TGF-β–converted CD4⁺ suppressor T cells inhibited antigen-specific expansion of transgenic CD4⁺ T cells in vivo. Freshly isolated CD4⁺ KJ1-26⁺ T cells (DO11.10 CD4⁺ cells) or with equal number of OVA only (Control cell), plus 2 ng/ml TGF-β (TGF-β cell) or plus 1 ng/ml IL-10–converted (IL-10 cell) CD25⁺ KJ1-26⁺ T cells were injected i.p into normal BALB/c mice. Some mice were injected with 2.5 × 10⁵ of Control cell, TGF-β cell, or IL-10 cell alone. Mice were immunized with Peptide 323-339 emulsified with IFA on day 2. On day 11, draining lymph nodes (inguinal) were harvested, and cells were stained with Tricolor–anti-CD4, FITC–KJ1-26, and PE–anti-CD25 and analyzed on FACSCalibur™. (A–D) Live cells were gated, and the profiles between CD4 versus KJ1-26 are displayed. The numbers represent CD4⁺KJ1-26⁺ cell frequency within lymph node cells. The numbers in the parentheses are injected cells. Each group contained two mice and the lymph node cells were pooled before staining. (a–d) CD4⁺KJ1-26⁺ T cells in corresponding A–D were gated, and CD25⁺ frequency is shown. (E) Total CD4⁺KJ1-26⁺ T cells in draining lymph nodes per mouse (percentage of CD4⁺KJ1-26⁺ × the number of total lymph node cells/2). (F) 2 × 10⁶ lymph node cells labeled with 3 μM CFSE were cultured with 1 μg/ml Peptide 323-339 in 24-well plates for 72 h. Cells were stained with Tricolor–anti-CD4 and PE–KJ1-26 and analyzed on FACSCalibur™. CD4⁺KJ1-26⁺ cells were gated, and the CFSE^hi cells are shown. The marker was set according to CFSE fluorescence of the live CD4⁺ KJ1-26⁺ T cells in parallel cultures with medium only. (G) 2 × 10⁶ lymph node cells were cultured with Peptide 323-339 in the presence of GolgiPlug™ for 5–6 h. Cells were stained with Tricolor–anti-CD4 and FITC–KJ1-26 antibodies before being fixed and intracellularly stained for IL-4 or IFN-γ cytokines with respective antibodies (PE-anti–IL-4 or PE-anti–IFN-γ, 0.5 μg/10⁶ cells). 40,000–80,000 cells were acquired on FACSCalibur™, and percentages of KJ1-26⁺ IL-4⁺ or IFN-γ⁺ cells within CD4⁺ T cells are shown. The experiments were repeated twice with similar results.