Dogma that the regulatory T cell (Treg) prevents catastrophic autoimmunity throughout the lifespan relies on the assumption that the FoxP3 locus is transcribed exclusively in Treg. To test the assumption, we used the Rag2−/− and the Rag2−/− mice with the Scurfy (sf) mutation (FoxP3sf/Y or FoxP3sf/sf) to evaluate FoxP3 expression outside of the lymphoid system. Immunohistochemistry and real-time PCR revealed FoxP3 expression in breast epithelial cells, lung respiratory epithelial cells, and prostate epithelial cells, although not in liver, heart, and intestine. The specificity of the assays was confirmed, as the signals were ablated by the Scurfy mutation of the FoxP3 gene. Using mice with a green fluorescence protein open reading frame knocked into the 3′ untranslated region of the FoxP3 locus, we showed that the locus is transcribed broadly in epithelial cells of multiple organs. These results refute an essential underlying assumption of the dogma and question the specificity of FoxP3-based Treg depletion.
The Forkhead transcription factor FoxP3 has been initially identified as a causative mutation of a fatal autoimmune disease in mouse and human (1, 2, 3, 4). More recently, FoxP3 was shown to be a key transcription factor for function of regulatory T cells (Treg)3 (5, 6), where it is expressed at levels comparable to a housekeeping gene in CD4+CD25+ Treg (5). In an attempt to test the physiological function of Treg, two groups have designed strategies to delete Tregs by treating transgenic mice expressing a diphtheria toxin (DT) receptor (DTR) under the control of the FoxP3 locus with DT (7, 8). The acute and fatal autoimmune diseases in adult and/or neonates were used to support a dramatic conclusion that the Treg cells prevent catastrophic autoimmunity throughout the lifespan of mice (7). For this conclusion to be valid, however, one must ascertain that Treg is the only cell type in mice that can express FoxP3 and, by inference, the DTR in the transgenic mice. Unfortunately, no serious effort has been made to confirm this assumption.
We have reported expression of FoxP3 protein in thymic (9) and breast epithelial cells (10). Although these data are inconsistent with the notion of Treg-exclusive expression of FoxP3, activation of the locus in these organs would in theory not affect DT-mediated fatal injury, as these organs are not vital for the survival of mice. In this study, we systemically investigated expression of FoxP3 by immunohistochemistry, real-time PCR, and the knockin mice with a bicistronic FoxP3 locus that coexpresses the enhanced GFP (EGFP) under the control of the endogenous FoxP3 promoter/enhancer elements (11). In this work we report a broad expression of the FoxP3 gene in epithelial cells of multiple lineages. Our data raised a serious concern about interpretation of the fatal inflammation in DT-treated FoxP3-DTR transgenic/knockin mice.
Materials and Methods
Wild-type (WT) or FoxP3EGFP mice that express both FoxP3 and EGFP under the endogenous regulatory sequence of the FoxP3 locus have been described (11). BALB/c RAG-2−/− and RAG-2−/− mice with the Scurfy (sf) mutation of the FoxP3 gene have also been described (9). All mice were used at 6–8 wk after birth.
Immunohistochemistry and immunofluorescence
Tissues from mice were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned according to the standard procedure. Endogenous peroxidase activity was quenched using 3% H2O2 for 30 min at room temperature; nonspecific binding sites in the sections were blocked using 5% normal BSA (Sigma-Aldrich) in PBS for 1 h at room temperature. Sections of 5 μm were made and reacted with affinity-purified anti-FoxP3 polyclonal Ab (9). The slides were washed in PBS and subsequently incubated with the second Ab, HRP-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology) for 1 h at room temperature. After being washed in PBS, slides were developed with 3, 3′-diaminobenzidine, and counterstained with hematoxylin.
The FoxP3EGFP mouse sections were incubated with rat anti-mouse K8 Ab (Troma I; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA) overnight at 4°C. This is followed by Cy3-conjugated donkey anti-rat secondary Abs (rabbit absorbed; Millipore). The expression of GFP is visualized by Alexa 488-conjugated rabbit anti-GFP IgG fraction (Molecular Probes) at 4°C.
Quantitative real-time PCR analysis
Real-time PCR for FoxP3 expression was performed as described previously (7). Samples were run in triplicate and the relative expression was determined by normalizing expression of each target to the endogenous reference, hypoxanthine phosphoribosyltransferase (Hprt) transcripts. The primer sequences were as follows: FoxP3, 5′-TACTTCAAGTTCCACAACATGCGACC-3′ (sense) and 5′-CGCACAAAGCACTTGTGCAGACTCAG-3′ (antisense); and Hprt, 5′-AGCCTAAGATGAGCGCAAGT-3′ (sense) and 5′-TTACTAGGCAGATGGCCACA-3′ (antisense).
