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International Immunology Advance Access originally published online on June 13, 2006
International Immunology 2006 18(8):1197-1209; doi:10.1093/intimm/dxl060
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© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Foxp3-dependent and -independent molecules specific for CD25+CD4+ natural regulatory T cells revealed by DNA microarray analysis

Naoshi Sugimoto1,2,*, Takatoku Oida1,*, Keiji Hirota1, Kyoko Nakamura1, Takashi Nomura1, Takashi Uchiyama2 and Shimon Sakaguchi1,3

1 Department of Experimental Pathology, Institute for Frontier Medical Sciences, Kyoto University, 53 Shogoin Kawahara-cho, Sakyo-ku, Kyoto 606-8507, Japan
2 Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto 606-8507, Japan
3 Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan

Correspondence to: S. Sakaguchi; E-mail: shimon{at}frontier.kyoto-u.ac.jp


   Abstract
Top
 Abstract
Introduction
 Methods
 Results and discussion
 Supplementary data
 References
 
Naturally occurring CD25+CD4+ regulatory T cells (Tregs) actively engage in the maintenance of immunologic self-tolerance and immunoregulation. They specifically express the transcription factor Forkhead box P3 (Foxp3) as a master control molecule for their development and function. Although several cell-surface molecules have been reported as Treg-specific markers, such as CD25, glucocorticoid-induced TNFR family-related gene/protein and CTL-associated molecule-4, they are also expressed on activated T cells derived from CD25–CD4+ naive T cells. To identify Treg-specific molecules controlled by Foxp3, we performed DNA microarray analysis by comparing the following pairs of cell populations: fresh CD25+CD4+ T cells versus fresh CD25–CD4+ T cells, activated CD25+CD4+ T cells versus activated CD25–CD4+ T cells and retrovirally Foxp3-transduced CD25–CD4+ T cells versus mock-transduced CD25–CD4+ T cells. We found that the Gpr83, Ecm1, Cmtm7, Nkg7, Socs2 and glutaredoxin genes are predominantly transcribed in fresh and activated natural Treg as well as in Foxp3-transduced cells, while insulin-like 7, galectin-1, granzyme B and helios genes are natural Treg specific but Foxp3 independent. G protein-coupled receptor 83 (Gpr83) expression on the cell surface of natural Treg was confirmed by staining with Gpr83-specific antibody. Retroviral transduction of either group of genes in CD25–CD4+ T cells failed to confer in vitro suppressive activity. Thus, there are several genes that are expressed in a highly Treg-specific fashion. Some of these genes are controlled by Foxp3, and others are not. These genes, in particular, Gpr83, Ecm1 and Helios, could potentially be used as specific markers for natural Treg.

Keywords: Ecm1, galectin-1, Gpr83, helios, immune tolerance


   Introduction
Top
 Abstract
 Introduction
Methods
 Results and discussion
 Supplementary data
 References
 
Naturally occurring CD25+CD4+ regulatory T cells (Tregs) actively maintain immunological self-tolerance (1, 2). This is illustrated by the finding that depletion of CD25+CD4+ T cells from normal animals leads to spontaneous development of various autoimmune diseases, such as autoimmune gastritis, thyroiditis and type 1 diabetes, as well as inflammatory bowel disease. Also, reconstitution of normal CD25+CD4+ T cells prevents the development of these aforementioned diseases (3–5). They also suppress a variety of physiological and pathological immune responses to quasi-self or non-self antigens. For example, depletion of CD25+CD4+ T cells provokes effective tumor immunity to autologous tumor cells in otherwise non-responding animals (6), enhances immune responses to invading or co-habiting microbes (7, 8), triggers allergic responses to innocuous environmental substances (9, 10) and breaks feto-maternal tolerance during pregnancy (11). This population can also be exploited to establish immunologic tolerance to non-self antigens (12, 13). Thus, the same Treg cell population naturally present in the immune system engages in negative control of various types of immune responses to self and non-self antigens.

Recent studies have shown that the transcription factor Forkhead box P3 (Foxp3) and CD25 (IL-2R {alpha}-chain) play essential roles for the development, function and maintenance of natural Treg (1). Foxp3 is specifically expressed in natural Treg in the thymus and periphery (14–16). Foxp3 deficiency abrogates the thymic development of natural Treg and produces severe autoimmune/inflammatory disease in humans and rodents (17–20). Ectopic expression of Foxp3 in CD25–CD4+ naive T cells by retroviral gene transfer can convert them to natural Treg-like cells functionally and phenotypically (14, 15), indicating that Foxp3 can be a master control gene for the development and function of natural Treg. CD25 was first shown to be a highly specific marker for natural Treg (3). Several recent studies have clearly shown that CD25 is not a mere marker for the chronically activated state of natural Treg but an indispensable molecule for their survival in the periphery as an essential component of the high-affinity IL-2R (21). For example, neutralization of circulating IL-2 for a limited period substantially reduces natural Tregs and elicits autoimmune disease similar to diseases produced by depletion of natural Tregs (22). Genetic CD25 deficiency produces severe autoimmune/inflammatory disease in humans and rodents (23, 24), and inoculation of normal CD25+CD4+ T cells prevents the disease in rodents (25). In addition, Foxp3 directly or indirectly controls the expression of the IL-2 and CD25 genes in natural Treg (14, 15).

