Figure 2. Depletion of tumor-specific T_reg cells does not prevent or treat relapsing melanoma.

(A) Depletion of tumor-specific Foxp3⁺ T_reg cells during recurrence (late) does not retreat melanoma. C57BL/6 lymphopenic RAG^−/− mice (5 mice/group) were inoculated with B16.F10 melanoma (2 × 10⁵ cells). Tumor-bearing mice were treated with 2 × 10⁵ naïve TRP-1 Foxp3-DTR CD4⁺ T cells by intravenous tail vein injection on day 10 after tumor inoculation. Tumors were followed until recurrence of melanoma. DT (diphtheria toxin) was injected i.p. at the prescribed concentration of 50 μg/Kg every other day for three doses total. PBS controls had no effect on T_reg cells (not shown). Experiments repeated 5 times. (B) Flow cytometry of Foxp3 eGFP expression in TRP-1 Foxp3-DTR CD4⁺ T cells from relapsing mice treated with or without DT at time of sacrifice. (No DT, 35.3%; +DT (late), ~2%). Experiments repeated at least five times. (C) Depletion of tumor-specific Foxp3⁺ T_reg cells before recurrence (early) does not prevent melanoma recurrence. C57BL/6 lymphopenic RAG^−/− mice (5 mice/group) were inoculated with B16.F10 melanoma (2 × 10⁵ cells). Tumor-bearing mice were treated with 2 × 10⁵ naïve TRP-1 Foxp3-DTR CD4⁺ T cells by intravenous tail vein injection on day 10 after tumor inoculation. Tumors were followed until recurrence of melanoma. At injection of T cells (day 10), DT (diphtheria toxin) was injected i.p. at the prescribed concentration of 50 μg/Kg every other day for three doses total. PBS controls had no effect on T_reg cells (not shown). Experiments repeated five times. (D) Functional characteristics of tumor-specific CD4⁺ T cells during recurrence with T_reg cells depleted early. Flow cytometry of IFN-γ and TNF-α expression in TRP-1 Foxp3-DTR CD4⁺ T cells from non-relapsing and relapsing mice. Data represent at least three experiments. (E) Percent of TRP-1 CD4⁺ T cells that are dual producers of IFN-γ and TNF-α during recurrence. P < 0.0156 for non-relapsing and relapsing groups. Experiments repeated four times.