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The Journal of Immunology, 2008, 181, 6898 -6905
Copyright © 2008 by The American Association of Immunologists, Inc.
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Human CD4+CD25+Foxp3+ Regulatory T Cells Do Not Constitutively Express IL-35

Emilie Bardel1,*, Frédérique Larousserie1,*,{dagger}, Pascaline Charlot-Rabiega*, Aurore Coulomb-L'Herminé{ddagger} and Odile Devergne2,*

* Centre National de la Recherche Scientifique Unité Mixte de Recherche 8147, Université Paris Descartes, Paris, France; {dagger} Service d’Anatomie Pathologique, Assistance Publique Hôpitaux de Paris, Université Paris Descartes, Hôpital Cochin, Paris, France; and {ddagger} Service d’Anatomie Pathologique, Assistance Publique Hôpitaux de Paris, Hôpital Trousseau, Paris, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
EBV-induced gene 3 (EBI3) can associate with p28 to form the heterodimeric cytokine IL-27, or with the p35 subunit of IL-12 to form the EBI3/p35 heterodimer, recently named IL-35. In mice, IL-35 has been shown to be constitutively expressed by CD4+CD25+Foxp3+ regulatory T cells (Treg cells) and suggested to contribute to their suppressive activity. In this study, we investigated whether human Treg cells express IL-35. Double-staining analysis of human thymuses showed that neither Foxp3+ nor CD25+ cells coexpressed EBI3. Similarly, Foxp3+ cells present in human lymph nodes, tonsils, spleens, and intestines did not express EBI3. Consistent with these in situ observations, Treg cells purified from blood or tonsils were negative for EBI3 by immunoblotting. Other human T cell subsets, including effector T cells, naive and memory CD4+ T cells, CD8+ and {gamma}{delta} T cells also did not constitutively express EBI3, which contrasts with IL-35 expression observed in murine CD8+ and {gamma}{delta} T cells. Furthermore, although CD3/CD28 stimulation consistently induced low levels of EBI3 in various CD4+ T cell subsets, no EBI3 could be detected in CD3/CD28-stimulated Treg cells. RT-PCR analysis showed that, whereas p35 transcripts were detected in both Teff and Treg cells, EBI3 transcripts were detected only in activated Teff cells, but not in resting or activated Treg cells. Thus, in contrast to their murine counterpart, human Treg cells do not express detectable amounts of IL-35.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
EBV-induced gene 3 (EBI3)3 is a member of the IL-12 family that was identified in B lymphocytes based on its induction following EBV infection (1). It codes for a 34 kDa-secreted glycoprotein homologous to the p40 subunit of IL-12. It can associate with p28, a peptide analogous to the p35 subunit of IL-12, to form the heterodimeric cytokine EBI3/p28, named IL-27 (2). It can also dimerize with IL-12 p35 to form another heterodimeric cytokine of the IL-12 family, EBI3/p35 (3), that was recently called IL-35 (4). IL-27 signals through a heterodimeric receptor composed of a specific component, IL-27R (also called TCCR, WSX-1 or IL-27R{alpha}) and the common chain, gp130 (5). It acts on multiple cell types including CD4+ and CD8+ T cells, B cells, NK cells, macrophages, neutrophils, mast cells, and endothelial cells, and plays important roles in the immune system, specially in the regulation of Th and inflammatory responses (reviewed in Refs. 6 , 7).

Although the in vivo association between EBI3 and p35 was originally evidenced in human placental extracts (3), it was recently shown that IL-35 is constitutively expressed by mouse CD4+CD25+Foxp3+ regulatory T (Treg) cells (4). Transcripts coding for EBI3 and p35 were observed to be constitutively coexpressed by mouse Treg cells and EBI3/p35 heterodimer was coprecipitated from the cell culture supernatant of these cells. In addition, in vitro and in vivo studies suggested that the expression of IL-35 by mouse Treg cells contributed to their suppressive function. In transfer experiments, Treg cells from EBI3–/– or p35–/– mice showed reduced capacity to control homeostatic expansion of CD4+CD25– effector T (Teff) cells and were less effective than wild-type Treg cells to cure mice in a model of inflammatory bowel disease. This defective function was ascribed to a lack of IL-35 production, as the alternative partners for EBI3 and p35, p28, and p40, respectively, are not expressed by mouse Treg cells. In addition, recombinant mouse IL-35 was shown to inhibit the proliferation of mouse Teff cells in vitro (4). In another recent study, a single chain mouse IL-35-Fc fusion protein was demonstrated to enhance the proliferation of mouse Treg cells, while inhibiting the development of Th17 cells (8). The signaling cascade induced by IL-35 and its receptor have not been described.

