Characterization of T_reg cells in MGUS and MM compared with healthy donors. (A) PBMCs were isolated, incubated with anti-CD4 and -CD25 antibodies, and analyzed by flow cytometry. Results are expressed as percentages of lymphocytes. Number of samples analyzed in each category is given in parentheses. ^*P ≤ .01 by Student t test analysis. MGUS and MM patients had significantly greater numbers of CD4⁺CD25⁺ T cells than healthy donors. (B) CD4- and CD25-expressing T cells were isolated as described in panel A, and cells expressing high levels of CD25 were then analyzed. Results are expressed as percentages of lymphocytes expressing CD4 and CD25^high. Number of samples analyzed in each category is given in parentheses. No significant increases in CD4⁺CD25^high T-cell numbers were observed in MGUS or MM patients compared with healthy donors. (C) Representative example of flow cytometry data using healthy donor cells and cells from patients with MGUS and MM. Frequency of CD25⁺ cells in the lymphocyte gates was analyzed using anti-CD25 PE antibody along with anti-CD4 FITC antibody. Quadrants were established using isotype controls, and stained cells were analyzed using Cytomics FC 500 (Beckman-Coulter, Fullerton, CA) and CXP software. (D) PBMCs were isolated and incubated with anti-FOXP3 antibodies (eBiosciences) for intracellular staining and then were analyzed by flow cytometry. Cells in the lymphocyte gates were used for the analysis. Results are expressed as percentages of lymphocytes expressing FOXP3, and the number of samples analyzed in each category is given in parentheses. A significantly decreased number of FOXP3⁺ (P < .01) T_reg cells were observed in MGUS and MM patients compared with healthy donors. (E) Frequency of FOXP3⁺ cells in the lymphocyte gates was analyzed using anti-FOXP3 PE antibody. Dual-color analysis (PE-FOXP3 and Pc5-CD4) was optimized and used in these studies. Quadrants were established using isotype controls, and stained cells were analyzed using Cytomics FC 500 (Beckman-Coulter) and CXP software. Data are representative of 5 separate experiments. (F) PBMCs were stained with anti-FOXP3 antibodies and then analyzed using multiphoton microscopy. (BioRad MRC 1024ES multiphoton system; Bio-Rad, Hercules, CA). A Zeiss Axiovert S 100 inverted microscope equipped with a high-quality water immersion 40×/1.2 numeric aperture C-Apochromat objective was used to obtain images (total magnification is 640×). Images were reconstructed using the Bio-Rad LaserSharp and/or MetaMorph software (MetaMorph Imaging Series, Universal Imaging, West Chester, PA). Higher frequencies of stained cells were observed in PBMCs from healthy donors than in PBMCs from MGUS and MM patients. Error bars in panels A, B, and D indicate SEM.