Struktur-Funktions-Analysen von IRF4 in der T-Zelldifferenzierung

The hematopoietic transcription factor interferon regulatory factor (IRF) 4 regulates the development and function of T, B and dendritic cells of the immune system. Within the T-cell compartment, this factor directs the differentiation of common precursor cells to the Th/c2, Th/c9 and Th/c17 cell subsets, which, apart from their multiple physiological functions, can cause severe autoimmune diseases like multiple sclerosis, inflammatory bowel disease and also asthma. On its own, IRF4 binds weakly to promoters. In order to function, it needs the interaction with other partner transkription factors, which override the effect of an autoinhibitory domain at the C-terminal region of IRF4 that prevents the binding of its DBD at the promoter. Therefore, the first part of this work deals with the question, whether the autoinhibitory domain affects the biological activity of IRF4 on T cell differentiation. For this, the IRF4 deletion mutant (d421-450) which selectively lacks the autoinhibitory domain, was cloned, overexpressed in IRF4 deficient T cells and compared in its effect on T cell differentiation with total IRF4. As characterized by subset-specific cytokine production and expression of chemokine receptors and master transcription factors, the mutant d421-450 enhanced overall Th/c-cell differentiation and in particular Th/c17-cell differentiation. In contrast, the mutant d411-450 which in addition lacks the 10 aa located upstream of the autoinhibitory domain, lost all examined activity during T - cell differentiation. These findings confirm that (1) the autoinhibitory domain has an inhibitory effect on T-cell differentiation, and (2) the 10 aa upstream of the autoinhibitory domain are important for T-cell differentiation. This is the first report of the biological effect of the autoinhibitory domain on T cell differentiation. IRF4 has a modular domain structure, in which the DBD is responsible for binding to the promoter, while the RD mediates the interaction with other transcription factors. Dependent on the respective partner, this interaction occurs at different positions within the RD. In this regard, aa 411-420 are not only involved in the differentiation of the Th/c2, Th/c9 and Th/c17-cell subsets (as shown in the first part), but are also important for the interaction with the transcription factors PU.1 and NFATc2. In the second part of this work, it was therefore analysed in detail, how the modification of this region affects the capacity to regulate T-cell differentiation. It was found that the proline exchange mutant P411-412 mainly inhibited Th/c9 and Th/c17 differentiation, while the P411-413 mutant totally lost activity in all studied T cell differentiation conditions. We hypothesize that the proline substitutions help to destroy the formation of an α-helical structure of the peptide chain in this region. Thus, in the second part of this work it has been successfully demonstrated for the first time that the aa 411-412 of IRF4 are essential for Th/c9 and Th/c17-cell differentiation.