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...

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The endothelial nitric-oxide synthase (eNOS) is activated by transient increases in intracellular Ca2؉ elicited by stimulation of diverse receptors, including bradykinin B 2 receptors on endothelial cells. eNOS and B 2 receptors are targeted to specialized signal-transducing domains in the plasma membrane termed plasmalemmal caveolae. Targeting to caveolae facilitates eNOS activation following receptor stimulation, but in resting cells, eNOS is tonically inhibited by its interactions with caveolin, the scaffolding protein in caveolae. We used a quantitative approach exploiting immunofluorescence microscopy to investigate regulation of the subcellular distribution of eNOS in endothelial cells by bradykinin and Ca 2؉. In resting cells, most of the eNOS is localized at the cell membrane. However, within 5 min following addition of bradykinin, nearly all the eNOS translocates to structures in the cell cytosol; following more protracted incubations with bradykinin, most of the cytosolic enzyme subsequently translocates back to the cell membrane. The bradykinin-induced internalization of eNOS is completely abrogated by the intracellular Ca 2؉ chelator BAPTA; conversely, Ca 2؉ -mobilizing drugs and agonists promote eNOS translocation. These results establish that eNOS targeting to the membrane is labile and is subject to receptor-regulated Ca 2؉ -dependent reversible translocation, providing another point for regulation of NO-dependent signaling in the vascular endothelium.