Results and Discussion
To avoid interference of FoxP3 from lymphocytes, we used RAG-2-deficient mice lacking T and B cells to analyze expression of FoxP3 in nonlymphoid organs. As control for Ab specificity, we generated Rag2−/− Scurfy mice that have a naturally occurring truncation mutation resulting in an early termination codon. Because of the non-sense-mediated decay of the mRNA, essentially no FoxP3 can be detected in the Scurfy mice (9). We performed immunohistochemical staining to detect expression of FoxP3 in various tissues using affinity-purified polyclonal Ab (9). As shown in Fig. 1, in FoxP3WTRag2−/− mice, FoxP3 was found in mammary epithelial cells, lung bronchial epithelial cells, and prostate epithelial cells. In contrast, no staining can be found in intestine (Fig. 1), kidney, liver, and heart (data not shown). Because none of the tissues from the FoxP3sf/Y or sf/sfRag2−/− mice show staining, we conclude that the staining is specific for the FoxP3 protein as it is largely diminished by a FoxP3 mutation that leads to disappearance of mRNA by a non-sense-mediated decay (Fig. 2).
In conjunction with immunohistochemistry, we investigated FoxP3 mRNA levels in various mouse tissues by real-time PCR. A typical positive amplification is shown in Fig. 2,A, and the summary data from multiple organs are shown in Fig. 2,B. These data demonstrated that FoxP3 was expressed at significant levels in mammary gland, lung, and prostate in FoxP3WT/YRag2−/− mice, but no expression could be detected in liver. Again, the FoxP3 transcript was reduced by 20- to 500-fold in FoxP3sf/Y or sf/sfRag2−/− mice. It is worth noting that the levels of the FoxP3 transcript are ∼3- to 100-fold lower in other organs in comparison to those in the spleen. The overall lower abundance makes it harder to detect FoxP3 expression in nonlymphoid organs. To determine whether the full-length FoxP3 open reading frame was transcribed, we used primers that encompass the whole open reading frame to amplify FoxP3 mRNA. The PCR products were analyzed by agarose gel electrophoresis. As shown in Fig. 2 C, the m.w. of the product was consistent with it being full-length mRNA. This is confirmed by DNA sequencing of the RT-PCR product (data not shown).
To test whether activation of the FoxP3 locus in the epithelial cells extends to the EGFP knocked into the 3′ untranslated region of the FoxP3 mRNA, we used anti-GFP Abs to evaluate the GFP expression in the FoxP3EGFP mice (11) with WT mice serving as control for nonspecific binding and autofluorescence. Consistent with the immunohistochemical findings, the GFP protein was detected in mammary epithelial cells, lung bronchial epithelial cells, and prostate epithelial cells of FoxP3EGFP mice as revealed by double staining with anti-GFP and anti-K8 Abs. Nonspecific anti-GFP binding and autofluorescence were not found in the corresponding tissue in wild-type mice (Fig. 3). We also observed no specific signal in kidney, liver, intestine, and heart in the FoxP3EGFP mice (data not shown). It is well established that immunofluorescence with anti-GFP Abs, a strategy used here, is more sensitive and better able to discriminate between authentic GFP expression and autofluorescence (12). Relying on direct GFP visualization may explain the failure of others to observe FoxP3 locus activation in the thymic epithelial cells (13). Our previous study also showed much lower expression of FoxP3 in Rag1-deficient thymic tissue (9). Given the demonstrated function of T cells in the development of thymic epithelial cells (14), it is plausible that the difficulty in detecting FoxP3 in Rag-deficient mice may be an indirect consequence of abnormal development of epithelial cells. However, it is unclear whether the different approaches explain the lack of report on the GFP in nonlymphoid organs, as others have not directly addressed the issue. Because the open reading frame of DTR was inserted in a similar fashion, it is intriguing that in the transgenic and knockin mice used by others for Treg deletion with DT treatments (7, 8) the epithelial cells may be among those targeted by DT treatment. Because epithelial cells of vital organs expressed FoxP3 at high levels, such targeting, either alone or in combination with Treg cell depletion, may contribute to lethality caused by DT treatment.
Finally, FoxP3 is an interesting transcription factor that binds to a large number of genes important for a number of fundamental cellular functions (10, 15, 16, 17). Our documentation of its broad expression in epithelial cells of multiple organs suggests that it may play a more broad function outside of Treg cells. In this regard, we reported critical roles for FoxP3 as a regulator of thymopoiesis when expressed in thymic epithelial cells and as an important tumor suppressor gene when expressed in mammary epithelial cells, likely through regulation of oncogenes such as ErbB2 and Skp2 (9, 10, 17).
We thank Dr. Lishan Su for anti-FoxP3 Abs.
The authors have no financial conflict of interest.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This study was supported by grants from the U.S. Department of Defense and the National Institutes of Health.
Abbreviations used in this paper: Treg, regulatory T cell; DT, diphtheria toxin; DTR, DT receptor; EGFP, enhanced GFP; Hprt, hypoxanthine phosphoribosyltransferase; sf, Scurfy; WT, wild type.