Transforming growth factor-ß inhibits human antigen-specific CD4+ T cell proliferation without modulating the cytokine response

Machteld M. Tiemessen1, Steffen Kunzmann2, Carsten B. Schmidt-Weber2, Johan Garssen3, Carla A. F. M. Bruijnzeel-Koomen1, Edward F. Knol1 and Els Van Hoffen1

1 Department of Dermatology/Allergology, University Medical Center, PO Box 85.500, 3508 GA Utrecht, The Netherlands 2 Swiss Institute of Allergy and Asthma Research (SIAF), CH-7270 Davos, Switzerland 3 Numico Research, PO Box 7005, 6700 CA Wageningen, The Netherlands

Correspondence to: M. Tiemessen; E-mail: m.tiemessen{at}azu.nl
Transmitting editor: S. L. Swain


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transforming growth factor (TGF)-ß has been demonstrated to play a key role in the regulation of the immune response, mainly by its suppressive function towards cells of the immune system. In humans, the effect of TGF-ß on antigen-specific established memory T cells has not been investigated yet. In this study antigen-specific CD4+ T cell clones (TCC) were used to determine the effect of TGF-ß on antigen-specific proliferation, the activation status of the T cells and their cytokine production. This study demonstrates that TGF-ß is an adequate suppressor of antigen-specific T cell proliferation, by reducing the cell-cycle rate rather than induction of apoptosis. Addition of TGF-ß resulted in increased CD69 expression and decreased CD25 expression on T cells, indicating that TGF-ß is able to modulate the activation status of in vivo differentiated T cells. On the contrary, the antigen-specific cytokine production was not affected by TGF-ß. Although TGF-ß was suppressive towards the majority of the T cells, insensitivity of a few TCC towards TGF-ß was also observed. This could not be correlated to differential expression of TGF-ß signaling molecules such as Smad3, Smad7, SARA (Smad anchor for receptor activation) and Hgs (hepatocyte growth factor-regulated tyrosine kinase substrate). In summary, TGF-ß has a pronounced inhibitory effect on antigen-specific T cell proliferation without modulating their cytokine production.

Keywords: CD25, IL-10, immunosuppression, Smad protein, tolerance


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
A natural state of tolerance is maintained by multiple mechanisms, such as clonal deletion, anergy and the presence of regulatory T cells (14). Regulatory T cells, also called Tr or Th3 cells, mediate their suppressive effect mainly by secretion of IL-10 and transforming growth factor (TGF)-ß (5,6). Many investigators have demonstrated the importance of the immunosuppressive cytokine TGF-ß in the homeostasis of T cell regulation. TGF-ß1 knockout mice develop severe multifocal inflammatory responses and die at 3–4 weeks of age (7). Evidence for a positive role of TGF-ß in tolerance induction is demonstrated in studies where administration of TGF-ß resulted in an improved clinical course in mice with experimental allergic encephalomyelitis and in delayed heart allograft reactions in rodents (8,9).

Although TGF-ß can exert its effect on many different cell types, CD4+ T cells may be the main target since anti-CD4 antibodies are protective in TGF-ß1 knockout mice (7). TGF-ß exerts its effect on T cells via binding to the TGF-ßRII, which recruits and phosphorylates TGF-ßRI. TGF-ßRI activates the ligand-specific Smad proteins (Smad2 and Smad3). Smad2 and Smad3 are kept in the cytoplasm where they are bound to the proteins SARA (Smad anchor for receptor activation) and Hgs (hepatocyte growth factor-regulated tyrosine kinase substrate) (10). Upon activation, Smad2 and Smad3 are released from SARA and Hgs, and are translocated to the nucleus where they activate TGF-ß target genes. Recent studies showed that SARA and Hgs can act as attenuators of TGF-ß responsiveness (11). The inhibitory Smad protein, Smad7, prevents activation and/or nuclear translocation of Smad2 and Smad3 (12). Disruption of TGF-ß signaling in T cells was observed in mouse models after induction of Smad7 overexpression. This resulted in the development of a hyper-reactive T cell response and antigen-induced airway inflammation after antigen inhalation (13). Moreover, in humans it was found that, in patients suffering from inflammatory bowel disease, Smad7 was up-regulated in mucosal T cells compared to T cells isolated from control subjects (14). These studies show that the effect of TGF-ß was limited due to the high Smad7 expression in antigen-specific T cells, which resulted in the development of inflammation.

Diminished expression of TGF-ßRII may also lead to unresponsiveness of T cells towards TGF-ß. Studies with mice expressing a dominant-negative form of TGF-ßRII showed a severe inflammation in the gut and lung, both sites where the immune system is constantly triggered by environmental antigens (1517). This demonstrates that the expression of TGF-ßRII is necessary in maintaining tolerance at mucosal sites. In human T cells it was recently shown that the expression of TGF-ßRII could be modulated via IL-10 (18). IL-10 could enhance the expression of TGF-ßRII, thereby increasing the suppressive effect of TGF-ß. Thus, the responsiveness of T cells towards TGF-ß depends on the differential expression of the Smad proteins as well as TGF-ßRII at the time of antigen-specific stimulation.

The effects of TGF-ß on established antigen-specific memory T cells have only been studied in murine models. TGF-ß was shown to be suppressive towards murine Th1 memory cells and to stimulate murine Th2 cells (19). In humans, the effect of TGF-ß was shown after mitogenic stimulation of T cells, isolated from healthy donors (20,21). However, the effect of TGF-ß on human antigen-specific T cell responses has not been investigated yet. Therefore, in this study cow’s milk-specific T cell clones (TCC) derived from in vivo differentiated peripheral blood T cells were used to investigate the effect of TGF-ß on the T cell itself. These TCC were maintained in vitro by repeated stimulation in an antigen-specific manner. The proliferative capacity and cytokine profiles of these TCC in the presence or absence of TGF-ß was investigated. To examine the mechanism via which TGF-ß could exert its function, analysis of the percentage of apoptotic cells, number of cell divisions and expression of T cell-surface markers was performed. In addition, the modulating effect of IL-10 on TGF-ß induced inhibition of T cell proliferation was examined, as well as the correlation between sensitivity of T cells for TGF-ß and mRNA expression of TGF-ß signaling molecules.

