I noticed that the lentiviral infection efficiency in primary human T cells decreased very fast and much more significantly than in normal cell lines (eg., HEK, Jurkat, tumor cells lines, etc). For example, I checked the infection efficiency by c-Myc antibody staining (FACS) of the infected T cells on Day3, Day5 and Day7 after lentiviral infection, and the c-Myc+ cell percentages were ~60%, ~40%, ~20%, respectively. I also infected primary human T cells with a different lentivirus (GFP+) and monitored the efficiency by fluorescence, and the GFP+ cell percentage also decreased from ~30% to ~10% during a week or two. The decrease is not so dramatic in other cell lines (eg., I used the same lentivirus in Jurkat and the efficiency maintained at >80% for weeks).
Is this normal? Has anyone observed thus dramatic decrease in primary T cells?
Integration of plasmid brought by lentiviral particles into the cell genome depends on the cell division status - if the cell is proliferating it will be successful, while in resting/non-actively dividing cells it will be less efficient. In cell lines such as Jurkats all cells are rapidly proliferating, so the infection efficiency stays the same irrespectively when the infection is done (and assuming that there was no problem with the virus). On the other hand, I suppose that primary human T cells were activated with anti-CD3 and -CD28 for first 2-3 days, when they increase their size start to rapidly dividing and thus become the most susceptible to the infection. Although T cells can be maintained proliferating for weeks in a medium with IL-2 and IL-15, the rate of proliferation and cell size reduces after removal from activation antibodies, which in turn leads to lower efficacy of lentiviral infection.
Hi Berislav Bošnjak thanks for the reply! I infected the primary T cells during the first 2-3 days after adding antiCD3/28 beads so they were still actively proliferating at the time of infection. (?)
Hi, it seems that I have misunderstood your problem. So, you lose transduced cells after you keep them longer in the culture. It would probably mean that transduced cells have some proliferative disadvantage over non-transduced cells. Have you considered sorting out transduced cells (if you have some fluorescent marker)? But I am not sure why it would happen only to primary T cells and not tumor cell lines.
Thanks for the reply Berislav Bošnjak !
Yes I also tried sorting out the positive cells (used MACS instead of FACS since its more gentle), and I still observed an decrease in the positive cell pencentage afterwards. Although the MACS sorting efficiency was not 100% and it was about 80% positive cells after sorting. And this number deceased to 50%, 35%, etc. afterwards in a period of a week or so. Yes we thought about the proliferative disadavantage too but it was just puzzling that it can make such a big difference and caused such fast decrease... (and only in primary human T cells)
I haven't worked with human cells, only with T cells from mice and haven't had such problems. Do you see the same effect when you tranduce the cells with control virus (e.g. only expressing a fluorescent dye)? Or exclusively with your virus? Maybe cells slience transduced protein, depending where it gets integrated - but as DNA should still be in them, you might check that with RT-PCR or DNA. Apart from that, I do not have any further suggestions.
Thanks Berislav Bošnjak . I am doing a GFP control right now. I will keep the RT-PCR idea in mind. Thank you!
Hi, Dr. Wu:
I wonder if you have solved your problem of decreasing lentiviral transduction efficiency and share me your thought. We encounter the same problem. Lv mediated primary T cell transduction goes down from 80% to 15% in 7-10 days. I will greatly appreciate if you have some insight about solving this problem.
regards
Yukai
because of suspension culture characteristic of primary T cell, tranditional lentiviruese are not suitable for transfection of primary, you could chose Ads, modified lentivirus, or drug screening firstly.
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