Crucial role of FOXP3 in the development and function of human CD25⁺CD4⁺ regulatory T cells

Abstract

Naturally occurring CD25⁺CD4⁺ regulatory T cells are engaged in the maintenance of immunological self-tolerance and down-regulation of various immune responses. Recent studies with mice showed that Foxp3, which encodes the transcription factor Scurfin, is a master regulatory gene for the development and function of CD25⁺CD4⁺ regulatory T cells. Here we examined the role of FOXP3 in human CD25⁺CD4⁺ regulatory T cells. The FOXP3 gene and its protein product were preferentially expressed in peripheral CD25⁺CD4⁺ T cells, in particular CD25⁺CD45RO⁺CD4⁺ T cells in normal individuals and, interestingly, in some human T cell leukemia virus type 1-infected T cell lines, which constitutively express CD25. TCR stimulation of CD25⁻CD45RO⁻CD4⁺ naive T cells failed to elicit FOXP3 expression at the gene or protein level. Ex vivo retroviral gene transfer of FOXP3, on the other hand, converted peripheral CD25⁻CD45RO⁻CD4⁺ naive T cells into a regulatory T cell phenotype similar to CD25⁺CD4⁺ regulatory T cells. For example, FOXP3-transduced T cells exhibited impaired proliferation and production of cytokines including IL-2 and IL-10 upon TCR stimulation, up-regulated the expression of regulatory T cell-associated molecules such as CD25 and CTL-associated antigen-4 and suppressed in vitro proliferation of other T cells in a cell–cell contact-dependent manner. Thus, human FOXP3 is a crucial regulatory gene for the development and function of CD25⁺CD4⁺ regulatory T cells, and can be used as their reliable marker. Furthermore, regulatory T cells de novo produced from normal naive T cells by FOXP3 transduction can be instrumental for treatment of autoimmune/inflammatory diseases and negative control of various immune responses.

autoimmunity, immune regulation, immunological tolerance, transcription factor

APC    antigen-presenting cells, ATL    adult T cell leukemia, CTLA-4    CTL-associated antigen-4, GITR    glucocorticoid-induced tumor necrosis factor receptor family-related gene, HPRT    hypoxanthine–guanine phosphoribosyl-transferase, HTLV-1    human T cell leukemia virus type 1, IPEX    immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome, ΔLNGFR    intra-cytoplasmatically truncated version of the human low-affinity nerve growth factor receptor, MACS    magnetic cell sorting, PMA    phorbol myristate acetate, RT    reverse transcription, TGF-β    transforming growth factor beta, T_R    naturally occurring regulatory T

Introduction

Immunological self-tolerance, i.e. unresponsiveness to self-constituents, is maintained not only by deletion of self-reactive lymphocytes in the central lymphoid organs but also by the control of their activation and expansion in the periphery. As a key mechanism of such peripheral self-tolerance, naturally occurring regulatory T (T_R) cells suppress the expansion/activation of self-reactive T cells which have escaped thymic negative selection (1–3). The majority of these T_R cells constitutively express CD25 (IL-2R α-chain), and constitute ∼5–10% of peripheral CD4⁺ T cells in mice and humans (4–10). Such CD25⁺CD4⁺ T_R cells are produced, at least in part, in the thymus as a functionally mature T cell sub-population in mice (4, 5). Depletion of murine CD25⁺CD4⁺ T_R cells or abrogation of their function evokes various organ-specific autoimmune diseases including autoimmune gastritis, thyroiditis and type 1 diabetes in otherwise normal mice (1, 4, 5). In humans, thymic and peripheral CD25⁺CD4⁺ T_R cells share many immunological characteristics with their murine counterpart (6–10). For example, human CD25⁺CD4⁺ T_R cells are hypo-responsive to TCR stimulation, and suppress the activation/proliferation of other T cells in a cell–cell contact-dependent manner, not via inhibitory humoral factors such as IL-10 or transforming growth factor beta (TGF-β), at least in vitro. However, in contrast to CD25⁺CD4⁺ T cells in normal naive mice, the majority of which are T_R cells, CD25⁺CD4⁺ T cells in human peripheral blood contain activated non-regulatory T cells as well, hampering clear delineation of T_R cells (11–13).

Recent studies showed that murine Foxp3 (FOXP3 in humans), which encodes a forkhead/winged-helix transcription repressor termed Scurfin, can be specifically expressed in CD25⁺CD4⁺ T_R cells and associated with their development and function (14–18). Foxp3/FOXP3 was originally reported to be the causative gene for an X-linked multi-organ autoimmune/inflammatory disease in mice and humans. FOXP3 mutations in humans cause immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), which is characterized by high incidences of autoimmune diseases including type 1 diabetes and thyroiditis, inflammatory bowel disease and allergic disease such as atopic dermatitis and food allergy (19–21). A Foxp3-mutant mouse strain known as scurfy develops similar X-linked multi-organ pathology accompanying massive lymphocytic infiltration and uncontrolled activation of CD4⁺ T cells (22–25). Immunological similarities between IPEX/scurfy and abnormalities produced by depletion of CD25⁺CD4⁺ T_R cells prompted several groups, including us, to study the role of Foxp3 in the development and function of CD25⁺CD4⁺ T_R cells in mice (16–18). The studies showed that Foxp3 is specifically expressed in CD25⁺CD4⁺ T_R cells; retroviral transduction or transgenenic expression of Foxp3 can convert CD25⁻CD4⁺ naive T cells to those with a regulatory phenotype. Furthermore, Foxp3–mutant scurfy mice and Foxp3–deficient mice produced by gene targeting generate few CD25⁺CD4⁺ T_R cells and inoculation of normal CD25⁺CD4⁺ T_R cells can prevent autoimmune/inflammatory diseases in these mice. These results taken together indicate that Foxp3 can be a master control gene for the development and function of CD25⁺CD4⁺ T_R cells in mice.

Here we show that FOXP3 is specifically and stably expressed in human CD25⁺CD4⁺ T_R cells as well, that TCR stimulation of naive CD25⁻CD4⁺ T cells fails to elicit FOXP3 expression and that such FOXP3-expressing T cells in human PBMC can be phenotypically best characterized as CD25⁺CD45RO⁺CD4⁺ T cells. Furthermore, ex vivo retroviral gene transfer of FOXP3 can convert naive CD4⁺ T cells in PBMC to a regulatory T cell phenotype similar to CD25⁺CD4⁺ T_R cells. We also show that some human T cell leukemia virus type 1 (HTLV-1)-infected T cell lines, which constitutively express the CD25 molecule, also express FOXP3. These results collectively indicate that FOXP3 can be a crucial regulatory gene for the development and function of CD25⁺CD4⁺ T_R cells in humans and is a reliable marker for human CD25⁺CD4⁺ T_R cells. Furthermore, T_R cells produced de novo from normal T cells by FOXP3 transduction could be instrumental for the treatment of various autoimmune/inflammatory diseases and the control of rejection in organ transplantation.

