed t test); NS p>0.05. All data in this figure are from one experiment (n=5 per group). All animal experiments used in this Figure were in accordance with the Institute Animal Care and Use Guidelines for the University of Pennsylvania.

Supplementary Figure 4 Activated CD4+ T cells can be enriched by Percoll density centrifugation. (Related to Figure 2a–c and 5a)

Polyclonal CD4+ T cells were prepared from C57/Bl6 spleens using EasySep Mouse CD4+ T Cell Isolation Kit (STEMCELLS) and stimulated in vitro with anti-CD3ɛ (final 1 μg/mL), anti-CD28 (final 0.5 μg/mL) antibodies and rIL-2 (final 100 U/mL) as shown in Fig.1. CD4+ T cells were spin-transduced with empty-GFP RV using Polybrene (final 4 μg/mL) and tissue culture treated plates at day 1 (a) or indicated timing (d) after stimulation without enrichment of blasting cells, and analyzed for GFP expression one day after RV-transduction. Some of CD4+ T cells were processed by density centrifugation with 30% and 60% Percoll layers at day 1 (b, c). Data are representative of two independent experiments (one technical replicate per experiment). (a) Small sized ‘resting’ CD4+ T cells are mostly RV negative. (b) Density centrifugation enriches activated ‘blasting’ CD4+ T cells at day 1. Flow plots that are gated on 7-AAD negative CD4 positive population show enrichment step from input (left) to shortly after Percoll separation at day 1 (right). (c) Graph showing recovery of small (‘resting’) and large (‘blast’) size CD4+ cells in each interface and bottom after Percoll centrifugation. (d) RV transduction efficiency to CD4+ cells peaks at 48 hours after in vitro stimulation.

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Kurachi, M., Kurachi, J., Chen, Z. et al. Optimized retroviral transduction of mouse T cells for in vivo assessment of gene function. Nat Protoc 12, 1980–1998 (2017). https://doi.org/10.1038/nprot.2017.083

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