Depletion of CD4⁺CD25^Hi Treg population reduces suppression of Teff response. Tregitope suppression is dependent on CD4⁺CD25^Hi T cells. A. PBMC from allergic individuals were stained with anti-CD4 and anti-CD25 antibodies and analyzed by flow cytometry. The CD4⁺CD25^Hi subset (gate 100) was sorted and discarded. B. CD4⁺CD25^Hi depleted and nondepleted PBMC were costimulated with HDM lysate with or without hTregitope 289. CD4⁺CD25^Hi-depleted PBMC were less able to suppress IFN-γ than were nondepleted PBMC. The levels of IFN-γ were reduced from 33.5 pg/mL to 11.8 pg/mL in the whole PBMC and from 16.5 pg/mL to 12.4 pg/mL in the CD4⁺CD25^Hi-depleted subset.