A Bacterial Artificial Chromosome Reporter System for Expression of the Human FOXP3 Gene in Mouse Regulatory T-Cells

Abstract

The transcription factor FOXP3 plays key roles in the development and function of regulatory T cells (Treg) capable of preventing and correcting immunopathology. There has been much interest in exploiting Treg as adoptive cell therapy in man, but issues of lack of nominal antigen-specificity and stability of FoxP3 expression in the face of pro-inflammatory cytokines have been a concern. In order to enable fundamental studies of human FOXP3 (hFOXP3) gene regulation and to provide preclinical tools to guide the selection of drugs that might modulate hFOXP3 expression for therapeutic purposes, we generated hFOXP3/AmCyan bacterial artificial chromosome (BAC) transgenic mice and transfectants, wherein hFOXP3 expression was read out as AmCyan expression. Using the transgenic mice, one can now investigate hFOXP3 gene expression under defined experimental conditions used for mouse Foxp3 (mFoxp3) studies. Here, we demonstrate that hFOXP3 gene expression in BAC transgenic mice is solely restricted to CD4+ T-cells, as for mFoxp3 gene expression, showing that hFOXP3 expression in Treg cells depends on fundamentally similar processes to mFoxp3 expression in these cells. Similarly, hFOXP3 expression could be observed in mouse T-cells through TCR stimulation in the presence of TGF-β. These data suggest that, at least in part, cell type-specific human and mouse foxp3 gene expression is regulated by common regulatory regions which for the human, are located within the 110-kb human FOXP3 BAC DNA. To investigate hFOXP3 gene expression further and to screen potential therapeutics in modulating hFOXP3 gene expression in vitro, we also generated hFOXP3/AmCyan expression reporter cell lines. Using the reporter cells and transcription factor inhibitors, we showed that, just as for mFoxp3 expression, inhibitors of NF-κB, AP1, STAT5, Smad3, and NFAT also block hFOXP3 expression. hFOXP3 induction in the reporter cells was also TGF-β dependent, and substantially enhanced by an mTOR inhibitor, Torin1. In both the reporter transgenic mice and cell lines, histone H4 molecules in the hFOXP3 promoter and enhancers located in human CNS1 and CNS2 regions were highly acetylated in natural Treg and TCR/TGF-β-induced Treg, indicating hFOXP3 gene expression is regulated by mechanisms similar to those previously identified for the mFoxp3 gene.

IPEX as a Consequence of Alternatively Spliced FOXP3

“…Although high FOXP3 expression levels are commonly attributed to CD4 + CD25 high Treg cells, FOXP3 is also induced by TCR stimulation in naive CD4 + CD25 − T cells and persists for several days in activated CD4 + T cells (20). Notably, activation-induced FOXP3 expression seems to depend on the CNS3 element, because generation of transgenic mice with a bacterial artificial chromosome that encodes for a reporter protein inserted into the human FOXP3 gene (consisting of >50 kb each side of the start codon, including CNS1 and CNS2, but not CNS3) demonstrated that TCR stimulation alone (i.e., without transforming growth factor-β activation) does not induce transgenic FOXP3 expression in murine CD4 + T cells (21).…”

Diminished circulating concentration of interleukin‐35 in Helicobacter pylori‐infected patients with peptic ulcer: Its association with FOXP3 gene polymorphism, bacterial virulence factor CagA, and gender of patients

“…The fork head box protein 3 (FOXP3) is considered as the main transcription factor of Treg cells that plays key roles in the development and function of Treg cells . The localization of FOXP3 gene in humans is on chromosome Xp11.23q13.3 .…”

“…22 The fork head box protein 3 (FOXP3) is considered as the main transcription factor of Treg cells that plays key roles in the development and function of Treg cells. 27 The localization of FOXP3 gene in humans is on chromosome Xp11.23q13.3. 27 The mutations in FOXP3 and Treg cell deficiency result in the development of various autoimmune diseases in humans as is well described in the Immunodysregulation Polyendocrinopathy Enteropathy X-linked (IPEX) syndrome.…”

“…27 The localization of FOXP3 gene in humans is on chromosome Xp11.23q13.3. 27 The mutations in FOXP3 and Treg cell deficiency result in the development of various autoimmune diseases in humans as is well described in the Immunodysregulation Polyendocrinopathy Enteropathy X-linked (IPEX) syndrome. 20 Moreover, some FOXP3 gene polymorphisms could affect the function and quantity of the FOXP3 protein and thus influence the Treg function, being associated with various inflammatory and autoimmune diseases.…”

Role of CNSs Conserved Distal Cis-Regulatory Elements in CD4 + T Cell Development and Differentiation

“…The resulting lack of control on pro-inflammatory cytokines boosts IL-17 production. Based on Foxp3 CNS1 similarities in human and mouse ( 69 ), it is plausible that FOXP3 CNS1 plays a role in the control of human infectious diseases.…”