MILs in myeloma patients contain few regulatory T cells. (A) CD25 expression. BM and peripheral blood from both myeloma patients and normal donors were stained for CD4 and CD25 expression. Mean fluorescent intensity for CD25 expression is indicated in parentheses in the right upper quadrant of each dot plot. (B) FOXP3⁺ expression on CD4⁺/CD25⁺ cells. Intracellular FOXP3 staining was performed on T cells. Graphed is the percentage of FOXP3 expression in the CD4⁺/CD25⁺ cells. Patients, N = 12; normal, N = 3. (C) Suppression assays with CD25⁺ cells. MILs from myeloma patients were stimulated with anti-CD3/CD28 beads alone, with the addition of unactivated CD4⁺/CD25⁺ autologous MILs at a 1:1 ratio of MILs to CD4⁺/CD25⁺ MILs, or with the addition of unactivated CD4/CD25 PBLs at the 1:1 ratio. After a 5-day stimulation, tumor-specific proliferation of MILs was determined by ³H-thymidine incorporation after 48 hours.