The p35 gene is constitutively expressed at low levels in many cell types and therefore displays an almost ubiquitous expression (9). In contrast, EBI3 gene expression is restricted to specific cell types and highly inducible. In previous in situ and in vitro studies in humans, we found that EBI3 was expressed at high levels in placental trophoblast cells and activated dendritic cells, and at lower levels in macrophages and endothelial cells. Regarding lymphocytes, EBI3 expression was readily detected in activated normal B cells as well as tumoral B and T cells, but was undetectable in normal resting CD3+ T cells (10, 11, 12, 13, 14, 15). However, because Treg cells constitute a minor population among total T cells, EBI3 expression by this specific T cell subset may have gone unnoticed. Therefore, in this study we specifically investigated EBI3 expression by human Treg cells to determine whether they can express IL-35. Our in situ and in vitro analyses indicate that these cells do not express IL-35.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Human tissues

Postnatal normal thymus or tonsil specimens were obtained from children who underwent corrective cardiac surgery or tonsillectomy, respectively, at Necker Hospital (Paris). Fetal thymus specimens were collected after miscarriage (Trousseau Hospital, Paris). Non-neoplastic spleens, lymph nodes, and intestinal tissues were previously described (12, 15). Fresh tissues, obtained with informed consent of the patient, and fixed tissues, collected for histological examination and diagnostic purposes, were studied in accordance with French ethical guidelines.

Immunohistochemistry and immunocytochemistry

Before staining, formalin-fixed paraffin-embedded tissues were subjected to Ag retrieval by heat pretreatment in citrate (pH 6) or Tris/EDTA (pH 9) buffer. Single immunostaining was performed on serial tissue sections using an indirect avidin-biotin peroxidase or alkaline phosphatase kit (BioGenex). In double immunostaining experiments, binding of the primary Ab in the first label was detected using peroxidase-conjugated anti-mouse EnVision+ reagent (DakoCytomation) and diaminobenzidine as chromogen. Binding of the primary Ab in the second label was detected using an indirect avidin-biotin alkaline phosphatase kit (BioGenex) and Fast Red (DakoCytomation) as chromogen. Sections were counterstained with Mayer hematoxylin. EBI3 was detected using 2G4H6 mAb (10) at 2–4 µg/ml, and Foxp3 was detected using 236A/E7 mAb (Abcam) at 10–20 µg/ml. CD25 mAb (clone 4C9, Novocastra) was used at a 1/100 dilution.

For immunostaining on methanol/acetone (1/1)-fixed cytospin preparation, slides were rehydrated in Tris-buffered saline, blocked with Tris-buffered saline containing 5% human Ig and 1% BSA, and then incubated with control rabbit IgG (Sigma-Aldrich) or rabbit anti-EBI3 IgG (11) (20 µg/ml) for 60 min. Ab binding was detected using anti-rabbit peroxidase-conjugated EnVision+ reagent and diaminobenzidine as chromogen.

Photomicrographs were taken on a Leica DMRB microscope using a 3 CCD color video camera (Sony DXC-950P) using TRIBVN ICS software.

Isolation of human B and T cells

B and T cells were purified from adult peripheral blood (14 donors) or pediatric tonsil (four donors) by magnetic separation using reagents from Miltenyi Biotec.

B cells were purified from tonsils by negative selection by depletion of tonsillar mononuclear cells from T/NK cells by incubation with CD2 microbeads as described (16).

CD4+ T, CD8+ T, or {gamma}{delta} T cells were purified from PBMC isolated by Ficoll-Paque Plus (Amersham Biosciences) gradient centrifugation by using CD4+ T cell isolation kit II, CD8+ T cell isolation kit II, or {gamma}{delta} microbead kit, respectively. In some cases, CD4+ T cells were further separated into naive or memory T cells by incubation with CD45RO microbeads and separation by using a LS column. The negative fraction (naive CD4+ T cells) was collected and the positive fraction was passed again over a MS column to enrich in CD45RO+ cells.

Treg and Teff cells were isolated from PBMC using the CD4+CD25+ Treg cell isolation kit. In brief, CD4+ T cells were first purified by negative selection and then incubated with CD25 microbeads followed by separation by using a MS column. The negative fraction (CD4+CD25– T cells) was collected and the positive fraction was passed over a second MS column to enrich in Treg cells. Purified Treg cells accounted for 1–3% (mean 1.8%) of the total CD4+ T fraction. In some cases, CD4+ T cells, Teff and Treg cells were purified from tonsils. In these cases, tonsillar mononuclear cells were first depleted of B cells by incubation with CD19 microbeads and separation on a LS column, and then submitted to purification using the CD4+CD25+ regulatory T cell isolation kit as described above.

In each case, purity of the cell separation was verified by immunostaining and FACS analysis (FACSCalibur or FACSCanto II, BD Biosciences), and was as follows: B cells: > 98% CD19+, CD4+ T cells: 95–99%, CD8+ T cells: 90–94%, {gamma}{delta} T cells: 90–94%, naive CD4+ T cells: 92–97% CD4+CD45RA+, memory CD4+ T cells: >97% CD4+CD45RO+, Teff cells: 95–99% CD4+CD25– and Treg cells: 95–99% CD4+CD25+.