To elucidate the molecular basis of Treg function, we have identified in this report which genes are expressed differentially between natural Treg and other T cells when globally assessed using a DNA microarray technique. A number of studies have compared global gene expression profiles between freshly prepared CD25+CD4+ Tregs and CD25–CD4+ naive T cells (26–32). In addition to a similar comparison, we have here attempted to compare activated Tregs with activated naive T cells, and Foxp3-transduced with mock-transduced naive T cells. The microarray system we applied is a dual-color type with 70mer- or 75mer-long oligo probes, by which genes transcribed at low levels can be detected with high sensitivity and specificity. Our specific aims of this study are to identify the molecules that are specifically expressed in Treg, to determine which genes are up- or down-regulated by Foxp3 in Treg and to assess whether the genes specifically expressed in natural Treg can confer a suppressive activity to normal naive T cells when the genes are retrovirally transduced to naive T cells. We show that several genes are expressed in a Treg-specific manner; some of these genes are Foxp3 controlled and others are not. Interestingly, we observed that G protein-coupled receptor 83 (Gpr83) was expressed specifically on the cell surface of natural Treg by FACS analysis and by fluorescence immunohistochemistry using a specific polyclonal antibody. Use of these molecules as Treg markers should be instrumental in further delineating Treg from other T cells and thereby characterizing the molecular basis of the development and function of Treg.


   Methods
Top
 Abstract
 Introduction
 Methods
Results and discussion
 Supplementary data
 References
 
Mice
Six- to ten-week old female BALB/c mice were purchased from CLEA Japan, Co. (Tokyo, Japan), and used according to the institutional guidelines for animal welfare.

Reagents
The following reagents were purchased from BD Biosciences (San Jose, CA, USA): purified anti-CD3 (145-2C11), purified anti-CD28 (37.51), biotinylated- or PE–anti-CD25 (7D4), FITC– or allophycoerythrin (APC)–anti-CD4 (RM4-5), PE–streptoavidin. Purified anti-human GPR83 rabbit polyclonal antibody (catalog ID: LS-A4954) was kindly provided by Medical & Biological Laboratories (Nagoya, Japan) and PE–anti-Foxp3 was obtained from eBioscience (San Diego, CA, USA). Purified anti-rabbit Ig–Alexa488 and anti-mouse Ig–Alexa546 were obtained from Molecular Probes (Eugene, OR, USA). Anti-CD8 (3.155) and anti-CD24 (J11D) were used as tissue culture supernatants from the respective hybridomas, as previously described (4). The anti-rat IgG used for panning was purchased from ICN Pharmaceuticals (Aurora, OH, USA). Murine recombinant IL-2 (3.89 x 106 U mg–1) was a gift from Shionogi Co. (Osaka, Japan). T cells were cultured in RPMI-1640 medium supplemented with 10% FBS, 100 U ml–1 penicillin, 100 µg ml–1 streptomycin and 50 µM 2-mercaptoethanol, which were all purchased from Sigma (St Louis, MO, USA).

Preparation of T cell sub-populations and retroviral transduction of Foxp3
Lymphocyte suspensions were prepared from spleens and lymph nodes, followed by RBC lysis with ammonium chloride-based buffer. To prepare CD25+ or CD25–CD4+ T cells, lymphocytes were treated with anti-CD8 (clone 3.155) and anti-CD24 (clone J11D) and panned on anti-rat IgG-coated petri dishes; this CD4+-enriched fraction was stained with anti-CD25–biotin, followed by PE–streptoavidin and anti-CD4–FITC and then sorted by a cell sorter (MoFlo, Dako Cytomation). For activation of T cells, cells were cultured for 3 days with plate-bound anti-CD3 (10 µg ml–1), soluble anti-CD28 (1 µg ml–1) and recombinant mouse IL-2 (100 U ml–1). For Foxp3 retroviral transduction, sorted CD25–CD4+ T cells were activated with anti-CD3 (0.5 µg ml–1) and IL-2 (100 U ml–1) in the presence of X-irradiated (18 Gy) BALB/c splenocytes as antigen-presenting cells (APCs) for 24 h, and were infected with the retroviral supernatant of Foxp3/MIGR1 or MIGR1 transfected Plat-E packaging cells as described previously (14). At 24 or 60 h after infection, green fluorescence protein cells were sorted by MoFlo; a portion of cells sorted 60 h after infection were washed and allowed to rest in medium for an additional 12 h.

