Expression of proteins associated with T_reg cells. CTLA4, GITR, CD62L, TGF-β1, and IL-10 were assessed on CD4⁺ T cells coexpressing CD25 by either cell surface or intracellular multicolor flow cytometry. Percent positive cells were determined using stringent gating criteria with less than 1% of events within the positive gate when analyzing respective isotype controls. At least 25,000 events per analysis were acquired. Each dot represents a single individual assessed in the respective group; mean expression (line) of all samples in each group is also shown. Significant differences (P < .05, Student t test) between controls and CLL samples are marked by an asterisk: (A) intracellular CTLA4 (P < .01), (B) extracellular CTLA4 (P < .05), (C) intracellular GITR (P < .001), (D) extracellular CD62L (P < .01), (E) intracellular TGF-β1(P < .001), and (F) intracellular IL-10 (P < .05). (G) Expression of FOXP3 by human CD4⁺CD25^hi T cells. CD4⁺CD25^hi T cells and CD4⁺CD25^- T cells were sorted by MACS from peripheral blood of healthy controls (n=7) and patients with CLL (n = 6). Real-time PCR for FOXP3 was performed and relative fold changes of CD4⁺CD25^hi T cells to CD4⁺CD25^- T cells were normalized to GAPDH as described.³⁸