Using an antigen-specific clonal CD4+ T cell model this report demonstrates that TGF-ß is an effective suppressor of antigen-specific proliferation without affecting the cytokine production of the in vivo differentiated T cells. T cells derived from different donors (allergic and non-allergic) as well as T cells with different cytokine profiles were equally inhibited by TGF-ß in their antigen-specific proliferation. This model suggests a possible modulating role of TGF-ß in T cell-mediated disorders to suppress the T cell response.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Cow’s milk-specific TCC
Peripheral blood mononuclear cells (PBMC) were isolated from venous blood of non-allergic children (mean age 4.5 years) and of age-matched children with a diagnosed cow’s milk allergy (CMA), after informed consent was obtained. The study was approved by the Medical Ethical Committee of the University Medical Center, Utrecht. The diagnosis for CMA was confirmed by positive cow’s milk-specific IgE levels as determined by the CAP system FEIA (Pharmacia Diagnostics, Uppsala, Sweden), positive skin prick test and a positive double-blind placebo-controlled food challenge for cow’s milk. The non-allergic children had normal total IgE levels and had a negative family history for atopy. Cow’s milk-specific TCC were established from blood as described previously (22). The antigen used for T cell stimulation was a mix of cow’s milk proteins (CMP), which consisted of equal quantities of total casein, {alpha}-lactalbumin and ß-lactoglobulin (50 µg/ml) (gift from Dr R. Floris, NIZO Food Research, Ede, The Netherlands). In short, PBMC were cultured using an antigen-specific culturing system with irradiated autologous Epstein–Barr virus-transformed B cells (EBV-B cells), pre-incubated overnight with CMP, as antigen-presenting cells (APC). If these cultures indicated a high CMP-specific T cell proliferation in the lymphocyte stimulation test (LST), T cells were cloned by limiting dilution. Established TCC were tested in LST to determine specificity for the various CMP and were re-stimulated every 14 days. The antigen specificity and IL-4:IFN-{gamma} ratio of the TCC used in this report are presented in Table 1.


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Table 1. Antigen-specificity and IL-4:IFN-{gamma} ratio of CMP-specific TCC derived from peripheral blood of non-allergic and allergic children
 
Cell culture media
EBV-B cells were cultured in RPMI 1640 (Gibco, Grand Island, NY) supplemented with 10% FCS. Established TCC were maintained in IMDM (Gibco) supplemented with 2% pooled human AB serum, 5% Yssel’s medium (23), and 50 U/ml IL-2 and IL-4 (a kind gift from Novartis Research Institute, Vienna, Austria). For the experiments in the presence and absence of TGF-ß, IL-2 and IL-4 were omitted from the medium. All media were supplemented with penicillin (100 IU/ml), streptomycin (100 mg/ml) and glutamine (1 mM) (Gibco).

LST and cytokine production
The LST were performed in triplicate in 96-well U-bottom plates (Greiner, Frickenhausen, Germany). Each well contained 4 x 104 T cells and 4 x 104 irradiated (55 Gy) autologous EBV-B cells, pre-incubated overnight with CMP, as APC. EBV-B cells, incubated without antigen, were used as negative controls. The various combinations of TGF-ß1 (1 ng/ml), IL-10 (25 ng/ml) and latency associated peptide (LAP) (800 ng/ml) were added directly to the co-culture. Recombinant human IL-10, TGF-ß1 and LAP were purchased from R & D systems (Abingdon, UK).

Proliferation at 24, 48, 72 and 96 h was measured by [3H]thymidine incorporation [1 µCi/well (Amersham, Little Chalfont, UK)], which was added during the last 18 h of culturing. Proliferative responses are expressed as the mean [3H]thymidine incorporation (c.p.m.) of triplicate wells ± SEM or as indicated. Prior to addition of [3H]thymidine, supernatant was collected from the triplicate wells and stored at –20°C. Cytokine production was measured by ELISA according to the manufacturer’s recommendations (for IL-4, IL-10 and IFN-{gamma}: Sanquin, Amsterdam, The Netherlands; and for IL-2: Diaclone, Besançon, France). The detection limit was 0.6 pg/ml for IL-4, 1.2 pg/ml for IL-10, 2 pg/ml for IFN-{gamma} and 15 pg/ml for IL-2.

Flow cytometry
To examine the effect of TGF-ß on apoptosis, flow cytometry was performed with phycoerythrin (PE)-conjugated Annexin-V according to the manufacturer’s recommendations (BD Biosciences, San Jose, CA). To examine the effect of TGF-ß on T cell activation markers, antibodies to CD4, CD25 and CD69 (all PE-conjugated), and FITC-conjugated CD28 were used for flow cytometry (BD Biosciences). Expression levels of CD25, CD28 and CD69 by each TCC were measured as mean fluorescence intensity (MFI) in the presence or absence of TGF-ß. The effect of TGF-ß on the expression of the co-stimulatory molecules on the B cells was measured with FITC-conjugated CD80 and CD86 antibodies (BD Biosciences). The expression levels of CD80 and CD86 were measured as MFI after culturing of the EBV-B cells in the presence or absence of TGF-ß. All the analysis was performed on a Becton Dickinson FACScan with WinMDI software.

Carboxy-fluorescein diacetate succinimidyl ester (CFSE) proliferation assay
To analyze the number of cell divisions, CFSE was used at a final concentration of 0.5 µM (Molecular Probes, Eugene, OR). T cells were resuspended at a concentration of 107 cells/ml in PBS and labeled with CFSE for 10 min at 37°C. Unbound CFSE was quenched by washing the cells twice in culture medium containing 10% FCS. Before co-culture, the labeled T cells were left in medium for 2 h at 37°C and washed once more to prevent non-specific labeling of the EBV-B cells. T cells were stimulated in a 1:1 ratio with the pre-incubated EBV-B cells (106:106), and harvested after 72, 96 and 120 h of co-culture. For analysis, the cells were washed, stained with PE-conjugated CD4 and analyzed with a FACScan (BD Biosciences). The number of cell divisions was correlated with the decrease in MFI.

