TGFß regulates the CD4+CD25+ T-cell pool and the expression of Foxp3 in vivo

Christoph Schramm1, Samuel Huber1, Martina Protschka1, P. Czochra1, Jürgen Burg2, Edgar Schmitt3, Ansgar W. Lohse1, Peter R. Galle1 and Manfred Blessing1,4

1 Department of Medicine, 2 Institute of Pathology and 3 Institute of Immunology, Faculty of Medicine, Johannes Gutenberg-University, Mainz, Germany
4 Faculty of Veterinary Medicine, Center for Biotechnology and Biomedicine, University of Leipzig, Leipzig, Germany

Correspondence to: C. Schramm; E-mail: schramm{at}uni-mainz.de


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Factors influencing the development of CD4+CD25+ T-cells in vivo are poorly understood. In order to investigate the contribution of TGFß1 to the development and function of CD4+CD25+ T-cells, we generated a gain of function mutation resulting in the overexpression of an active form of TGFß1 in T-cells under control of the human CD2 promoter. In peripheral lymphoid organs and in the thymus, the frequency of CD4+CD25+ T-cells was increased in transgenic mice. This appeared to be due to an autocrine effect of TGFß on T-cells, since concomitant impairment of TGFß-signaling in double transgenic mice resulted in a phenotype similar to wild type. In contrast, in single transgenic mice with impaired TGFß-signaling in T-cells, CD4+CD25+ T-cell numbers were reduced in peripheral lymphoid organs but not in the thymus. In addition, TGFß was found to regulate the expression of Foxp3 in vivo, a transcription factor essential for the generation and function of regulatory T-cells. In CD4+CD25+ T-cells, TGFß1 increased the expression of Foxp3, whereas a decreased expression was seen in CD4+CD25+ T-cells with impaired TGFß-signaling. TGFß1 induced the expression of IL-10 in transgenic T-cells, but the increased in vitro suppressive capacity observed in transgenic CD4+CD25+ T-cells was due to the secretion of TGFß and not IL-10. Therefore, our study provides in vivo evidence for a role of TGFß in the homeostasis of CD4+CD25+ T-cells.

Keywords: IL-10, regulatory T-cells, TGFß-signalling, thymus, tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
It is now well accepted that dominant T-cell mediated regulation of effector cells in the periphery is required in order to maintain immunological homeostasis (1). CD25 is one of several markers that are used to investigate the nature of regulatory T-cells. The thymus appears to be able to generate fully developed CD4+CD25+ regulatory T-cells, which constitute ~5% of CD4+ mature thymocytes and 5–10% of CD4+ T-cells in the periphery (1). In experiments using neonatal thymectomy, CD4+CD25+ T-cells were shown to leave the mouse thymus beginning on day 3 after birth and to reach adult levels at the age of 2 weeks (2). Recently, evidence has arisen that regulatory T-cells can also be generated in peripheral lymphoid organs (3). Factors influencing the central and peripheral generation of CD4+CD25+ T-cells are of considerable interest and so far poorly understood.

One of the factors recently described to influence T-cell development and to be essential for the generation and function of CD4+CD25+ regulatory T-cells is Foxp3, a member of the forkhead transcription factors (47). However, the regulation of Foxp3 in vivo is largely unknown.

Transforming growth factor beta (TGFß) is a pleiotropic factor with central functions in the regulation of cell proliferation and differentiation as well as in the maintenance of immune homeostasis (8). The contribution of TGFß to the development and function of CD4+CD25+ T-cells is less clear. In neonatal TGFß knockout mice, a normal thymic T-cell development including the in vitro function of CD4+CD25+ T-cells has been published (9). These results have just been confirmed in adolescent TGFß knockout mice but in vivo, a reduced suppressive capacity of CD4+CD25+ T-cells was seen (10). However, one should keep in mind that T-cells from TGFß knockout mice have an activated phenotype and that these mice constitute a rather unsuitable model to study the development of regulatory T-cells due to their multifocal inflammatory disease (11,12). Interestingly, it has been suggested that TCR activation in the presence of TGFß converts naive mouse CD4+CD25– T-cells to regulatory CD4+CD25+ T-cells through the induction of Foxp3 (3). In addition, human CD4+CD25+ T-cells could be expanded by TGFß in vitro (13) and the induction of TGFß in the pancreas of transgenic mice lead to an increase in local CD4+CD25+ T-cell numbers (14).

