2.5. Isolation of CD3+ T cells and naïve CD4+ T cells
After 8-week-old C57BL/6J mice were anesthetized with isoflurane, the spleen was taken out, and the spleen single-cell suspension was prepared following the previous method (Flaherty and Reynolds, 2015), and the spleen cells were treated following the standard protocol of Naïve CD4+ T Cell Isolation Kit (19765A, STEMCELL) to isolate naïve CD4+ T cells. Then they were cultured in a plate coated with CD3 antibody (100339, Biolegend), and continued to be cultured in the presence of CD28 antibody (102115, Biolegend) until be used. For GFP mouse spleen CD3+ T cells, after obtaining spleen single cell suspension in the same manner as above, Biotin anti-mouse CD3 Antibody (100303, Biolegend) and Anti-Biotin MicroBeads (5190902231, Miltenyi Biotec) were added successively according to the product protocals. The LS column magnetic bead sorting method was used to obtain CD3+ T cells with a purity of more than 90%.