Targeting consensus Nef protein to the secretory/endosomal pathway enhances presentation of class II–restricted epitopes to CD4⁺ T cells. (A) Diagram of genetic constructs to target Nef expression to the lysosome, the secretory pathway, or the cytoplasm. Constructs consist of an N-terminal ER-translocation signal sequence, the consensus nef ORF, and a C-terminal LAMP-1 lysosomal targeting motif, as shown. (B) To measure the effect of secretory/endosomal targeting on the presentation of a class II–restricted Nef epitope, BLCLs were transfected with mRNA encoding each of the constructs shown in panel A or were mock transfected. The response of a Nef-specific CD4⁺ T-cell line (recognizing the consensus epitope KFDSRLAFHHMARELH) was measured by ICS. BLCL and CD4 line were derived from the same subject. As a positive control for antigen expression, the same BLCLs, which are HLA B57⁺, were used to stimulate a nonautologous B57-restricted CD8⁺ clone specific for the HW9 epitope HTQGYFPDW. Results are representative of 3 experiments. (C) Rapid and prolonged presentation of a class II–restricted epitope by APCs transfected with lysosome-targeted Nef antigen. BLCLs were transfected with lysosome-targeted Nef chimera, mock transfected, or loaded with 10 μg/mL of cognate peptide or irrelevant peptide and washed 3 times. APCs were then cultured for the indicated time period before being used to stimulate a Nef-specific CD4⁺ T-cell line in an ICS assay. Value on the x-axis indicates the length of time between transfection of the target and addition of BFA to the ICS assay. Results are representative of 2 experiments.