Spatiotemporal resolution of germinal center Tfh cell differentiation and divergence from central memory CD4+ T cell fate

Transcriptome analysis of shPlekho1 and shCD19 GC-Tfh cells responding to influenza infection. Overall design: Purified CD45.1+CD45.2+ and CD45.2+ OT-II T cells were activated with 0.5 µg/ml anti-CD3 and 1 µg/ml anti-CD28 antibodies in plates pre-coated with 20 µg/ml goat anti-hamster IgG under Th0 condition. The activated cells were transduced at 18 and 48 hours with shCD19 and shPlekho1 virus-containing media, respectively. After the second transduction, cells were expanded for 2 days in complete T cell medium containing 20 U/ml recombinant human IL-2. 2 × 104 retrovirally infected shPlekho1 OT-II cells were co-transferred with shCD19 control OT-II cells intravenously into CD45.1+ SMARTA recipient mice followed by intranasal infection with PR8-OVA 2 days after cell transfer. The donor OT-II cells from the medLN of the recipient mice were analyzed by RNA-seq at day 14 p.i.