Specificity of tetramer staining and detection of HSV-specific cells in PBMCs. (a) Clone 5491.2000.48, specific for HSV-2 VP22 a.a. 49–57, stained with phycoerythrin-conjugated tetramer B7-RPR (B7-RPR–PE) and anti-CD8α. (b) Similar analysis of control clone 5491.2000.48, specific for ICP0 a.a. 743–751. (c) Similar analysis of PBMCs from subject 7282 stimulated for 12 days with VP22 a.a. 49–57. (d) Cytotoxicity of a typical clone, 7282.12, derived after sorting the cells in c for high expression of CD8α and tetramer binding. Targets were autologous EBV-LCLs either untreated (diamonds), infected with HSV-2 (squares), or pulsed with peptide VP22 a.a. 49–57 (circles). (e) Clone 5491.2000.81 stains with tetramer B7-APA-PE. (f) Control clone 5491.2000.48 does not stain with tetramer B7-APA-PE. (g) Clone 5491.2000.81 kills autologous EBV-LCLs infected with HSV-2 (triangles) but not HSV-1–infected (diamonds) or –uninfected EBV-LCLs (squares). (h) Lysis of autologous EBV-LCLs pulsed with peptide ICP0 a.a. 743–751 by clone 5941.2000.81. (i) CD8α-high cells in whole PBMCs of HLA B7–expressing subjects analyzed for binding of tetramer B7-RPR-PE. Integers above bars show number of replicate aliquots of PBMCs stained. Bar heights are means, and error bars represent standard deviations. For some subjects, staining used PBMCs thawed on two separate days (A and B) but obtained at a single phlebotomy. Controls (con) 1–3 are HSV-2–infected but HLA B7-negative; controls 4–6 are HSV-2–uninfected and HLA B7-positive.