Multiparameter ICS Assay for SIV Gag/pol Responses

Antigen-specific cellular immune responses were assessed in multiparameter ICS assays as described previously^{57 (link),58 (link)}. Briefly, thawed cryopreserved PBMCs were co-cultured for 6 h with autologous herpes virus papio-immortalized B-LCL cells that had been infected with a recombinant vaccinia virus expressing SIV Gag/pol. They were incubated with brefeldin A (BD) for the final 5 h of stimulation. Then immunostaining was performed using a CytofixCytoperm kit (BD) and the following monoclonal antibodies: anti-CD3 (clone SP34-2, Alexa700; BD), anti-CD4 (clone L200, APC-H7; BD), anti-CD8 (clone DK25, APC; Dako), anti-IFN-γ (clone 4 S.BS, PE; BD), anti-TNF-α (clone MAM11 PE-Cy7), and anti-IL-2 (clone MQ-1-17H12). A fixable-dead-cells stain kit (Invitrogen) was used to exclude dead cells from the analysis. Samples were fixed with 1% freshly prepared paraformaldehyde for at least 1 h and then analyzed in a FACSCanto II flow cytometer (BD). Data were analyzed using FACSDiVa (BD) software.