In vitro culture

T cell subsets (2.5 x 106/ml) were stimulated for 2 days with beads coated with CD3 and CD28 Abs (10 µg/ml each, T cell activation/expansion kit, Miltenyi Biotec), or PHA (4 µg/ml, Roche Diagnostics), in RPMI 1640 medium supplemented with 10% FBS, L-glutamine, and antibiotics (complete RMPI medium) or in X-Vivo 15 medium (BioWhittaker), in the absence or presence of IL-2 (Roche Diagnostics, 20–500 U/ml). B cells were cultured for 2–3 days in complete RPMI 1640 medium in the presence of goat polyclonal CD40 Ab (0.5 µg/ml, R&D Systems). In some cases, T cells were treated with brefeldin A (Sigma-Aldrich, 10 µg/ml) for the last 4 h of the culture.

Proliferation assay

To evidence the suppressive activity of Treg cells, Teff cells (5 x 104 per well) were cultured in triplicate for 4 days in X-Vivo 15 medium in 96-well plates coated with anti-CD3 (UCHT1, R&D Systems, 2.5 to 5 µg/ml) in the presence of 5 x 104 irradiated (5000 rad) PBMC and of variable numbers of Teff cells. Proliferation was measured by adding 0.5 µCi per well of [3H]thymidine (Amersham Biosciences) for the last 8 h of the fourth day of incubation.

Western blot analysis and ELISA

Cells were washed in ice-cold PBS and lysed for 1 h on ice in lysis buffer (1% Nonidet P-40, 50 mM Tris (pH 7.4), 150 mM NaCl, 3% glycerol, 1.5 mM EDTA) supplemented with protease inhibitors (1 mM PMSF, 1 µg/ml pepstatin, 1 µg/ml leupeptin). Cell lysate was centrifuged for 15 min at 13,000 x g and the supernatant was assayed for protein concentration using the microBCA protein assay (Pierce). Lysates (from 25 to 50 µg) were subjected to SDS-PAGE and transferred to nitrocellulose for immunoblotting. EBI3 and Foxp3 were detected using 2G4H6 and 236A/E7 mouse mAbs, respectively. Actin was detected using goat polyclonal Abs (I19, Santa Cruz Biotechnology). Binding of primary Abs was detected with HRP-conjugated anti-mouse (Amersham Biosciences) or anti-goat (Santa Cruz Biotechnology) Abs. Peroxidase reaction was developed with chemiluminescence reagents (Pierce). EBI3 ELISA was performed as previously described (10). It detects both free EBI3 and EBI3 complexed with either p28 or p35 (detection limit, 1 ng/ml). In some cases, cell culture supernatants from CD4+ T, Teff, or Treg cells stimulated for 2 days with beads coated with CD3 and CD28 mAbs were concentrated using Amicon Ultra centrifugal filter unit (Millipore) before being tested by ELISA.

RNA extraction and RT-PCR analysis

RNA was isolated by TRIzol extraction, followed by DNase I digestion and reverse transcription using M-MLV reverse transcriptase and oligo(dT) primer (all reagents from Invitrogen). EBI3 primers were as follows: 5'-GCAGACGCCAACGTCCAC-3' (sense) and 5'-CCAGTCACTCAGTTCCCCGT-3' (antisense). p35 and β2-microglobulin primers were as previously described (11, 17). The thermal cycle profile was as follows: 1 min 94°C, 30 s at 55°C, and 20 to 35 s at 72°C for 30 (β2-microglobulin), 32 (EBI3), or 34 (p35) cycles.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Treg cells present in the thymus do not express IL-35

First, we investigated by immunohistochemistry whether naturally occurring Treg cells developing in the thymus express EBI3. Treg cells can be identified in situ based on their constitutive expression of Foxp3 and high expression of CD25. Because the expression of EBI3 had not been previously analyzed in the human thymus, we first stained serial sections from two fetal thymuses (18 and 22 wk of gestation) and six pediatric thymuses (aged 7 days to 8 mo) with anti-EBI3 and anti-Foxp3 mAbs to determine whether EBI3+ cells could be detected in the same compartments as Foxp3+ cells (Fig. 1, A and B). Numerous Foxp3+ or EBI3+ cells were detected in all cases and their distribution did not vary with the age of the thymus. Both Foxp3+ or EBI3+ cells were predominantly located in the medulla, although some positive cells were also scattered in the cortex (Fig. 1, A and B). However, while Foxp3+ cells had a lymphoid morphology, most EBI3+ cells were morphologically consistent with dendritic cells. Double-staining experiments performed in the eight cases of thymus demonstrated that all Foxp3+ cells were negative for EBI3 (Fig. 1, C–E). It also showed that some Foxp3+ cells in the medulla were in close association with EBI3+ dendritic cells (Fig. 1E). Similarly, when thymic sections were double-stained with anti-EBI3 and anti-CD25 mAbs, no CD25+ cell showed positivity for EBI3 (Fig. 1, F–H). As observed for Foxp3+ cells, some medullar CD25+ thymocytes could be observed in direct contact with EBI3+ dendritic cells (Fig. 1H). These findings are in agreement with a previous in situ analysis showing that human thymic CD25+CTLA4+ Treg cells preferentially localized within the thymic medulla in close association with activated dendritic cells (18). Importantly, they indicate that Treg cells developing in the human thymus do not express IL-35, independently of the age of the thymus.