Methods

Antibodies and flow cytometry

The following FITC-, PE- and/or PC5-conjugated mAbs were used: anti-CD4 (13B8.2), anti-CD8 (B9.11), anti-CD19 (J4.119), anti-CD38 (T16), anti-CD45RO (UCHL1), anti-CD69 (TP1.55.3), anti-CD103 (2G5), anti-CD122 (CF1), anti-CTL-associated antigen-4 (CTLA-4) (BNI3), anti-HLA-DR (Immu-357) and respective isotype controls (purchased from Immunotech, Marseille, France). Anti-CD25 mAb (M-A251) and anti-CCR4 mAb (1G1) were from BD PharMingen (San Diego, CA, USA) and anti-glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) mAb (110416) was from R&D Systems (Minneapolis, MN, USA). Cells were analyzed on an Epics-XL flow cytometer (Beckman Coulter, Miami, FL, USA). For intracellular staining, cells were fixed and permeabilized with Cytofix/Cytoperm (BD PharMingen).

Sorting of lymphocyte sub-populations

With approval from the human ethics committee of the Institute for Frontier Medical Sciences, Kyoto University, human peripheral blood was drawn from healthy adult volunteers under written informed consent. PBMC were prepared by Ficoll gradient centrifugation. Sorting of lymphocyte sub-populations was performed by magnetic cell sorting (MACS) (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. Briefly, CD4⁺, CD8⁺, pan T cells or CD19⁺ cells were sorted with Cell Isolation Kit II. In some experiments, CD4⁺ T cells were subsequently split into CD25⁺ and CD25⁻ sub-populations with CD25 MicroBeads. Similarly, with CD45RO or CD45RA MicroBeads, CD4⁺ T cells were separated into CD45RO⁺ and CD45RO⁻ sub-populations. T cell-depleted autologous PBMC were used as antigen-presenting cells (APC) after irradiation (18 Gy). Purity of sorted populations was always >95%.

T cell activation

RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin (Sigma, St Louis, MO, USA) was used for T cell culture. In some real-time quantitative reverse transcription (RT)-PCR and western blotting experiments, sorted T cells (5 × 10⁵ per well) were stimulated with immobilized anti-CD3 mAb (UCHT1, BD PharMingen), in the presence of recombinant human IL-2 (20 U ml⁻¹, R&D Systems) or soluble anti-CD28 mAb (5 μg ml⁻¹; clone CD28.2, BD PharMingen) in 24-well plates (1.0 ml per well). For immobilization, anti-CD3 mAb (5 μg ml⁻¹ in PBS) was coated overnight onto plates at 4°C and then washed twice. For retroviral infection, peripheral CD25⁻CD45RO⁻CD4⁺ T cells (2.5 × 10⁶ per well) were activated for 48 h with soluble anti-CD3 mAb (20 ng ml⁻¹; HIT3a, BD PharMingen) and IL-2 (20 U ml⁻¹) in the presence of autologous APC (5.0 × 10⁶ per well) in six-well plates (2.5 ml per well).

Production of retrovirus

The cDNA encoding full-length human FOXP3 (amino acids 1–432) was amplified by RT-PCR from cDNA of adult peripheral CD25⁺CD4⁺ cells using specific primers (5′-GCG-GCC-GCA-TGC-CCA-ACC-CCA-GGC-CTG-GC-3′ and 5′-CTC-GAG-TCA-GGG-GCC-AGG-TGT-AGG-GTT-G-3′). The cDNA was cloned into the retroviral vector pGCsamIN, which has a long terminal repeat derived from murine stem cell virus and internal ribosomal entry site followed by an intra-cytoplasmatically truncated version of the human low-affinity nerve growth factor receptor (ΔLNGFR), so that the cloned vector could simultaneously express FOXP3 and ΔLNGFR (26). To produce the recombinant retrovirus, the plasmid DNA was co-transfected into 293 gp-packaging cells (containing the gag and pol genes but lacking the env gene) along with pCL-10A1 (10A1 env-expressing plasmid) by calcium phosphate transfection using a Profection Mammalian Transfection System (Promega, Madison, WI, USA) (27). Culture supernatant containing virus was collected, and virus titer was confirmed by infection of CD4⁺ Jurkat cell lines, where gene transfer efficiency reached at least 70%.

Retroviral transduction

Peripheral CD25⁻CD45RO⁻CD4⁺ T cells were activated as described above and re-suspended to the concentration of 1.0 × 10⁵ cells ml⁻¹. RetroNectin (Takara Bio, Otsu, Japan), recombinant human fibronectin fragment CH-296, was coated and retrovirus supernatant was preloaded on six-well plates according to the manufacturer's instructions, then activated cells were applied (2.5 ml per well) for transfection. After transfection, cells were maintained in medium supplemented with 20 U ml⁻¹ IL-2. On day 7 post-transfection, ΔLNGFR was stained with biotin-labeled anti-nerve growth factor receptor mAb (NGFR5; Lab Vision, Fremont, CA, USA), and FITC- or PE-conjugated streptavidin (BD PharMingen). In some experiments, ΔLNGFR⁺ cells were sorted by MACS with anti-FITC or anti-PE MicroBeads (Miltenyi Biotec).

Proliferation assays

A total of 1.0 × 10⁴ sorted T cells with 2.0 × 10⁴ APC were cultured (0.2 ml per well, U-bottomed 96-well plate) for 96 h in the presence of soluble anti-CD3 mAb (20 ng ml⁻¹; HIT3a). [³H]Thymidine (37 kBq per well) incorporation (cpm) for the last 18 h of co-culture was measured as an indicator of cell proliferation and expressed as the mean (±SD) of triplicate cultures. Background counts in the wells with APC alone were always <1000 cpm. Transwell experiments were carried out in 24-well plates (0.8 ml per well) with 1.0 × 10⁵ fresh CD25⁻CD45RO⁻CD4⁺ cells, 1.0 × 10⁵ APC and soluble anti-CD3 mAb (20 ng ml⁻¹; HIT3a) in the absence or presence of 1.0 × 10⁵FOXP3-transduced CD25⁻CD45RO⁻CD4⁺ cells added directly to the culture or to the Transwell inserts (pore size 0.4 μm, Corning Costar, Cambridge, MA, USA). For blocking experiments, 10 μg ml⁻¹ neutralizing anti-IL-10R mAb (3F9; BD PharMingen) and/or 50 μg ml⁻¹ anti-TGF-β_1,2,3 (1D11; R&D Systems) were used, with doses determined according to the results of others (6, 16).

