PROJECT SUMMARY
Regulatory T cells (Treg) play a critical role in maintaining immune system homeostasis and preventing
autoimmunity and immunopathology. Defective Treg function is linked to multiple autoimmune diseases including
type 1 diabetes and multiple sclerosis. On the other hand, enrichment of Treg cells within tumors is thought to
be a barrier to effective anti-tumor immune responses. The development and maintenance of the Treg cell
lineage are dependent on the transcription factor Foxp3, as loss of function mutations lead to severe
lymphoproliferative disease in mice and humans. Thus, understanding the mechanisms that govern Foxp3
induction and stability may lead to the development of novel therapies for autoimmune disease and cancer. Dr.
Zheng and colleagues recently developed a system to perform genome-wide CRISPR/Cas9 knockout screens
to identify Foxp3 regulators in mouse Tregs. The unbiased screen results not only confirmed a number of known
Foxp3 regulators but also revealed many novel factors that control Foxp3 expression. Gene ontology analysis
of newly identified Foxp3 regulators revealed eIF5A itself and two genes involved in its hypusination, indicating
a previously unknown role for hypusinated eIF5A (eIF5AH) in controlling Foxp3 expression and Treg function.
Hypusination is a post-translational modification of lysine residue by the action of two enzymes, deoxyhypusine
synthase (DHPS) and deoxyhypusine hydroxylase (DOHH) using spermidine as a substrate. A conserved lysine
residue in eIF5A is the only known hypusination target site in eukaryotes. Upon hypusination, eIF5AH functions
as a facilitator that promotes mRNA translation initiation, elongation and termination. Although eIF5AH is known
for regulating cell growth, how hypusination pathway maintains cell identity and cell type specific function is
unclear. The unbiased genome-wide Treg screen identified eIF5A, DHPS and DOHH as negative regulators of
Foxp3 expression. Deletion of each of these genes by CRISPR knockdown leads to significantly increase of
Foxp3 expression, and surprisingly, reduced Treg suppressor function. This unusual decoupling of high Foxp3
expression with increased suppressor function suggests that hypusination pathway adds a new layer of
regulatory mechanism controlling Treg function. The overall objective of this study is to define the role of
hypusinated eIF5A in Foxp3 expression and Treg function. This goal will be accomplished by elucidating
mechanistically on how eIF5AH regulates expression of Foxp3 and other signature genes in Tregs (Aim 1), and
exploring the in vivo consequence of blocking hypusination in conditional knockout mouse models (Aim 2). The
outcomes of the proposed studies are expected to fundamentally advance the understanding on how
hypusinated eIF5A regulates Foxp3 expression and Treg function. The outcomes of this research could provide
evidence to support targeting hypusination pathway to enhance anti-tumor immune response.