Characterization of mucosal FOXP3⁺ cells. Counterstaining of FOXP3 (red; detection with SA-Cy2 or SA-Cy3), CD3 (green; anti–Dig-Cy5) with surface markers of T_regs (blue; anti–goat Cy3) CTLA-4 (A), GITR(C), and CD25 (blue; anti–mouse Alexa 488) by immunofluorescence (B, bottom panel) or of FOXP3 (brown; immunoperoxide) with CD25 (red; APAAP) by immunohistochemistry (B, top panel) revealed clear coexpression. On the other hand, counterstaining of FOXP3 (red), CD3 (green), and T-lymphocyte activation markers (blue; anti–mouse Alexa488) HLA-DR (D) and CD38 (E) did not detect any cells positive for both markers. Depicted are representative sections from duodenal biopsies of HIV-infected patients. Original magnification ×600 (A-C) and ×400 (D-E). Microscope: Olympus AX70 (Olympus, Hamburg, Germany). Numerical aperture of objective lenses: ×40, 0.95 mm; ×60, 1.40. Camera: JVC KY-F70 (JVC, Yokohama, Japan). Acquisition software: DISKUS (Carl H. Hilgers, Königswinter, Germany). Software used for image processing: Adobe Photoshop 7.0 (Adobe, San Jose, CA).