cDNA preparation and microarray analysis
RNA was prepared using an RNAeasy mini kit (Qiagen, Hilden, Germany) with on-column DNase treatment. Total RNA (20 ng) was reverse transcribed to cDNAs and amplified using a Super SMART PCR cDNA Synthesis Kit (BD Biosciences). cDNAs were labeled with Cy3 or Cy5 Mono-reactive Dye (GE Healthcare Bio-Sciences, Piscataway, NJ, USA) and hybridized to the dual-color type microarrays with mouse genome 32K and TF1560 BMR chips (Bio Matrix Research, Chiba, Japan). The former carry 32 000 oligo probes of 70mer long for unbiased genes based on sequences from Ensembl database, and the latter carry 1560 probes of 75mer long for mouse transcription factors and signaling molecules or mouse homologues of relevant human genes depicted from Mouse Genome Informatics and Online Mendelian Inheritance in Man databases. The dual-color type chips can only compare between two samples, but have high sensitivity and specificity even for those genes expressed at low levels. The TF1560 chip microarray was further optimized for transcription factors and signaling molecules that are generally expressed at low levels. Fluorescence intensities in these microarrays were quantified using QuantArray 3.0 software (Perkin Elmer, Wellesley, MA, USA). The signal intensity data were imported into Genespring 6.0 (Silicon Genetics, Redwood City, CA, USA). Gene transcripts with expression levels lower than negative control spots in both dye-swapped conditions were considered below the cutoff and were removed. The ratios of the signal intensities between the two cell types were calculated by Lowess normalization according to manufacturer's instructions (Bio Matrix Research).

Real-time PCR analysis
Real-time quantitative reverse transcription (RT)–PCR was performed to measure gene expression levels of selected genes using Platinum SYBR Green qPCR Super Mix (Invitrogen, San Diego, CA, USA) and TaqMan ABI Prism 7700 (Applied Biosystems, Foster City, CA, USA) with the following primer pairs: hypoxanthine guanine phosphoribosyl transferase (HPRT): 5'-TGAAGAGCTACTGTAATGATCAGTCAAC-3' and 5'-AGCAAGCTTGCAACCTTAACCA-3', Gpr83: 5'-GAAGATGCTGGTGCTTGTGGTAGTC-3' and 5'-AAGTGGTGATTAGGTAGTGGAGCCC-3', Ecm1: 5'-ACTACCTGCTCCGACCCTGC-3' and 5'-CCTGTTCTGGATATGGAAGCTCG-3', Cmtm7: 5'-CAGATGGTCACCCTGCTGATTG-3' and 5'-GGTATGAAGAAAGGCGTGTCTATGC-3', Nkg7: 5'-CAAGCCAAGAGACTCAAGTAGCAGG-3' and 5'-AGAGGAAGAGGATGAAGGAGACCC-3', insulin-like 7: 5'-TCGCTGATGGAGAAGCCAATAC-3' and 5'-AGAAAGCCTGGGGGATTTG-3', Emp3: 5'-TCCCAGACAAAGAGTCCCTGAACC-3' and 5'-TCCTCGGTGTGGATGGCATAGATG-3', galectin-1: 5'-TCGCCAGCAACCTGAATCTC-3' and 5'-GGTCCCATCTTCCTTGGTGTTAC-3', granzyme B: 5'-5'-CTCCACGTGCTTTCACCAAA-3' and 5'-GGAAAATAGTACAGAGAGGCA-3', Socs2: 5'-TCTGGGGACTGCCTTTACCAAC-3' and 5'-CCTCTGGGTTCTCTTTCACATAGC-3', glutaredoxin: 5'-GGACATCACAGCCACTAACAACACC-3' and 5'-ATCTGCTTCAGCCGAGTCATCAG-3' and helios: 5'-ACTCCTCAGAAGTTTGTGGGGG-3' and 5'-GCTGGGCTTTGTTTCCTCTTG-3'. Expression level of each gene was normalized to HPRT.

FACS analysis and cytospin staining
For FACS staining, purified spleen and lymph node cells were stained with anti-human GPR83 antibody followed by Alexa488–anti-rabbit antibody. Cells were then stained with APC–anti-CD4 together with PE–anti-CD25 or, after fixation with the fixation/permeabilization buffer, with PE–anti-Foxp3 (eBioscience). Stained cells were analyzed using FACSCaliber with CellQuest Pro software (BD Biosciences). For cytospin staining, freshly prepared purified spleen and lymph node cells or spleen cells activated for 3 days by addition of anti-CD3 (0.5 µg ml–1) and IL-2 (100 U ml–1) were spanned onto slide glasses by Shandon Cytospin 4 Cytocentrifuge (Thermo Electron Corporation, Waltham, MA, USA) and fixed with 4% PFA following permeabilization with 0.1% Triton X-100 with each step for 10 min. The fixed and permeabilized samples were incubated with PBS/5% BSA for 30 min, and were subsequently incubated with purified anti-human GPR83 and PE–anti-Foxp3 for 1 h. Samples were then stained with Alexa488–goat anti-rabbit IgG and Alexa546–goat anti-mouse IgG. All incubation steps were performed at room temperature. Stained samples were scanned and analyzed using an Axiovert200 microscope and AxioVision software (Carl Zeiss, Oberkochen, Germany).