Cytokine assays

Cells were re-suspended at 1 × 10⁶ cells ml⁻¹ and re-stimulated with 50 ng ml⁻¹ phorbol myristate acetate (PMA) and 500 ng ml⁻¹ ionomycin for 4 h, and GolgiStop (BD PharMingen), containing monensin, was added 2 h before cell harvest. Cytokine production was assessed by intracellular staining with PE-conjugated anti-IFN-γ (45.15; Immunotech), anti-IL-2 (MQ1-17H12), anti-IL-4 (8D4-8), anti-IL-10 (JES3-9D7) and respective mouse/rat isotype controls (BD PharMingen). In some experiments, to measure IL-2 production, 2.5 × 10⁴ purified T cells with 5.0 × 10⁴ APC were cultured in the presence of soluble anti-CD3 mAb (20 ng ml⁻¹; HIT3a) and culture supernatant was collected after 72 h of culture. Concentration of IL-2 was measured by ELISA with a Quantikine human IL-2 immunoassay (R&D Systems).

Quantitative RT-PCR

Total RNA was extracted from sorted cells and first-strand cDNA was synthesized according to standard protocols (16). FOXP3 mRNA levels were quantified by real-time PCR with the ABI/PRISM 7700 sequence detection system (PE Applied Biosystems, Foster City, CA, USA), and the QuantiTect Probe PCR Kit (Qiagen K.K., Tokyo, Japan). FOXP3–specific primers and an internal fluorescent TaqMan probe were designed as follows: FOXP3 primers, 5′-CAG-CAC-ATT-CCC-AGA-GTT-CCT-C-3′ and 5′-GCG-TGT-GAA-CCA-GTG-GTA-GAT-C-3′; FOXP3 probe, 5′-FAM-TCC-AGA-GAA-GCA-GCG-GAC-ACT-CAA-TG-TAMRA-3′. Pre-developed TaqMan Assay Reagent for human hypoxanthine–guanine phosphoribosyl-transferase (HPRT) (PE Applied Biosystems), 20× pre-mixture of HPRT-specific primers and an internal fluorescent TaqMan probe, was utilized for measurement of HPRT mRNA levels as an internal control. These primers were designed to span exons (8 and 9) so as not to anneal to contaminating genomic DNA. Each PCR sample contained 0.4 μM primers and 0.2 μM TaqMan probe in a final volume of 50 μl, and amplification was carried out via 15 min at 95°C denaturation step followed by 45 cycles of 15 s at 94°C and 60 s at 60°C. Standard curves of cDNAs from CD25⁺CD4⁺ T cells obtained from donor I were used to calibrate the threshold cycle to relative quantities of FOXP3 and HPRT cDNAs in each sample. Relative FOXP3 expression was calculated by dividing the relative quantity of FOXP3 by the relative quantity of HPRT in each sample and normalized by setting that of CD25⁺CD4⁺ cells from donor I at 100. Normalized scores <0.1 were considered negligible. All samples were run in triplicate and expressed as the mean (±SD).

Western blotting

Sorted cells were lysed in Laemmli sample buffer, separated on 10% SDSP gels (28), and transferred to Immobilon-P transfer membrane (Millipore, Billerica, MA, USA). The membrane was incubated with 2 μg ml⁻¹ of goat anti-human FOXP3 polyclonal antibody (Abcam, Cambridge, UK) overnight at 4°C in Tris-buffered saline with 0.1% Tween 20 plus 5% freeze-dried milk. HRP-labeled secondary antibody (1 : 1000 anti-goat IgG, Cappel, Aurora, OH, USA) and the ECL system (Amersham, Arlington Heights, IL, USA) were used for detection of FOXP3 protein. Actin was subsequently detected with mouse anti-actin antibody (Chemicon, Temecula, CA, USA) and HRP-labeled secondary antibody (Cappel) as an internal control.

Cell lines

ATL-43Tb(−), ED-40515(−), ED-70423C(−), ATL-55T(−) and ATL-35T(−) are HTLV-1-infected T cell lines (29, 30). ATL-35T(−) is a clone of non-leukemic cell origin, while the other four lines were derived from leukemic cells. ED-40515(−) and ED-70423C(−) are identical leukemic clones originated from the same donor with adult T cell leukemia (ATL) at different periods. HUT102 (31), MT2 (32), SY (33) (HTLV-1-infected T cell lines), Jurkat (acute lymphoblastic T cell leukemia), Karpas 299 (anaplastic large-cell T cell lymphoma) and Ramos (Burkitt's B cell lymphoma) were kindly provided by K. Imada, H. Ohno and M. Nishikori, Kyoto University, Kyoto, Japan. SKW-3 (chronic lymphocytic T cell leukemia) and MOLT-4 (acute lymphoblastic T cell leukemia) were kindly provided by Tetsuji Naka, Osaka University, Osaka, Japan.

Results

Human peripheral CD25⁺CD4⁺ T cells are composed of CD45RO⁺ regulatory cells and CD45RO⁻ non-regulatory cells

We first assessed the regulatory function of the CD45RO⁺ or CD45RO⁻ fraction of CD25⁺CD4⁺ T cells prepared from PBMC of healthy adult individuals (11, 12). Figure 1A shows that approximately two-thirds of CD25⁺CD4⁺ T cells, which constituted ∼10% of CD4⁺ T cells (see also Fig. 1E), were CD45RO⁺. When CD25⁺CD45RO⁺CD4⁺ T cells prepared were stimulated with soluble anti-CD3 mAb in the presence of autologous APC, they exhibited hypo-proliferative responses to TCR stimulation and suppressed the proliferation of co-cultured CD25⁻CD4⁺ T cells; in contrast, CD25⁺CD45RO⁻CD4⁺ T cells prepared from the same samples did not show hypo-proliferation or suppressive activity, shown as a representative experiment (Fig. 1B) and total results of several independent experiments (Fig. 1C).

Fig. 1.

