In several murine models of autoimmune arthritis, Th17 cells are the dominant initiators of inflammation. In human arthritis the majority of IL-17–secreting cells within the joint express a cytokine phenotype intermediate between Th17 and Th1. Here we show that Th17/1 cells from the joints of children with inflammatory arthritis express high levels of both Th17 and Th1 lineage-specific transcription factors, RORC2 and T-bet. Modeling the generation of Th17/1 in vitro, we show that Th17 cells “convert” to Th17/1 under conditions that mimic the disease site, namely low TGFβ and high IL-12 levels, whereas Th1 cells cannot convert to Th17. Th17/1 cells from the inflamed joint share T-cell receptor (TCR) clonality with Th17 cells, suggesting a shared clonal origin between Th17 and Th17/1 cells in arthritis. Using CD161, a lectin-like receptor that is a marker of human Th17, we show synovial Th17 and Th17/1 cells, and unexpectedly, a large proportion of Th1 cells express CD161. We provide evidence to support a Th17 origin for Th1 cells expressing CD161. In vitro, Th17 cells that convert to a Th1 phenotype maintain CD161 expression. In the joint CD161+ Th1 cells share features with Th17 cells, with shared TCR clonality, expression of RORC2 and CCR6 and response to IL-23, although they are IL-17 negative. We propose that the Th17 phenotype may be unstable and that Th17 cells may convert to Th17/1 and Th1 cells in human arthritis. Therefore therapies targeting the induction of Th17 cells could also attenuate Th17/1 and Th1 effector populations within the inflamed joint.

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 ...

Background: Growing knowledge about cellular interactions in the immune system, including the central role of cytokine networks, has lead to new treatments using monoclonal antibodies that block specific components of the immune system. Systemic cytokine concentrations can serve as surrogate outcome parameters of these interventions to study inflammatory pathways operative in patients in vivo. This is now possible due to novel technologies such as multiplex immunoassays (MIA) that allows detection of multiple cytokines in a single sample. However, apparently trivial underappreciated processes, (sample handling and storage, interference of endogenous plasma proteins) can greatly impact the reliability and reproducibility of cytokine detection.