Proliferation assays
Purified CD25–CD4+ cells (2.5 x 104 per well in a U-bottomed 96-well plate) and irradiated APCs (5.0 x 104 per well) were cultured for 72 h in the presence of 0.5 µg ml–1 anti-CD3 mAb. [3H]thymidine (1 µCi per well, DuPont/NEN) was added during the last 6 h of culture. Retrovirally gene-transduced cells, prepared as described previously in Foxp3 transduction, were added for indicated numbers. Results are expressed as the mean of triplicate cultures. Background counts in the wells with APCs alone were always <200 counts per minute.


   Results and discussion
Top
 Abstract
 Introduction
 Methods
 Results and discussion
Supplementary data
 References
 
Microarray analysis
To screen for the genes specifically expressed in natural CD25+CD4+ Treg and Foxp3-induced Treg-like cells, we performed microarray analysis with the dual-color type mouse 32K and TF1560 BMR chips (see Methods), and compared the following pairs of cell populations (Fig. 1A): (i) CD25+CD4+ T cells and CD25–CD4+ T cells freshly prepared from normal naive BALB/c mice, (ii) CD25+CD4+ T cells and CD25–CD4+ T cells activated in vitro, (iii) fresh and activated CD25+CD4+ T cells, (iv) fresh and activated CD25–CD4+ T cells, (v) Foxp3- and mock-transduced CD25–CD4+ T cells 12 h after infection, (vi) Foxp3- and mock-transduced cells 60 h after infection and (vii) similarly treated cells rested for an additional 12 h. The threshold lines for significantly high or low expressions of the genes in the 32K or TF1560 microarray were set on 2- or 1.5-fold higher or lower expression, respectively, based on their distributions (Fig. 1B and C).


Figure 1
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  		Fig. 1 DNA microarray scatter plots revealing expression of activation induced-genes in fresh CD25+CD4+ Treg and Foxp3-transduced CD25–CD4+ T cells. Total RNA was extracted from fresh or activated CD25+ or CD25–CD4+ T cells from BALB/c spleens and lymph nodes, and was transcribed to cDNA, amplified, labeled and hybridized to mouse 32K and TF1560 chips. Four combinatory comparisons, (1–4) shown in (A), from freshly prepared or activated CD25+ or CD25–CD4+ cell populations were subjected to each chip assay. Foxp3- or mock-transduced cells collected after 24, 60 or 72 h with a rest for the last 12 h were also compared [comparisons (5–7) in A]. The expression value of each gene transcript was plotted as a scatter plot, and threshold lines were drawn at 2- and 1.5-fold expression change, respectively (B and C). The values for Foxp3 expression are shown for comparison of two chip assays. Gene transcripts up- or down-regulated in activated versus fresh CD25–CD4+ T cells are colored red or green, respectively, for fresh Treg versus CD25–CD4+ T cells (D), Foxp3- versus mock-transduced CD25–CD4+ T cells at rested state (E), and activated versus fresh CD25–CD4+ T cells (F). The numbers 1–7 in B–F correspond to the comparison pairs (1–7) in (A).

 
When we marked the up-regulated gene transcripts in red on the scatter plot of activated versus fresh CD25–CD4+ T cells with the 32K chip (Fig. 1F), we noticed that these red spots predominantly fell into the up-regulated side in the scatter plot of fresh CD25+CD4+ T cells versus fresh CD25–CD4+ T cells (Fig. 1D). A similar pattern was also observed with Foxp3- versus mock-transduced CD25–CD4+ T cells at resting state (Fig. 1E). Although the degrees of difference in each set were generally below the threshold, these distinctly discernable patterns suggest that there is a basal level of Treg activation in their default steady state in the periphery of normal naive mice, and that the activation is controlled by Foxp3.