Figure 1
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  		FIGURE 1. Immunohistochemical analysis of EBI3 expression by human thymic Treg cells. A and B, Serial sections from a pediatric thymus stained with anti-Foxp3 (A) or anti-EBI3 mAbs (B). Foxp3+ or EBI3+ cells are abundant and preferentially located in the medulla. C–E, Double staining with anti-Foxp3 and anti-EBI3 Abs in sections from pediatric thymuses of various ages. Foxp3+ cells (indicated by arrows, brown nuclear staining) located in the cortex (C) or in the medulla (D and E), are negative for EBI3 (red cytoplasmic staining). In E, a Foxp3+ cell in direct contact with a EBI3+ cell having extended cytoplasmic processes and morphologically consistent with a dendritic cell is shown (*). F–H, Double staining with anti-EBI3 and anti-CD25 mAbs in sections from pediatric thymuses. CD25+ cells (indicated by arrows, red plasma membrane staining) present in the cortex (F) or in the medulla (G and H) are negative for EBI3 (brown cytoplasmic staining). In H, two examples of direct interaction between a CD25+ lymphoid cell and an EBI3+ dendritic cell are shown. In one case, this interaction takes place close to a Hassall’s corpuscle consistent with a previous study showing its role to instruct dendritic cells to induce Treg cells (18 ). On each picture, the age of the thymus is indicated at the bottom left. Co, Cortex; Me, medulla; Ha, Hassall’s corpuscle. Objective, x10 in A and B, x40 in C–H.

 
Lack of IL-35 expression by Foxp3+ cells in peripheral tissues

Next, we analyzed the in situ expression of EBI3 by Treg cells present in peripheral tissues. To this end, various human tissues that had been previously analyzed for EBI3 expression by immunohistochemistry and included lymph nodes (n = 5), tonsils (n = 3), spleens (n = 5), and digestive tissues (n = 4) (12, 15) were subjected to double staining with anti-Foxp3 and anti-EBI3 Abs. In lymph nodes and tonsils, Foxp3+ cells were mainly located in the T cell zones, but were also present at the border of follicles and inside follicles in agreement with previous studies (19). However, whatever their localization, Foxp3+ cells were not costained with anti-EBI3 Ab (Fig. 2, A–D, and data not shown). In spleens, Foxp3+ cells were largely restricted to the peri-arteriolar T cell areas and similarly were negative for EBI3 (data not shown). In digestive tissues (large and small bowel, appendix), Foxp3+ cells were detected in the T cell zones surrounding B cell follicles and were also scattered in the lamina propria. Again, these cells were in all cases negative for EBI3 (Fig. 2, E–H). This lack of EBI3 detection in Foxp3+ cells was not due to a lack of sensitivity of EBI3 immunohistochemistry, because many other cell types including dendritic cells, macrophages, B cell blasts, and plasma cells were positive for EBI3 in these tissues (Fig. 2). Thus, as observed for natural thymic Treg cells, mature Treg cells present in peripheral tissues do not express detectable amounts of EBI3. In these tissues, no double-staining with anti-EBI3 and anti-CD25 mAbs was performed to investigate the expression of EBI3 by Treg cells because of the presence of many CD25+ cells others that Treg cells that could express EBI3, such as activated B cells.


Figure 2
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  		FIGURE 2. Immunohistochemical analysis of EBI3 expression by Foxp3+ cells in peripheral human tissues. Double staining with anti-Foxp3 and anti-EBI3 mAbs in sections from a reactive lymph node (A–D), an appendix (E), and small and large bowel (F–H). Foxp3+ cells (brown nuclear staining) are indicated by arrows. In lymph nodes, these cells are abundant in interfollicular areas (A and D), but are also found in follicles, crossing the mantle zone (B) and scattered inside follicles (C). In the intestine, Foxp3+ cells are located around B follicles of the GALT (E–G) or scattered in the lamina propria (H). In all cases, Foxp3+ cells are negative for EBI3, although numerous EBI3+ cells (red cytoplasmic staining) including dendritic cells and macrophages (A, D–G), follicular B cells (B), and plasma cells (H) are detected. IF, Interfollicular area; Ma, mantle zone; Fo, follicle. Objective, x20 in A, x40 in B–H.

