CD4 cell depletion enhanced the expansion and differentiation of transferred tumor-sensitized CD8 T cells. (A) CFSE staining profiles of gated CD8 cells isolated from the spleens (top panels) or lymph nodes (LN; bottom panels) from groups of tumor-bearing mice treated with 6 × 106 CFSE-labeled tumor-sensitized CD8 T cells (Sen T), AGN2a-4P vaccine (Vac), or anti-CD4 mAb treatment. The anti-CD4 mAb was administered 1 day before HSCT, and the AGN2a-4P vaccine was given 2 days after HSCT. Cells were harvested for flow cytometric analysis 4 days after HSCT. (B) Splenocytes (top panels) or LN (bottom panels) were collected from recipient mice 14 days after adoptive transfer and stained with fluorescently labeled anti-CD8, anti-CD44, and anti-CD62L mAbs. Shown are 2-color flow cytometric histograms depicting the CD44 (x-axis) and CD62L (y-axis) expression on gated CD8 T cells. Cells from normal, nontransplanted mice were included for comparison. (C) Spleens were collected 11 days after HSCT, CD8 T cell numbers counted (left panel), and absolute numbers of tumor-reactive IFN-γ–secreting CD8 T cells (right panel) were determined. **P < .01 compared with the other treatment groups. (D) Immunohistochemical staining of tumors for infiltrating CD8 T cells. Frozen sections of tumors from the indicated experimental groups were stained with CD8-specific antibody (200× magnification). All results are representative of 2 or 3 separate experiments in which splenocytes and LN cells were pooled from 3 or 4 individual mice.