With the 32K chip, 38 gene transcripts (37 actual genes) were up-regulated in fresh Treg compared with fresh CD25–CD4+ T cells. In addition, 76 transcripts (45 genes) were up-regulated in fresh Treg with TF1560 chip (Fig. 2A and Supplementary Tables 1 and 5; Supplementary Tables available at International Immunology Online). By combination of two assay chips, 76 genes were categorized as fresh Treg specific. Similarly, when activated Tregs were compared with activated CD25–CD4+ T cells in 32K and TF1560 chips, a total of 65 genes were categorized as activated Treg specific by combination of the two assay chips (Fig. 2A and Supplementary Tables 1 and 5; Supplementary Tables available at International Immunology Online). Among these fresh or activated Treg-specific genes, 13 genes were shared. They were Ccdc22 (coding for coiled coil domain containing 22), Cmtm7 [for CKLF-like MARVEL transmembrane domain containing 7], Ctla4 (for cytotoxic T lymphocyte associated molecule-4), Ecm1 (for extracellular matrix protein 1), Foxp3, Insl7 (for insulin-like 7), Lgals1 (for galectin-1), Ltb (for lymphotoxin-ß), Dusp14 (for dual specificity phosphatase 14), Ephx1 (for epoxide hydrolase 1, microsomal), S100a10 (for calpactin), Socs2 (for suppressor of cytokine signaling 2) and Tieg (for transforming growth factor-ß inducible early growth response). Other reports with DNA microarray data showed that genes such as glucocorticoid-induced TNFR family-related gene/protein (GITR) and neuropilin-1 were also expressed at higher levels in natural Treg compared with CD25–CD4+ T cells whether in a fresh or activated state. In the present experiments, GITR and neuropilin-1 were indeed expressed at higher levels in natural Treg when compared between freshly prepared CD25+ and CD25– cells. This trend was also seen after activation, although the difference was below the threshold (Fig. 2A and Supplementary Table 1; Supplementary Table available at International Immunology Online). This discrepancy, compared with other reports, could be attributed to different microarray platforms and the method of in vitro activation: we stimulated cells for 3 days with plate-bound anti-CD3, soluble anti-CD28 and IL-2, while others used only two of these stimulations for shorter time periods.


Figure 2
Figure 2
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  		Fig. 2 Gene transcripts up- or down-regulated in CD25+CD4+ T cells compared with CD25–CD4+ T cells. Each Venn diagram consists of two circles representing a group of up-regulated (A) or down-regulated gene transcripts (B): fresh CD25+CD4+ T cells versus fresh CD25–CD4+ T cells (left circle) and activated CD25+CD4+ T cells versus activated CD25–CD4+ T cells (right circle), with mouse 32K or TF1560 chips (left and right diagram, respectively). Normalized expression ratios for each gene transcript are shown as histograms, and genes overlapping between fresh and activated pairs are depicted with shaded names.

 
Since retroviral transduction of Foxp3 in CD25–CD4+ T cells confers a Treg phenotype and suppressive activity, we also compared Foxp3-transduced CD25–CD4+ T cells with mock-transduced CD25–CD4+ T cells at three time points after transduction: 24, 60 and 60 h followed by a 12-h resting period. There were a total of 90 genes expressed at higher levels in Foxp3-transduced cells than in mock-transduced cells at various time points after transduction with either one or both of the assay chips. Interestingly, however, only 27 genes out of 90 Foxp3-specific genes were shared with Treg-specific genes (Fig. 3A and Supplementary Tables 2 and 6; Supplementary Tables available at International Immunology Online). These genes were Ltb, Folr4 (for folate receptor 4), Gpr83 (for G protein-coupled receptor 83), S100a6 (coding for calcyclin), Glrx (for glutaredoxin), Igh-6 (for Ig {gamma}-1 chain C region), Kif24 (for kinesin family member 24), Pld3 (for phospholipase D3), Ephx1, S100a10, Socs2, Cdkn1a (for cyclin-dependent kinase inhibitor 1A), Hif1a (for hypoxia inducible factor 1{alpha}), Il10 (for IL-10), Il2ra (for IL-2R{alpha}), Nfkbia (for nuclear factor-{kappa}B inhibitor {alpha}), Rab11a (for RAS oncogene family 11a), Sdc4 (for syndecan 4), Tgif (for TG interacting factor), Ubce8 (for ubiquitin-conjugating enzyme 8), Anxa6 (for annexin A6), Ccl4 (for C-C chemokine ligand 4), Creb3 (for cAMP responsive element binding protein 3), Maged1 (for melanoma antigen family D1), Rgs2 (for regulator of G protein signaling 2), Stat1 (for signal transducer and activator of transcription 1), Tcn2 (for transcobalamin 2) and one unknown gene transcript.


Figure 3
Figure 3
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  		Fig. 3 Gene transcripts commonly up- or down-regulated in Foxp3-transduced cells and D25+CD4+ T cells. For up-regulated (A) or down-regulated (B) genes, each Venn diagram consists of three circles representing groups of genes up (down)-regulated in fresh Treg versus CD25–CD4+ T cells, activated Treg versus CD25–CD4+ cells and Foxp3- versus mock-transduced cells at 24, 60 or 72 h with last 12 h at rest after transduction. Tables show an expression change for each gene transcript overlapping between D25+CD4+ T cells and Foxp3-transduced cells. Values (in fold) above threshold expression are shaded.

