Retroviral Transduction of T Cells

The detailed protocol was described previously^{51 (link)}. PLAT-E cells were transfected with shRNAmir using TransIT-LT1 Reagent (Mirus). Retrovirus-containing supernatant was harvested after 48 h and mixed with 2-mercaptoethanol and polybrene (Millipore) for subsequent transductions. Purified naive OT-I CD8⁺ T cells were in vitro activated by anti-CD3 (145-2C11) and anti-CD28 (37.51) (eBioscience) for at least 18 h and then spinfected at 805 × g with retrovirus for 1 h at 37 °C. After 4 h incubation, the retrovirus-containing medium was replaced by T cell medium. Transduction efficiency were measured by flow cytometric analysis of ametrine-reporter after 24 h and 1 × 10⁴ shRNA transduced cells were transferred into host mice followed by Lm-OVA infection. For Ncor1 shRNA knockdown, purified P14 CD8⁺ T cells were in vitro activated and transduced by shRNA retrovirus similarly to OT-I CD8⁺ T cells. Transduced P14 CD8⁺ T cells (5 × 10⁵) were transferred into host mice followed by 1.5 × 10⁵ pfu LCMV-C13 i.p. infection, which results in an acute infection^{51 (link)}. The full hairpin sequence for shRNA: shYy1: 5′-TGCTGTTGACAGTGAGCGCCCTCCTGATTATTCTGAATAATAGTGAAGCCACAGATGTATTATTCAGAATAATCAGGAGGTTGCCTACTGCCTCGGA-3′, shNr3c1: 5′-TGCTGTTGACAGTGAGCGAATGCATGATGTGGTTGAAAAATAGTGAAGCCACAGATGTATTTTTCAACCACATCATGCATGTGCCTACTGCCTCGGA-3′.