Effects of emTh-1 cells on antitumoral T lymphocytes and Tregs in vivo. (A) EmTh-1 plus CRCL immunotherapy induces tumor-specific killer T lymphocytes. B16 tumor–bearing mice were injected with control PBS or were treated with B16-derived CRCL and allogeneic emTh-1 cells as indicated in “Tumor growth in vivo and combination immunotherapy.” Seven days after the last vaccination, splenocytes were harvested and incubated for 3 days with CRCL (25 μg/mL) and 50 U/mL IL-2. T lymphocytes were then purified on a nylon wool column and incubated for 36 hours with either B16 tumor cells or irrelevant 4T1 breast cancer cell targets as indicated (effector T cells to target tumor cells ratio = 20:1). Cytotoxicity was determined as previously reported.^28,50  + Control T, Tumor cells cultured with T lymphocytes from mice injected with PBS; +T [emTh-1], tumor cells cultured with T lymphocytes from mice treated with CRCL plus emTh-1. *P < .005, a significant difference compared with control T cells cultured with B16 melanoma cells. (B) EmTh-1 cells skew the differentiation of CD4⁺CD25⁻FoxP3⁻ naive T lymphocytes toward CD4⁺CD25⁺FoxP3⁻ effector T cells rather than Tregs in vivo. CD4⁺CD25⁻FoxP3⁻ naive T lymphocytes isolated from Thy1.2 FoxP3EGFP transgenic BALB/c mice (10⁷ cells) were transferred to 12B1 tumor–bearing congenic Thy1.1 BALB/c mice. Animals were treated with emTh-1 cells (+emTh-1 cells) or with control PBS (No emTh-1 cells). Endogenous T cells of recipient Thy1.1 mice express the Thy1.1 but not the Thy1.2 antigen, which allows the specific tracking and identification of Thy1.2 T lymphocytes in Thy1.1 mice. Spleens were harvested and dissociated, and conversion of transferred naive Thy1.2 CD4⁺CD25⁻FoxP3⁻ T cells into GFP+ (FoxP3-expressing) Treg in vivo was determined by evaluating the frequency of Thy1.2⁺GFP⁺ cells after gating on the CD4⁺ T-cell population using flow cytometry. (C) EmTh-1 cells impair Treg suppressive function in vivo. Responder CD4⁺CD25⁻ T lymphocytes were stimulated with anti-CD3/anti-CD28 T-cell expander beads in the absence (CD25⁻) or presence of Tregs isolated from the draining lymph nodes of untreated tumor-bearing mice (CD25⁻ + [Treg]_untreated) or of tumor-bearing mice treated with emTh-1 (CD25⁻ + [Treg]_emTh-1). Responder CD4⁺CD25⁻ T-lymphocyte proliferation was determined after 48 hours using BrdU incorporation assays. NS, nonsignificant; *P < .02; **P < .001.