Human peripheral CD25⁺CD4⁺ T cells are composed of CD45RO⁺ regulatory cells and CD45RO⁻ non-regulatory cells. (A) Representative flow cytometric profiles of adult human PBMC for CD4, CD25 and CD45RO. (B and C) Comparison of proliferative response and suppressive activity upon TCR stimulation with soluble anti-CD3 mAb and autologous APC between the CD45RO⁺ fraction and the CD45RO⁻ fraction among CD25⁺CD4⁺ T cells. [³H]Thymidine incorporation of CD45RO⁺CD25⁺CD4⁺ and CD45RO⁻CD25⁺CD4⁺ T cells, alone or mixed with the same number of CD25⁻CD4⁺ T cells, were measured. One representative result of independent experiments from six donors is shown in (B), and relative responses ([³H]thymidine incorporation of each culture was divided by that of CD25⁻CD4⁺ T cells) in the total six experiments are shown in (C). (D) The ratio of CD25⁺CD4⁺ cells and CD25⁺CD45RO⁺CD4⁺ cells among CD4⁺ cells in 26 donors (donors A–Z). Cell number of each population was analyzed as shown in (A). Values (%) of CD25⁺CD4⁺ among CD4⁺ T cells and those of CD25⁺CD45RO⁺CD4⁺ T cells among CD4⁺ T cells are indicated as total bar length and black bar length, respectively. The average ratio of CD25⁺CD4⁺ cells (12.0%) and CD25⁺CD45RO⁺CD4⁺ cells (6.5%) is shown by the solid line and the dotted line, respectively. (E) The ratio of CD25⁺CD45RO⁺CD4⁺ cells among CD25⁺CD4⁺ cells in the same 26 samples. Values (%) are indicated as total bar length. The average ratio (54.5%) is shown by the dotted line.

Given the finding that the expression of CD45RO can operationally, if not specifically, define T_R cells in the CD25⁺CD4⁺ T cell population in human PBMC, we assessed the ratio of the number of CD25⁺CD4⁺ T cells or CD25⁺CD45RO⁺CD4⁺ cells to that of CD4⁺ cells in 26 individual healthy adult donors (Fig. 1D). The percentages of CD25⁺CD4⁺ cells or CD25⁺CD45RO⁺CD4⁺ cells among CD4⁺ cells varied from 6.7 to 20.6% (mean ± SD: 12.0 ± 3.6%) and from 3.0 to 16.4% (mean ± SD: 6.5 ± 3.0%), respectively. The ratios of CD25⁺CD45RO⁺CD4⁺ cells among CD25⁺CD4⁺ cells (Fig. 1D) showed a wide distribution, from 30.3 to 88.3% (mean ± SD: 54.5 ± 16.4%). Thus, the proportion of CD25⁺CD45RO⁺CD4⁺ T_R cells among CD4⁺ T cells or CD25⁺CD4⁺ T cells is variable among individuals, indicating that human peripheral CD25⁺CD4⁺ T cells may contain varying numbers of regulatory and non-regulatory T cells.

FOXP3 is preferentially expressed by CD25⁺CD45RO⁺CD4⁺ T cells

We examined the expression pattern of FOXP3 mRNA in various subsets of adult PBMC by real-time quantitative RT-PCR. The specific amplification of FOXP3 was verified by gel electrophoresis and sequence analysis of the replicon (data not shown). FOXP3 was specifically expressed in CD4⁺ T cells but minimally in CD8⁺ T cells and absent in CD19⁺ B cells in PBMC from six randomly selected donors, B, I, J, P, Q and S (Fig. 2A). CD25⁺CD4⁺ cells exhibited an ∼60-fold higher level of FOXP3 expression than CD25⁻CD4⁺ cells. Among CD25⁺CD4⁺ cells, the CD45RO⁺ population specifically transcribed the FOXP3 gene at a 12-fold higher level compared with the CD45RO⁻ population (Fig. 2B). Among CD25⁻CD4⁺ cells, FOXP3 mRNA levels were also higher in the CD45RO⁺ fraction than the CD45RO⁻ fraction, which expressed very low but detectable levels of FOXP3 (Fig. 2B). These results, together with those showing that the CD45RO⁺ fraction among CD25⁺CD4⁺ T cells predominantly possessed regulatory activity (Fig. 1B), indicate that FOXP3 expression correlates well with the regulatory activity in human T_R cells (16). The normalized levels of FOXP3 expression by CD25⁺CD4⁺ cells were, however, variable among individuals, e.g. the levels ranged from 49.0 to 156.0 (mean ± SD: 94.0 ± 38.1) among the six individuals (Fig. 2C), as the ratio of CD25⁺CD45RO⁺CD4⁺ T cells among CD25⁺CD4⁺ cells varied among donors (Fig. 1E). Interestingly, however, there was a strong positive correlation between the levels of FOXP3 expression and the ratio of CD25⁺CD45RO⁺CD4⁺ T cells to CD25⁺CD4⁺ cells (r = 0.96). Thus, human CD25⁺CD4⁺ T cells are not a homogeneous population of T_R cells and, among CD25⁺CD4⁺ T cells, expression of CD45RO correlates well with expression of FOXP3 and hence regulatory activity.

Fig. 2.

FOXP3 is preferentially expressed by CD25⁺CD45RO⁺CD4⁺ T cells. (A) FOXP3 mRNA expression of CD19⁺, CD8⁺ and CD4⁺ lymphocytes from adult PBMC. CD4⁺ lymphocytes were further separated into CD25⁺ or CD25⁻ cells. Relative FOXP3 expression was measured by real-time quantitative RT-PCR and normalized as described in Methods. Asterisk represents negligible FOXP3 expression. (B) CD25⁻CD4⁺ cells (left) and CD25⁺CD4⁺ cells (right) were further sorted into CD45RO⁻ and CD45RO⁺ compartments, respectively, for FOXP3 mRNA expression analyses. The average (±SD) of normalized scores among the six donors are shown in (A) and (B). (C) Correlation between the ratio of CD25⁺CD45RO⁺CD4⁺ T_R cells among CD25⁺CD4⁺ cells (X axis) and the FOXP3 mRNA expression level of CD25⁺CD4⁺ cells (Y axis) was assessed with PBMC from the six donors, B, I, J, P, Q and S. Dotted line represents regression line.

TCR stimulation of naive CD25⁻CD4⁺ T cells is unable to elicit FOXP3 expression

Next, we examined whether the expression of FOXP3 in various subsets of CD4⁺ T cells was altered upon TCR stimulation. FOXP3 mRNA expression levels were not much altered in either the CD25⁺ or CD25⁻CD4⁺ population following stimulation by immobilized anti-CD3 in the presence of soluble anti-CD28 or IL-2, the former being sustained high and the latter low (Fig. 3A and B), which is at variance with the results reported by others (28).

Fig. 3.

