Figure 4

In spite of high sensitivity, not all antigen-specific cells are detected by IA^s/PLP 139-151 dextramers. (A) Pre-FC-sorting. PLP-reactive cells generated from SJL mice immunized with PLP 139-151 were stained with PLP 139-151 or control tetramers or dextramers, followed by anti-CD4 and 7-AAD. After analyzing the cells as negative or positive for tetramers and dextramers, tet^- and dext^- CD4 cells were sorted by FC, and the cells were divided into two aliquots. One of these was immediately stained with PLP 139-151 and control dextramers, and percentages of dext⁺ cells were evaluated in the live (7-AAD^-) subset. (B) Post-FC-sorting. The second aliquot of tet^- and dext^- cells was rested for two weeks and the cells were stimulated with PLP 139-151 in the presence of irradiated antigen-presenting cells; after two days, growth medium was supplemented. Viable cells were harvested on day 6 poststimulation and stained with PLP 139-151 or control dextramers and anti-CD4 and 7-AAD. After analyzing the cells by FC, dext⁺ and dext^- cells were enumerated in the live CD4 subset. Control, TMEV 70-86. Data from one of the two individual experiments each involving three mice are shown.