Anti cd8 fitc clone t8

PBMCs were stained using HLA-A^*02:01 Dextramer-RPE [GPC3_144–152 (FVGEFFTDV), HIV_19–27 (TLNAWVKVV) or negative control; Immudex] and HLA-A^*24:02 Dextramer-RPE [GPC3_298–306 (EYILSLEEL), HIV_583–591 (RYLKDQQLL); Immudex] for 15 min at room temperature, followed by anti-CD8-FITC (clone T8, Beckman Coulter), anti-PD-1-APC (clone EH12.2H7, BioLegend), or isotype control-APC (clone MOPC-21, BioLegend) for 20 min at 4°C. Flow cytometry was performed using a FACSCanto II (BD Biosciences).

MHC class I tetramers specific to survivin peptide epitopes were used to assess the presence of survivin-specific T cells generated by survivin vaccine (SurVaxM) stimulation, as described above. PBMC from vaccinated animals were isolated following immunization with SurVaxM. Becton Dickinson MHC class I tetramers (Chicago, IL) were designed to recognize T cell receptors that bind the core CTL epitope of SurVaxM [21] (link). Survivin-specific R-PE-labeled tetramers were incubated with PBMC for 10 minutes at 25°C. Samples were incubated with anti-CD8 FITC (clone T8, Beckman Coulter, Brea, CA) for 20 minutes at 4°C. Cells were fixed for analysis by flow cytometry. CD8+ cells that were doubly labeled were considered positive for tetramer, ovalbumin loaded tetramers were utilized as a negative control. Data were analyzed using FCS Express software. Analysis was based upon gating on CD8+ T cells only.