TCR stimulation of naive CD25⁻CD4⁺ T cells is unable to elicit FOXP3 expression. (A and B) CD25⁺CD4⁺ or CD25⁻CD4⁺ cells were activated for the indicated hours with immobilized anti-CD3 in the presence of soluble anti-CD28 (A) or IL-2 (B) and assessed for the mRNA expression of FOXP3 by quantitative RT-PCR. One representative result from three separate donors is shown. (C and D) CD25⁻CD4⁺ cells from donors B, I and P were further separated into CD45RO⁺ and CD45RO⁻ cells, and activated for the indicated hours with immobilized anti-CD3 plus soluble anti-CD28. FOXP3 mRNA expression by CD45RO⁺ population (C) or CD45RO⁻ population (D) was assessed by quantitative RT-PCR. (E) CD25⁺CD4⁺, CD25⁻CD45RO⁺CD4⁺ or CD25⁻CD45RO⁻CD4⁺ cells were activated for the indicated hours with immobilized anti-CD3 plus soluble anti-CD28 and assessed for FOXP3 protein expression by western blotting. One representative result (donor I) from three separate donors (B, I and P) is shown.

Then, to further evaluate the possibility of FOXP3 induction upon TCR stimulation in the CD25⁻CD4⁺ cells, CD25⁻CD4⁺ cells from three randomly selected donors (B, I and P) were sorted into CD45RO⁺ and CD45RO⁻ populations, and each was stimulated with immobilized anti-CD3 plus soluble anti-CD28. CD25⁻CD45RO⁺CD4⁺ cells from donor I showed up-regulation of FOXP3 transcription to some extent, especially in the early phase of activation, while CD25⁻CD45RO⁺CD4⁺ cells from donors B and P did not (Fig. 3C). CD25⁻CD45RO⁻CD4⁺ cells from the three donors failed to increase FOXP3 transcription upon TCR stimulation (Fig. 3D). Compatible with FOXP3 mRNA expression, expression of FOXP3 protein was confined to the CD25⁺CD4⁺ population, and CD25⁻CD45RO⁺CD4⁺ or CD25⁻CD45RO⁻CD4⁺ T cells from the three donors failed to produce detectable amounts of FOXP3 protein whether they were stimulated or not (Fig. 3E). Consistent with the results of others (28), two bands (∼55 and 50 kDa) were observed in this FOXP3 protein detection. The results collectively indicate that FOXP3 is preferentially and stably expressed in CD25⁺CD45RO⁺CD4⁺ T_R cells in human PBMC at the gene and protein level, and that mere activation cannot induce its expression in naive/non-regulatory T cells.

FOXP3 expression in HTLV-1-infected T cell lines

It is known that HTLV-1-infected T cell lines constitutively express CD25 and can be expanded ex vivo in the presence of IL-2 (6, 34, 35). To determine whether they also express FOXP3, we examined FOXP3 expression of eight HTLV-1-infected cell lines, all of which expressed CD25 (data not shown), and five other T/B cell lines. Three out of eight HTLV-1-infected cell lines, MT2, ATL-55T(−) and ATL-35T(−), expressed FOXP3 mRNA at high levels whereas others did not (Fig. 4A). All the five HTLV-1-non-infected lines tested were FOXP3 negative. At the protein level, the FOXP3 proteins of 50 and 55 kDa were detected in MT2 (Fig. 4B), as observed in fresh CD25⁺CD4⁺ T cells (Fig. 3E), but not in Jurkat (Fig. 4B).

Fig. 4.

FOXP3 expression in HTLV-1-infected T cell lines. (A) FOXP3 mRNA expression among 13 lymphocyte-derived cell lines was analyzed by real-time quantitative RT-PCR. Note that the first eight cell lines were HTLV-1 infected and expressed CD25. For comparison, FOXP3 expression levels of CD25⁺CD45RO⁺CD4⁺ cells and CD25⁻CD4⁺ cells from donor I were measured at the same time. Asterisk represents negligible FOXP3 expression. (B) FOXP3 protein expression of MT2 and Jurkat cell lines was assessed by western blotting. One representative result of three independent experiments is shown in (A) and (B).

The two FOXP3 proteins of 55 and 50 kDa correspond to the products of the native form and the splice isoform of Foxp3 mRNA, respectively; the latter lacks exon 2 (36; supplementary figure 1, available at International Immunology Online). It is of note that throughout the present study the native form and splice isoform of FOXP3 mRNA were not distinguished in the quantitative analyses of FOXP3 mRNA expression, in which primers were designed to span exons 8 and 9, i.e. outside exon 2. Also of note is that the peptide recognized by the anti-human FOXP3 antibody is not encoded by exon 2.

Retroviral transduction of FOXP3 to peripheral CD25⁻CD45RO⁻CD4⁺ naive T cells

Our previous work showed that murine CD25⁻CD4⁺ naive T cells were converted to cells with regulatory features following retrovirally enforced expression of Foxp3 (16). This prompted us to determine whether retrovirus-mediated ectopic expression of FOXP3 could confer similar regulatory properties to human CD25⁻CD45RO⁻CD4⁺ naive T cells as well. Retroviral vector expressing both FOXP3 and ΔLNGFR (FOXP3/pGCsamIN) or ΔLNGFR alone (pGCsamIN) as a control was generated (Fig. 5A). The vector was used to infect CD25⁻CD45RO⁻CD4⁺ cells that were purified from PBMC of healthy adults and stimulated for 48 h with anti-CD3 and IL-2 in the presence of autologous APC. One week after infection, the transduction efficiency of the gene was estimated by assessing the expression level of ΔLNGFR. Gene transfer efficiency, with or without introduction of FOXP3, reached ∼20% (Fig. 5B, upper row) by application of RetroNectin, with which high efficiency of gene transfer to T lymphocytes could be achieved (37). It was noted that, compared with ΔLNGFR⁺ cells from pGCsamIN-infected cultures, a fewer number of ΔLNGFR⁺ cells, especially ΔLNGFR^high ones, were obtained from FOXP3/pGCsamIN-infected cultures (Fig. 5B, upper row). Even though we added IL-2 to the cultures based on the fact that CD25⁺CD4⁺ T_R cells can be expanded in vitro without loss of function in the presence of IL-2 (6, 34), ΔLNGFR^high cells, i.e. FOXP3^high cells, from FOXP3/pGCsamIN-infected cultures showed less expansion presumably because FOXP3 suppresses proliferation (16–18, see below).

Fig. 5.

