Figure 6

IA^s/PLP 139-151 dextramers detect PLP-specific cells ex vivo. (A) LNC. Groups of SJL mice were immunized with PLP 139-151 and killed 10 days later; the draining LN were harvested to prepare single cell suspensions. CD4 cells were enriched from LNC by negative selection based on magnetic bead separation. The cells were treated with or without NASE, followed by staining with PLP 139-151 or control tetramers or dextramers, followed by a cocktail containing anti-CD4 and 7-AAD and anti-CD25 or anti-CD44. After tet⁺ and dext⁺ live cells (7-AAD^-) cells were acquired by FC, they were analyzed with respect to CD4, CD25 or CD44 subsets. Top panel shows representation of flow cytometric analysis, whereas the lower left panel indicates mean ± SEM values obtained from five individual experiments each involving two mice. (B) MNC from CNS tissues. Groups of three mice were immunized with PLP 139-151 in CFA to induce EAE, and animals showing paralytic signs were killed to harvest brains and spinal cords. The tissues were processed to obtain MNC by percoll density-gradient separation, and the cells were stained with PLP 139-151 or control tetramers or dextramers and anti-CD4 and 7-AAD. After acquiring the cells by FC, percentages of tet⁺ and dext⁺ cells were determined in the live CD4 population. Representative data from two individual experiments, each involving 2 to 3 mice, are shown. Control, TMEV 70-86.