Frequency of CD4⁺CD25^highFoxP3⁺ T cells. (A) Flow cytometric analysis of CD4 and CD25 on peripheral blood–derived T cells from a healthy individual (top panel) and a multiple myeloma (MM) patient (bottom panel). Samples were stained with the appropriate isotype controls (data not shown) or CD4 and CD25 mAbs. CD4⁺CD25⁺ T cells were divided into CD25^low and CD25^high cells according to previously published data. ^22,47  Numbers represent percentage of events within the respective rectangle. Settings shown here were used for the analysis of all samples under study. (B) Frequency of CD4⁺CD25^high T cells in 26 healthy donors and 9 MGUS patients, 8 untreated and 2 treated stage I MM patients, 3 untreated and 11 treated stage II MM patients, and 43 treated stage III MM patients. Shown here are median, 75th percentile (box), standard deviation (whiskers), and outliers (dots) (*P < .05, Student t test). (C) Frequency of CD4⁺CD25^high T cells in peripheral blood (PB) and bone marrow (BM) of 6 MM patients. (D) Serial analysis of CD4⁺CD25^high T cells in MM patients at 2 different time points separated by at least 6 months (Tx indicates conventional chemotherapy [n = 3]; SCT, autologous stem cell transplantation [n = 5]; and after SCT, after autologous SCT [n = 5]). (E) Expression of FOXP3 by human CD4⁺CD25^high T cells. CD4⁺CD25^highT cells (dark gray bars) and CD4⁺CD25^– T cells (light gray bars) were sorted by magnetic activated cell sorting (MACS) from peripheral blood in healthy controls (n = 7) and MM patients (n = 6). Real-time RT-PCR for FOXP3 was performed, and relative expression of FOXP3 in CD4⁺CD25^highT cells as well as CD4⁺CD25^– T cells was normalized to GAPDH as described. ²²  Error bars represent standard deviation (SD). (F) Flow cytometric analysis of FoxP3 in CD4⁺CD25^highT_reg cells in MGUS (n = 8) as well as untreated (n = 7) and treated (n = 13) MM patients compared with healthy donors (n = 14). Shown are the median, 75th percentile (box), standard deviation (whiskers), and outliers (dots). *P < .05, Student t test.