Retroviral transduction of FOXP3 to peripheral CD25⁻CD45RO⁻CD4⁺ naive T cells. (A) Retroviral constructs of FOXP3/pGCsamIN and pGCsamIN. (B) Retroviral transduction efficiency of FOXP3/pGCsamIN or pGCsamIN on day 7 post-infection was estimated by staining for ΔLNGFR (upper row). ΔLNGFR⁺ cells were sorted from FOXP3/pGCsamIN-infected or pGCsamIN-infected cultures by MACS (lower row). (C) FOXP3 mRNA expression of ΔLNGFR⁺ cells sorted from FOXP3/pGCsamIN- or pGCsamIN-infected cultures was assessed by quantitative RT-PCR and compared with freshly isolated CD25⁺CD45RO⁺CD4⁺ cells and CD25⁻CD45RO⁻CD4⁺ cells from the same donor. (D) FOXP3 protein expression of ΔLNGFR⁺ cells sorted from FOXP3/pGCsamIN- or pGCsamIN-infected cultures was assessed by western blotting. One representative result of three independent experiments from three donors is shown in (B)–(D).

ΔLNGFR⁺ cells were sorted from FOXP3/pGCsamIN-infected or pGCsamIN-infected cultures (Fig. 5B, lower row) and their levels of FOXP3 mRNA were assessed by quantitative RT-PCR. The expression level of FOXP3 mRNA by ΔLNGFR⁺ cells sorted from FOXP3/pGCsamIN-infected cultures was ∼30% that of freshly prepared CD25⁺CD45RO⁺CD4⁺ T_R cells, while the expression level by ΔLNGFR⁺ cells sorted from control pGCsamIN-infected cultures was comparable with that of freshly prepared CD25⁻CD45RO⁻CD4⁺ naive T cells (Fig. 5C), also indicating that activation of naive T cells per se cannot induce FOXP3 expression (Fig. 3). The possibility that FOXP3 cDNA integrated into retroviral genomic DNA was amplified in the quantitative PCR experiments was ruled out because control samples without reverse transcriptase showed no significant FOXP3 expression (data not shown). At the protein level, FOXP3 was specifically detected in ΔLNGFR⁺ cells from FOXP3/pGCsamIN-infected cultures (Fig. 5D). Interestingly, only one band (55 kDa) of FOXP3 protein, corresponding to the native form of FOXP3, was detected in FOXP3-transduced cells, in contrast with two bands observed in CD25⁺CD4⁺ cells and MT2 line (Figs 3E and 4B,and supplementary figure 1, available at International Immunology Online).

FOXP3-transduced naive CD4⁺ T cells are hypo-responsive to stimulation

To evaluate regulatory properties of FOXP3-transduced cells, we initially assessed proliferative responses and cytokine production of FOXP3-transduced T cells upon stimulation. FOXP3/pGCsamIN-infected T cells stimulated with soluble anti-CD3 and autologous APC proliferated as poorly as freshly isolated CD25⁺CD45RO⁺CD4⁺ T_R cells (Fig. 6A). In contrast, proliferative responses of pGCsamIN-infected T cells were comparable with those of freshly isolated CD25⁻CD45RO⁻CD4⁺ cells. Such hypo-responsiveness of FOXP3-transduced T cells or freshly isolated CD25⁺CD45RO⁺CD4⁺ T_R cells was abrogated by exogenous IL-2 in a dose-dependent fashion (Fig. 6B). Upon stimulation with PMA and ionomycin, FOXP3/pGCsamIN-infected T cells did not produce IL-2, IFN-γ, IL-4 or IL-10 to an extent detectable by intracellular cytokine staining, while pGCsamIN-infected T cells produced IL-2 and IFN-γ (Fig. 6C). These results indicate that transduction of FOXP3 renders non-regulatory CD4⁺ cells hypo-responsive and suppresses the formation of cytokines by them.

Fig. 6.

FOXP3-transduced naive CD4⁺ T cells are hypo-responsive to stimulation. (A) Upon TCR stimulation with soluble anti-CD3 and autologous APC, the proliferative response of ΔLNGFR⁺ FOXP3/pGCsamIN-infected T cells or pGCsamIN-infected T cells was compared with freshly isolated CD25⁺CD45RO⁺CD4⁺ cells or CD25⁻CD45RO⁻CD4⁺ cells from the same donor. (B) Titrated doses of IL-2 were added at the beginning of the stimulation with soluble anti-CD3 and autologous APC. Proliferative response of ΔLNGFR⁺ FOXP3/pGCsamIN-infected T cells or pGCsamIN-infected T cells, freshly isolated CD25⁺CD45RO⁺CD4⁺ cells or CD25⁻CD45RO⁻CD4⁺ cells from the same donor were estimated. (C) Cytokine production of FOXP3/pGCsamIN-infected T cells or pGCsamIN-infected T cells was assessed by intracellular staining after stimulation with PMA and ionomycin. One representative result of independent experiments from three separate donors is shown in (A)–(C).

Up-regulation of T_R-associated molecules by FOXP3 transduction of naive CD4⁺ T cells

It has been reported that CD25, CD45RO, GITR, CD122, HLA-DR, CCR4 and CTLA-4 are constitutively expressed by T_R cells, while they are inducibly expressed by non-regulatory T cells upon activation (6, 7, 11, 13, 38, 39). CCR4 has been reported to be expressed by T_R cells and to guide T_R cells to the sites of antigen presentation in secondary lymphoid tissues and the sites of inflammation to attenuate T cell activation (39). We therefore examined the expression of these surface/intracellular molecules by FOXP3-transduced T cells (Fig. 7). Expression levels of CD25 became higher in proportion to the level of ΔLNGFR on FOXP3/pGCsamIN-infected T cells, contrasting with pGCsamIN-infected T cells, in which the level of CD25 was not elevated even in the ΔLNGFR^high population (Fig. 7A). Expression of CD45RO, GITR, CD122, HLA-DR, CCR4 and CTLA-4 was also higher in ΔLNGFR^high FOXP3/pGCsamIN-infected T cells compared with pGCsamIN-infected T cells (Fig. 7B). It was unlikely that such up-regulation of T_R-associated molecules in FOXP3-transduced T cells was due to excessive cell activation for retroviral infection, since the expression level of other activation-related markers such as CD4, CD38 or CD69 was not altered by the expression of FOXP3. No expression of CD103 was observed either on FOXP3/pGCsamIN-infected T cells, pGCsamIN-infected T cells or freshly isolated CD25⁺CD45RO⁺CD4⁺ T_R cells (data not shown), contrasting with murine CD103, which is preferentially expressed on CD25⁺CD4⁺ T_R cells and on FOXP3-infected T cells (16). Thus, FOXP3 transduction to naive CD4⁺ cells results in specific up-regulation of many T_R-associated molecules.

Fig. 7.

