Novel Molecules in Dendritic Cells

Immune responses are initiated in the T-cell areas of secondary lymphoid organs, where recirculating naive T-cells encounter antigen presenting dendritic cells (DC). DC are unusually effective in taking up, processing and presenting antigen to lymphocytes. DC need to detect microbial products or cytokines to transform from immature DC to mature DC with a reduced capacity to take up antigen but now with the capacity to induce primary specific T-cell mediated immune responses in lymphoid organs. Many of the steps leading to mature DC are integrin and chemokine dependent. Integrin mediated contacts and chemokine gradients are necessary to travel from the periphery to lymphatic ducts or blood vessels, for transendothelial intra- and extravasation and for cell to cell contact in the induction of immune responses in the T-cell areas of secondary lymphoid organs. In a previous project we have isolated a molecule termed CYTIP (Cytohesin-1 interacting protein) which is expressed in hematopoietic cells and upregulated in DC during the maturation process. We have functionally characterized CYTIP by overexpressing it in Jurkat cells. We found a negative regulatory role in integrin mediated cell adhesion. The binding of CYTIP to its binding partner cytohesin-1 leads to a complete membrane detachment of the complex, which causes the loss of integrin mediated adhesion. In preliminary experiments with CYTIP transfected Jurkat T-cells we found that coculture with DC leads to the translocation of CYTIP into the nucleus, indicating that a DC-T-cell interaction site is probably controlled by this molecule. Cytohesin-1 has also been shown to be involved in the signalling of chemokine receptor mediated migration. Here we apply for support to continue our work on the role of CYTIP in DC. After the construction of suitable tools (CYTIP-specific monoclonal antibodies, fusion proteins) we will monitor the localization of CYTIP in T-cells and DC upon contact of both cells and interfere with the contact by a pannel of relevant antibodies to find out which contact structures are responsible for the translocation of CYTIP. In migration assays we will examine whether CYTIP binding to cytohesin-1 regulates cell migration in response to chemokines. Using dominant negativ mutants we hope to get insight in the importance of the regulatory role of CYTIP/cytohesin-1 in DC and T-cells. The elucidation of a control point of DC adhesion and migration may help to design a DC adjusted for clinical use.

This project lead to the further functional characterization of a molecule induced during the maturation of dendritic cells. The molecule CYTIP was shown to be part of the regulatory equipment used by dendritic cells to regulate T- cell activation, or, more specifically, the duration of dendritic cell T-cell contact. Dendritic cells are pivotal regulators of immunity and tolerance. They are critical for the organism`s capacity to defend itself against pathogens. A key event in the successful induction of an immune response is the antigen specific activation of T-cells by antigen presenting cells. The initial contact of T-cells and dendritic cells takes place in the lymph nodes, is antigen-independent and is intended for the scanning of a high number of T-cell receptors for antigen to find one of the few that are specific for a given antigen presented by dendritic cells. If antigen recognition occurs, the interaction is productive, leading to a strengthening of the interaction. Otherwise the interaction between T-cell and dendritic cells is loose and transient. In both cases the interaction needs to disolve, so that T-cells can either further search for a relevant antigen or differentiate into effector cells devoted to the elimination of the original antigen. Within this proposal CYTIP has been schown to play a role in the regulation of the disolving of the interaction between dendritic cells and T-cells. Dendritic cells that do not express CYTIP keep longer in T-cell contact than dendritc cells expressing the molecule. Functionally, the inhibition of CYTIP expression in dendritic cells leads to an impaired activation of T-cells, probably because their scanning procedure is hampered by the longer contact times with dendritic cells. Therefore, a mechanism allowing the regulation of the time of interaction between dendritic cells and T-cells is certainly of importance for the regulation of antigen specific T-cell activation, an event central to adaptive immunity.