TMZ-mediated lymphodepletion induces homeostatic proliferation and enhances vaccine-induced immune responses. (A-B) Mice were given increasing doses of TMZ before vaccination as described in “Preclinical temozolomide treatment, OT-I transfer, vaccination and PC61 administration.” The frequency and absolute number of OVA-specific T cells were monitored in peripheral blood using an OVA-specific tetramer and anti-CD8 mAb. For all murine tetramer FACS analyses, cells were gated with the following strategy: total lymphocytes were gated from forward and side scatter, CD8⁺ T cells were selected from the lymphocyte gate, and the population of OVA⁺ T cells was selected from total CD8⁺ lymphocytes (supplemental Figure 11). No vaccine, n = 3; untreated, n = 5; 50 mg/kg TMZ, n = 5; 75 mg/kg TMZ, n = 5; and 200 mg/kg TMZ, n = 5. Increasing TMZ dose was associated with both increasing frequencies of OVA-specific T cells (Pearson correlation = 0.69; P = .0008) and increasing absolute numbers of OVA-specific T cells (Pearson correlation = 0.54; P = .014). This experiment was repeated twice with similar results. (C) Untreated (n = 3), TMZ-lymphodepleted (n = 3), or 5-Gy TBI-treated (n = 3) C57BL/6 mice received CFSE-labeled OT-I T cells and OVA vaccination. Proliferation of CD4⁺, CD8⁺, and CD8⁺OVA⁺ T cells was monitored by CFSE dilution. Representative FACS plots are shown.