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Immunology. Oct 2006; 119(2): 203–211.
PMCID: PMC1782350

Splice variants of human FOXP3 are functional inhibitors of human CD4+ T-cell activation

Abstract

FOXP3 has been identified as a key regulator of immune homeostasis. Mutations within the FOXP3 gene result in dysregulated CD4+ T-cell function and elevated cytokine production, leading to lymphoproliferative disease. FOXP3 expression in CD4+ T cells is primarily detected with the CD4+ CD25+ regulatory T-cell population. In humans the protein is detected as a doublet following immunoblot analysis. The lower band of the doublet has been identified as a splice isoform lacking a region corresponding to exon 2. The aim of this study was to investigate whether the splice variant form lacking exon 2 and a new novel splice variant lacking both exons 2 and 7, were functional inhibitors of CD4+ T-cell activation. The data generated showed that full-length FOXP3 and both splice variant forms of the protein were functional repressors of CD4+ T-cell activation.

Keywords: human T lymphocytes, chimeric receptors, FOXP3, splice variants, inhibition of T-cell activation

Introduction

FOXP3 is a member of the forkhead (FKH)-winged helix family of transcription factors and it is believed to act as a transcriptional repressor. Evidence that FOXP3 plays a role in regulating T-cell activation came initially from studies with CD4+ T cells isolated from the scurfy mouse. The scurfy mouse has a 2 base-pair (bp) insertion within the Foxp3 gene, located just upstream of the FKH domain, resulting in a frameshift mutation and a premature stop codon.1 This results in a truncated gene product lacking the functional C-terminal FKH domain. As a direct consequence of this mutation, the CD4+ T cells are hyperresponsive to T-cell receptor (TCR) stimulation, resulting in the production of elevated levels of a number of cytokines, including granulocyte–macrophage colony-stimulating factor, interleukin (IL)-2, IL-4, interferon-γ (IFN-γ) and tumour necrosis factor-α (TNF-α).2,3 The hyper-responsive CD4+ T cells are the effector cells responsible for the development of the severe lymphoproliferative disease, which is characteristic of this mutation. Conversely, characterization of CD4+ T cells isolated from transgenic mice overexpressing the Foxp3 gene product, showed reduced proliferative responses to various stimuli and low levels of IL-2 production.4

Studies using the Jurkat T-cell line have also shown that transient expression of FOXP3 resulted in an attenuation of activation-induced IL-2 production.5 A recent study has shown that human CD4+ T cells transduced with FOXP3-expressing retrovirus, exhibit decreased levels of IL-2, IL-4 and IFN-γ production following activation.6 These studies go on to show that FOXP3 is a specific repressor for nuclear factor of activated T cells and nuclear factor-κB.

In humans, mutations in the orthologous gene result in a genetic disorder known as immune dyresgulation polyendocrinopathy, enteropathy, X-linked syndrome (IPEX).7 Patients with IPEX syndrome exhibit similar phenotypic features to those observed in the scurfy mouse and often succumb to early onset diabetes, eczema and suffer severe enteropathy. A number of mutations in the FOXP3 gene have been identified in IPEX patients and these mutations are located throughout the gene. The severity of the phenotype of both the scurfy mouse and patients with IPEX highlights the essential role of this protein in maintenance of immune homeostasis.

An insight into a role for FOXP3 in regulatory T-cell function has arisen from the observation that the scurfy phenotype can be rescued following the adoptive transfer of wild type T-cell enriched splenocytes.8 Female carriers of the FOXP3 mutation are also apparently healthy. Even though they display random X chromosome inactivation, the presence of normal, in addition to mutated FOXP3 alleles, expressed in the peripheral CD4+ T cells of IPEX carriers, appears sufficient to prevent disease.9 In addition, CD4+ CD25+ T cells from scurfy mice lacked regulatory activity whereas the CD4+ CD25+ T cells from the FOXP3 overexpressing transgenic mice had suppressive activity.10 Complementary to these studies, retroviral transduction of mouse CD4+ CD25 T cells with the Foxp3 gene results in the generation of regulatory T-cell activity.11,12 A subsequent study has shown that Foxp3-transduced CD4+ T cells with specificity for islet antigen were able to stabilize and reverse disease in mice with recent-onset diabetes.13 Overexpression of FOXP3 in human CD4+ CD25 T cells following retroviral transduction, was also sufficient to convert the cells into a regulatory phenotype.14

