IntroductionT regulatory (T reg ) cells play an important role in the maintenance of self-tolerance, control of auto-immunity, and regulation of T-cell homeostasis, and they modulate overall immune responses against infectious agents and tumor cells. 1 Natural T reg cells develop during normal T-cell maturation in the thymus and represent 5% to 10% of the CD4 ϩ cell compartment in the peripheral blood. 2 These cells express CD4 and CD25 surface antigens as well as CTLA-4, GITR, CD103, CD62L, CD69, CD134, CD71, CD54, and CD45RA. 3 The suppressive activity of T reg cells is associated with the overexpression of FOXP3, a member of the forkhead/winged helix family, which acts as a transcriptional repressor. 4 T reg cells suppress CD25 Ϫ CD4 ϩ T-cell proliferation on the basis of cell-cell contact and suppress immune responses by secreting immunosuppressive cytokines such as IL-10 and TGF-. 5 A significant impairment of T-cell function is observed in patients with multiple myeloma (MM) and patients with monoclonal gammopathy of undetermined significance (MGUS). Although phenotypic and functional aberrations in CD4 and CD8 cells have been described in MM and MGUS, 6-9 the biologic basis for these abnormalities remains unclear. Because T reg cells play an important role in modulating normal immune responses, the abnormal T reg -cell activity in myeloma patients could contribute to immune dysfunction in MM and could provide a new target to enhance immune responses. Therefore, in this study we evaluated natural T reg -cell number and function in patients with MGUS and MM and compared them with those of healthy donors.
Study design
Phenotypic characterizationCD4 ϩ CD25 ϩ T reg -cell numbers were analyzed by flow cytometric analyses in peripheral-blood mononuclear cells (PBMCs) collected from healthy donors, patients with MGUS, and patients with newly diagnosed MM. Approval for these studies was obtained from the institutional review board of the Dana-Farber Cancer Institute and the Veterans Administration (VA) Boston Healthcare System. Informed consent was provided according to the Declaration of Helsinki.
Measurement of FOXP3 expressionAs FOXP3 is specifically expressed by T reg cells and is required for their suppressive activity, we analyzed the proportion of PBMCs expressing intracellular FOXP3 using anti-FOXP3 antibody (eBiosciences, San Diego, CA) using dual-color flow cytometry and multiphoton microscopy. Level of protein expression was quantitated by Western blotting and by real-time reverse transcription-polymerase chain reaction (RT-PCR) using previously described methods. 10
Suppressive activity of T regulatory cellsTo evaluate the function of T reg cells, PBMCs were first depleted of CD25 ϩ T cells (which contain T reg cells) by positive selection using anti-CD25-coated microbeads (Miltenyi Biotech, Auburn, CA), according to the manufacturer's instructions. 11 PBMCs depleted of CD25 ϩ cells and control PBMCs containing CD25 ϩ cells were stimulated with anti-CD3 antibody for 3 days, and proliferation was measu...