 
Treg cells purified from adult peripheral blood do not constitutively express IL-35

To further investigate the expression of IL-35 by human Treg cells, we examined whether Treg cells present in peripheral blood express EBI3. For this purpose, CD4+ T cells were isolated from PBMC and further separated into CD4+CD25– T cells and CD4+CD25+ T cells by magnetic cell separation. Treg cells can be distinguished from activated Teff cells transiently expressing CD25 by surface staining with CD127 (IL-7R{alpha}), a marker expressed at high levels on activated Teff cells, but at low levels on Treg cells (20, 21). FACS analysis of purified CD4+CD25+ cells showed a ≥95% purity based on CD4 and CD25 expression and the presence of 4–6% CD127high cells, indicating that at least 94% of CD4+CD25+ T cells purified from blood have a Treg phenotype (Fig. 3A).


Figure 3
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  		FIGURE 3. Analysis of EBI3 expression in purified Treg cells. A, Cell surface phenotype of purified Treg cells. CD4+ T cells were isolated from PBMC and separated into CD25+ and CD25– fractions by magnetic cell sorting. Dot plot analysis (log fuorescence intensity on both axes) of the different fractions labeled with CD4-FITC, CD25-PE, and CD127-Alexa Fluor 647 Ab are shown. The percentage of cells in each quadrant or gate is indicated. A representative experiment is shown. B, Functional characterization of purified Treg cells. CD4+CD25– Teff cells and CD4+CD25+ Treg cells were cultured in triplicates in CD3 Ab-coated 96-well plates, either separately or mixed together at the indicated ratio, in the presence of irradiated PBMC. Proliferation was measured by adding [3H]thymidine for the last 8 h of the fourth day of incubation. Results are expressed in cpm (mean of triplicates ± SD) and are representative of three experiments. C, Western blot analysis. Cell lysates from whole CD4+ T cells, Teff cells and Treg cells purified from blood (two different donors are shown) or tonsils were analyzed by immunoblotting with anti-Foxp3 and anti-EBI3 mAbs. Anti-actin Abs were used as a control for equal total protein loading. As positive control for EBI3, cell lysates from tonsillar B cells (a different donor is shown on each blot), either freshly isolated (B cells, lanes 1 and 6) or stimulated with anti-CD40 Abs (act. B cells, lanes 2 and 7) were used. For Foxp3, two major bands corresponding to the two isoforms present in human are detected. A barely detectable Foxp3 signal was occasionally seen in the cell lysate from total CD4+ T cells (data not shown).

 
In vitro functional studies confirmed that purified CD4+CD25+ T cells were bona fide Treg cells. Indeed, consistent with a functional Treg phenotype, CD4+CD25+ T cells were anergic in response to a polyclonal stimulation as shown by their proliferation level that corresponded to 2–9% of that of Teff cells tested in parallel, and inhibited Teff cell proliferation in a dose dependent-manner, with very strong suppression (from to 81–92% inhibition) when Teff cells and Treg cells were cocultured at a 1:1 ratio (Fig. 3B).

Cell lysates from blood-purified Treg cells were then analyzed by Western blot for Foxp3 and EBI3 expression, in parallel with cell lysates from whole CD4+ T and Teff cells (Fig. 3C) (three different donors tested). As a positive control for EBI3, purified tonsillar B cells, either freshly isolated or stimulated with anti-CD40 Abs, were used (four different donors). B cells were chosen as a positive control, because in mice the level of EBI3 gene expression in Treg cells is comparable to that observed in B cells (4). In cell lysates from CD4+CD25+ T cells (lanes 5 and 10), a strong signal for Foxp3 was specifically observed further assessing their Treg phenotype. Consistent with a previous study (15), EBI3 was readily detected in the lysates of freshly isolated B cells (lanes 1 and 6) and its expression increased upon in vitro B cell stimulation (lanes 2 and 7). In contrast, EBI3 was undetectable in cell lysates from whole CD4+ T cells, Teff and Treg cells, even after a prolonged exposure of the blot (Fig. 3C, lanes 3–5, 8–10) (detection limit for EBI3 blot, 0.2 ng/lane). Similarly, when Treg cells were purified from tonsils (two different donors tested) and their cell lysates analyzed by Western blot, expression of Foxp3, but not of EBI3, was observed (Fig. 3C, lane 13). Thus, in vitro analysis of purified CD4+CD25+Foxp3+ Treg cells confirmed that they do not constitutively express EBI3.

