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Intra-assay variance proved to be less then 10%, whereas interassay variability ranged between 10 and 22%. This multiplex system proved to be a powerful tool in the quantitation of cytokines. It will provide a more complete picture in differences between activated lymphocyte cytokine profiles from healthy individuals and those from patients with chronic inflammatory diseases.Cytokines are soluble proteins that are secreted by cells of the immune system. These proteins can alter the behavior and properties of different cell types. Although cytokine functions are complex, cytokine profiles are highly relevant parameters of an immune response. Different cytokines possess biological overlapping functions, and they have the ability to regulate production of other cytokines. Therefore, analysis of the function of the complete set of cytokines expressed within microenvironments (e.g., a site of inflammation) is often of more value than analysis of a single isolated cytokine (13).Cytokines can be quantitated at various levels. mRNA can be detected by real-time PCR; intracellular proteins can be measured by fluorescence-activated cell sorter staining of permeabilized cells, and secreted cytokines can be quantified with bioassays, enzyme-linked immunosorbent assays (ELISAs), radioactive immunosorbent assays, and microarrays. Multiplex assays for detection of cytokines at the mRNA (6) and cellular levels (16, 18) are commonly used. However, these assays have one or more limitations, like the need for a large sample volume or detection of precursor proteins rather than native secreted proteins. In addition, these techniques are time-consuming and laborious.Recent advances concerning applications for the simultaneous detection of proteins have resulted in different particlebased flow cytometric assays. These assays have proven to be very useful in the simultaneous detection of cytokines in body fluids. Unfortunately, at present, either the number of different microspheres or the availability of predefined kits limits these assays (1, 3). The Bio-Plex system employing the Luminex multianalyte profiling technology (Lab-MAP) allows individual and multiplex analysis of up to 100 different analytes in a single microtiter well (20).Our laboratory focuses on immunoregulation and immunotherapy of children with autoimmune diseases-in particular, juvenile idiopathic arthritis (JIA). Sample volumes are relatively small due to our patient population. For a number of cytokines, ready-to-use beads are available, but not for the full spectrum. To overcome these limitations, we chose to develop and validate our own multiplex assays with the Bio-Plex system. With this assay, we were able to detect human cytokines in antigen-stimulated peripheral blood mononuclear cell (PBMC) culture supernatants from both autoimmune and healthy individuals. We showed that it is a reliable, fast, and reproducible technique with a sensitivity that is comparable to that of conventional ELISAs. MATERIALS AND METHODSCell isolation and cultures. Heparinized blood ...

Regulatory T cells (Tregs) are a specific subset of lymphocytes that are critical for the maintenance of self-tolerance. Expression levels of the transcription factor Foxp3 have been causally associated with Treg differentiation and function. Recent studies show that Foxp3 can also be transiently expressed in effector T cells; however, stable Foxp3 expression is required for development of a functional Treg suppressor phenotype. Here, we demonstrate that Foxp3 is acetylated, and this can be reciprocally regulated by the histone acetyltransferase p300 and the histone deacetylase SIRT1. Hyperacetylation of Foxp3 prevented polyubiquitination and proteasomal degradation, therefore dramatically increasing stable Foxp3 protein levels. Moreover, using mouse splenocytes, human peripheral blood mononuclear cells, T cell clones, and skin-derived T cells, we demonstrate that treatment with histone deacetylase inhibitors resulted in significantly increased numbers of functional Treg cells. Taken together, our data demonstrate that modulation of the acetylation state of Foxp3 provides a novel molecular mechanism for assuring rapid temporal control of Foxp3 levels in T cells, thereby regulating Treg numbers and functionality. Manipulating Foxp3 acetylation levels could therefore provide a new therapeutic strategy to control inappropriate (auto)immune responses.