RT-PCR
Total RNA was isolated from several cow’s milk-specific TCC using the RNeasy mini kit (Qiagen, Hilden, Germany) according to the manufacturers protocol. The RNA was eluted in 40 µl water and subjected to reverse transcription. Approximately 5 µg total RNA (12 µl) was reverse transcribed by addition of 500 µg/ml oligo(dT)12 primer (Roche, Basel, Switzerland), RNase Inhibitor (Roche) (10 U/µl), dNTP (5 mM each dNTP; Qiagen) and Omniscript transcriptase (Qiagen) (0.2 U/µl) for 1 h at 37°C. The cDNA was denatured at 90°C for 5 min and used for PCR amplification. All PCR reactions were performed with Taq polymerase (Qiagen). RT-PCR was performed using the following primers: GAPDH forward 5'-CTT CGC TCT CTG CTC CTC CT-3', GAPDH reverse 5'-GCT GAT GAT CTT GAG GCT GTT G-3', IL-10 forward 5'-ATG CCC CAA GCT GAG AAC CAA GAC-3', IL-10 reverse 5'-CCC AGA GCC CCA GAT CCG ATT TTG-3', TGF-ßRII forward 5'-TGA CCC CAA GCT CCC CTA CCA TGA-3', TGF-ßRII reverse 5'-TGA TGT CAG AGC GGT CAT CTT CCA-3', Smad3 forward 5'-CGT CCA TCC TGC CTT TCA CTC-3', Smad3 reverse 5'TGG CAC CAA CAC AGG AGG TAG-3', Smad7 forward 5'-AAG TCA AGA GGC TGT GTT GC-3', Smad7 reverse 5'-TCT CGT AGT CGA AAG CCT TGA TGG AGA AA-3', SARA forward 5'-AAA ATG CTG TTG CAG AAG ACC A-3', SARA reverse 5'-TTT AAT TGT GAA GGG GAA CAG ACA-3', Hgs forward 5'-GCG TCT CCT AGA CAA GGC GA-3', Hgs reverse 5'-GTC ATC ACC CCG AAC TGC AC-3'. The primers for IL-10 and TGF-ßRII were used as described before (18,24). PCR products were loaded next to a standard (1kbplus; Life Technologies) and analyzed on 1% agarose gels. Image analysis was performed using a fluorescent imager analyzer FLA 3000 (Fuji, Dielsdorf, Switzerland) and quantified using the AIDA software (Raytest, Urdorf, Switzerland). The expression of IL-10, TGF-ßRII, Smad3, Smad7, SARA and Hgs was quantified as percentages compared to GAPDH expression.

Statistical analysis
Statistical analysis of the effect of LAP on TGF-ß-induced inhibition was performed with a Mann–Whitney U-test. Non-parametric paired analysis (Wilcoxon test) was performed to examine differences in the expression of cell-surface markers (CD25, CD28 and CD69), distribution over the cell cycles induced by TGF-ß and differences in the expression of TGF-ß-signaling molecules 96 h after stimulation. Differences associated with P values of less than 0.05 were considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TGF-ß inhibits antigen-specific T cell proliferation
To determine the optimal effective dose of TGF-ß administered, the effect of several concentrations TGF-ß on antigen-specific T cell proliferation was tested (Fig. 1A). The optimal dose for suppression appeared to be 1 ng/ml, which was used throughout the following experiments. TGF-ß-induced inhibition of antigen-specific proliferation was most pronounced at 96 h after stimulation, therefore the results from 96 h are shown unless the results on earlier time points deviate from the result at 96 h (Fig. 1B). The effectiveness of tolerance induction has been demonstrated to be dependent on the dose of the antigen administered. Therefore it was examined whether TGF-ß was able to suppress antigen-specific T cell proliferation in TCC stimulated with low (2 µg/ml) and high (50 µg/ml) doses of antigen. Since the low dose of antigen (2 µg/ml) results in an average reduction in proliferation of 55% (data not shown), this dose was considered to be a partial stimulation. As is shown in Fig. 1(C), the suppressive effect of TGF-ß on antigen-specific proliferation was comparable between partial and full antigen stimulation of T cells. Addition of a TGF-ß neutralizing peptide (LAP) in the culture system significantly reversed the inhibitory effect of TGF-ß (Fig. 1D, Mann–Whitney U-test, *P < 0.01). Inhibition of antigen-specific proliferation of TCC by TGF-ß derived from both non-allergic as well as allergic donors was not significantly different (mean percentage proliferation in the presence of 1 ng/ml TGF-ß; non-allergic 38.5 ± 7.0% and allergic 46.1 ± 10.6%). Only two out of 17 TCC tested in an antigen-specific LST were not inhibited by TGF-ß (Fig. 1E).



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Fig. 1. TGF-ß inhibits antigen-specific T cell proliferation independent of the antigen dosage used and its effect can be reversed by LAP. (A) CMP-specific TCC were stimulated with irradiated autologous EBV-B cells, pre-incubated with 50 µg/ml CMP and cultured in the presence of the indicated concentrations of TGF-ß for 96 h. [3H]Thymidine was added during the last 18 h of culture. Results are expressed as proliferation (mean [3H]thymidine incorporation ± SEM). (B) CMP-specific TCC were stimulated with irradiated autologous EBV-B cells, pre-incubated with 50 µg/ml CMP, and cultured in the presence of TGF-ß (1 ng/ml) for 24, 48, 72 and 96 h. The results are expressed as mean proliferation (± SEM) compared to CMP-induced proliferation (100%). (C) CMP were used as antigen at a concentration of 2 or 50 µg/ml. TCC were stimulated in the presence or absence of TGF-ß (1 ng/ml). The results (mean [3H]thymidine incorporation ± SEM) are shown for one representative TCC. (D) Addition of LAP (800 ng/ml) together with TGF-ß during stimulation significantly reverses the inhibitory effect of TGF-ß at 96 h (Mann–Whitney U-test, *P < 0.01). The graph shows the results of at least two independent experiments per TCC (n = 5). Results are expressed as mean proliferation (± SEM) compared to CMP-induced proliferation (100%). (E) CMP-specific TCC of both non-allergic and allergic donors were tested for their proliferation at 96 h in the presence of TGF-ß (1 ng/ml). Proliferation in the presence of TGF-ß is expressed as percentage compared to CMP-induced proliferation (100%).