Analysis of constitutive EBI3 expression in various human T cell subsets

In mice, EBI3 and p35 mRNA have been shown to be constitutively coexpressed, not only by Treg cells, but also, albeit at a lower level, by CD8+ and {gamma}{delta} T cells (4). Thus, to determine whether the discrepancy observed between humans and mice for IL-35 expression is restricted to Treg cells or also extends to other T cell subsets, we analyzed by Western blot the expression of EBI3 in various human T cell subsets purified from peripheral blood by magnetic cell separation (three to five donors tested for each subset). In the cell lysates from {gamma}{delta} T cells, CD8+ T cells, or naive or memory CD4+ T cells, no constitutive expression of EBI3 was detected (Fig. 4, lanes 1–7). Thus, in contrast to the situation observed in mice, none of the human T cell subsets we analyzed (Treg and Teff, naive and memory CD4+ T, CD8+ T, and {gamma}{delta} T cells) expressed detectable amounts of EBI3 constitutively.


Figure 4
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  		FIGURE 4. Analysis of constitutive EBI3 expression in various human T cell subsets. Cell lysates (40 µg/lane) from various purified T cell subsets (indicated at the top of each lane) were analyzed by Western blot with anti-EBI3 mAb and subsequently with anti-actin Abs to verify equal total protein loading. For {gamma}{delta} and CD8+ T cells, lysates from two different donors are shown. As positive controls, lysates from tonsillar B cells, either freshly isolated (lane 8) or stimulated for 2 days with anti-CD40 Abs (lane 9) were used.

 
Analysis of EBI3 expression in in vitro-stimulated CD4+ T cell subsets

In a previous study, we showed that low levels of EBI3 could be induced in human T cells upon in vitro stimulation with PHA and IL-2 (14). Therefore, to further analyze the expression of EBI3 in human T cells, whole CD4+ T cells or subsets of naive or memory CD4+ T, Treg, and Teff cells were stimulated for 2 days with beads coated with anti-CD3 and anti-CD28 Abs (a more physiological stimulus than PHA) in the absence or presence of IL-2, and cell lysates were analyzed for EBI3 expression by immunoblotting (Fig. 5) (six different donors tested for non-Treg CD4+ T cells and five for Treg cells). In all CD4+ T cell subsets, except Treg cells, CD3/CD28 stimulation resulted in EBI3 induction. In these cells, EBI3 levels were higher than those observed following PHA/IL-2 stimulation (Fig. 5A, lanes 12 and 13) and were not further enhanced by the addition of IL-2 (Fig. 5A, left panel and 5B, right panel). EBI3 levels in CD3/CD28-activated CD4+ T cells were variable depending on the donor (Fig. 5A, left blot), and were consistently much lower than those observed in activated B cells (Fig. 5, A and B). In contrast, no EBI3 induction was observed in CD3/CD28-activated Treg cells from all donors tested (Fig. 5B, lanes 2 and 7). Addition of IL-2 to Treg cell culture resulted in increased cell viability (data not shown) and enhanced expression of Foxp3 (Fig. 5B, right blot), in keeping with previous data (22), but still no detectable EBI3 expression (Fig. 5B, lane 8). Thus, in both resting or CD3/CD28-stimulated Treg cells, no expression of EBI3 was detected by Western blot.


Figure 5
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  		FIGURE 5. Analysis of EBI3 expression in activated CD4+ T cell subsets. A, Cell lysates from whole, naive, or memory CD4+ T cells, either freshly isolated (–) or stimulated for 2 days with beads coated with anti-CD3 and anti-CD28 Abs (+), alone or in presence of 20 U/ml IL-2 (+ IL-2) or with PHA plus IL-2, were analyzed by Western blot with anti-EBI3 and anti-actin Abs. As positive controls, cell lysates from B cells, either freshly isolated or stimulated for 2 days with anti-CD40 Abs, were used. For CD4+ T cells, lysates obtained from two different donors are displayed to show the variable magnitude of EBI3 induction. B, Cell lysates from Teff and Treg cells purified from the same donor (two different donors are shown) and cultured in parallel as indicated in A, were analyzed by Western blot with anti-EBI3, anti-actin, and in some cases anti-Foxp3 Abs. No induction of EBI3 was observed in activated Treg cells independently of the donor. Similar results were observed when Treg cells were cultured with higher doses of IL-2 (100 to 500 U/ml). Also, no EBI3 signal was detected in Treg cells when they were treated with brefeldin A (10 µg/ml) for the last 4 h of the culture (data not shown).

