Fig. S3.

(A and C) Naive OT-I CD8 T cells (5 × 10⁵) were purified by negative selection and adoptively transferred into TRAMP/POET mice (day 0). Mice were stimulated with 5 µg anti–DEC-205–OVA and 50 µg anti-CD40 on day 28, along with mono- or combination therapy: 50 µg aOX40 or control rat IgG on days 28 and 29, and 200 µg aCTLA-4 on days 28, 30, and 3. (A) Model of treatment. (B) Naive OT-I CD8 T cells (2.5 × 10⁶) were purified by negative selection and adoptively transferred into TRAMP/POET or wild-type C57BL/6 mice. Three days later, lymph nodes were processed, and OX40 and CTLA-4 expression was analyzed by flow cytometry on donor OT-1 cells. For CTLA-4 expression on donor OT-1 cells, the unstained control is shown in gray; the wild-type control is shown as a solid black line; TRAMP/POET mice are shown as a purple line. For OX40 expression (Middle and Right), images represent OT-I⁺ (Thy1.1⁺) CD8 T cells versus OX40 expression; the numbers shown are the percentage of OX40⁺ OT-I cells. (C) Splenocytes and prostate-draining lymph nodes (pooled for each group) were analyzed on day 35, and donor cell phenotype was determined by flow cytometry. Donor OT-I cells were gated on live CD8⁺ Thy1.1⁺ events. Graphs depict the mean ± SEM for one of three independent experiments (n = 4 or 5 per group).