The molecular mechanism by which FOXP3 modifies development and regulatory activity of regulatory T-cells
Zusammenfassung der Projektergebnisse
T regulatory (TR)-cells represent a heterogeneous class of T-cells with the ability to avoid, to suppress or to stop antigen specific or unspecific immune responses. In most cases the transcription factor FOXP3 (forkhead family transcription factor3) is involved in these processes. Thus, controlling their immune-regulatory potential holds promise for the treatment of immune mediated diseases such as type 1 diabetes. Unlike murine TR, human TR coexpress equal amounts of two isoforms of FOXP3 one corresponding to the canonical full-length sequence; and the other lacking the sequence encoded by exon 2. In in vitro models it was shown that FOXP3Δ2+ T cells were less hyporesponsive, less efficient in blocking RORγt-directed IL-17 expression and produced more IL-2 than FOXP3+ T cells suggesting that the two isoforms may have distinct functions in human. Therefore we decided to generate a conditional knockout of exon 2 in mice to investigate the human situation in vivo. The generation of a mouse that posses the conditional knockout of exon 2 in the Foxp3 gene bore the difficulty that not a whole gene is knocked out but just one exon. Using conventional cloning strategy this goal is not feasible since range for the insertion of the loxp sites, which allow the deletion, is very limited and the open reading frame has to stay intact to ensure proper protein production. A new form of chromosome engineering, termed recombineering makes it possible to introduce loxP sites and selectable markers anywhere in a gene. Efficient homologous recombination in E. coli is made possible by the use of the Red genes of bacteriophage λ, which permits linear double-strand DNA fragments (i.e., those carrying loxP sites and selection markers) to be inserted into DNA cloned on plasmids. The conditional knockout was successfully cloned, transformed into ES cells and injected and chimeras have been already obtained. As a reflection of the human immune regulation via insert of the spliced variant of the full length Foxp3 that exist in human but not mice, this cko Foxp3ΔEx2 mouse will be very important for the further understanding of regulation of the immune system in humans and will give valuable insights of the interaction of important transcriptional factors that have been associated with autoimmune diseases.