It is widely accepted that FOXP3 is highly expressed in both mouse and human CD4+ CD25+ regulatory T cells and plays a key role in the functionality of these cells. However, one clear difference between mouse and human FOXP3 expression arises following immunoblotting for FOXP3 protein. Mouse FOXP3 is detected as a single discrete band, whilst human FOXP3 protein is consistently detected as a doublet.1416 The lower band of the doublet has been identified as a splice isoform lacking a region corresponding to exon 2.14,16 A recent study has shown that this splice isoform is capable of functioning as a transcriptional repressor.17 The aim of this study was to investigate whether the splice variant form lacking exon 2 and a new novel splice variant identified in these studies which lacks both exons 2 and 7, were both functional inhibitors of CD4+ T-cell activation.

Materials and methods

Medium and reagents for cell culture

The medium used throughout was RPMI-1640 (Invitrogen, Paisley, UK) supplemented with 5% (v/v) human AB serum (Cambrex Biosciences, Verviers, Belgium), 2 mm l-glutamine, 50 U/ml penicillin and 50 µg/ml streptomycin (all Invitrogen). OKT-3 (anti-human CD3) was purified from hybridoma supernatant. Soluble CD33 extracellular region was purified from hybridoma supernatant, as described previously.25

Constructs

The expression vector pcDNA3.1 (Invitrogen) containing cDNA encoding full-length FOXP3 (amino acids 1–431) with a C-terminal flag tag was a gift from Dr R. Khattri. A mutant form of FOXP3 lacking the forkhead-domain (amino acids 1–327) was cloned from human peripheral blood mononuclear cells (PBMC) cDNA using the following primers:

  • 5′-gagagagaattcgccaccatgcccaaccccaggcctggcaa-3′
  • 5′-gagagaaagcttctacttgtcatcgtcgtccttgtagtcgttgtggaggaactctgggaatgtg-3′

The FKH mutant was cloned into pcDNA3·1- via EcoRI/HindIII restriction sites. This construct also contains a C-terminal flag tag. Flag-tagged FOXP3 was used in the experiment illustrated in Fig. 2. Flag-tagged FOXP3 and untagged FOXP3 gave identical results in the cotransfection system. All cotransfection data shown is with untagged FOXP3. Flag-tagged FKH mutant was used throughout these studies. Additionally, human FOXP3 was cloned from human PBMC cDNA using the following primers:

  • 5′-gagagagaattcgccaccatgcccaaccccaggcctggcaa-3′
  • 5′-gagagagcggccgctcaggggccaggtgtagggttg-3′
Figure 2
Reduced CD4+ T-cell proliferation in response to anti-CD3 stimulation following overexpression of full-length FOXP3 in human CD4+ T cells. (a) Western blot analysis of human CD4+ T cells transfected with increasing amounts of control vector (pcDNA3·1) ...

The polymerase chain reaction (PCR) product was cloned into pcDNA3.1+ via EcoRI and NotI restriction sites. The chimeric receptor used contains the signalling regions from CD28 fused to the ζ signalling region from the TCR/CD3 complex, TCRζ.20

Isolation of human CD4+ T cells

Human peripheral blood was obtained from normal healthy donors and PBMCs were isolated via density gradient centrifugation on Ficoll-Paque (Amersham Biosciences, Chalfont St Giles, UK). CD4+ T cells were purified via negative selection using magnetic-activated cell sorting human CD4+ T cell isolation kits, according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany).

Western blot analysis

Transfected CD4+ T cells were lysed in 20 µl lysis buffer (9 ml of membrane prep buffer (20 mm Hepes, 10 mm MgCl2, 100 mm NaCl in distilled water, pH 7·4), 1 ml of 1% Triton-X-100 and a protease inhibitor tablet (Roche, Lewes, UK)). A micro BCA protein assay was then carried out (Pierce Biotechnology, Rockford, IL) to determine total protein concentration and to ensure equal loading. Samples were separated on 10% (w/v) Tris-Glycine gels (Invitrogen) and transferred to polyvinylidene fluoride membrane. The blots were blocked in phosphate-buffered saline (PBS) containing 2% (w/v) milk powder for a minimum of 1 hr and then probed with polyclonal rabbit antisera raised against recombinant human Foxp3 1 : 1000 followed by goat anti-rabbit Fc-horseradish peroxidase diluted 1 : 10 000 (Stratech Sci. Ltd, Luton, UK).

