CD4⁺ and CD8⁺ T-cell specificity of a DC-expanded T-cell line. MDDCs derived from an HIV⁺ subject were mock transfected or transfected with LysoNef mRNA and matured with cytokine cocktail. CFSE-stained autologous PBMCs were cocultured with transfected MDDCs for 14 days. The resulting T-cell lines were tested by ELISpot with an array of 27 OLPs spanning the consensus Nef sequence (not shown): T cells expanded with nef RNA responded to 6 of the OLPs, whereas no significant response was expanded by mock-transfected MDDCs. For each of the 6 peptides that elicited a strong response by ELISpot, the T-cell line was tested in an ICS assay to determine the percent specificity and degree of proliferation of CD4⁺ and CD8⁺ T cells. A peptide that elicited no response by ELISpot was used as the irrelevant control.