In vitro T-cell activation

Spleen or lymph node single-cell suspensions were prepared from EF4.1 or EVα3 mice and 5 × 10⁵ cells per well were stimulated in 96-well plates with the indicated amount of the index env_122–141 epitope (DEPLTSLTPRCNTAWNRLKL), the shorter env_124–138L epitope (PLTSLTPRCNTAWNR) or the altered peptides epitopes env_124–138Y (PLTSYTPRCNTAWNR) and env_124–138I (PLTSITPRCNTAWNR). Hybridoma cell lines were generated and stimulated as previously described²³. T-cell activation was assessed 18 h later by flow cytometric detection of CD69 or CD44 (eBiosciences).