We therefore investigated TGFß as one of the factors influencing the maintenance and peripheral development of CD4+CD25+ T-cells in vivo.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation and maintenance of transgenic mice
The cDNA of a mutated simian TGFß1 was inserted in the SmaI site of the human CD2 (hCD2) minilocus expression vector (15). The mutation consists of the insertion of two serine residues at position 223 and 225 of the simian TGFß1 cDNA, giving a biologically active form of TGFß1 (16). The expression cassette was excised by NotI/SalI digestion, gel purified and used for pronuclear microinjection of fertilized eggs of strain FVB/N, essentially as described (17). Offspring was biopsied at ears or tails and analyzed for genotype by PCR using an hCD2 specific primer [5'-TTTGTAGCCAGCTTCCTTCTG-3'; corresponding to position 243–263; GenBank accession no. X07871; (18)] and a simian TGFß1 specific primer [5'-AGCCGCAGCTTGGACAGGATC-3', corresponding to position 437–417; GenBank accession no. M16658; (19)].

All transgenic lines were established and maintained as heterozygotes on a FVB/N background. For all experimental procedures age matched non-transgenic littermates were used as controls. Animal care was in accordance with all governmental and institutional guidelines.

RNA isolation and RT–PCR
RNA was isolated from mouse tissue using TRI Reagent (Sigma-Aldrich, Steinheim, Germany) according to the manufacturer's instructions. Reverse transcription was performed using the RevertAidTM H minus First strand cDNA Synthesis Kit (MBI Fermentas, St-Leon-Rot, Germany).

The above mentioned primer pair was used for PCR and the source of DNA could be determined by the size of the PCR product (354 bp for cDNA and 431 bp for genomic DNA).

Real time quantitative PCR
cDNA samples were subjected to real-time quantitative PCR analyses using primers and an internal fluorescent probe specific for Foxp3 or HPRT essentially as described (5). The relative quantity of Foxp3 in each sample was normalized to the relative quantity of HPRT.

Histological assessment
Several organs were assessed histologically. Five-micrometre sections were stained with hematoxylin and eosin (H&E) or May Grünwald Giemsa. Fibrosis was assessed using Elastica van Gieson or Sirius Red staining.

Separation of lymphoid cells
For the separation of B- and T-lymphocytes, spleen cells from 8- to 9-week-old mice were marked with anti-mouse CD3–FITC, anti-mouse CD19–PE mAb, anti-mouse CD4–FITC/microbeads or anti-mouse CD8–FITC (BD Pharmingen, Heidelberg, Germany). MACS beads conjugated with anti-FITC/PE antibodies were added and cells separated using LS+ cell separation columns (Miltenyi Biotech, Bergisch Gladbach, Germany). For the isolation of CD4+CD25+ T-cells, cells were labeled with anti-mouse CD25–FITC (clone 7D4, BD Pharmingen) and isolated with anti-FITC Multisort-Kit (Miltenyi Biotech). In a second step, cells were marked with anti-mouse CD4-beads. Purity of cell preparation ranged around 95% as assessed by flow cytometry. For the analysis of Foxp3 expression, cells were isolated with a FACSVantage cell sorter (Becton Dickinson, Heidelberg, Germany) reaching a purity of >97%.

Flow cytometry
Spleen cells and thymocytes from transgenic mice and non-transgenic littermates were analyzed using anti-mouse CD4–FITC/PE, CD8–PE/FITC/antigen presenting cell (APC), CD62L–PE, CD44–PE/FITC, CD25–FITC and CD19–PE mAb (all from BD Pharmingen). Flow cytometry was performed with a FACSCalibur or FACSVantage SE using CELL-quest software (Becton Dickinson, Heidelberg, Germany). At least 10 000 cells were analyzed.

