ABSTRACT
Immunotherapy is rapidly evolving as an effective treatment option for many cancers. With the emerging fields of cancer vaccines and adoptive cell transfer therapies, there is an increasing demand for high-throughput in vitro cytotoxicity assays that efficiently analyze immune effector functions. The gold standard (51)Cr-release assay is very accurate but has the major disadvantage of being radioactive. We reveal the development of a versatile and nonradioactive firefly luciferase in vitro transcribed (IVT) RNA-based assay. Demonstrating high efficiency, consistency, and excellent target cell viability, our optimized luciferase IVT RNA is used to transfect dividing and nondividing primary antigen presenting cells. Together with the long-lasting expression and minimal background, the direct measurement of intracellular luciferase activity of living cells allows for the monitoring of killing kinetics and displays paramount sensitivity. The ability to cotransfect the IVT RNA of the luciferase reporter and the antigen of interest into the antigen presenting cells and its simple read-out procedure render the assay high-throughput in nature. Results generated were comparable to the (51)Cr release and further confirmed the assay's ability to measure antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. The assay's combined simplicity, practicality, and efficiency tailor it for the analysis of antigen-specific cellular and humoral effector functions during the development of novel immunotherapies.
Subject(s)
Antigen-Presenting Cells/immunology , Cytotoxicity, Immunologic , Immunity, Cellular , Immunity, Humoral , Immunoassay , T-Lymphocytes, Cytotoxic/immunology , Antigen-Presenting Cells/cytology , Antigens/genetics , Antigens/immunology , Cell Line, Tumor , Cell Survival , Coculture Techniques , Genes, Reporter , Humans , Luciferases/genetics , Luciferases/immunology , Plasmids/chemistry , Plasmids/metabolism , RNA, Messenger/genetics , RNA, Messenger/immunology , T-Lymphocytes, Cytotoxic/cytology , Transcription, Genetic , TransfectionABSTRACT
BACKGROUND: IL-10-treated dendritic cells (DCs) have been shown to inhibit T-cell responses through induction of anergy and regulatory T cells in various model systems, including allergic inflammation, but the factors being involved in this inhibition are still unclear. OBJECTIVE: This study set out to analyze such factors produced or induced by IL-10-treated DCs by using gene expression profiling and to explore their function. METHODS: CD4(+) T cells from allergic donors were stimulated with autologous monocyte-derived allergen-pulsed mature DCs or IL-10-treated DCs. After 24 hours, the transcriptional profile was analyzed by using Affymetrix technology. Results were validated by using quantitative real-time PCR, protein expression, and functional in vitro and in vivo studies. RESULTS: In CD4(+) T-cell/IL-10-treated DC cocultures the expression of several known genes, such as IL13, IL5 and OX40, was suppressed. Interestingly, there was only one factor that was strongly upregulated: the DC-derived chemokine CCL18. In vitro addition of CCL18 to cocultures of CD4(+) T cells and allergen-pulsed DCs resulted in a similar inhibition of T(H)2 cytokine production as induced by allergen-pulsed IL-10-treated DCs without exogenous CCL18, whereas T(H)1 cytokine production, IL-10 production, and proliferation were not affected. Furthermore, in a humanized mouse model of allergy using PBMC-engrafted NOD-scid-γc(-/-) mice, CCL18, but not another T(H)2-associated chemokine, CCL17, inhibited airway reactivity and lung inflammation. Chemotaxis assays revealed that CCL18 preferentially attracted regulatory T cells and, less efficiently, T(H)2 cells. CONCLUSION: These data demonstrate that CCL18 might represent a molecule of significant importance in immunoregulation and might be a therapeutic target in patients with allergic airway diseases.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , Chemokines, CC/immunology , Dendritic Cells/immunology , Respiratory Hypersensitivity/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cells, Cultured , Coculture Techniques , Dendritic Cells/transplantation , Disease Models, Animal , Humans , Immune Tolerance , Interleukin-10/immunology , Mice , Mice, Inbred NOD , Mice, Knockout , Microarray Analysis , TranscriptomeABSTRACT
A multitude of antigens has been recently identified by screening of cDNA expression libraries derived from human tumors with autologous sera. Using a phage autoantibody assay and small panels of sera derived from cancer patients or controls it has been shown that some of these antigens display cancer-associated autoantibody responses. The diagnostic and prognostic significance of these potentially cancer-related autoantibodies remains unclear until large-scale assays are developed and serological data are available for hundreds of cancer patients and controls. The major bottleneck for the development of large-scale assays are the cloning, expression and the purification of each of the respective antigens. Due to these limitations and despite the potential clinical relevance large-scale autoantibody tests are established for only a few of these tumor antigens. Here we describe an enzyme-linked immunosorbent assay, Crude lysate ELISA (CrELISA), suitable for antigens identified by expression screening based on crude lysates of antigen-expressing bacteria. This assay permits sensitive and specific autoantibody seroscreening without the need of laborious and time-consuming cloning, expression and purification of recombinant proteins. CrELISA is robust and provides a versatile high throughput procedure for the rapid evaluation of multiple antigens in large-scale serology.
