RESULTS Transfer of small numbers of CD4+Trp1+ T cells in combination with radiation and CTLA-4 blockade produces potent rejection of established B16/BL6 melanoma tumors

To determine whether activation and differentiation of small numbers of naive tumor-reactive CD4+ T cells in vivo could drive rejection of established tumors, we used naive CD4+Trp1+ cells isolated from Trp1 Tg mice. C57BL/6 mice were challenged with a lethal dose (2.5× 105 cells) of the poorly immunogenic B16/BL6 mouse melanoma line. 10–12 d after challenge, when tumors were clearly visible (Fig. 1 b), mice received 5 Gy of irradiation (radiation therapy [RT]) followed by adoptive transfer of naive CD4+Trp1+ cells (CD4-ACT). To assess the potential combinatorial efficacy of CTLA-4 blockade and CD4-ACT, a cohort of mice also received anti–CTLA-4 mAb. RT by itself failed to induce regression of established tumors (Fig. 1 a). Additional controls including anti–CTLA-4+ RT or anti–CTLA-4+ CD4+Trp1+ also failed to induce regression (unpublished data). Remarkably, transfer of as few as 50,000 CD4+Trp1+ T cells into irradiated hosts was sufficient to induce initial regression of large established melanoma tumors and depigmentation in all mice (Fig. 1 a). However, in the absence of anti–CTLA-4, the combination of RT and CD4-ACT failed to eradicate tumors completely, and tumors recrudesced in up to 60% of mice (Fig. 1 a). The addition of anti–CTLA-4 produced long-term protection in all mice (Fig. 1 a), as well as development of disseminated depigmentation (Fig. 1 b, far right). Analysis of T cell dynamics in the blood of treated mice showed rapid expansion of CD4+Trp1+ cells after transfer into irradiated mice (Fig. 1 c). CTLA-4 blockade increased expansion by two-to threefold, reaching up to 40% of blood lymphocytes (Fig. 1 c), while simultaneously preventing the expansion and accumulation of CD4+Trp1+Foxp3+ cells (Fig. 1 d).