 
To further investigate EBI3 regulation in resting and in vitro activated human Treg and Teff cells, EBI3 expression was analyzed by immunocytochemistry and by ELISA of the cell culture supernatant. Intracellular staining for FACS analysis could not be performed because of the lack of suitable anti-human EBI3 Abs. By immunostaining of cytospin samples, EBI3 was detected only in CD3/CD28-activated Teff cells (Fig. 6b), but not in freshly purified Teff or Treg cells (Fig. 6, a and d, respectively) nor in activated Treg cells (Fig. 6, e and f), in agreement with immunoblotting data. By ELISA, levels of secreted EBI3 in the cell culture supernatants of T cells were undetectable, unless supernatants were concentrated by 8-fold. Under these conditions, from 3 to 4.4 ng/ml EBI3 (mean, 3.5 ng/ml) were detected in the supernatants of CD3/CD28-activated CD4+ T cells or Teff cells, but concentrated supernatants from activated Treg cells remained negative for EBI3 (two to three donors tested for each T cell subsets). Thus, at both intracellular or secreted levels, no EBI3 was detected in resting or activated human Treg cells.


Figure 6
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  		FIGURE 6. EBI3 immunostaining of purified Teff and Treg cells. Cytospin preparations of Teff (a—c) and Treg (d—f) cells, either freshly purified or stimulated for two days with beads (appearing as small brown spheres on the figure) coated with CD3/CD28 mAbs were stained with rabbit anti-EBI3 IgG or control rabbit IgG (Col) as indicated on each panel. Only activated Teff cells are positive for EBI3 (brown cytoplasmic staining). Objective: x100.

 
In murine Treg cells, EBI3 expression was investigated both at the mRNA and protein levels (4). Thus, to verify that the lack of EBI3 detection in human Treg cells is not due to a technical issue (lower sensitivity of the protein analysis compared with the mRNA analysis) or to dissociation between EBI3 mRNA and protein expression in humans, we tested various CD4+ T cells subsets for EBI3 expression by RT-PCR. In parallel, p35 gene expression was analyzed (Fig. 7). In CD4+ T cells, EBI3 gene expression was undetectable before stimulation, but was consistently induced upon CD3/CD28 stimulation, in keeping with our protein data. In contrast, p35 gene expression was detected in both unstimulated or stimulated CD4+ T cells (Fig. 7A). Also in agreement with the protein data, EBI3 transcripts were undetectable in freshly purified Teff or Treg cells (Fig. 7B, lanes 3 and 4) and were detected in activated Teff cells, but not activated Treg cells (Fig. 7B, lanes 6–9). Interestingly, p35 signal was much stronger in resting Treg cells than in resting Teff cells (Fig. 7B, compare lane 4 with lane 3), this up-regulation being specifically observed in resting but not activated Treg cells. This latter finding is in keeping with the data described in mouse. Indeed, constitutive expression of p35 gene has been shown to be much higher in mouse Treg cells than in mouse Teff cells, and to be down-regulated in Treg cells upon CD3/CD28 stimulation (4). Taken together, these data showed no dichotomy for EBI3 between mRNA and protein expression, and confirmed the lack of EBI3 expression in human Treg cells. They also pointed out that while EBI3 gene expression appeared to differ between human and mouse Treg cells, p35 gene seemed to be similarly regulated among human and mouse Treg cells.


Figure 7
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  		FIGURE 7. RT-PCR analysis of EBI3 and p35 gene expression in various human CD4+ T cell subsets. Purified CD4+ T cells (A), Treg and Teff cells (B), either freshly purified (–) or stimulated for 40 h with CD3/CD28 Ab coated beads (+) were analyzed for EBI3 and p35 gene expression by RT-PCR. The lane denoted cDNA (–) corresponds to the negative control where no cDNA was added in the PCR. β2-microglobulin (β2m) was used as an internal control for equal material in each reaction. cDNA from freshly isolated B cells (lane 1) was used as a positive control. Each braket indicates cells from a same donor processed in parallel (for example, two different donors are shown for resting and activated CD4+ T cells on A, and for activated Teff and Treg cells, on B). The lower mobility band present in p35 PCR in resting CD4+ T cells on A, was not consistently detected. On B, lane 7, a faint p35 band was visible on the gel, but is barely detectable on the scanned image.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we investigated whether human Treg cells can express IL-35 protein. Because immunoassays to detect EBI3 expression are more sensitive than those to detect p35, we focused our analysis on EBI3 expression and initially used immunohistochemistry and Western blotting, two assays that in our hands were the most sensitive to detect EBI3 expression. Although in this and previous studies (10, 11, 12, 13, 14, 15), we could readily detect EBI3 expression in many normal or tumoral cell types, we failed to detect significant expression of EBI3 by human Treg cells in all compartments analyzed (thymus, spleen, lymph node, tonsil, intestine, and blood). Other protein assays (ELISA, immunocytochemistry) or RT-PCR analysis similarly did not show EBI3 expression in human Treg cells, constitutively or upon stimulation. Therefore, these cells are unlikely to express substantial amounts of the two EBI3-associated heterodimeric cytokines, IL-35 or IL-27.

