Soluble α cd3 and α cd28 antibodies

Spleens were collected from mice at the time of perfusion. Spleens were mashed and red blood cells were lysed using Red Cell Lysis Buffer (Sigma, St. Louis, MO) to obtain a single-cell suspension of splenocytes. Splenocytes were stimulated with either Phytohaemagglutinin (PHA, Sigma, 4 μg/ml) or with 1 μg/mL soluble α-CD3 and α-CD28 antibodies (BD Biosciences) and suspended in complete RPMI 1640 media (Lonza) supplemented with 10% FBS (Gemini Bio Products, West Sacramento, CA), 1% penicillin/streptomycin (Sigma), and 20 U/mL IL-2 (NIH AIDS Reagent Program). Cultures were maintained for 14 days. To determine if harvested splenocytes released replication competent virus supernatant was collected through centrifugation at 1500 rpm for 5 min, further centrifuged at 5000 rpm for 5 min to clear off cell debris and incubated with new PBMCs from same donor used for mouse reconstitution. These cells were stimulated with α-CD3 and α-CD28 in complete RPMI overnight prior to splenocyte supernatant addition and maintained for 7 days.

PBMCs were isolated from HIV sero-negative human donor venous blood and isolated using SepMate PBMC Isolation (STEMCELL Technologies). Cells were cultured overnight with 20u/ml IL-2 (NIH AIDS Reagent Program) and 1 μg/mL soluble α-CD3 and α-CD28 antibodies (BD Biosciences) in Roswell Park Memorial Institute medium 1640 (RPMI; Thermo Fisher) supplemented with 10% HI-FBS serum (HI-FBS; Sigma) and 1% penicillin-streptomycin (Thermo Fisher). Cells were maintained in a 5% CO₂ humidified atmosphere at 37°C. PBMCs were then infected with 2ng/1×10⁶ cells/ml of HIV-1-GFP. Cells were washed and maintained for one week in culture before reconstitution of 2 × 10⁷ cells per neonatal animal xenotransplanted with uninfected NHAs.