Monoclonal antibodies to human interferon-α and interferon-γ : Characterization and application with focus on interferon-γ as a T cell cytokine

• The major functions of the immune system are to combat infections and to control that normal auto-immunity does not turn into auto-aggression. Antibodies, together with T cell receptors and MHC molecules, dictates the immunological specificity. However, soluble factors (like cytokines and enzymes) and interactions between leucocytes and endothelium (via adhesion molecules) are decisive for the qualitative and quantitative nature of the immune response to a provocation.The aims of this study were to establish and characterize mouse monoclonal antibodies (mAbs) to the human cytokines interferon alpha (IFNa) and interferon gamma (IFNy), to develop and evaluate the applicability of immunoassays based on these mAbs, and to study T cell secretion of IFNy in response to antigen and in extracellular matrix interactions in vitro.Reactivity of the established mAbs with their respective natural antigen was ascertained and mAbs binding to distinct epitopes identified. We found individual binding patterns of the mAbs to IFNa variants. These varied if the IFNa subtypes were in solution or bound to different solid supports, underlining the importance of thorough characterization of mAbs. The applicability of the anti-IFNa mAbs in ELISAs and their use for characterization of natural IFNa mixtures was demonstrated.Of the mAbs established to IFNy two had suitable characteristics for a sensitive sandwich ELISA, which was also adapted for determinations of IFNy in body fluids as evaluated with serum and plasma. This ELISA combination was also applied in an ELISPOT assay for determination of IFNy secretion on the single cell level. Using the ELISA we could show that IFNy is a quantitative but also qualitative marker for cellular immunity in vitro as evaluated with Francisella tnlarensis as a model antigen (IFNy is required for clearance of this infection). Furthermore, appropriate culture conditions for future studies of IFNy as a marker for immunological memory in short term cultures were defined.Using purified human T cells suboptimally activated through the TcR/CD3 complex with immobilized OKT3 we found that co-immobilized fibronectin (Fn) could potentiate their secretion of IFNy as measured in ELISA and on the single cell level with the ELISPOT. This potentiation was inhibited with antibodies to the integrin VLA-5. IFNy has been shown to be an important factor in lymphocyte migration and Fn is an extracellular matrix component and abundant in most tissues.The finding that Fn can augment IFNy secretion may thus provide new insights into the migration dependent effector functions of T cells.

Ämnesord

NATURVETENSKAP  -- Biologi -- Immunologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Immunology (hsv//eng)

Nyckelord

immunologi

Immunology

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