For the analysis of apoptosis, cells were stained with anti-mouse CD4–PE, CD8–APC, annexin-V–FITC (all from BD Pharmingen) and propidium iodide (Sigma, Deisenhofen, Germany) at a concentration of 5 µg/ml according to the manufacturer's instructions and analyzed within 1 h after staining. For the analysis of activation induced cell death, 30 µg of purified anti-CD3{varepsilon} (clone 145-2C11, BD Pharmingen) was injected i.p. into 4-week-old mice. Cell proliferation was measured using specific, FITC-conjugated anti-Ki67 mAb (BD Pharmingen).

Cell culture
Cells were cultivated in serum free medium (PAN Biotech, Aidenbach, Germany) with penicillin (100 U/ml) and streptomycin (100 µg/ml; Life Technologies, Eggenstein, Germany). 2 x 106 cells/ml were plated onto precoated 24-well plates (Greiner, Frickenhausen, Germany). For precoating, plates were incubated at 4°C with 10 µg/ml anti-mouse CD3 mAb in 0.1 M sodium phosphate buffer, pH 8.5, overnight. Anti-mouse CD28 mAb (10 µg/ml; BD Pharmingen) was added to the medium. For allogenic stimulation, irradiated CD3-depleted spleen cells from C57/Bl6 mice were used as antigen-presenting cells (APC) at a ratio of 1:10 and 3 µg/ml anti-mouse CD3 mAb was added to the culture. Cells were grown at 37°C in a water-saturated atmosphere with 5% CO2 in air. Supernatants were collected after 48–96 h and frozen in liquid nitrogen.

Cell proliferation assays
For the analysis of suppressor function, various numbers of CD4+CD25+ were added to 1x105 CD4+CD25– wild-type or transgenic T-cells or CD4+CD25– T-cells from hCD2-{Delta}kTßRII mice (20). Assessment of antigen-presenting function was performed with 1 x 104–1 x 105 APC isolated from wild-type or transgenic mice stimulating 1 x 105 C57/BL6 CD4+ T-cells. Cells were stimulated allogenically in 96-well flat-bottom plates (Greiner) for 96 h in serum free medium as described above. Transwell experiments were performed in 24-well plates (Greiner) using 0.4 µm filters (Millicell, Millipore, Molsheim, France), the concentration of cells being the same as in experiments using 96-well plates. Cells were pulsed with 2.5 µCi/well [3H]thymidine for the last 12 h of culture. Samples were harvested and counted in a Betaplate liquid scintillation counter (Wallac, Freiburg, Germany).

Cytokine assays
Concentrations of IL-2, IFN{gamma}, IL-4, IL-10 (Mouse BD OptEIA ELISA Sets, BD Pharmingen) TNF{alpha} and IL-13 (R+D Systems) in cell culture supernatants were measured according to the manufacturer's instructions. Concentrations of TGFß1 were measured using chicken anti-human TGFß1 (R+D Systems) in a standard ELISA protocol.

Statistical analysis
Mean ± SEM are given. For comparison of groups the two-sided Wilcoxon Rank sum test was applied and a P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Generation of CD2-TGFß1 mice
In order to constitutively express biologically active TGFß1 in T-cells, we inserted the cDNA of a mutated simian TGFß1 into the human CD2 minilocus expression vector (Fig. 1A). The hCD2 promoter/enhancer drives the copy number dependent expression in T-cells (15). We obtained 12 founder animals which were fertile. Of these, three transgenic lines were further characterized and were designated according to ILAR guidelines as TgN(CD2TGF)1Mbl (abbreviated as line Q), TgN(CD2TGF)2Mbl (abbreviated as line X) and TgN(CD2TGF)3Mbl (abbreviated as line Y).



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Fig. 1. Generation and characterization of transgenic mice. (A) Schematic representation of the CD2-TGFß1 construct. The cDNA for the constitutively active modified simian TGFß1 was inserted into the second exon of the hCD2 minilocus expression vector. Localization of the primers used for transgene detection is shown. (B) Transgenic mRNA expression was analyzed using RT–PCR spanning an intron in order to differentiate between genomic DNA and RNA expression. The expected size of the RT–PCR product is 354 bp, whereas the product generated on unspliced DNA has a size of 430 bp. The same pattern was seen in all three transgenic lines tested. (C) Production of active TGFß1 by CD3+ T-cells isolated from the spleens of transgenic and wild-type mice was analyzed using ELISA after 48 h of culture (P < 0.01 for unstimulated cells and for cells costimulated with anti-CD3/CD28 mAb). This experiment was performed with CD3+ and CD4+ T-cells for lines Q, Y and X with similar results.