 
TGF-ß inhibits cell cycle progression of antigen-specific T cells
To investigate the inhibitory mechanisms of TGF-ß on the antigen-specific T cell proliferation, the percentage of apoptotic cells was examined. Annexin-V+ cells were determined in the presence and absence of TGF-ß at 24, 48, 72 and 96 h after stimulation. Addition of TGF-ß appeared to have no significant effect on the percentage of Annexin V+ cells at all time points (Fig. 2A).



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Fig. 2. Effect of TGF-ß on the amount of apoptotic cells and number of cell divisions. (A) Antigen-specific T cells were stimulated in the absence (open symbols) or presence of TGF-ß (closed symbols). The graph shows the percentage of Annexin-V+ cells (mean ± SEM) of five different TCC from at least two independent experiments for each TCC. (B) The number of cell divisions was analyzed at 96 h after stimulation of CFSE-labeled T cells in either the absence or presence of TGF-ß. The percentage of cells in each cell division was calculated by setting arbitrary boundaries for each cell division (numbered 1–5). The CFSE intensity at the time of labeling is represented as the white peak in both panels. The results are a representative example of three independent experiments for one TCC of which the inhibition of CMP-specific proliferation by TGF-ß was 70%.

 
In contrast, the number of cell divisions, measured as a decrease in CFSE fluorescence, was clearly diminished at 96 h after antigen-specific stimulation in the presence of TGF-ß. Figure 2(B) shows that the T cells, which were stimulated in the absence of TGF-ß, undergo more cell divisions compared to T cells stimulated in the presence of TGF-ß (MFI = 120.38 versus 170.45 respectively, P < 0.05). The amount of cells in each cell division demonstrates that cells, cultured in the presence of TGF-ß, are more abundant in the first two cycles compared to cells cultured without TGF-ß (29 and 37% versus 15 and 29%). In contrast, T cells cultured without TGF-ß are more abundant in the later two cell cycles (21 and 13% versus 9 and 5% of the cells cultured with TGF-ß).

TGF-ß has a differential effect on T cell activation and co-stimulation markers (CD25, CD69 and CD28), but does not influence the expression of CD80 and CD86 on APC
In concordance with the demonstrated inhibition of antigen-specific proliferation, TGF-ß significantly decreased the expression level of the IL-2 receptor, CD25 (P < 0.05). In contrast, the expression level of CD69 was significantly up-regulated in the presence of TGF-ß (Fig. 3A and B). The effect of TGF-ß on the expression of CD25 and CD69 was present at 24, 48, 72 and 96 h after antigen-specific stimulation (data not shown). To examine whether TGF-ß directly affected the CD25 expression or indirectly via a down-regulation of IL-2 production, IL-2 production in the culture supernatant was measured using ELISA. Although the amounts of IL-2 production were very low, addition of TGF-ß did not affect the production of IL-2 by T cells (mean production ± SEM at 24 h: no TGF: 221 ± 111 pg/ml, TGF: 212 ± 101 pg/ml; at 96 h: no TGF: 22 ± 2.6 pg/ml, TGF: 25 ± 2.8 pg/ml). To analyze the possibility that the effect of TGF-ß was mediated via changes in co-stimulatory molecules, the expression levels of CD28 on T cells, and CD80 and CD86 on the APC (EBV-B cells), were measured in the presence and absence of TGF-ß. TGF-ß did not affect the expression levels of CD80 and CD86 on the EBV-B cells. In contrast it did decrease the expression level of CD28 on T cells (P < 0.05) (Fig. 3C).



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Fig. 3. TGF-ß has a differential effect on the T cell markers CD25, CD69 and CD28, and no effect on the B cell markers CD80 and CD86. TCC were stimulated with irradiated autologous EBV-B cells, which were pre-incubated with 50 µg/ml CMP. Flow cytometry was performed 96 h after stimulation of the cells in the presence or absence of TGF-ß (1 ng/ml). (A) The MFI for CD25 and CD69 in the presence (black line) and absence of TGF-ß (grey line) is shown for one representative TCC (TCC 9.12), and as (B) independent experiments for TCC from non-allergic donors (circles) and TCC from allergic donors (squares). TGF-ß significantly decreased the expression of CD25 and CD28, and significantly increased the expression of CD69 (Wilcoxon test, *P < 0.05). (C) The expression of CD80 and CD86 on EBV-B cells and of CD28 on T cells was measured in the absence (open bars) and presence (black bars) of TGF-ß. The results are expressed as the MFI (mean ± SEM) of three independent experiments. The expression of CD28 was significantly decreased in the presence of TGF-ß (Wilcoxon test, *P < 0.05).

 
IL-10 has no enhancing effect on TGF-ß induced inhibition of antigen-specific T cell proliferation
In Fig. 4(A) the effect of IL-10 and/or TGF-ß on T cell proliferation is shown for three representative TCC. An additive inhibitory effect of TGF-ß and IL-10 was only observed in TCC that were inhibited by IL-10 itself (TCC 9.6). The more pronounced inhibition found in TCC 9.6 seemed to be due to a cumulative rather than a synergistic inhibitory effect on proliferation of IL-10 and TGF-ß. Most TCC were not inhibited in their antigen-specific proliferation by IL-10 (15 out of 17 tested). In these TCC, IL-10 did not enhance the inhibitory effect of TGF-ß. It was examined whether addition of IL-10 could enhance the expression of TGF-ßRII on antigen-specific T cells. Figure 4(B) demonstrates that mRNA levels of TGF-ßRII of a representative CMP-specific TCC before and 96 h after antigen-specific stimulation with CMP were not affected in the presence of TGF-ß or IL-10.



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Fig. 4. IL-10 does not enhance the sensitivity of antigen-specific TCC for TGF-ß. (A) Of all the TCC tested, three representatives are shown. The TCC were stimulated with irradiated autologous EBV-B cells, pre-incubated with CMP, either in the presence of TGF-ß (1 ng/ml) (open bars) or IL-10 (25 ng/ml) (grey bars) or in the presence of both cytokines (black bars), and cultured for 96 h. Proliferation was measured by [3H]thymidine incorporation during the last 18 h of incubation. Results are expressed as percentages of proliferation (mean ± SEM) relative to CMP-specific proliferation (set at 100%) from three independent experiments. (B) The mRNA expression of different TCC for TGF-ßRII was analyzed prior to stimulation (lane 1) and 96 h after CMP-specific stimulation (lane 2) in the presence of TGF-ß or IL-10 (lane 3 and 4 respectively). The results are shown for one representative TCC.