 
On the other hand, 15 gene transcripts with the 32K chip and 42 transcripts (27 genes) with the TF1560 chip were down-regulated in fresh Treg compared with fresh CD25–CD4+ T cells. In combination, 40 genes were categorized as down-regulated in fresh Treg in either one or the both of the chips (Fig. 2B and Supplementary Tables 3 and 7; Supplementary Tables available at International Immunology Online). Similarly, 56 genes were down-regulated in activated Treg compared with activated CD25–CD4+ T cells in either one or both of the chips. Among them, five genes were commonly down-regulated in fresh and activated Treg, which were Lrrc57 (coding for leucine-rich repeat containing 57), F2R (for coagulation factor II receptor), Il4ra (for IL-4R {alpha}), Pde4b (for phosphodiesterase 4B) and Satb1 (for special AT-rich sequence binding protein 1).

For Foxp3-transduced CD25–CD4+ T cells, a total of 107 genes were shown to be down-regulated compared with mock-transduced cells at various time points in either one or both of the assay chips (Fig. 3B and Supplementary Tables 4 and 8; Supplementary Tables available at International Immunology Online). These genes included the Il4 gene, transcription of which has been predicted to be directly repressed by Foxp3 using human CD4+ T cell line (33). Merging the assay results, 18 down-regulated genes were shared with Foxp3-transduced cells and fresh or activated CD25+CD4+ T cells. These genes were Tmem23 (coding for transmembrane protein 23), Akr1c18 (for aldo–keto reductase family 1, member C18), Il4 (for IL-4 precursor), Il24 (for IL-24), Pim2 (for proviral integration site 2), Kc1a18-like (for similar to keratin complex 1, acidic gene 18), Rbssip2-like (for similar to RNA-binding motif, single-stranded interacting protein 2), F2R, Il4ra, Pde4b, Ccl4, Cdc2l2 (for cell division cycle 2 homologue-like 2), Cd53 (for CD53 antigen), Cdkn1a, Idb2 (for inhibitor of DNA binding 2), Il13 (for IL-13), Jak2 (for Janus kinase 2) and Tarbp2 (for TAR RNA binding protein 2).

Real-time quantitative RT–PCR analysis
Based on the results of the microarray analyses in Fig. 1, we selected several genes that were up-regulated in fresh or activated CD25+CD4+ Tregs and Foxp3-transduced CD25–CD4+ T cells, and quantitatively assessed by real-time RT–PCR their mRNA expression levels in thymocyte sub-populations [i.e. CD4–CD8–, CD4+CD8+, CD4+CD8– (CD4 single positive (SP)) and CD4–CD8+ (CD8 SP) thymocytes], CD25+ or CD25–CD4 SP thymocytes, CD4+ or CD8– peripheral T cells and CD25+ or CD25–CD4+ T cells (Fig. 4). The assessed genes were those encoding putative cell-surface proteins, such as Gpr83, Ecm1, Cmtm7, Nkg7 (natural killer cell protein 7), Insl7 and Emp3 (epithelial membrane protein 3), putative secreted proteins, such as galectin-1 and granzyme B, and transcription factors or signaling molecules, such as Socs2, glutaredoxin, and helios (Znfn1a4).


Figure 4
Figure 4
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  		Fig. 4 Real-time quantitative RT–PCR analysis of Treg-specific genes selected from microarray analyses. Gpr83 (A), Ecm1 (B), Cmtm7 (C), Nkg7 (D), insulin-like 7 (E), Emp3 (F), galectin-1 (G), granzyme B (H), Socs2 (I), glutaredoxin (J) and helios (K). RNA was prepared either freshly from thymus, spleen/lymph node cells or after activating spleen/lymph node cells. Foxp3- or mock-tranduced CD25–CD4+ T cells were sorted for high green fluorescence protein expression 60 h after infection. The means with SDs of triplicate experiments are shown. DN: CD4–CD8– double-negative cells, DP: CD4+CD8+ double-positive cells, CD8: CD8+ SP cells, CD4: CD4+ SP cells, 25–: CD25–CD4+ T cells, 25+: CD25+CD4+ T cells.

 
Gpr83 is a putative G protein-coupled receptor whose function, cellular localization and its ligand are completely unknown. It was originally reported to be expressed predominantly in the brain (34), but several recent microarray analyses revealed that it was also expressed in Treg (28, 35). It is not yet known, however, whether Gpr83 transcription is under the control of Foxp3. In our present study, the Gpr83 gene was expressed higher in CD25+CD4 SP thymocytes and CD25+CD4+ peripheral T cells than in CD25–CD4 SP thymocytes/T cells, although CD25–CD4 SP thymocytes expressed Gpr83 at a low level (Fig. 4A). Gpr83 expression in peripheral CD25+CD4+ T cells was lower than CD25+CD4 SP thymocytes, but much higher than peripheral CD25–CD4+ T cells, which scarcely expressed the gene in similar fashion to CD8+ T cells and CD3– cells. This higher expression of Gpr83 in Treg was maintained after activation. Interestingly, the expression of Gpr83 appeared to be under the control of Foxp3, since Foxp3-transduced CD25–CD4+ T cells expressed Gpr83 mRNA at much higher levels than mock-transduced cells. Thus, the Gpr83 protein can be a specific and stable surface marker for natural Treg that is Foxp3 controlled.

