An accelerated loss of tumor-reactive CD8 T cells in CD4-depleted hosts correlated with the failure to generate antitumor T-cell memory. Tumor-bearing mice were treated with TBI, HSCT, tumor-sensitized T-cell transfer, vaccination, and anti-CD4 mAb as depicted in Figure 4A. (A) Spleens were collected on days 7, 11, 18, 30, 45, and 65 after HSCT. The arrowheads indicate the vaccination times (2, 7, and 14 days after HSCT). Some mice were revaccinated (“Recall” vaccine) on day 65 after HSCT, and spleens collected 5 days later. Splenic CD8 T cells were isolated by immunomagnetic sorting and tested for tumor reactivity in IFN-γ ELISPOT assays. The figure shows the absolute numbers of tumor-reactive IFN-γ–secreting CD8 T cells (± SD) per spleen. Experimental values on day 70 after HSCT were adjusted by subtracting background values that were determined from mice given naive T cells at the time of HSCT and a single vaccine 65 days after HSCT. **P < .01. (B) Annexin V staining of CD8 T cells isolated 21 days after HSCT from the indicated groups of mice. The experiment is representative of 3 separate experiments in which splenocytes were pooled from 3 individual mice.