Functional analyses of CYTIP in dendritic cells

To initiate T-cell-dependent immune responses dendritic cells take up antigen in peripheral tissues and migrate to the draining lymph nodes, where they interact with antigen-specific T-cells. To efficiently prime nave T-cells dendritic cells need to undergo a maturation process that implies differential regulation of function-related genes, e.g. up-regulation of major histocompatibility complex and costimulatory molecules at the surface. Interactions between mature dendritic cells and potentially reactive T-cells during priming events have been extensively studied by 2-photon microscopy and found to occure in 3 pases each characterized by contact intervals between dendritic cells and T-cells. These contacts between antigen presenting cells and T-cells are mediated by a number of molecules, including MHC/peptide complexes-T-cell receptor, costimulatory molecules and adhesion molecules organized in the supra molecular activation cluster (SMAC) or immunological synapse. We have shown, that also the de-attachment of the dendritic cells-T-cells contacts are actively regulated by dendritic cells through the intracellular molecule CYTIP, which is induced during maturation. When CYTIP expression is silenced in mature dendritic cells, T-cell contacts with dendritic cells last longer and T-cell activation is impaired, probably due to reduced screening capacity in search of matching MHC-peptide -T-cell receptor-pairs. In this proposal we want to futher investigate the mode of action of CYTIP especially in the context of T-cell priming and activation with in vitro studies using human monocyte derived dendritic cells to investigate the contact times of dendritic cells silenced for CYTIP to T-cells in time lapse experiments with confocal microscopy. We want to examine the phosphorylation status of CYTIP after dendritic cell-T-cell contact as a possible control element for CYTIP function and check several more adhesion structures for their potential to initiate CYTIP translocation. With the newly available k.o. mice we want to investigate whether the published impairment of immunological responses in these mice are at least partially due to the described functional alterations in dendritic cells and for the newly identified binding partner of CYTIP, SOCS1, we want to investigate the role of this interaction in the functions of CYTIP and SOCS1. With the identification of the mechanism underlying the de- attachment of dendritic cells and T-cells we define a novel point of regulation of specific immune responses which may eventually allow for an additional tool to manipulate immune responses.

Dendritic cells are used for immunotherapies in clinical studies to combat cancer, inflammation and autoimmunity. To enhance their performance in the clinical settings a variety of regulatory interventions in dendritic cell functions are available but more are still desirable. The aim of our studies was to identify possible targets for the fine tuning of dendritic cell preparation for clinical applications. In the course of this project we were able to assign regulatory functions to two proteins produced by dendritic cells during their maturation. Dendritic cells start a maturation process upon encounter with an antigen and acquire the ability to shape immunity in a way to elicit the best possible protection against this antigen. We compared the proteins produced by dendritic cells before and after this maturation process and identified CYTIP as one of the proteins produced during maturation. In a former project we showed that CYTIP is involved in the regulation of the interaction of dendritic cells and T cells by interacting with the adhesion molecule LFA1. This project was aimed to identify additional functions of this molecule in human dendritic cells and applying a mouse model for in vivo studies. Our efforts lead to the insight that in human monocyte derived dendritic cells SOCS1 interacting with CYTIP induces the degradation of CYTIP by the proteasome, and that CYTIP regulates dendritic-cell function in contact hypersensitivity in a mouse model.Additional work was dedicated to another molecule found to be induced during dendritic cell maturation, Bam32, originally identified in B cells as an adaptor molecule involved in the signalling of their antigen receptor. For this molecule we could identify a specific function in human monocyte- derived dendritic cells, its participation in the regulation of MHC class I-induced CD8+ T cell activation.Together, our findings add to the knowledge of dendritic cell function and therefore offer further possible regulatory interventions in preparing dendritic cells for clinical applications.