Recognition by the T cell receptor(TCR)/CD3 complex of antigenic peptide presented by major histocompatibility complex molecules is a prerequisite for antigen-specific T-cell activation. Full activation and subsequent maturation/differentiation of resting T-cells, however, requires a variety of other signals provided by the highly specialized microenvironment of the secondary lymph organs. Among these signals are cell contact-mediated costimulatory signals and cytokines that can be provided by activated antigen presenting cells (APC). These and other accessory signals, such as those from growthfactors and extra cellular matrix components, are all part of the local microenvironment. This complex interaction of costimulatory signals plays a determining role in the subsequent maturation/differentiation of the antigen-stimulated CD4+ T-cell. Helper T-cells can differentiate into distinct functional subsets based on the profile of secreted cytokines (i.e. ThO, Th1 & Th2). Another aspect of maturation/differentiation is the acquisition of specific adhesion/homing properties (i.e. skin-, mucosal- and peripheral lymph node-homing). A fundamental understanding of the factors involved in the processes determining what type of immune response is generated and how those helper T-cells are recruited to the relevant area to be optimal effective is a crucial first step to design potential successful immunomodulation therapies. Our major aim is therefore is to study the maturation/differentiation of human "naive" CD4+ T-cells. This will be examined by: A) studying the role of accessory pathways involved in interaction between "naive" CD4+ T-cells and the antigen presenting cell (APC), utilizing established cell lines (e.g. myeloid and B-cell lineage) and primary cells (e.g. monocytes, B- cells, dendritic cells). Further direct analysis of the pathways involved will be performed in studies with blocking antibodies and by stable transfection of cell lines with genes encoding for relevant accessory molecules; B) Analyzing the contribution of cytokines and growth factors, which will be examined with the use of recombinant proteins, such as IL-1, -2, -4, -6, -7, -10, -12, IFN-gamma and TGF-beta1, as well as with neutralizing antibodies against these cytokines/growthfactors or their cell surface receptors. Different TCR signalling systems will be used in the various models, by utilizing CD3-specific mAb, and allo- and superantigen stimulation. To study the role of these factors on the generation of functional distinct CD4+ T-cell subsets we will analyze the various T-cell cultures for the following three aspects: 1) Cytokine profile (ELISA, PCR); 2) Cell surface phenotype (FACS); 3) Adhesion/homing phenotype (FACS, adhesion assays). This multi-facetted approach may provide us with in vitro models in which one can manipulate both the generation of functional and phenotypically distinct helper T-cell subsets and moreover also direct the specific homing capabilities of these subsets. Detailed insight in the many aspects of the maturation/differentiation of naive CD4+ T-cells will be of real relevance for new approaches in immunomodulation to prevent transplant rejection and autoimmunity.