CD8⁺ T cells are detected in subjects with low CD4⁺ counts after in vitro expansion. (A) Sample negative tetramer stain in subject D19-03-29 with HLA A*0101 allele; tetramer binds A1-NS3-ATDALMTGY. This subject did not have an ELISpot response to this epitope. (B) Immunodominant ex vivo HCV-specific response in subject D3-01-49, tetramer binds A1-NS3-ATDALMTGY, representing 3.6% of CD8⁺ cells, corresponding with an ELISpot response of 3000 SFC/10₆ PBMCs. (C) This panel demonstrates that tetramer binding is specific to this antigen because after a 6-hour incubation more than 30% of tetramer^hi cells secrete IFN-γ in the presence of appropriate peptide (gated on CD8^hi). (D) Tetramer stain after 14 days of in vitro stimulation in subject C9-02-05 (CD4 = 207 cells/mm³) bearing A*0201 allele with peptide 1406-1415, KLVALGINAV. No ex vivo response by ELISpot or tetramer was detected. (E) Expansion of cells specific to novel epitope contained within E1-TQLRRHIDDLVGSATL in subject C41-03T (CD4 = 92 cells/mm³) after 14 days, demonstrated by IFN-γ ICS on cell line. Ex vivo ELISpot response equaled 25 SFC/10⁶ PBMCs, representing an extremely low level response initially below the cutoff of significance for earlier ex vivo analyses but capable of proliferation in vitro to 3.7% of CD8^hi cells.