FIG. 4.

Combination T-cell transfer can enhance therapy of established 7-day tumors in combination with TBI. (A–D) C57BL/6 mice were challenged with 5×10⁵ B16ova cells and administered 5 Gy TBI on day 6. Activated T cells (1×10⁷) were administered on day 7. (A) Mice were monitored for tumor growth. One experiment representative of four is shown. (B, C) Spleens were harvested from mice with 10-mm tumors. Total splenocytes were analyzed by FACS for OT-I T cells (B; Vα2⁺Vβ5⁺) and Pmel T cells (C; Thy1.2⁻). Graphs represent percentage of total splenic CD8⁺CD3⁺ cells staining as each clone. (D) RNA was isolated from 10-mm tumors. RT-PCR was performed for ova and gp100. (E) OT-I mice were treated as in Fig. 3A. DNA was isolated from 10-mm escape tumors and tested for ova gene content by PCR. (F) Tumors and spleens were harvested from OT-I mice with 10-mm tumors. Pmel T cells were identified by FACS of single-cell suspensions. The graph indicates the percentage of CD8⁺CD3⁺ cells staining positive for Thy1.1. (G) Surviving mice were inspected after 2–3 months for persistent tumor burden. Black masses at the tumor challenge site were analyzed by histology. One representative picture is shown; it is from a wild-type C57BL/6 mouse treated with 5 Gy TBI followed by OT-I and Pmel cotransfer. Original magnification was 20×. Color images available online at www.liebertonline.com/hum