Transfection of human CD4+ T cells

Transfections were carried out using Amaxa's NucleofectorTM technology (Amaxa Biosystems, Cologne, Germany). Three µg of chimeric receptor DNA and 9 µg of plasmid DNA containing a variant form of FOXP3 were cotransfected per 4 × 106 CD4+ T cells using NucleofectorTM human T-cell solution, program U-13. Cells were rested at 37° for 4 hr post-transfection. Cells were then counted and seeded at 5 × 105 cells/well in either 96-well flat-bottomed CD33-coated plates (5 µg/ml) or uncoated plates (stimulated versus unstimulated). Cells were incubated for a further 24 hr. Supernatants were then removed for cytokine analysis and cells were removed for analysis of cell viability, chimeric receptor expression levels, FOXP3 expression levels and the levels of CD69 and CD25 expression. For single transfections 9 µg of FOXP3 or control plasmid DNA was used, per 4 × 106 cells.

Cytokine assays

The commercially available enzyme-linked immunosorbent assay Duoset kits for human cytokines IL-2, IL-10 and TNF-α (R & D Systems, Abingdon, UK) were used as indicated by the manufacturer.

Flow cytometric analysis

Cells were pelleted in PBS supplemented with 5% (v/v) FCS and 0·1% (w/v) sodium azide (fluorescence-activated cell sorting (FACS) buffer) and incubated with the appropriately diluted antibody for 30 min on ice. The cells were then washed twice in FACS buffer and analysed on a FACScalibur flow cytometer equipped with CellQuest software (BD Biosciences, San Jose, CA.). The following antibodies from BD Pharmingen were used: anti-human CD25-phycoerythrin (PE; clone M-A251, mouse immunoglobulin G1 (IgG1)), anti-human CD69-PE (clone FN50, mouse IgG1) and mouse IgG1-PE control (MOPC-21). CD33-fluoroscein isothiocyanate (FITC) and CD33-Alex 488 were conjugated in-house and used at a final concentration of 1 µg/ml. Propidium iodide (PI) (BD Pharmingen) was used at a final concentration of 0·5 µg/ml.

Intracellular staining

Intracellular staining was carried out using Immunotech's IntraPrepTM permeabilization reagent, according to the manufacturer's instructions (Beckman Coulter, High Wycombe, UK). The following antibodies from eBioscience were used: anti-human FOXP3–PE (clone PCH101, rat IgG2a) and rat IgG2a–PE control.

Proliferation assays with transfected cells

Irradiated CD4+ T-cell depleted PBMCs (5 × 104) were incubated in 96-well U-bottomed plates with 2·5 × 105FOXP3 or control vector transfected cells and 1 µg/ml soluble anti-CD3. On day 3, 0·5 µCi/well 3H-thymidine was added for 4–6 hr. Cultures were harvested and radionuclide uptake measured by liquid scintillation counting.

Results

Cloning of full-length human FOXP3 and splice variant isoforms

Amplification of full-length human FOXP3 from human PBMC cDNA was carried out by PCR. The PCR product was cloned into the expression vector, pcDNA3·1+ and analysis of restriction fragments revealed clones containing three different sized inserts. An alignment of the sequences from the different clones (Fig. 1a) showed that the largest fragment represented full-length FOXP3. The other two fragments sizes represented FOXP3 lacking a 105 bp region (Δ2) and FOXP3 lacking both the 105 bp region and an additional 81 bp region (Δ2 Δ7).

Figure 1
Identification of variant forms of the human FOXP3 gene. (a) Alignment of the published human FOXP3 sequence with the three different-sized clones. (b) Human FOXP3 exon structure. Exons are depicted in alternating bold and non-bold type with exons 2 and ...