 
TGF-ß does not affect cytokine production of antigen-specific T cells
To analyze the effect of TGF-ß on cytokine production, supernatants were analyzed at 24, 48, 72 and 96 h of culturing with and without TGF-ß. One representative TCC is shown to demonstrate that the production of cytokines during culture decreased over time (Fig. 5A). However, no effect on cytokine production was observed by TGF-ß during the 96 h of stimulation. In Fig. 5(B), the production of IL-4 and IFN-{gamma} as well as the ratio of IL-4:IFN-{gamma} is shown for all TCC at 96 h after stimulation in the presence and absence of TGF-ß. There is no significant effect of TGF-ß on the production of either IL-4 or IFN-{gamma}. Also, the production of IL-10 measured in the supernatant was not altered in the presence of TGF-ß (Fig. 5C). There was no correlation between the inhibitory effect of TGF-ß on the antigen-specific proliferation and the cytokine production observed.



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Fig. 5. TGF-ß does not affect the antigen-specific cytokine production nor affect the IL-4:IFN-{gamma} ratio by antigen-specific T cells. (A) IL-4, IFN-{gamma} and IL-10 production was measured in culture supernatants 24, 48, 72 and 96 h after antigen-specific stimulation. The results show the production of each cytokine (mean ± SEM, pg/ml) in the presence and absence of TGF-ß of three independent experiments of one representative TCC. (B) The production of IL-4 and IFN-{gamma} (pg/ml), the IL-4:IFN-{gamma} ratio, and (C) the production of IL-10 are shown after antigen stimulation alone or in the presence of TGF-ß at 96 h. Each dot represents the mean of an individual TCC.

 
mRNA expression of TGF-ß signaling molecules is not related to susceptibility for TGF-ß
Two out of the 17 tested TCC were found to be unresponsive towards TGF-ß-induced inhibition of proliferation. It was examined whether the decreased sensitivity of these TCC (TCC 9.7 and 2.2) for TGF-ß was a result of an increased Smad7 expression (inhibitory Smad protein) or a decreased Smad3 expression (stimulatory Smad protein). Moreover, since it was recently demonstrated that a high expression of SARA and Hgs reduced the sensitivity of T cells towards TGF-ß (11), the mRNA levels of SARA and Hgs from TGF-ß-sensitive and -insensitive TCC were also analyzed. The mRNA expression of Smad3, Smad7, SARA and Hgs of several TCC was analyzed prior to stimulation, and showed no correlation with the sensitivity of TCC towards TGF-ß (Fig. 6A). Antigen-specific stimulation of TCC in the presence of TGF-ß resulted in a significantly higher mRNA expression of SARA compared to TCC stimulated in the absence of TGF-ß (Fig. 6B). Expression of Hgs also tended to be higher in the presence of TGF-ß (not significantly, P = 0.08). The expression levels of Smad3 and Smad7 were not significantly different in the presence of TGF-ß.



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Fig. 6. Smad3, Smad7, SARA and Hgs mRNA levels do not differ between TGF-ß-sensitive and -insensitive TCC, whereas the expression of SARA is significantly higher after stimulation in the presence of TGF-ß. (A) The mRNA expression for Smad3, Smad7, SARA and Hgs was analyzed in two insensitive TCC (black bars) and three sensitive TCC (open bars) prior to stimulation. (B) The mRNA expression of the four different TGF-ß signaling molecules was also analyzed 96 h after stimulation in the presence of absence of TGF-ß. The same two insensitive TCC (circles) and three sensitive TCC (squares) were analyzed. The mRNA expression of SARA was significantly higher in the presence of TGF-ß (Wilcoxon, *P < 0.05). The results are expressed as mRNA expression (mean percentage ± SEM) relative to GAPDH expression (set at 100%).

 

    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TGF-ß is an immunosuppressive cytokine, which has an important role in the maintenance of tolerance to self-antigens and environmental antigens (25). Disruption of TGF-ß signaling in T cells or a diminished TGF-ß production is involved in the development of several immune disorders (1315,26). In murine, as well as human, disease models it was shown that induction of peripheral tolerance can be achieved via an enhancement of TGF-ß production by CD4+ T cells (2732). To characterize TGF-ß as a potential therapeutic agent in human disease models it is necessary to evaluate the effect of TGF-ß on differentiated human T cells.

To study the effect of TGF-ß on individual CD4+ T cells, an antigen-specific model of cow’s milk-specific TCC was used. This model provides the opportunity to analyze the effect of TGF-ß in homogeneous T cell populations that are fully antigen-specific, without the disturbance of non-specific T cell reactivity. It was demonstrated that the suppressive effect of TGF-ß is independent of the amount of antigen used and does not discriminate between T cells derived from non-allergic or allergic donors. The inhibition of the antigen-specific proliferation by TGF-ß was not correlated with an increase in apoptosis (Fig 2). In a study with mitogen-stimulated human PBMC it was demonstrated that TGF-ß could have a protective effect on apoptosis (33). This discrepancy could be explained by the use of mitogenic stimulation versus antigen-specific stimulation. However, another study with antigen-specific murine effector CD4+ T cells also showed the protective effect of TGF-ß on apoptosis (34). In this study, antigen-specific Th2 effector cells were generated from naive CD4+ T cells by polarization in vitro with the usage of IL-4 and anti-IFN-{gamma}. These effector T cells were generated in 4 days and cultured for short periods (7 days). The TCC used in our study were polarized and differentiated in vivo, and maintained in culture for longer periods (>30 days). The variation in generation and polarization may result in different stages of T cell differentiation. This may suggest that the effect of TGF-ß on T cell apoptosis is associated with the differentiation status of the T cells. The present study is the first to describe that, in an antigen-specific system where TGF-ß clearly inhibits T cell proliferation, a pronounced difference is observed in the distribution of cells over the different cell divisions between T cells cultured with and without TGF-ß (Fig 2). TGF-ß has been shown in other cell types to inhibit proliferation via an up-regulation of cell-cycle inhibitors such as p21 or p27 (35,36). The same mechanism may play a role in the TGF-ß induced suppression of an antigen-specific T cell response. Together, our results suggest that TGF-ß mainly exerts its suppressive function not by induction of apoptosis, but by reducing the cell-cycle rate of antigen-specific T cells.