 
Analysis of transgene expression
Tissue-specific transgene expression was assessed by RT–PCR spanning an intron in order to differentiate mRNA expression from genomic DNA contamination. Transgene-specific mRNA expression was seen in CD4+ and CD8+ T-cells, but not in B-cells, heart, kidney, liver or skin. Results for line Y are shown in Fig. 1(B). Protein expression was determined using ELISA for active TGFß1. In contrast to wild-type cells, unstimulated transgenic T-cells from all three lines were shown to secrete active TGFß1 at low levels (for line Y: 55 ± 11 vs 0 pg/ml, P < 0.01; Fig. 1C). Upon costimulation, significantly more active TGFß1 was produced by transgenic T-cells as compared to wild-type T-cells (for line Y: 104 ± 9 vs 17 ± 3 pg/ml, P < 0.01; Fig. 1C).

Autocrine TGFß1 selectively affects T-cell development in the thymus with a relative increase in CD4+CD25+ cells
Heterozygous animals of all three lines were born at the expected frequency and showed no macroscopic abnormalities at birth. However, thymic atrophy was noted in transgenic mice beginning at the age of 2 weeks. Histological analysis of thymic sections revealed that the atrophy seen in transgenic animals was more pronounced in the thymic cortex than in the medulla, maybe due to the fact that the cortex is the main site of thymocyte proliferation (Fig. 2A). By the age of 2 weeks, thymocyte numbers began to decrease in transgenic mice, and by the age of 6 weeks, the total thymocyte number was reduced by ~45% in transgenic mice of all three lines (for line Y: 98 ± 5 vs 177 ± 7, P < 0.02; Fig. 2B). Using flow cytometric analysis, it was shown that the reduction in cell number was mainly due to a significant reduction in CD4+CD8+ thymocyte numbers and to a lesser extent in CD4–CD8– and CD4+CD8– cell numbers (Fig. 2C).



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Fig. 2. Effects of active TGFß1 on thymic T-cell development. (A) Thymic atrophy was detected in all three transgenic lines that histologically seemed to primarily affect the thymic cortex. Representative thymic sections from an animal of line Y and a control littermate is shown at the age of 6.5 weeks (H&E). Bars represent 5 mm for the macroscopic and 500 µm for the microscopic view. (B) Total thymic cell numbers were assessed at different time points and depicted as the percentage relative to cell numbers of wild-type littermates. P < 0.01 at the age of 4 weeks and P < 0.02 at the age of 6 weeks. (C) A significant reduction in CD4+CD8+, CD4–CD8– and CD4+CD8– cell numbers from CD2-TGFß1 transgenic mice as compared to wild-type mice was detected by flow cytometric analysis of thymocyte cell suspensions. Data are shown from one representative experiment out of two including 20 mice at the age of 7 weeks (**P < 0.01, *P < 0.03). (D) Apoptosis was measured using annexin-V and propidium iodide staining of various thymocyte populations. Representative results for annexin-V expression of transgenic and wild-type CD4+CD8+ thymocytes are given at the age of 6 weeks. The gate was set on CD4+CD8+ cells excluding propidium iodide positive cells. Twenty-four animals were analyzed per group in four separate experiments. (E) The expression of Ki-67 was analyzed by flow cytometry in various thymocyte subpopulations. A significant reduction in expression of Ki-67 was detected in CD4–CD8– thymocytes (P < 0.05). Results are shown for line Y. The experiments were repeated for lines Q, X and Y with similar results.

 
Since TGFß influences both proliferation and apoptosis in T-cells (21), we assessed whether these effects were due to increased apoptosis of thymocyte subsets using propidium iodide and annexin-V staining. A significant increase in annexin-V highly positive CD4+CD8+ thymocytes was seen in transgenic as compared to wild-type mice between the age of 4 and 6 weeks (4.6 ± 0.2 vs 3.6 ± 0.2%, P < 0.03, mean fluorescence intensity for annexin-V: 133 ± 4 vs 111 ± 5, P < 0.03 at the age of 6 weeks; Fig. 2D). No difference was detected in all other thymocyte subsets using annexin-V or propidium iodide staining.

