Regulatory cells specific for p24 antigen can be found among the CD4⁺CD25⁺ T-subset in HIV-infected patients. (A) Specific induction of IL-10 production following stimulation of CD4⁺CD25⁺ cells with p24 antigen. Cells (5 × 10⁵/well) were incubated for 48 hours alone or in the presence of 5 μg/mL plate-bound anti-CD3 (α-CD3) and 5 μg/mL soluble anti-CD28 (α-CD28) mAb or with 5 μg/mL HIV p24 antigen before assessment of intracellular IL-10 production by flow cytometry. Intracellular staining was performed using anti–IL-10–PE mAb or isotype-matched control mAb. Results from 5 patients studied are presented. The numbers in the top corner of each histogram indicate the percent of cells positive for IL-10 (gray histograms) compared with cells cultured in media alone (outlined histograms). (B) Induction of TGF-β1 mRNA expression in CD4⁺CD25⁺ cells following stimulation with p24 antigen. RNA was extracted from unfractionated CD4⁺ cells or purified CD4⁺CD25^– and CD4⁺CD25⁺ T cells incubated for 48 hours alone (lane 1) or in the presence of plate-bound anti-CD3 and soluble anti-CD28 mAbs (5 μg/mL each; lane 2) or 5 μg/mL HIV p24 protein (lane 3). Data from 4 patients studied are presented. (C) Induction of TGF-β1 mRNA expression in CD4⁺CD25⁺ cells following stimulation with p24, CMV, or PPD antigens. Purified CD4⁺CD25⁺ T cells from 3 other patients were cultured for 48 hours alone (lane 1); in the presence of anti-CD3 and anti-CD28 (lane 2); in 1:50 dilution CMV antigen (lane 3); or 5 μg/mL PPD antigen (lane 4) or p24 antigen (lane 5).