In addition to a remarkable inhibitory effect on antigen-specific T cell proliferation, TGF-ß also modulated T cell activation markers. After 96 h, a significant decrease in the expression of CD25 (IL-2 receptor) and an increase in the expression of CD69 (early activation marker) could be demonstrated (Fig. 3). Since it was previously suggested that a decrease in CD25 expression might be the direct result of a decrease in IL-2 production (37), the levels of IL-2 in the supernatant were also measured. Although the levels of IL-2 were low, the production of IL-2 was not diminished in the presence of TGF-ß, indicating that the decrease in CD25 expression was a direct effect of TGF-ß on the T cell and not indirectly via influence of IL-2 levels. This is in agreement with a previous study by Kehrl et al., who described that CD25 expression was decreased in the presence of TGF-ß (20). In contrast, others showed that the expression levels of CD25 and CD69 were not affected by TGF-ß in T cells after mitogenic stimulation (38). However, that study also showed a promoting effect of TGF-ß on mitogen-induced T cell proliferation. A possible explanation for the discrepancy between their study and the presented data in our report might be the use of mitogenic stimulation instead of antigen-specific stimulation and the use of PBMCs instead of established TCC.

A recent study showed that induction of tolerance in mice, marked by less responsiveness of antigen-specific T cells at 96 h, was preceded by the activation of antigen-specific T cells, which was determined by an enhanced CD69 expression at 6 h (39). Surprisingly, the expression of CD69 in our study was slightly enhanced by TGF-ß even at 96 h. This may indicate that the enhancement in CD69 expression demonstrated by Sun et al. (39) was also induced by TGF-ß, suggesting a role for TGF-ß in tolerizing antigen-specific T cells.

The effect of TGF-ß on antigen-specific T cell proliferation and activation might be mediated via the APC, e.g. by affecting the expression of the co-stimulatory molecules (40). However, the expression level of CD80 and CD86 on the EBV-B cells was not decreased by TGF-ß, indicating that TGF-ß exerts its function on the T cell itself during the antigen presentation by the APC (Fig 3B). Moreover, TCC stimulated with mitogen in the absence of APC were equally suppressed by TGF-ß (unpublished observation). A direct effect on the T cell was further confirmed by the observation that the expression of CD28 on the T cells was decreased by TGF-ß. This suggests that the antigen-specific T cells are rendered less responsive towards stimulation by APC, via a direct effect of TGF-ß on the T cell.

IL-10 and TGF-ß both have been demonstrated to be major cytokines in controlling the process of peripheral tolerance. Both cytokines are well known for their suppressive effect on T cells. However, in this study IL-10 only slightly decreased the antigen-specific T cell response in a few TCC. It was suggested before by Cottrez et al. (18) that IL-10 might even enhance the suppressive effect of TGF-ß via up-regulation of the TGFßRII. In this report, IL-10 did not enhance the suppression of proliferation by TGF-ß and the mRNA expression of TGFßRII seemed not elevated in the presence of IL-10.

In mice and humans, sensitivity of antigen-specific T cells towards TGF-ß was recently shown to depend on the expression of the inhibitory Smad7 protein (13,14). Both studies demonstrate that diminished TGF-ß signaling in cells with a high Smad7 expression results in inflammation. The present study showed that, prior to and after stimulation, the TCC that are insensitive towards TGF-ß do not contain significantly higher levels of Smad7 mRNA compared to TGF-ß-sensitive TCC. In addition, for other TGF-ß signaling molecules (Smad3, SARA and Hgs), no differences were also observed between sensitive and insensitive TCC prior to or after stimulation (Fig 6A and B). However, this does not exclude differences in protein phosphorylation and/or translocation or differences in protein levels of TGF-ß signaling molecules between sensitive and insensitive TCC. Addition of TGF-ß during stimulation resulted in a significantly higher mRNA expression of SARA compared to T cells stimulated without TGF-ß. A trend towards higher expression of Hgs was observed in the presence of TGF-ß. It was recently demonstrated that high levels of SARA and Hgs are correlated with a reduced sensitivity of T cells towards TGF-ß (11). This may indicate that the high expression of SARA and Hgs after stimulation in the presence of TGF-ß leads to decreased sensitivity of TCC towards TGF-ß, suggesting a possible negative feedback mechanism in TGF-ß signaling.

It was demonstrated in the present paper that TGF-ß is a good candidate to suppress antigen-specific CD4+ T cell proliferation without altering the cytokine production of these T cells. Although a decrease in cytokine production was observed over time, which is most likely due to consumption by the T cells, TGF-ß was not able to induce a decrease or increase in the cytokines measured. This indicates that a shift from a Th1 to a Th2 profile or vice versa is not accomplished in vitro. Also, an increase in IL-10 production could not be achieved via the addition of TGF-ß, suggesting that TGF-ß will not induce differentiated Th1 or Th2 cells to adapt a more suppressive phenotype. Several investigators have indicated a possible beneficial role for TGF-ß in treatment of patients with autoimmune diseases, such as systemic lupus erythematosus, reduction in acute graft versus host disease or other chronic inflammatory diseases (2729). In allergic diseases, such as asthma, beneficial effects of TGF-ß were demonstrated in several mouse models (30,31,41). The results of the present study demonstrate that TGF-ß is a potent suppressor of the antigen-specific T cell response and does not modulate the cytokine production of an in vivo differentiated T cell in an in vitro system. T cells derived from both non-allergic as well as allergic donors are equally suppressed in their antigen-specific proliferation by TGF-ß, irrespective of the cytokine profile of the T cells. This suggests that TGF-ß might not only be beneficial in allergy, but may be able to suppress T cell responses in both Th1- and Th2-skewed chronic inflammatory diseases. An enhancement of the TGF-ß production in vivo at sites where the disease-related antigens are encountered might be of importance in the suppression of the local immune response, inducing a more tolerogenic environment to prevent a detrimental response against innocuous antigens.


    Acknowledgements
 
The authors wish to thank Dr R. Floris (NIZO Food Research, Ede, The Netherlands) for the purified cow’s milk proteins and the Bloodbank Utrecht for providing us with human AB serum.