To confirm this, we attempted to stain live or fixed mouse spleen and lymph node cells for cell-surface expression of the Gpr83 protein by utilizing an anti-human GPR83 rabbit polyclonal antibody. Fresh CD25+CD4+ T cells or Foxp3+CD4+ T cells indeed showed Gpr83-specific staining of the cell surface with the antibody (Fig. 5A). Furthermore, the specific staining was maintained after activation of the spleen cells (Fig. 5B).


Figure 5
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  		Fig. 5 Gpr83 is specifically expressed on the cell surface of natural Treg. Purified spleen and lymph node cells were stained with anti-human GPR83 followed by Alexa488–anti-rabbit IgG. Cells were then stained with APC–anti-CD4 together with PE–anti-CD25 or, after fixation, with PE–anti-Foxp3 and subjected to FACS analysis. The gates for each histogram are shown as squares in the scatter plot. Gray shades: second antibody only. Lines: Gpr83 staining (A). Alternatively, cells that were freshly prepared or activated for 3 days were spanned onto slide glasses by cytospin, fixed and permeabilized. The samples were stained with purified anti-human GPR83 and PE–anti-Foxp3 followed by Alexa488–goat anti-rabbit IgG and Alexa546–goat anti-mouse IgG and examined by a laser scanning microscope. Red, Foxp3; green, Gpr83; yellow, merged (B).

 
Ecm1 is an extracellular protein suggested to serve as a transporter of, or binds to, soluble factors and matrix substrates. Mutation of Ecm1 results in lipid proteinosis, characterized by skin and mucosal infiltration and scarring, indicating its important roles in skin physiology and homeostasis (36). Similarly to Gpr83, Ecm1 mRNA was transcribed at much higher levels in CD25+CD4+ T cells in the thymus and periphery; its higher expression than other T cells was maintained after activation, and its expression was also induced in naive T cells by Foxp3 transduction (Fig. 4B).

Cmtm7 is a recently cloned gene that shares homology with chemokine-like factor (CKLF), but has an unknown function (37). This gene was also expressed at higher levels in fresh and activated CD25+CD4+ T cells compared with fresh or activated CD25–CD4+ T cells (Fig. 4C), although the differences were relatively small compared with the expression of Gpr83 or Ecm1. Transcription of Cmtm7 mRNA was efficiently induced in naive T cells by Foxp3 transduction.

Nkg7 is a component of cytotoxic granules of NK cells and cytotoxic CD8+ T cells (38, 39). Nkg7 transcription was slightly higher in Treg, and efficiently induced in naive T cells by Foxp3 transduction (Fig. 4D).

Transcription of the insulin-like 7 gene, which was reported to be expressed nearly exclusively in the brainstem (40), was highly specific for CD25+CD4+ T cells in both fresh and activated conditions (Fig. 4E). Contrary to Gpr83 and Ecm1, however, its mRNA transcription was not induced in naive T cells by Foxp3 transduction.

Emp3, which belongs to the peripheral myelin protein 22-kDa gene family and is thought to be involved in the regulation of cell proliferation and cell interactions (41), was expressed at higher levels in Treg only in an activated state, and was not induced by Foxp3 transduction (Fig. 4F).

Galectin-1 and granzyme B are putative secreted proteins. The former is a potential immune regulator, and capable of suppressing Th1-mediated autoimmune diseases such as collagen-induced arthritis (42). The latter was reported to be one of the mediators of Treg suppression (31). Although real-time RT–PCR confirmed that both genes were expressed at higher levels in Treg than in CD25–CD4+ T cells in the thymus and the periphery at fresh or an activated state, neither was induced by ectopic Foxp3 expression in naive T cells (Fig. 4G and H), indicating that neither of them is an indispensable mediator of Treg-mediated suppression.

Socs2 is a member of the Socs negative regulator family (43, 44), and deficiency of the Socs2 gene was recently reported to result in uncontrolled inflammation and elevated mortality (44). Socs2 is expressed at higher levels in fresh Treg in the thymus and periphery, but this difference was not observed after activation, although the initial microarray analysis suggested that Socs2 expression was also higher at an activated state (Fig. 4J). Since Foxp3-transduced CD25–CD4+ T cells express higher levels of Socs2 than mock-transduced CD25–CD4+ T cells, Socs2 seems, at least in part, to be under the control of Foxp3. Whether Socs2 is involved in conferring the Treg phenotype remains to be determined.

Glutaredoxin is a redox-controlling enzyme (45), which was expressed slightly at higher levels in Treg and was induced by Foxp3 (Fig. 4K).

