Fig. 3. Lack of detectable conversion of FoxP3⁻ CD4⁺ T cells to induced T_regs in B16 melanoma in vivo.

After tumor inoculation of B16 cells, Rag2^−/− mice were injected with either 3 × 10⁶ total splenocytes or GFP-depleted splenocytes of a FoxP3-GFP reporter mouse. On day 7 tumor, tumor-draining lymph node (TdLN) and spleen were analyzed for the amount of GFP⁺/CD25⁺/FoxP3⁺ cells within their CD4⁺ T cell compartment. (A) Representative example for results obtained with total splenocytes (top row) or GFP-depleted splenocytes injected in vivo (lower row). Cells were pregated on living cells and CD3⁺CD4⁺ cells. (B) Statistical analysis of six mice shown as mean ± SEM and two-sided Mann-Whitney U test to determine significance. ****P < 0.0001; ***P < 0.001).