    Abbreviations
 
APC—antigen-presenting cell

CFSE—carboxy-fluorescein diacetate succinimidyl ester

CMA—cow’s milk allergy

CMP—cow’s milk protein

EBV-B cells—Epstein–Barr virus-transformed B cell

Hgs—hepatocyte growth factor-regulated tyrosine kinase substrate

LAP—latency associated peptide

LST—lymphocyte stimulation test

MFI—mean fluorescence intensity

PBMC—peripheral blood mononuclear cell

PE—phycoerythrin

SARA—Smad anchor for receptor activation

TCC—T cell clone

TGF—transforming growth factor


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Friedman, A. and Weiner, H. L. 1994. Induction of anergy or active suppression following oral tolerance is determined by antigen dosage. Proc. Natl Acad. Sci. USA 91:6688.[Abstract]
  2. Shevach, E. M. 2002. CD4+ CD25+ suppressor T cells: more questions than answers. Nat. Rev. Immunol. 2:389.[ISI][Medline]
  3. MacDonald, T. T. 1998. T cell immunity to oral allergens. Curr. Opin. Immunol. 10:620.[CrossRef][ISI][Medline]
  4. Schmidt-Weber, C. B. and Blaser, K. 2002. T-cell tolerance in allergic response. Allergy 57:762.[CrossRef][ISI][Medline]
  5. Roncarolo, M. G., Bacchetta, R., Bordignon, C., Narula, S. and Levings, M. K. 2001. Type 1 T regulatory cells. Immunol. Rev. 182:68.[CrossRef][ISI][Medline]
  6. Weiner, H. L. 2001. Oral tolerance: immune mechanisms and the generation of Th3-type TGF-beta-secreting regulatory cells. Microbes Infect. 3:947.[CrossRef][ISI][Medline]
  7. Letterio, J. J. and Roberts, A. B. 1998. Regulation of immune responses by TGF-beta. Annu. Rev. Immunol. 16:137.[CrossRef][ISI][Medline]
  8. Kuruvilla, A. P., Shah, R., Hochwald, G. M., Liggitt, H. D., Palladino, M. A. and Thorbecke, G. J. 1991. Protective effect of transforming growth factor beta 1 on experimental autoimmune diseases in mice. Proc. Natl Acad. Sci. USA 88:2918.[Abstract]
  9. Wallick, S. C., Figari, I. S., Morris, R. E., Levinson, A. D. and Palladino, M. A. 1990. Immunoregulatory role of transforming growth factor beta (TGF-beta) in development of killer cells: comparison of active and latent TGF-beta 1. J. Exp. Med. 172:1777.[Abstract]
  10. Miura, S., Takeshita, T., Asao, H., Kimura, Y., Murata, K., Sasaki, Y., Hanai, J. I., Beppu, H., Tsukazaki, T., Wrana, J. L., Miyazono, K. and Sugamura, K. 2000. Hgs (Hrs), a FYVE domain protein, is involved in Smad signaling through cooperation with SARA. Mol. Cell Biol. 20:9346.[Abstract/Free Full Text]
  11. Kunzmann, S., Wohlfahrt, J. G., Itoh, S., Asao, H., Komada, M., Akdis, C. A., Blaser, K. and Schmidt-Weber, C. B. 2003. SARA and Hgs attenuate susceptibility to TGF-ß1-mediated T cell suppression. FASEB J. 17:194.[Abstract/Free Full Text]
  12. Massague, J. 2000. How cells read TGF-beta signals. Nat. Rev. Mol. Cell Biol. 1:169.[CrossRef][ISI][Medline]
  13. Nakao, A., Miike, S., Hatano, M., Okumura, K., Tokuhisa, T., Ra, C. and Iwamoto, I. 2000. Blockade of transforming growth factor beta/Smad signaling in T cells by overexpression of Smad7 enhances antigen-induced airway inflammation and airway reactivity. J. Exp. Med. 192:151.[Abstract/Free Full Text]
  14. Monteleone, G., Kumberova, A., Croft, N. M., McKenzie, C., Steer, H. W. and MacDonald, T. T. 2001. Blocking Smad7 restores TGF-beta1 signaling in chronic inflammatory bowel disease. J. Clin. Invest. 108:601.[Abstract/Free Full Text]
  15. Gorelik, L. and Flavell, R. A. 2000. Abrogation of TGFbeta signaling in T cells leads to spontaneous T cell differentiation and autoimmune disease. Immunity 12:171.[ISI][Medline]
  16. Lucas, P. J., Kim, S. J., Melby, S. J. and Gress, R. E. 2000. Disruption of T cell homeostasis in mice expressing a T cell-specific dominant negative transforming growth factor beta II receptor. J. Exp. Med. 191:1187.[Abstract/Free Full Text]
  17. Hahm, K. B., Im, Y. H., Parks, T. W., Park, S. H., Markowitz, S., Jung, H. Y., Green, J. and Kim, S. J. 2001. Loss of transforming growth factor beta signaling in the intestine contributes to tissue injury in inflammatory bowel disease. Gut 49:190.[Abstract/Free Full Text]
  18. Cottrez, F. and Groux, H. 2001. Regulation of TGF-beta response during T cell activation is modulated by IL-10. J. Immunol. 167:773.[Abstract/Free Full Text]
  19. Ludviksson, B. R., Seegers, D., Resnick, A. S. and Strober, W. 2000. The effect of TGF-beta1 on immune responses of naive versus memory CD4+ Th1/Th2 T cells. Eur. J. Immunol. 30:2101.[CrossRef][ISI][Medline]
  20. Kehrl, J. H., Wakefield, L. M., Roberts, A. B., Jakowlew, S., Alvarez-Mon, M., Derynck, R., Sporn, M. B. and Fauci, A. S. 1986. Production of transforming growth factor beta by human T lymphocytes and its potential role in the regulation of T cell growth. J. Exp. Med. 163:1037.[Abstract]
  21. Ahuja, S. S., Paliogianni, F., Yamada, H., Balow, J. E. and Boumpas, D. T. 1993. Effect of transforming growth factor-beta on early and late activation events in human T cells. J. Immunol. 150:3109.[Abstract/Free Full Text]
  22. Schade, R. P., Ieperen-Van Dijk, A. G., Van Reijsen, F. C., Versluis, C., Kimpen, J. L., Knol, E. F., Bruijnzeel-Koomen, C. A. and Van Hoffen, E. 2000. Differences in antigen-specific T-cell responses between infants with atopic dermatitis with and without cow’s milk allergy: relevance of TH2 cytokines. J. Allergy Clin. Immunol. 106:1155.[CrossRef][ISI][Medline]