Helios is a member of the ikaros transcription factor family (46). Ikaros is important for T cell development, and hence, helios may also be important for Treg lineage development. It was previously reported that all immature thymocytes expressed helios, and only a small fraction of mature T cells maintain helios expression in the periphery (47). In accordance with this report, helios was expressed in CD4+CD8+ thymocytes in the present study; however, CD25+CD4 SP thymocytes also expressed helios at higher levels than DP cells, and the expression was maintained in the periphery (Fig. 4I). This Treg-specific expression pattern was also maintained after activation. Notably, however, helios was not induced in naive T cells by Foxp3 transduction. Since helios was expressed earlier (at the CD4–CD8– stage) than Foxp3 expression (at CD4 SP stage) (14), it is tempting to speculate that constant helios expression after maturation may trigger Treg lineage commitment at the late DP stage.

We then attempted to assess whether retroviral expression of some of the genes depicted as Treg specific by our microarray analysis could confer in vitro suppressive activity to naive T cells (Fig. 6). The forced gene expression resulted in variable in vitro survival depending upon which gene was introduced (data not shown). For instance, Helios- and to a lesser extent Socs2-transduced cells had significant low survival after transduction. From the experiments conducted with genes that did not hamper cell survival significantly, expression of the Gpr83, Ecm1 and galectin-1 genes failed to confer a suppressive function and a clear Treg phenotype to CD25–CD4+ T cells, contrasting with Foxp3-transduced CD25–CD4+ T cells which exerted potent in vitro suppression even in small numbers.


Figure 6
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  		Fig. 6 In vitro suppression assay of retrovirally gene-transduced cells co-cultured with fresh CD25–CD4+ T cells. Gpr83, Ecm1, Galectin-1, Socs2 or Foxp3 was retrovirally transduced to CD25–CD4+ T cells, and then green fluorescence protein+ cells were sorted and mixed with freshly prepared CD25–CD4+ T cells at indicated ratios. Cells were stimulated with anti-CD3 (0.5 µg ml–1) and irradiated APC for 3 days, and [3H]-thymidine incorporation during the last 6 h of culture was measured. The means with SDs of triplicate experiments are shown.

 
In summary, our global gene expression profiling of fresh Treg, activated Treg and Foxp3-transduced CD25–CD4+ T cells by DNA microarray analysis revealed several candidates of Treg-specific molecules, which were confirmed by real-time RT–PCR analysis. Interestingly, some of them (Gpr83, Ecm1, Cmtm7, Nkg7, Socs2 and glutaredoxin) appear to be under control of Foxp3, which is a ‘master control’ gene for the development and function of natural Treg, and the others (insulin-like 7, galectin-1, granzyme B and helios) appear to be independent of Foxp3 in their expression. It is likely that the expression of these molecules could be controlled by other Treg-specific mechanisms, which could act in a Foxp3-independent fashion or at an upstream step of Foxp3 action. Forced expression of these genes in naive T cells failed to confer a Treg-like function or phenotype. We surmise that Foxp3-independent expression of some Treg-specific genes might be related to their unique mode of activation in the thymus and periphery (e.g. possible co-stimulation via CTL-associated molecule-4) and their high self-reactivity, rather than Foxp3-regulated suppression (1). The expression of helios appears to be upstream of Foxp3 expression since helios is transcribed in more immature thymocytes than those expressing Foxp3 (47) (Fig. 6). Further study is needed to clarify how these genes contribute to the differentiation and function of natural Treg. Some of them, such as Gpr83, are highly specific for Treg and preparation of an antibody specific for the products of such genes will facilitate more reliable delineation and specific manipulation of Treg in clinical settings.


   Supplementary data
Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Supplementary data
References
 
Supplementary data are available at International Immunology Online.


   Acknowledgements
 
We thank Yasushi Onishi for technical advice, Toshio Kitamura for reagents and Fiona Rawle for critically reading the manuscript. 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.


   Abbreviations
 
APC, antigen-presenting cell
CKLF, chemokine-like factor
Cmtm7, CKLF-like MARVEL transmembrane domain containing 7
Ecm1, extracellular matrix protein 1
Emp3, epithelial membrane protein 3
Foxp3, Forkhead box P3
GITR, glucocorticoid-induced TNFR family-related gene/protein
Gpr83, G protein-coupled receptor 83
HPRT, hypoxanthine guanine phosphoribosyl transferase
Nkg7, natural killer cell protein 7
Treg, naturally occurring CD25+CD4+ regulatory T cell
RT, reverse transcription
Socs2, suppressor of cytokine signaling 2
SP, single positive

   Notes
 
* These authors contributed equally to this work. Back

Transmitting editor: K. Yamamoto

Received 1 March 2006, accepted 10 May 2006.


   References
Top
 Abstract
 Introduction
 Methods
 Results and discussion
 Supplementary data
 References
 

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