The proliferative activity of various thymocyte subsets was determined by flow cytometric analysis of Ki67-expression. A significantly reduced Ki67 expression was detected only in the transgenic CD4–CD8– cell population (22.0 ± 1.7 vs 28.1 ± 1.8%, P < 0.05; Fig. 2E). Therefore, it seems that a reduced proliferation of CD4–CD8– cells as well as an increased rate of apoptosis in CD4+CD8+ cells might contribute to the reduced numbers of CD4+CD8+ cells observed.

The CD4+CD8– cell population was then further analyzed and a relative increase in CD4+CD25+ T-cells in transgenic mice was found (5.4 ± 0.2 vs 4.0 ± 0.2%, P < 0.02; Fig. 3A). We were then interested in the target cell population of TGFß1. We therefore crossed TGFß1-overexpressing mice with transgenic mice which overexpress a dominant negative TGFß type II receptor under control of the hCD2 promoter. In these mice, TGFß signalling is impaired specifically in T-cells and they were previously shown to have a normal thymic T-cell development (20). In double transgenic mice the number of CD4+CD25+ T-cells was similar to wild type. Similarly, the changes in thymic cell populations induced by TGFß1 were almost completely abolished in double transgenic mice, suggesting that TGFß1 selectively affects the development of T-cells in the thymus in an autocrine manner (Fig. 3B).



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Fig. 3. TGFß alters thymic T-cell development in an autocrine manner. The frequency of thymic CD4+CD25+ T-cells (A) and the distribution of thymic cell populations (B) were analyzed using flow cytometry in single transgenic and CD2-TGFß1xCD2-{Delta}kTßRII double transgenic mice (dTG). (A) Overexpression of TGFß1 resulted in an increased frequency of CD4+CD25+ cells in the thymus. (B) The autocrine action of TGFß is demonstrated in double transgenic mice with an impaired TGFß-signaling in T-cells (dTG) which show a thymic cell distribution similar to wild type. The results are representative of 20 mice at the age of 7 weeks (*P < 0.02).

 
TGFß1 affects CD4+CD25+ T-cells in peripheral lymphoid organs
A progressive decline in total splenic cell numbers was observed in transgenic mice beginning at the age of 4 weeks (Fig. 4A). This decrease resulted from a selective reduction of CD4+ T-cells (11.6 ± 2 vs 25.8 ± 1.8 x 106 at the age of 8 weeks, P < 0.02; Fig. 4B), whereas CD8+ T-cell numbers and B-cell numbers were not affected. The CD4+ T-cell pool was further analyzed and an increased frequency of CD4+CD25+ T-cells was found in mice overexpressing TGFß1 (spleen: 11.8 ± 0.2 vs 7.5 ± 0.2%, P < 0.02; Fig. 4C). The same pattern was observed in lymph nodes, arguing against alterations in the homing properties of lymphocyte subsets (data not shown). In double transgenic mice with additional impairment of TGFß signalling, the frequency of CD4+CD25+ T-cells was similar to wild type, again suggesting an autocrine effect of TGFß.



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Fig. 4. TGFß1 affects the peripheral CD4+CD25+ T-cell pool. (A) Total splenic cell numbers were assessed at various ages and the relative amount of CD4+ T-cells determined using flow cytometric analysis. Total CD4+ T-cell numbers are depicted relative to cell numbers of wild-type littermates. Reduced CD4+ T-cell numbers were detected in CD2-TGFß1 transgenic animals beginning at the age of 4 weeks with a further decrease by the age of 6 and 8 weeks. These experiments were performed at various ages for lines Q, X and Y yielding similar results. (B) A significant reduction in the number of CD4+ T-cells, but not of CD8+ T-cells or B-cells was seen in the spleens of CD2-TGFß1 transgenic mice at the age of 8 weeks. (C) The frequency of CD4+CD25+ T-cells in the spleen was increased in CD2-TGFß1 single transgenic animals and decreased in single transgenic mice with impaired TGFß-signaling. In double transgenic mice, the frequency was similar to wild type. *P < 0.02.