  23. Yssel, H., de Vries, J. E., Koken, M., Van Blitterswijk, W. and Spits, H. 1984. Serum-free medium for generation and propagation of functional human cytotoxic and helper T cell clones. J. Immunol. Methods 72:219.[CrossRef][ISI][Medline]
  24. Jonuleit, H., Schmitt, E., Stassen, M., Tuettenberg, A., Knop, J. and Enk, A. H. 2001. Identification and functional characterization of human CD4+CD25+ T cells with regulatory properties isolated from peripheral blood. J. Exp. Med. 193:1285.[Abstract/Free Full Text]
  25. Gorelik, L. and Flavell, R. A. 2002. Transforming growth factor-beta in T-cell biology. Nat. Rev. Immunol. 2:46.[CrossRef][ISI][Medline]
  26. Arkwright, P. D., Chase, J. M., Babbage, S., Pravica, V., David, T. J. and Hutchinson, I. V. 2001. Atopic dermatitis is associated with a low-producer transforming growth factor beta(1) cytokine genotype. J. Allergy Clin. Immunol. 108:281.[CrossRef][ISI][Medline]
  27. Andersson, P. O., Stockelberg, D., Jacobsson, S. and Wadenvik, H. 2000. A transforming growth factor-beta1-mediated bystander immune suppression could be associated with remission of chronic idiopathic thrombocytopenic purpura. Ann. Hematol. 79:507.[CrossRef][ISI][Medline]
  28. Horwitz, D. A., Gray, J. D. and Zheng, S. G. 2002. The potential of human regulatory T cells generated ex vivo as a treatment for lupus and other chronic inflammatory diseases. Arthritis Res. 4:241.[CrossRef][ISI][Medline]
  29. Hirayama, Y., Sakamaki, S., Matsunaga, T., Kuroda, H., Kusakabe, T., Akiyama, T., Kato, J., Kogawa, K., Koyama, R., Nagai, T., Ohta, H. and Niitsu, Y. 2002. Granulocyte-colony stimulating factor enhances the expression of transforming growth factor-beta mRNA in CD4-positive peripheral blood lymphocytes in the donors for allogeneic peripheral blood stem cell transplantation. Am. J. Hematol. 69:138.[CrossRef][ISI][Medline]
  30. Hansen, G., McIntire, J. J., Yeung, V. P., Berry, G., Thorbecke, G. J., Chen, L., DeKruyff, R. H. and Umetsu, D. T. 2000. CD4+ T helper cells engineered to produce latent TGF-beta1 reverse allergen-induced airway hyperreactivity and inflammation. J. Clin. Invest. 105:61.[Abstract/Free Full Text]
  31. Haneda, K., Sano, K., Tamura, G., Shirota, H., Ohkawara, Y., Sato, T., Habu, S. and Shirato, K. 1999. Transforming growth factor-beta secreted from CD4+ T cells ameliorates antigen-induced eosinophilic inflammation. A novel high-dose tolerance in the trachea. Am. J. Respir. Cell Mol. Biol. 21:268.[Abstract/Free Full Text]
  32. Terui, T., Sano, K., Shirota, H., Kunikata, N., Ozawa, M., Okada, M., Honda, M., Tamura, G. and Tagami, H. 2001. TGF-beta-producing CD4+ mediastinal lymph node cells obtained from mice tracheally tolerized to ovalbumin (OVA) suppress both Th1- and Th2-induced cutaneous inflammatory responses to OVA by different mechanisms. J. Immunol. 167:3661.[Abstract/Free Full Text]
  33. Cerwenka, A., Kovar, H., Majdic, O. and Holter, W. 1996. Fas- and activation-induced apoptosis are reduced in human T cells preactivated in the presence of TGF-beta 1. J. Immunol. 156:459.[Abstract]
  34. Zhang, X., Giangreco, L., Broome, H. E., Dargan, C. M. and Swain, S. L. 1995. Control of CD4 effector fate: transforming growth factor beta 1 and interleukin 2 synergize to prevent apoptosis and promote effector expansion. J. Exp. Med. 182:699.[Abstract]
  35. Datto, M. B., Li, Y., Panus, J. F., Howe, D. J., Xiong, Y. and Wang, X. F. 1995. Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism. Proc. Natl Acad. Sci. USA 92:5545.[Abstract]
  36. Polyak, K., Kato, J. Y., Solomon, M. J., Sherr, C. J., Massague, J., Roberts, J. M. and Koff, A. 1994. p27Kip1, a cyclin-Cdk inhibitor, links transforming growth factor-beta and contact inhibition to cell cycle arrest. Genes Dev. 8:9.[Abstract]
  37. Brabletz, T., Pfeuffer, I., Schorr, E., Siebelt, F., Wirth, T. and Serfling, E. 1993. Transforming growth factor beta and cyclosporin A inhibit the inducible activity of the interleukin-2 gene in T cells through a noncanonical octamer-binding site. Mol. Cell. Biol. 13:1155.[Abstract]
  38. Sillett, H. K., Cruickshank, S. M., Southgate, J. and Trejdosiewicz, L. K. 2001. Transforming growth factor-beta promotes ‘death by neglect’ in post-activated human T cells. Immunology 102:310.[CrossRef][ISI][Medline]
  39. Sun, J., Dirden-Kramer, B., Ito, K., Ernst, P. B. and Van Houten, N. 1999. Antigen-specific T cell activation and proliferation during oral tolerance induction. J. Immunol. 162:5868.[Abstract/Free Full Text]
  40. Takeuchi, M., Alard, P. and Streilein, J. W. 1998. TGF-beta promotes immune deviation by altering accessory signals of antigen-presenting cells. J. Immunol. 160:1589.[Abstract/Free Full Text]
  41. Schramm, C., Herz, U., Podlech, J., Protschka, M., Finotto, S., Reddehase, M. J., Kohler, H., Galle, P. R., Lohse, A. W. and Blessing, M. 2003. TGF-Beta regulates airway responses via T cells. J. Immunol. 170:1313.[Abstract/Free Full Text]




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