Analysis of HCV-specific and other T-cell responses in hypogammaglobulinemic cohort. (A) Percentage of positive responses to different antigens tested using a range of techniques. Numbers on top of each bar show absolute numbers of responding individuals tested for the various antigens. The numbers include those tested from the whole cohort, both PCR⁺ and PCR^–. Matrix ELISpot indicates antigens tested were 58 pools containing 301 overlapping peptides (P1-P58) spanning the whole HCV genome; TT, tetanus toxoid, CMV-L, cytomegalovirus lysate; A2-EBV, Epstein Barr virus peptide HLA-A2–GLCTLVAML, A2-HCV, HCV peptide HLA-A2–CINGVCWTV; and A1 HCV, HCV peptide HLA-A1–ATDALMTGY. (B) Mean magnitude of antigen-specific T-cell responses in ELISpot. Magnitude is shown as IFN-γ spot-forming cells (SFCs)/10⁶ peripheral blood mononuclear cells (PBMCs). For matrix ELISpot, mean number of reactive pools is 11 (range, 4-26 pools); mean magnitude is 137 SFC/10⁶ PBMCs (range, 45-360 SFC/10⁶ PBMCs). (A-B) The vertical line divides each graph into control antigens (left) and HCV antigens (right). (C) HCV-specific CD4⁺ proliferative responses as determined by the CFSE assay. Results are shown for 2 PCR^– hypogammaglobulinemic individuals previously exposed to HCV (first patient in top panels; second patient in middle and bottom control panels). PBMCs were stimulated for 6 days using HCV core peptide pools and HCV nonstructural proteins NS3/4 and NS5 as described. Responses represent the percentage of proliferating CD4⁺ T cells after subtraction of the background (negative). Undivided CD4⁺ T cells are detected in the top right quadrants of each FACS plot, and the CFSE signal is diluted with each cell division as the dye is distributed to the daughter cells. Numbers in the top left quadrants of each plot represent the percentage of HCV peptide– or protein-specific CD4⁺ T cells that have proliferated during the 6-day culture. One negative and 1 positive (phytohemagglutinin [PHA]) control are shown in the bottom panel. Cells are gated on CD4⁺ and Viaprobe-negative (live) cells (Viaprobe; Becton Dickinson, San Diego, CA).