 
Endogenous TGFß promotes the maintenance of CD4+CD25+ T-cells
Since overexpression of TGFß was found to influence CD4+CD25+ T-cells in an autocrine manner, we were interested whether endogenous levels of TGFß were involved in the development or maintenance of CD4+CD25+ T-cells. We therefore analyzed transgenic mice with impaired TGFß-signalling in T-cells which were previously shown to have a grossly normal thymic T-cell development (20). The frequency and number of CD4+CD25+ cells was unchanged in the thymus as compared to wild-type littermates, but in the spleen a significant reduction in the frequency and number of CD4+CD25+ T-cells was observed (6.2 ± 0.2 vs 7.5 ± 0.2%, P < 0.01; Fig. 4C). These results suggest that endogenous TGFß might not be essential for thymic development of CD4+CD25+ T-cells but might play a role in their maintenance or generation in peripheral lymphoid organs.

TGFß regulates the expression of Foxp3 in vivo
The expression of Foxp3 has been linked to the generation and function of regulatory T-cells (46), but the in vivo regulation of Foxp3 is largely unknown. We therefore analyzed the effect of TGFß on the expression of Foxp3 in the mouse models presented. As compared to wild-type CD4+CD25+ T-cells, the expression of Foxp3 was markedly increased in highly purified CD4+CD25+ T-cells ex vivo analyzed from mice overexpressing active TGFß1. In contrast, a reduced expression of Foxp3 was seen in CD4+CD25+ T-cells isolated from mice with impaired TGFß-signalling (Fig. 5). A low level of Foxp3 expression was seen in CD4+CD25– T-cells without consistent and significant differences between wild-type and either transgenic cells, whereas no expression was detected in B-cells. These data suggest a role for TGFß in the regulation of Foxp3 expression in vivo.



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Fig. 5. TGFß regulates the expression of Foxp3 in vivo. The expression of Foxp3 relative to HPRT expression was analyzed using real time quantitative PCR from ex vivo highly purified T-cell subsets. The experiment was repeated four times for CD2-TGFß1 and three times for CD2-{Delta}kTßRII transgenic mice giving similar results.

 
TGFß1 induces the expression of IL-10 in T-cells
Cytokine production of transgenic CD4+ and CD4+CD25– T-cells overexpressing TGFß1 was analyzed in comparison to wild type after allogenic stimulation or costimulation with anti-CD3/CD28 antibodies. In addition to increased levels of active TGFß1, transgenic CD4+, CD4+CD25– and CD4+CD25+ T-cells secreted more IL-10 after costimulation as compared to wild-type T-cells (for CD4+ see Fig. 6; CD4+CD25– wild type: 413 ± 53 vs transgenic: 974 ± 184 pg/ml; CD4+CD25+ wild type: 6 vs transgenic: 98 pg/ml), whereas in double transgenic mice, IL-10 production was similar to wild-type T-cells (wild type: 1318 ± 201 vs double transgenic: 1393 ± 87 pg/ml). No significant differences were observed for the secretion of IL-2, IL-4, IL-13, IFN{gamma} and TNF{alpha} after costimulation of CD4+ or CD4+CD25– T-cells (for CD4+ see Fig. 6; CD4+CD25–, IL-4: 30 ± 5 vs 43 ± 4; IL-13: 1529 ± 75 vs 1530 ± 117, IFN{gamma}: 12280 ± 1399 vs 10139±637 pg/ml, wild type vs transgenic cells).



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Fig. 6. Overexpression of active TGFß1 induces the expression of IL-10. CD4+ T-cells were isolated from the spleens of CD2-TGFß1 transgenic and wild-type mice. Cytokines were analyzed in the supernatants after 48 h of costimulation using ELISA. CD2-TGFß1 transgenic CD4+ T-cells produced significantly more IL-10 than cells isolated from wild-type littermates (P < 0.02). This experiment included 11 mice analyzed independently at the age of 8 weeks.