The human full-length FOXP3 gene is 1296 bp in size and has been reported to consist of 11 different exons.18 The Ensembl Exonview mapping program used to predict the exon structure of the FOXP3 protein, also suggested the existence of 11 coding exons (Fig. 1b). This exon prediction programme also enabled the missing nucleotide regions within the splice variants to be mapped to specific regions within the protein. The missing 105 bases corresponded to exon 2 and the missing region of 81 bases corresponded to exon 7.

Based on a proposed structure of the human FOXP3 protein published by Gambineri et al. it appeared that the clones lacking the 105 bp region (Δ2) were lacking a proportion of the proline-rich domain, a region important in protein–protein interactions.19 The clones with the smallest insert (Δ2 Δ7) lacked both a proportion of the proline-rich domain and a large proportion of the putative leucine-zipper.

Reduced CD4+ T-cell proliferation following overexpression of full-length FOXP3

The full-length FOXP3 gene was transiently expressed in resting human CD4+ T cells. Expression of the protein was confirmed by Western blot analysis (Fig. 2a). To assess whether overexpression of full-length FOXP3 had a functional effect, human CD4+ T cells were transfected with empty vector DNA or vector DNA encoding the FOXP3 gene and then incubated with autologous irradiated CD4-depleted PBMCs, in the presence of soluble anti-CD3. Proliferation was determined 3 days later by incorporation of 3H-thymidine. As is shown in Fig. 2(b), overexpression of FOXP3 appeared to result in a decrease in CD4+ T-cell proliferation following stimulation with soluble anti-CD3, as compared to the control cells. However, the transfection protocol used typically produces a transfection efficiency of approximately 60% of the viable cells and consequently a significant proportion of cells are not transfected. These cells would be responsive to stimulation by anti-CD3 and could be masking the inhibitory effect of FOXP3 overexpression.

Inhibition of CD28/TCRζ chimeric receptor induced cytokine production following overexpression of splice variant forms of FOXP3

A cotransfection system was devised in which the FOXP3 gene was cotransfected with a gene that would allow specific activation of transfected T cells but not non-transfected cells. The activation system made use of chimeric receptors, previously reported by Finney et al.20 The chimeric receptor construct consists of the intracellular signalling region of the TCR zeta chain (TCRζ) fused to the intracellular signalling region of CD28. The extracellular region of the chimeric receptor is an antibody single chain Fv (scFv) that binds to CD33 (Fig. 3a). Finney et al. reported that the addition of chimeric receptor transfected human T cells to CD33-coated antigen plates, induced a strong activating signal into the transfected cells which could be measured through cytokine release.20

Figure 3
Measurement of FOXP3 expression, CD4+ T cell viabilities and chimeric receptor expression following cotransfection with different variant forms of FOXP3 DNA or FKH mutant DNA. (a) Diagram of a chimeric receptor consisting of a single chain Fv linked to ...

Preliminary experiments involved cotransfection of the CD28/TCRζ chimeric receptor with either full-length FOXP3 or a mutant form of the FOXP3 gene with the forkhead region deleted (FKH mutant). These data confirmed that the transfection efficiency and the viabilities were equivalent between the two transfected populations (data not shown). Expression studies showed that overexpression of FOXP3 in human CD4+ T cells significantly inhibited CD69 up-regulation (data not shown). There was also a significant inhibition of IL-2 and TNF-α production as compared to the FKH mutant (data not shown). Subsequent experiments investigated the ability of the two splice variants to inhibit chimeric receptor-induced T-cell activation.

Purified CD4+ T cells were cotransfected with 3 µg of CD28/TCRζ chimeric receptor DNA and 9 µg of either full-length FOXP3, FOXP3 lacking exon 2 (Δ2), FOXP3 lacking exons 2 and 7 (Δ2 Δ7), or FKH mutant DNA. Figure 3(b) shows the viability of the cells on the basis of PI uptake. The percentage of cells expressing both FOXP3 and the chimeric receptor are shown in Fig. 3(c). Levels of chimeric receptor expression alone are shown in Fig. 3(d). The combined results for seven different donors are illustrated in Fig. 3(e, ,f).f). Figure 3(e) shows that the viabilities of the four transfected populations were equivalent with viability levels of around 60–70% being achieved. The levels of CD28/TCRζ chimeric receptor expression were also equivalent between the different groups (Fig. 3f) as were the proportions of cells expressing both FOXP3 and the chimeric receptor (Fig. 3c). Indeed, it appears that all the chimeric receptor positive cells were also expressing FOXP3.

