Domain requirements for the diverse immune regulatory functions of foxp3

Foxp3 is responsible for the major immunological features of Treg cells, including hypoproliferation in vitro, immune suppression of conventional T cells and resistance to Th2 cell differentiation. In addition to the Forkhead domain, the Foxp3 protein contains the N-terminal, zinc finger and leucine zipper domains. To understand how these domains contribute to Foxp3 functions, we systematically compared the roles of these domains in determining the 3 major immunological features of Treg cells. We designed a bridge-mediated mutagenesis method to generate Foxp3 mutants with complete deletion of each of the domains. CD4 T cells expressing the Foxp3 mutant with deletion of the N-terminal, leucine zipper or the forkhead domain showed robust TCR dependent proliferation in vitro, differentiated into Th2 cells, and lost immune suppressive activities in vitro and in vivo, demonstrating a complete loss of all 3 functions of Foxp3. In contrast, deletion of the zinc finger domain only partially impaired these functions of Foxp3. This result suggests that mutations in the zinc finger domain could lead to nonlethal autoimmune and allergic diseases, in which reduction rather than complete loss of Foxp3 functions is expected. In any case, deletion of a particular domain showed similar effects on all 3 functions of Foxp3. Therefore defining each of the immunological features of Treg cells requires intact Foxp3 proteins.