 
Increased suppressor function of transgenic CD4+CD25+ T-cells is mediated by TGFß and not IL-10
We were then interested in the suppressive capacity of transgenic T-cells. As compared to wild type, CD2-TGFß1 transgenic CD4+CD25+ T-cells demonstrated a higher overall suppressive capacity when allogenically stimulated in the presence of CD4+CD25– responder cells (Fig. 7A). In order to differentiate between membrane-bound TGFß and soluble cytokines in the suppressive effects observed, we prohibited cell contact between suppressor and responder cells by performing transwell experiments. There was no suppressive capacity in wild-type CD4+CD25+ T-cells in the transwell system. In contrast, suppression in transgenic CD4+CD25+ T-cells was mediated by soluble cytokines (Fig. 7B).



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Fig. 7. The increased suppressive capacity of CD2-TGFß1 transgenic CD4+CD25+ T-cells depends on soluble TGFß1 and not on IL-10. CD4+CD25+ T-cells were isolated from the spleens of CD2-TGFß1 transgenic and wild-type mice and titrated to CD4+CD25– responder cells at different concentrations in the presence of allogenic APC and anti-CD3 mAb. (A) Transgenic CD4+CD25+ T-cells show an increased suppressive capacity as compared to respective cells from wild-type littermates (P < 0.01). These experiments were performed twice for line Y and three times for line Q with similar results. (B) The role of soluble cytokines was analyzed in transwell experiments. CD2-TGFß1 transgenic, but not wild-type cells demonstrate suppressive capacity. These experiments were performed for lines Q and Y with similar results. (C) CD4+CD25– responder cells isolated from CD2-{Delta}kTßRII transgenic mice with impaired TGFß-signalling in T-cells were used in transwell experiments. Suppression by CD2-TGFß1 transgenic CD4+CD25+ T-cells was completely abrogated. Experiments were performed for lines Q and Y with similar results. (D) The role of TGFß in cell contact dependent suppression was analyzed using CD4+CD25– responder cells isolated from CD2-{Delta}kTßRII transgenic mice. No significant difference was detected between CD2-TGFß1 transgenic and wild-type cells. These experiments were performed three times for line Q and repeated for line Y giving similar results.

 
To determine whether soluble TGFß1 or other factors such as IL-10 were responsible for the suppression observed in the transwell experiments, we used CD4+CD25– T-cells isolated from transgenic mice with impaired TGFß-signalling as responder cells. In these transwell experiments, suppression by wild-type or transgenic cells was completely abolished by the block of TGFß-signalling in the responder cell population (Fig. 7C), arguing against a contribution of IL-10 to the suppressive effects observed. In contrast, when cell contact was allowed, wild-type and CD2-TGFß1 transgenic CD4+CD25+ T-cells suppressed responder cells with impaired TGFß-signaling to a similar degree (Fig. 7D), indicating that TGFß might not be the main factor involved in cell contact-dependent suppression.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A great effort has been made to define markers and subpopulations of regulatory T-cells, such as CD4+CD25+ T-cells (1). However, little is known about factors influencing their in vivo generation and maintenance. We here report that active TGFß1 selectively influences the development and function of CD4+CD25+ T-cells in an autocrine manner. The effect of TGFß on the maintenance of peripheral CD4+CD25+ T-cell numbers could be confirmed in mice with impaired TGFß-signaling in T-cells. In addition, TGFß seems to be involved in the regulation of Foxp3 in vivo, a transcription factor recently described to be essential for the development and function of CD4+CD25+ regulatory T-cells (46).

The thymus is able to generate fully developed CD4+CD25+ regulatory T-cells, but factors controlling their generation in the thymus are largely unknown. Our data suggest an autocrine promoting effect of active TGFß1 on the frequency of CD4+CD25+ T-cells in peripheral lymphoid organs and to a lesser extent in the thymus. TGFß1- and Smad3-knockout mice have no obvious defects in thymocyte development at birth (11,12,22,23), but it is difficult to study the development of regulatory T-cells in these mice due to their multifocal inflammatory disease. In addition, in several models of impaired TGFß-signaling in T-cells and thymocytes, no abnormality in thymic T-cell development was observed (20,2426). However, the development of CD4+CD25+ T-cells has not been studied in detail in these mice. We here report a normal thymic development of CD4+CD25+ T-cells in adult mice with impaired TGFß-signaling in T-cells, arguing against a relevant effect of endogenous TGFß on the central development of these cells. However, it cannot be excluded that other TGFß family members such as activins may compensate for the lack of TGFß-signaling as was suggested for BMP-2 and -4 (27).

