Foxp3 lacking exons 2 and 7 is unable to confer suppressive ability to regulatory T cells in vivo
Introduction
The immune system is delicately regulated to allow responses against foreign- but not self-antigens. Several distinct mechanisms uphold such immunological tolerance, including deletion of highly self-reactive lymphocytes during their development, lymphocyte hypo-responsiveness when antigens are encountered in the absence of co-stimulatory signals, and suppression of immune responses by cells with regulatory capacity. Among these tolerance mechanisms is the CD4+FOXP3+ regulatory T (Treg) cell population, which comprises a subset of T cells that suppresses immune activation in a dominant manner [1]. Abolished Treg cell function is the direct cause of the lymphoproliferative disease seen in immunodysregulation polyendocrinopathy enteropathy X-linked syndrome (IPEX) patients and scurfy mice [2], [3], [4]. In addition, a large number of studies have suggested that deficiencies in Treg cell function are an underlying cause for disease conditions ranging from infections to chronic inflammatory disorders [5].
Treg cells are characterized by expression of the forkhead/winged-helix transcription factor FOXP3 [6], [7], [8]. FOXP3 contains an N-terminal repressor domain, centrally located C2H2 zinc finger and leucine zipper domains and a forkhead domain at the C-terminus [9], [10], [11], [12]. The N-terminal repressor domain, partially encoded by exon 2, allows FOXP3 to interact with transcriptional activators and repressors to modulate gene expression [13]. The function of FOXP3's C2H2 zinc finger domain remains unknown and no IPEX causative mutations have been found in this domain. The leucine zipper, encoded by exon 7, domain is required for formation of FOXP3 multimers as well as for FOXP3 to be able to confer suppressor ability [9], [11], [14], [15]. The C-terminal forkhead domain mediates DNA-binding by FOXP3 and it is a hotspot mutation region in IPEX [12], [16].
The expression and function of FOXP3 is normally regulated at a transcriptional, post-transcriptional, and post-translational level. At the transcriptional level, alternative splicing appears to play a key role in FOXP3 regulation. The two most abundant FOXP3 isoforms, full-length FOXP3 (FOXP3fl) and FOXP3 lacking exon 2 (FOXP3Δ2), confer a suppressive ability to Treg cells [17], [18]. In contrast, FOXP3 lacking exons 2 and 7 (FOXP3Δ2Δ7) has been suggested to inhibit other FOXP3 isoforms in a dominant negative manner [19]. A recent study has also demonstrated that exon 7 of FOXP3 is required for proper Treg cell function, as two different point mutations, located near the intron 7 splice donor site, were found to result in exon 7 excision and IPEX syndrome [20]. However, despite the importance of FOXP3 in Treg cells, the regulation and functional consequences of FOXP3 isoform expression remains poorly understood.
Up to date all experiments addressing the function of FOXP3 isoforms have been performed in vitro as mice do not utilize alternative splicing of coding exons for FOXP3 transcripts [21]. However, FOXP3 is highly conserved and can function over species barriers [19], [22]. Here, we characterized the in vivo function of the mouse counterpart to FOXP3Δ2Δ7 by generating mice exclusively expressing Foxp3 lacking exon 2 and 7 (Foxp3δ2δ7). We show that mice exclusively expressing Foxp3δ2δ7 in essence phenocopy scurfy mice and that Foxp3δ2δ7 is unable to confer a suppressive ability to mouse Treg cells.
Section snippets
Mice
A targeting vector was constructed such that a Foxp3 cDNA lacking exons 2 and 7 was inserted at the translation initiation ATG codon of Foxp3 and the TGA stop of Foxp3 was replaced by a 2A peptide-GFP cassette followed by a double polyadenylation signal (pA) and an FRT flanked Neo cassette (Fig. 1a and Supplementary Fig. 1). The Foxp3δ2δ7-2A-GFP cassette was synthesized and the sequence was verified using DNA sequencing prior to subcloning it into the targeting vector. The
Mice exclusively expressing Foxp3 lacking exons 2 and 7 succumb to lymphoproliferative disease early in life
In vitro suppression assays have been helpful to study the function of mouse and human Treg cells, yet it still remains uncertain as to what extent these assays mimic suppression in vivo. Mice do not express different Foxp3 isoforms [21], which makes it challenging to study the functional consequences of alternative splicing of FOXP3 in vivo. However, FOXP3 is highly conserved and both FOXP3 and Treg cell-mediated suppression can function over species barriers [19], [22]. To define the function
Discussion
The regulation and functional consequences of FOXP3 isoform expression remains poorly understood. In this study we demonstrate that mice exclusively expressing an artificial FOXP3 isoform FOXP3δ2δ7, which correspond to human FOXP3Δ2Δ7, succumbs early in life to lymphoproliferative disease. Thus our in vivo findings corroborate previous in vitro studies that have suggested that FOXP3Δ2Δ7 is unable to confer a suppressive ability to Treg cells.
In 2005 Allen et al. described the existence of the
Conclusions
Mice that exclusively expressing an artificial FOXP3 isoform FOXP3δ2δ7, which correspond to human FOXP3Δ2Δ7, succumbs early in life to lymphoproliferative disease characterized by multi-organ inflammation, activation of T cells and APCs, increased cytokine expression, autoantibody production and severely impaired B cell development. In fact, these Foxp3δ2δ7/Y mice appear to phenocopy scurfy mice suggesting that the Foxp3δ2δ7 isoform is completely unable to confer a suppressive phenotype to
Funding
This work was supported by the Swedish Research Council (projects: 2008-3055 and 2012-1851), the Swedish Cancer Foundation, the Swedish Heart-Lung Foundation, the European Union (FP7 Marie Curie IRG program), the Swedish Society of Medicine, Sigurd and Elsa Goljes minne and Ruth och Richard Julin's foundation.
Authorship contributions
A.-L.J., S.L., R.M. and J.A. designed the research; A.-L.J., S.L., C.D. and R.M. performed experiments; A.-L.J., S.L., L.W and J.A. wrote the manuscript and all authors discussed the results and their implications and commented on the manuscript.
Acknowledgments
We thank Ellen Chen at Ingenious targeting Laboratories, Meimei Shan and Cindy Gutzeit at the Icahn School of Medicine at Mount Sinai for their help as well as Professor Robert A. Harris at Karolinska Institutet for linguistic advice.
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These authors contributed equally to this work.