Lysosomal targeting enhances the expansion of Nef-specific CD4⁺ T cells from PBMCs. MDDCs were derived from blood of 9 chronic HIV⁺ subjects, transfected with consensus LysoNef or CytoNef constructs, matured, and cocultured with autologous PBMCs for 12 to 14 days. To determine percent specificity of expanded T cells, resulting T-cell lines were tested in an ICS assay with the consensus pool of overlapping Nef peptides or with no added antigen. Results from a representative subject are shown in panel A; values within the gate represent the percentage IFN⁺ of CD4⁺ or CD8⁺ T cells. To determine the net response, the background response was subtracted from the response to peptide pool; thus for LysoNef and CytoNef, respectively, the net responses in panel A are 1.9% and 0.1% of CD4⁺ T cells and 0.9% and 2.9% of CD8⁺ T cells. (B) CD4 and CD8 responses are shown for all 9 subjects; each subject is represented by one symbol. The LysoNef construct expanded a greater CD4 response than the CytoNef construct for 9 of 9 subjects. The magnitudes of responses to LysoNef and CytoNef were compared using the Wilcoxon matched-pairs test (P values as shown). Lysosomal targeting produced a significant increase in CD4 responses compared with cytoplasmic targeting. Conversely, there was no significant difference in the magnitudes of the CD8 responses.