Figure 5. Isolation and in vitro activation of Thy1.1⁺/CD4⁺ memory cells.

A. Spleens were harvested 276 days after adoptive transfer 4×10⁷ Thy1.1⁺/CD62L^low/CD4⁺ effector T cells to tumor-free Thy1.2 mice. Thy1.2⁺ cells were depleted, and the negative fraction was separated into T_CM (CD62L^high) and T_EM (CD62L^low) subsets with MACS columns before culture activation. The percentage of Thy1.1⁺ T_EM or T_CM is indicated on the right of the dot-plot quadrants. B. On days 2, 7, 14 of culture, Thy1.1⁺ cells in each subset further were purified using MACS columns and on day 14, CD8⁺ T cells were depleted. At the completion of the in vitro activation with anti-CD3/IL-2/IL-7, cultures originally derived from T_CM or T_EM cells were analyzed for purity and phenotype.