Up-regulation of T_R-associated molecules by FOXP3 transduction of naive CD4⁺ T cells. (A) CD25 expression along with ΔLNGFR expression on FOXP3/pGCsamIN- or pGCsamIN-infected T cells derived from CD25⁻CD45RO⁻CD4⁺ T cells. Gated cell population was defined as ΔLNGFR^high. (B) Expression of T_R-associated or activation-associated markers compared between FOXP3/pGCsamIN-infected T cells and pGCsamIN-infected T cells. Histograms were gated on ΔLNGFR^high. One representative result of independent experiments from three donors is shown in (A) and (B).

FOXP3-transduced naive CD4⁺ T cells acquire cell–cell contact-dependent suppressive activity

FOXP3-transduced T cells exhibited in vitro suppressive activity similar to that of CD25⁺CD4⁺ T_R cells. Upon TCR stimulation with soluble anti-CD3 and autologous APC, FOXP3-transduced T cells as well as freshly prepared CD25⁺CD45RO⁺CD4⁺ T_R cells suppressed proliferation and IL-2 production of freshly prepared CD25⁻CD45RO⁻CD4⁺ responder cells in a dose-dependent manner (Fig. 8A and B). Exogenous IL-2 and TCR stimulation abrogated the suppression (Fig. 8C). We then examined whether the suppression by FOXP3-transduced T cells was mediated by a cell–cell contact mechanism or was dependent on inhibitory humoral factors such as IL-10 or TGF-β. FOXP3-transduced T cells were not able to suppress the activation of responder CD25⁻CD45RO⁻CD4⁺ cells when these two populations were separated by a semi-permeable membrane (Fig. 8D). Moreover, blockade of IL-10R and/or neutralization of TGF-β by a high concentration of specific mAbs failed to abrogate suppression by FOXP3-transduced T cells or by freshly sorted T_R cells (Fig. 8E). Thus, transduction of FOXP3 can confer suppressive potential to non-regulatory CD4⁺ cells in human PBMC, and the in vitro suppression exerted by FOXP3-transduced CD4⁺ T cells may require cell–cell contact and is not mediated by IL-10 or TGF-β, as is the case with T_R cells in humans and mice.

Fig. 8.

FOXP3-transduced naive CD4⁺ T cells acquire cell–cell contact-dependent suppressive activity. (A) Graded numbers of FOXP3/pGCsamIN-infected T cells, pGCsamIN-infected T cells or freshly isolated CD25⁺CD45RO⁺CD4⁺ cells from the same donor, were cultured with 1.0 × 10⁴ freshly prepared autologous CD25⁻CD45RO⁻CD4⁺ cells with soluble anti-CD3 and 2.0 × 10⁴ autologous APC. (B) Freshly prepared CD25⁻CD45RO⁻CD4⁺ T cells alone or mixed with the same number of ΔLNGFR⁺ FOXP3/pGCsamIN- or pGCsamIN-infected T cells, or freshly isolated CD25⁺CD45RO⁺CD4⁺ cells from the same donor, were stimulated with soluble anti-CD3 and autologous APC. IL-2 concentration of each culture supernatant was measured by ELISA. (C) Titrated doses of IL-2 were added at the beginning of the co-culture as in (B). (D) Freshly prepared CD25⁻CD45RO⁻CD4⁺ T cells and an equal number of ΔLNGFR⁺ FOXP3/pGCsamIN-infected T cells from the same donor were cultured unseparated, or separated by a semi-permeable membrane (Transwell), with TCR stimulation by soluble anti-CD3 and autologous APC. (E) A high concentration of anti-IL-10R mAb, anti-TGF-β mAb or the combination of both was added to the culture of freshly prepared CD25⁻CD45RO⁻CD4⁺ T cells alone, or as a mixture with ΔLNGFR⁺ FOXP3/pGCsamIN-infected T cells or with freshly prepared CD25⁺CD45RO⁺CD4⁺ cells from the same donor. One representative result of independent experiments from three donors is shown in (A)–(E).

Discussion

The present study showed that FOXP3 is preferentially and stably expressed in CD25⁺CD4⁺ T_R cells in humans, and that ex vivo retroviral gene transfer of FOXP3 can convert human naive CD4⁺ T cells into a regulatory T cell phenotype similar to CD25⁺CD4⁺ T_R cells. Thus, FOXP3 transduction alone is sufficient to induce CD25⁺CD4⁺ T_R cell function in naive T cells, indicating that FOXP3 may be a master regulatory gene for the function of CD25⁺CD4⁺ T_R cells in humans, as shown in rodents (16–18). The result also indicates that FOXP3 is currently the best molecular marker for CD25⁺CD4⁺ T_R cells. In addition, our results substantiate the notion that mutations of the FOXP3 gene affect the development and/or function of CD25⁺CD4⁺ T_R cells, thereby causing IPEX, and suggest that polymorphism of FOXP3 may contribute to determining genetic susceptibility to autoimmune disease including type 1 diabetes by affecting the development and functions of CD25⁺CD4⁺ T_R cells (19, 40, 41).

Given that FOXP3 is a highly reliable marker for T_R cells, a key issue then in defining T_R cells is which cell-surface molecule has a good correlation with FOXP3 expression. Although the majority of T_R cells appears to express CD25, activated T cells in general also express CD25. Among CD25⁺CD4⁺ T cells in normal healthy individuals, T_R cells are enriched in the CD25^highCD4⁺ population as in normal mice (42, 43). Here we have shown that CD45RO⁺CD25⁺CD4⁺ T cells, which constituted ∼55% of CD25⁺CD4⁺ T cells, predominantly expressed FOXP3. In addition, even though the proportion of CD45RO⁺CD25⁺CD4⁺ T cells was variable among individuals, the ratio of CD25⁺CD45RO⁺CD4⁺ T cells to CD25⁺CD4⁺ T cells was proportional to FOXP3 expression by CD25⁺CD4⁺ T cells (i.e. the higher the ratio, the higher the FOXP3 level of CD25⁺CD4⁺ T cells) and hence the strength of regulatory activity. Furthermore, retroviral transduction of FOXP3 enhanced CD45RO expression in naive CD4⁺ T cells whereas mere activation without FOXP3 transduction did not (Fig. 7B). These results collectively indicate that CD45RO expression is a good marker for FOXP3-expressing regulatory T cells contained in the CD25⁺CD4⁺ T cell population in human PBMC.