In peripheral lymphoid organs, the frequency of CD4+CD25+ T-cells was increased by the overexpression of TGFß1 and decreased by the impairment of TGFß-signaling in T-cells. These data suggest that TGFß might play a role in the regulation of the peripheral CD4+CD25+ T-cell pool. In humans, it has been reported that TGFß promotes the maintenance of CD4+CD25+ T-cells (13). Very recently, TGFß was described to convert naive mouse CD4+CD25– T-cells to suppressive CD4+CD25+ T-cells through the induction of Foxp3 (3) and the expression of TGFß in pancreatic islets lead to an increase in CD4+CD25+ T-cell numbers with regulatory property (14). Together these data suggest that there might be different CD4+CD25+ regulatory T-cell populations. Cells generated in the thymus might be less susceptible to the effects of TGFß, whereas TGFß might play a role in the regulation of the peripheral CD4+CD25+ T-cell pool. Further research is necessary to define possible mechanisms underlying these observations.

In addition, TGFß might be important for the in vivo regulation of Foxp3, a transcription factor that has been shown to be essential for the development and function of CD4+CD25+ regulatory T-cells (46). An increased expression of Foxp3 was found in CD4+CD25+ T-cells from mice overexpressing active TGFß1, whereas the expression was reduced in cells with impaired TGFß-signaling. In vitro, TGFß has recently been reported to induce the expression of Foxp3 in mouse CD4+CD25– T-cells (3), but there have been no in vivo data so far.

The mechanism of suppression of CD4+CD25+ T-cells remains controversial. In most in vitro studies, cell contact was required for suppression and this seemed independent of cytokines such as IL-10 or TGFß (28). In addition, functional CD4+CD25+ T-cells have been isolated from neonatal and adolescent TGFß knockout mice and a similar in vitro suppressive capacity to wild type has been reported (9,10). However, membrane-bound TGFß and even latency-associated peptide were implicated in the suppressive function of CD4+CD25+ T-cells (29,30) but this could not be confirmed by others (9). A small but significant effect on suppressor function was reported for soluble TGFß, but not for IL-10, in the analysis of human CD4+CD25+ T-cell clones (31). In the analysis of suppressor function, the in vitro setting might not resemble the in vivo situation closely enough. Recently, a reduced in vivo suppressive capacity was noted in CD4+CD25+ T-cells isolated from TGFß knockout mice (10). In our model, T-cells overexpress active TGFß1 as well as IL-10. However, the increased in vitro suppressive capacity observed in transgenic CD4+CD25+ T-cells could clearly be attributed to the action of TGFß and not IL-10 using responder cells with impaired TGFß-signaling. Experiments are under way to clarify the in vivo role of TGFß and IL-10 in our model.

The expression of IL-10 was induced by overexpression of active TGFß1 in T-cells. This might be due to a binding site for Smad4 on the IL-10 promoter (32). Other Th-2 cytokines were not induced, arguing against an interaction of Smad3 with GATA-3, the main transcription factor for Th2 development in our model (33).

In summary, the results presented suggest TGFß as a factor influencing the CD4+CD25+ T-cell pool in peripheral lymphoid organs and the expression of Foxp3 in vivo. Furthermore, a model is presented to dissect the in vivo effects of TGFß and IL-10 on inflammation and fibrogenesis in models of autoimmune disease.


    Acknowledgements
 
This study was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 548 and by MAIFOR, Faculty of Medicine, University of Mainz, and the Boehringer Ingelheim Foundation. We thank Marina Snetkova for technical assistance, Helmut Jonuleit for helpful discussions and Steffen Schmitt for assistance in cell sorting.


    Notes
 
The first two authors have contributed equally to this work.

Transmitting editor: A. Radbruch

Received 26 March 2004, accepted 14 June 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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