For the in situ analysis of EBI3 expression by natural or peripheral Treg cells, we used Foxp3 to identify these cells. In mice, Foxp3 expression is strictly restricted to Treg cells and therefore constitutes an exclusive marker of murine Treg cells (23). In contrast, in humans, Foxp3 is constitutively expressed at high levels by Treg cells, but is also transiently expressed at low levels by in vitro-activated Teff cells (Fig. 5B, and Ref. 24, 25, 26, 27, 28). Although some authors observed a conversion to a Treg phenotype (24), others did not (25, 26, 27). Therefore, although little information is available on the extent of Foxp3 expression by in vivo activated Teff cells in tissues, it is likely that all Foxp3+ cells detected in situ are not Treg cells. Notably, in human tonsils, Foxp3+ cells have been found not only among resting CD69–CD4+CD25+ T cells, but also, with a lower frequency, among activated CD69+CD4+CD25+ T cells comprising activated Teff cells (19). Nevertheless, because in the different tissues we analyzed, none of the Foxp3+ cells expressed EBI3, regardless of their intensity of Foxp3 staining, we can conclude that Foxp3+ Treg cells do not express EBI3 and therefore IL-35. Also, in the human thymus, both CD4+CD25+ and CD8+CD25+ regulatory thymocytes expressing Foxp3 mRNA have been described (29, 30). Our data suggest that neither of these two Foxp3+ suppressive subsets expressed EBI3.

This study also highlights the difference between humans and mice regarding EBI3 expression in T cells. In mice, gene microarrays identified EBI3 as a gene selectively overexpressed in Treg cells (4, 31). Accordingly, EBI3 protein was specifically detected by intracellular staining in mouse Treg but not Teff cells (4). In addition, transduction of mouse Teff cells with a Foxp3 retroviral vector resulted in strong EBI3 gene induction (4), while conversely, conditional ablation of Foxp3 in mature peripheral Treg cells resulted in the down-regulation of EBI3 gene expression (32), indicating that mouse EBI3 was a Foxp3 target gene. Our findings suggest that in humans, Foxp3 expression is not sufficient to induce significant EBI3 expression, which further emphasizes the differences observed between human and mouse Foxp3 (23).

This differential expression of EBI3 between humans and mice is not restricted to Treg cells, but is also observed in other T cell subsets. Whereas low levels of EBI3 transcripts were detected in mouse CD8+ and {gamma}{delta} T cells, no EBI3 expression was detected in both subsets in humans. Conversely, whereas CD3/CD28 stimulation failed to induce EBI3 expression in murine Teff cells and down-regulated its constitutive expression in Treg cells (4), it consistently induced EBI3 expression in human Teff cells. In contrast, similar to mouse Treg cells, human Treg cells expressed high amounts of p35 transcripts, constitutively. In addition, RT-PCR analysis showed that both EBI3 and p35 genes were expressed in activated human Teff cells, which therefore may produce IL-35. However, coimmunoprecipitation experiments performed from the cell lysate of activated Teff cells using our anti-EBI3 Abs and various commercial anti-p35 Abs failed so far to show substantial association between both proteins (our unpublished observations).

The recent identification of immunosuppressive functions for IL-35 led to the hypothesis that this novel cytokine may represent a new target for immunotherapy, notably in inflammatory diseases (4, 8). However, because of the differential regulation of EBI3 expression in T cells, IL-35 may play different roles in human and mouse. In humans, various cell types other than Treg cells might constitute potential sources of IL-35. Indeed, strong expression of EBI3, in the absence of detectable expression of p28, has been detected in tumoral cells of various forms of lymphomas (14, 15). Production of IL-35 by these cells might constitute a mechanism to evade the anti-tumoral immune response. Also, in previous studies, we showed that EBI3 and p35 were expressed by syncytiotrophoblast and extravillous trophoblast cells at the human fetal-maternal interface (10), a site of strong immunomodulation. Further studies of IL-35 expression profile in human and mouse, in both normal and pathological conditions, will be necessary to delineate its role in the regulation of the immune response and its potential interest for therapeutic manipulation.


   Acknowledgments
 
We thank the Departments of Otorhinolaryngology, Pediatric Cardiac Surgery, Hematology, and Pathology of Necker Hospital (Paris, France) for tissues and blood samples, and the Department of Pathology of Cochin Hospital (Paris, France) for technical assistance. We also thank Amédée Renan for technical advice.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 E.B. and F.L. share first authorship. Back

2 Address correspondence and reprint requests to Dr. Odile Devergne, Centre National de la Recherche Scientifique Unité Mixte de Recherche 8147, Hôpital Necker, Bâtiment Sèvres, 161 rue de Sèvres, Paris, France. E-mail address: odile. devergne{at}inserm.fr Back

3 Abbreviations used in this paper: EBI3, EBV-induced gene 3; Treg cell, regulatory T cell; Teff cell, effector T cell. Back

Received for publication May 5, 2008. Accepted for publication September 6, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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