Once it had been established that the transfection efficiency and the viabilities were broadly equivalent for the four different transfected cell populations, subsequent experiments concentrated on investigating whether the splice variants had any functional effect on signalling through the CD28/TCRζ chimeric receptor. Human CD4+ T cells were cotransfected with DNA encoding the CD28/TCRζ chimeric receptor and DNA encoding either a variant form of the FOXP3 gene or FKH mutant and seeded onto CD33-coated plates. The culture supernatants were harvested 24 hr post-transfection and assayed for human IL-2. Cells were removed and stained for cell surface CD69 and CD25.

Figure 4(a) shows that there was a significant inhibition of IL-2 production by both FOXP3 variants and the full-length version of the protein, as compared to the FKH mutant. Figure 4(b) shows that both splice variant forms of the human FOXP3 protein were also able to inhibit the up-regulation of CD69 expression following activation through the chimeric receptor. The levels of CD25 expression on the surface of cells expressing either a variant form of the FOXP3 protein or the FKH mutant were not significantly different (Fig. 4c). Together these data suggest that the two splice variant forms of the human FOXP3 protein are functional inhibitors of CD4+ T cell activation.

Figure 4
Splice variant forms of human FOXP3 are functional suppressors of CD28/TCRζ chimeric receptor-induced IL-2 production. Human CD4+ T cells were cotransfected with CD28/TCRζ chimeric receptor DNA and either DNA encoding a variant form of ...

Discussion

During the cloning of the full-length version of the human FOXP3 gene, two splice variant forms of the gene were identified. Mapping studies showed that the isoform referred to as Δ2, lacked exon 2, a region mapping within the proline-rich part of the gene. The other isoform, referred to as Δ2 Δ7, lacked both exons 2 and 7. Exon 7 maps to a region forming part of a leucine-zipper structure. In an attempt to determine if the two isoforms of FOXP3 were functional inhibitors of CD4+ T-cell activation, a cotransfection system was developed whereby T-cell activation is induced following specific signalling through the CD28/TCRζ chimeric receptor.

Preliminary experiments investigated the effect of overexpression of full-length FOXP3 on subsequent T-cell activation. Stimulation of the transfected cells for 24 hr through the chimeric receptor resulted in a significant inhibition of CD4+ T-cell activation, as measured by the release of both IL-2 and TNF-α and the levels of cell surface CD69 expression (data not shown). These data confirmed a role for FOXP3 in regulating human CD4+ T-cell activation and also confirmed that the FKH mutant was unable to suppress CD4+ T-cell activation. This is believed to be because the FKH mutant is unable to translocate to the nucleus.5

To investigate whether the missing domains in the splice variants were important for function, studies were undertaken to investigate whether the splice variants were also able to inhibit T-cell activation induced through the CD28/TCRζ chimeric receptor. The data generated from the functional studies showed that both variant forms of the FOXP3 protein significantly inhibited chimeric receptor induced CD4+ T-cell activation, as measured by the release of IL-2 and the expression of cell surface CD69. Interestingly, overexpression of FOXP3 did not affect the levels of CD25 cell surface expression. However, the levels of CD69 were inhibited and so it would not have been surprising for the CD25 levels to also be inhibited. The most likely explanation for this difference is that measuring CD25 expression 24 hr postactivation, was too early a time point to see any difference in expression levels. CD25 expression typically peaks around 3 days after activation unlike CD69, which is an early activation marker. There is data suggesting that CD25 expression is linked to FOXP3 expression. Retroviral transduction of FOXP3 into both mouse and human CD4+ CD25 resulted in elevated levels of CD25 expression compared to control transduced cells.11,14 Again it is possible that 24 hr post-activation may have been too early a time point to observe this effect in these studies. However it is also important to note that retrovirally transduced cells are preactivated, therefore it is conceivable that FOXP3 does not induce CD25 expression but rather maintains its expression.

