CD4+ T Cell Infection Assay

Peripheral blood mononuclear cells (PBMC) were obtained through leukophereses from healthy donors as previously described [25] (link). The written consent was obtained from the donors and the study was approved by the Duke University Institutional Review Board. PBMCs were isolated using the Ficoll-Hypaque density gradients and lymphocytes were isolated by elutriation using standard techniques. CD4⁺ T cells were negatively selected from PBMCs or lymphocytes on an autoMACS Pro Separator using the CD4⁺ T cell Isolation Kit II (Miltenyi Biotec, Auburn, CA). Purified CD4⁺ T cells were stimulated for 3 days in RPMI1640 containing 10% fetal bovine serum (FBS), interleukin 2 (IL-2) (32 IU/ml; Advanced Biotechnologies, Columbia, MD), soluble anti-CD3 (0.2 µg/ml; eBioscience, San Diego, CA) and anti-CD28 (0.2 µg/ml; BD Bioscience, San Diego, CA). The stimulated CD4⁺ T cells (1×10⁶) were infected with 1000 TCID₅₀ of each virus (m.o.i≈0.001). After an incubation at 37°C for 4 hours, the cells were washed 3 times with RPMI 1640, and cultured in a 24-well plate in 600 µl of RPMI 1640 containing 10% FBS and IL-2 (32 IU/ml). Each virus was cultured in triplicates. Viral replication was monitored daily for 5 days by measuring the p24 concentration in the culture supernatant with the p24 ELISA kit (PerkinElmer, Waltham, MA).