Naive CD4+ T Cell Isolation and Polarization

Naïve CD4⁺ T cells were obtained from pooled spleen and peripheral lymph nodes as previously described (18 (link)). Briefly, cells were purified by nylon wool and percoll density gradient separation. CD4 T cells were isolated by positive CD4 MACS selection (Miltenyi). Resulting CD4 cells routinely expressed a characteristic naive phenotype (small size, CD62L^hi, CD44^lo and CD25^lo) >97% TcR⁺. T_H1-polarized effectors were generated in vitro as described (20 (link)). Briefly, naïve Il2^−/− CD4 T cells were cultured with an equal number of irradiated APC (2x10⁵ per mL) in the presence of exogenous IL-2 (20 ng per mL), 2 ng per mL IL-12 (Peprotech), 10 μg per mL anti-IL-4 antibody (11B11; Bioxcell), and 5 μM OVA_II peptide. In vitro-primed memory cells were obtained by thoroughly washing effector cultures at 4 days and re-culturing the cells in fresh media for at least 3 days in the absence of Ag and exogenous cytokines. Live cells were isolated by Lympholyte separation (Cedarlane). All donor CD4 T cells were adoptively transferred in 200 μl phosphate buffered saline (PBS) by intravenous (i.v.) injection. A number of donor cells previously determined to be detectable at the memory phase, 2 x 10⁶, was transferred. Donor cell injection and viral infection occurred on the same day.