Nevertheless, these data demonstrated that the lack of exons 2 and 7 did not prevent FOXP3-dependent inhibition of T-cell activation. This was surprising because the missing exons encoded protein domains that might have been expected to be key to the function of FOXP3. Indeed, gene analysis of an IPEX patient has revealed an in-frame three base-pair deletion (E251) within the leucine zipper.21 In this study, the authors speculated that this deletion prevented dimerization, thus resulting in the failure of effector function. There has also been a reported mutation within exon 2. However, in this case, the mutation was a single base deletion resulting in a frame-shift and a predicted premature termination codon, resulting in a truncated protein lacking the functional FKH domain.22

It is still not clear if FOXP3 actually needs to dimerize in order to bind to DNA. It had been reported that FKH proteins bind to DNA as monomers.23 However, studies with Foxp1, Foxp2 and Foxp4, three FKH proteins that act as repressors of lung-specific gene transcription24 have shown that these FKH family members do require dimerization before DNA binding and transcriptional repression can occur. Interestingly this group showed that the zinc-finger domain of these proteins was not essential for transcriptional repression.

Clearly, in these studies described, the absence of a portion of the leucine-zipper did not prevent the FOXP3 protein from inhibiting T-cell activation, suggesting that dimerization is not required for this function. The absence of a region of the proline-rich region is also not vital for inhibition to occur.17 The molecular interactions and targets of FOXP3 are still unknown. It is possible that the signalling pathway that leads to inhibition of T-cell activation is distinct from the signalling pathway that generates regulatory activity. It is feasible that the FKH domain is the critical domain for DNA binding and this domain alone is sufficient to inhibit T-cell activation, potentially via the active repression of target promoters in proinflammatory genes.

The identification of splice variant forms of the protein suggests an additional level of complexity surrounding the biology of FOXP3. The functional relevance of these proteins has yet to be determined, although these studies have shown that each splice variant was capable of inhibiting T-cell activation as measured by inhibition of IL-2 production. It may be that the isoform lacking exon 2, which is naturally expressed in the human CD4+ CD25+ T-cell population, plays a role in limiting T-cell activation without also playing a role in the signalling pathways required to generate regulatory activity.

Experiments conducted to investigate whether CD4+ T cells cotransfected with DNA encoding CD28/TCRζ and full-length FOXP3 were able to suppress CD4+ T cells transfected with CD28/TCRζ alone, proved negative (data not shown). This was not because CD28/TCRζ transfected cells are resistant to suppression, as naturally occurring CD4+ CD25+ FOXP3+ regulatory cells were able to inhibit the CD28/TCRζ transfected cells (data not shown). Moreover, naturally occurring CD4+ CD25+ FOXP3+ regulatory cells transfected with the chimeric receptor, were successful at inhibiting IL-2 production from CD28/TCRζ transfected cells, following stimulation through the chimeric receptor (data not shown).

Thus, the ability of the splice variants to induce regulatory activity could not be investigated in this study. Attempts to show that the isoform lacking exons and 7 is naturally expressed in the CD4+ CD25+ regulatory population have also proved somewhat inconclusive. However, this variant form was cloned from human PBMC cDNA supporting the concept that it is expressed naturally. In addition, the missing region mapped to a complete exon, which is highly unlikely to have arisen from a PCR artefact. Future studies will hopefully determine the cell subtype specificity of this new novel FOXP3 variant.

In conclusion, these studies have confirmed that splice variant forms of the human FOXP3 protein exist. Whether these isoforms each have distinct functions is unclear. Transient expression of the isoforms in human CD4+ T cells showed that both variants significantly inhibited CD28/TCRζ chimeric receptor-induced T-cell activation. However, the apparent absence of these isoforms in the mouse suggests additional functions for these isoforms, which may have evolved over time. Clearly, as the signalling pathways involving FOXP3 are elucidated, the significance of these splice variants will become apparent.

Acknowledgments

We thank Drs Patrick Slocombe and Gill Holdsworth for advice on molecular biology and immunoblotting techniques, repectively. We also thank Drs Tim Bourne, Roli Khattri and Fiona Powrie for helpful comments and discussions on this work and manuscript.

Abbreviations

IPEX
immune dysregulation polyendocrinopathy, enteropathy, X-linked syndrome
FKH
forkhead
PI
propidium iodide
bp
base-pairs

References

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