Assuming that FOXP3 plays a key role in the development and function of CD25⁺CD4⁺ T_R cells, it is controversial whether naive T cells can also give rise to FOXP3-expressing regulatory T cells in the periphery upon antigenic stimulation. Walker et al. (28) have reported that, in humans, CD25⁺CD4⁺ T cells with regulatory function and expressing FOXP3 were generated as a consequence of stimulating CD25⁻CD4⁺ T cells with plate-bound anti-CD3 and soluble anti-CD28. In our present experiments, however, FOXP3 mRNA or protein expression did not significantly increase in CD25⁻CD4⁺ T cells, in particular CD25⁻CD45RO⁻CD4⁺ T cells, upon stimulation with immobilized anti-CD3 in the presence of soluble anti-CD28 or IL-2 (Fig. 3). Similar to our result in humans, TCR stimulation of CD25⁻CD4⁺ T cells failed to elicit Foxp3 expression in mice (16), and Levings et al. reported that human CD25⁺CD4⁺ T cells generated by activating CD25⁻CD4⁺ T cells with anti-CD3 and anti-CD28 did not acquire regulatory properties in response to allo-antigens (6). These contradictory results suggest two possibilities regarding peripheral development of FOXP3-expressing CD25⁺CD4⁺ regulatory T cells from CD25⁻CD4⁺ T cells. One is that naive T cells can differentiate to CD25⁺CD4⁺ regulatory T cells upon TCR stimulation in certain situations of T cell activation. The other is that committed regulatory T cells with the CD25⁻, and presumably CD45RO⁺, phenotype may be present in the periphery as a minor fraction of CD4⁺ T cells and retain suppressive function, and may become CD25⁺ upon activation (44). Supporting the latter, CD25⁻CD4⁺ cells in human PBMC, especially the CD45RO⁺ population, expressed a low but detectable level of FOXP3 in our experiments (Fig. 2B), and CD25⁻CD45RO⁺CD4⁺ cells of one donor showed up-regulation of FOXP3 at the mRNA level (Fig. 3C). Furthermore, CD25⁺CD45RB^lowCD4⁺ T cells in normal naive mice express Foxp3 at a low level (16); such CD25⁻CD45RB^lowCD4⁺ T cells can become Foxp3^highCD25⁺CD4⁺ T_R cells ex vivo (M. Ono et al., unpublished results). Analysis with T cell clones from naive T cells is required to determine whether low-level expression of FOXP3 in CD25⁻CD4⁺ T cells can be attributed to a small number of committed regulatory T cells that do not express CD25, or really naive T cells can become FOXP3-expressing regulatory T cells. In addition, recent studies have shown that, only in the presence of TGF-β (humans and mice) or mature autologous dendritic cells (humans), ex vivo TCR stimulation can convert peripheral CD25⁻CD4⁺ naive T cells to FOXP3/Foxp3-expressing T_R cells (45–47). TGF-β possibly contained in FCS used for cell culture, or the condition of APC, might account for the variance between our results and those by Walker et al. (28), regarding the generation of FOXP3-expressing T_R cells by TCR stimulation.

HTLV-1-infected T cells share some immunological similarities with CD25⁺CD4⁺ T_R cells, which are dependent on IL-2 for their generation and survival (6, 34). For example, the majority of HTLV-1-infected T cells, whether leukemic clones or not, is CD4⁺ and constitutively express CD25, and grow ex vivo in an IL-2-dependent manner, though some clones ultimately lose this dependency (29, 35, 48). We therefore assessed FOXP3 expression of HTLV-1-infected cell lines to determine whether FOXP3 can be another marker for HTLV-1-infected T cells. Some HTLV-1-infected T cell lines indeed expressed significant levels of FOXP3 (Fig. 4). In addition, fresh leukemia T cells from many ATL patients expressed FOXP3 (49, our unpublished results). According to this result, some of HTLV-1-infected T cells could be CD25⁺CD4⁺ T_R cells in origin, and therefore immunosuppression could occur in ATL patients (50). It is possible that Tax, an HTLV-1-encoded viral protein, could activate FOXP3, since Tax activates transcription of virus RNA, and in turn, cellular genes (51). However, among FOXP3-producing lines, ATL-55T(−), which was derived from a leukemic clone, produced Tax, while ATL-35T(−) and MT2, which are non-leukemic clones, did not show Tax production (30), indicating no close relationship between Tax production and FOXP3 expression. It remains to be determined whether the moderate degree of association between FOXP3 and HTLV-1 infection represents a selective up-regulation of FOXP3 by the virus or merely hints at underlying viral tropism for CD25⁺CD4⁺ T_R cells. Attempts are being made to assess the regulatory function of FOXP3-expressing HTLV-1-infected leukemia T cells.

The mechanism by which human CD25⁺CD4⁺ T_R cells suppress the activation and expansion of other T cells is obscure at present. We here showed that FOXP3-transduced T cells exerted suppression in a cell–cell contact-dependent manner and not via far-reaching humoral factors. In contrast with Tr1 cells, which secrete IL-10 and can be induced by antigenic stimulation in the presence of IL-10 (52–54), FOXP3-transduced CD4⁺ T cells failed to produce IL-10 upon TCR stimulation and their suppressive activity was independent of IL-10, indicating that they are functionally similar to CD25⁺CD4⁺ T_R cells but different from Tr1 cells. Supporting this, murine Tr1 cells have been reported not to express Foxp3 (55). There is a possibility, however, that FOXP3-transduced T cells, and CD25⁺CD4⁺ T_R cells for that matter, may secrete IL-10 and other cytokines in certain in vivo situations and thereby contribute to negative control of in vivo immune responses as shown in rodents (56).

Human CD25⁺CD4⁺ T_R cells have been demonstrated to suppress antigen-specific T cell responses and to recognize a wide variety of antigens. Allopeptide-specific human CD25⁺CD4⁺ T_R cells can also be induced ex vivo (34). In mice, CD25⁺CD4⁺ T_R cells as well as Foxp3-transduced T cells prevented autoimmune and inflammatory disease. These findings indicate that ex vivo introduction of FOXP3 to naive T cells has the potential to produce ‘made-to-order regulatory T cells’, such as CD25⁺CD4⁺ T_R-like cells with specificity to particular autologous or allogeneic antigens, and such T_R-like cells can be used not only for gene therapy of IPEX but more generally for treatment of other autoimmune and inflammatory diseases or induction of transplantation tolerance.

Transmitting editor: T. Hirano

We thank Zoltan Fehervari, Takeshi Takahashi and Masahiro Ono for critically reading the manuscript, Makoto Otsu for advice on gene transduction to human lymphocytes, Kazunori Imada, Hitoshi Ohno, Momoko Nishikori and Tetsuji Naka for providing us with leukemia cell lines. This work was supported by grants-in-aid from the Ministry of Education, Sports and Culture, the Ministry of Human Welfare of Japan and Japan Science and Technology Agency.

Author notes

2Department of Gynecology and Obstetrics and 3Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan