Anti-CD28-induced co-stimulation and TCR avidity regulates the differential effect of TGF-ß1 on CD4+ and CD8+ naïve human T-cells

Brynja Gunnlaugsdottir1,2, Solrun M. Maggadottir1,2,3 and Björn R. Ludviksson1,2,3

1 Center for Rheumatology Research and 2 Institute for Medical Laboratory Sciences, Department of Immunology, Landspitali-University Hospital and 3 Department of Medicine, National University of Iceland, Reykjavik, Iceland

Correspondence to: B. R. Ludviksson; E-mail: bjornlud{at}landspitali.is


    Abstract
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
TGF-ß1 is a powerful regulator of various T-cell functions. However, it has been unclear how the T-cell responsiveness towards TGF-ß1 is affected by its phenotype or signaling intensity. In the present study, we demonstrate that the phenotype and the TCR-signaling intensity of the responding T-cell as well as the presence of anti-CD28 co-stimulation markedly affects how naïve human cord blood T-cells respond to TGF-ß1. In this report we demonstrate that the strength of the stimulatory signal modifies the T-cell response towards TGF-ß1. Thus, the greatest anti-proliferative effect of TGF-ß1 was observed during weak stimulatory conditions (low dose of anti-CD3 with no co-stimulatory signal). However, such anti-proliferative effect was reduced during strong stimulatory signal (high dose of anti-CD3 with a CD28-directed co-stimulatory signal). In addition, our results indicate that CD8+ T-cells are generally more responsive towards TGF-ß1 than CD4+ T-cells. To our surprise, naïve T-cells had a skewed Th1/Tc1 cytokine secretion pattern with high amounts of IL-2, IFN{gamma} and TNF{alpha}, but low amounts of IL-4, IL-5 and IL-10. TGFß1 significantly reduced the secretion of IL-2 and IFN{gamma}, but such suppression was partially prevented by anti-CD28-induced co-stimulation. In contrast, the inhibitory effect on IL-5 secretion was unaffected by anti-CD28 co-stimulation. Interestingly, TGF-ß1 induced IL-10 and TNF{alpha} secretion. However, the induction of IL-10 secretion was reduced during optimal stimulatory conditions while TGF-ß1 further induced TNF{alpha} secretion. These data demonstrate that the duration, intensity and type of signaling alters the sensitivity of T-cells to powerful immunological modifying agents like TGF-ß1.

Keywords: TGF-ß1, T Lymphocytes, cellular proliferation, costimulation, anergy


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Transforming growth factor-ß1 (TGF-ß1) is a multifunctional homodimeric protein of 25 kDa. It represents a family of structurally related polypeptides termed the TGF superfamily (1). TGF-ß is secreted by most cells of the immune system, including B- and T-cells and dendritic cells, and is found in large quantities in platelets (24). TGF-ß is known to be involved in gene expression, cellular differentiation and proliferation of a variety of cell types. Thereby, it plays a role in many different multicellular processes such as embryogenesis, wound healing, oncogenesis, fibrosis and immune regulation (5). TGF-ß is known to inhibit many T-cell functions including proliferation (3,6,7) and effector cell development of both CD4+ (8) and CD8+ T-cells (9). However, TGF-ß has also been reported to stimulate T-cells, partly by preventing apoptosis (10,11), but also by inducing proliferation (12,13). These contradictory findings reflect the fact that many factors affect the interactions between TGF-ß and T-cells. Thus, it seems that the phenotype, developmental stage and the cytokine milieu of the responding T-cell may strongly influence its sensitivity to TGF-ß1 and the type of response that is generated. TGF-ß1, 2 and 3 are three closely related isoforms expressed in mammals. TGF-ß2 and ß3 knockout mice die during embryo genesis while TGF-ß1 knockout mice suffer from severe immune dysregulation and develop several multifocal autoimmune diseases (14,15). Therefore, TGF-ß1 is believed to be more involved in immunological processes while the other two isoforms are vital for cellular differentiation and embryogenesis. Animal models with impaired TGF-ß receptors on T cells have shown that TGF-ß is vital for T-cell homeostasis (16). The lack of TGF-ß1 signaling leads to hypersensitivity of T-cells and the location of the inflammation indicates a lost tolerance towards non-pathogenic antigens (16). This imbalance also leads to multifocal autoimmune disorders (16).

The TGF-ß1 signaling pathway includes the phosphorylation and activation of members of the Smad (Sma- and Mad-related protein) family. Ligation to the receptor complex leads to phosphorylation of Smad2 and/or Smad3 inducing their binding to Smad4 and translocation to the nucleus (17). There the activated Smad complex is competent to access several target genes with the aid of DNA binding co-factors which act as transcription co-activators or transcription co-repressors. An increasing body of evidence indicates that the balance between available co-activators and co-factors determines whether the gene transcription is down regulated or up regulated by TGF-ß (17). This may explain why TGF-ß has been reported to both induce and inhibit several T-cell functions.

Naïve T-cells have been described as being more responsive towards TGF-ß than memory T-cells (18,19). The reduced TGF-ß sensitivity of memory T-cells has been shown to coincide with the loss of the TGF-ßRII (20). The responsiveness of CD8+ and CD4+ T-cells towards TGF-ß1 has, to our knowledge, not been compared in humans, but one study in mice showed that TGF-ß1 markedly enhanced co-stimulated TCR-driven proliferation of CD8+ T-cells while CD4+ T-cells were unresponsive (19). It has also been reported that Th1 cells are more susceptible towards TGF-ß1 than Th2 cells, both in mice and men (8,21).

Despite intensive research during the last decade, the effect of TGF-ß1 on T-cells and the interaction with the T-cell stimulation are unknown. The aim of this study was to evaluate if the type, strength and duration of primary T-cell stimulation affected the sensitivity towards TGF-ß1-driven regulation. We examined the effect on expansion, proliferation, and activation-induced cell death and cytokine secretion.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Study population
Human cord blood was collected from umbilical cords immediately following Caesarean or normal deliveries. Mothers with a history of autoimmune disorders were excluded from the study. Participating mothers gave their informed consents and the Landspitali bioethics committee approved the study.

Isolation of cord blood T-cells
Cord blood was diluted with 0.9% saline (1/3) and centrifuged over Histopaque-1077 (Sigma, Stockholm, Sweden) at 1200 g for 25 min at room temperature. The mononuclear cell layer was removed and washed twice with PBS. Nucleated red blood cells were coated with mouse anti-human glycophorin A mAb (GA-R2, Becton Dickinson, San Jose, CA) and magnetically removed with Dynabeads pan mouse IgG magnetic beads (110.22, Dynal, Oslo, Norway) as described (22). T-cells were isolated from the cord blood mononuclear cells (CBMCs) with a T-cell negative isolation kit (113.11, Dynal) as described by the manufacturer's protocol. Briefly, B-cells, activated T-cells, monocytes, NK cells and granulocytes were removed by adding mAbs against HLA Class II DR/DP, CD14, CD16 and CD56. The coated cells were then removed with depletion dynabeads. The purity of the T-cells (CD3, CD8 and CD4) and contamination of red blood cells (glycophorin A), B-cells (CD19), mononuclear cells (CD14) and NK-cells (CD56) was examined by flow cytometry. The purity of naïve human cord blood CD3+ T-cells ranged from 85 to 93% following the selection procedure.

CD4+ and CD8+ T-cells were positively selected from the cell suspension with CD8 or CD4 MACS Microbeads (Miltenyi Biotec, Bergish Gladbach, Germany) and MS separation columns in a magnetic separator (Mini MACS separation Unit, Miltenyi Biotec) following the producer's manual. The purity was consistently of >93% for CD4+ T-cells and >86% for CD8+ T-cells. CD14/CD4lo monocytes were <2% for CD4+ T-cells while CD56+/CD8+ NK cells were <11% of purified CD8+ T-cells. Less than 1% of selected cells were CD45RO positive.

Surface marker staining
The phenotypes of T-cells were evaluated by flow cytometry using the following conjugated mAbs: anti-CD3–FITC/PE/PerCP (SK7), anti-CD4–FITC/PE/PerCP (SK3), anti CD8–FITC/PE/PerCP (SK1), anti-CD25–PE (2A3), anti-CD14–PE (M{phi}P9), anti-CD19–FITC (4G7), anti-CD28–PE (L293), anti-CD45–PerCP (2D1), anti-45RA–FITC (Leu-18), anti-45RO–PE (UCHL-1), anti-CD56–PE (NCAM16.2), anti-CD62L–Cy-Chrome (Dreg-56), anti-CD69–PE (L78) and anti-CTLA-4–Cy-Chrome (BNI3). All were purchased from Beckton Dickinson. Anti-glycophorin A–FITC (JC159) and anti-CD16–FITC (DJ130c) were purchased from DAKO (Glostrup, Denmark). Anti-hTGFß RII–PE (25508) was purchased from R&D systems (Minneapolis, MN). Appropriate matched FITC, PE, PerCP IgG1/IgG2a isotype controls were purchased from Becton Dickinson. T-cells were stained for 20 min on ice. After washing with PBS, cells were resuspended in 400 µl of 0.5% formalin in PBS and analysed by flow cytometry (FACScan, Becton Dickinson) and the data were analysed by Cell Quest software (Becton Dickinson).

Stimulation and assessment of proliferation
Negatively (106/ml) and positively selected (5 x 105/ml) T-cells were cultured in a serum free medium Aim-V (12055-091, Gibco BRL, Paisley, UK) with or without 10 ng/ml of human recombinant TGF-ß1 (240-B-002, R&D Systems). The cells were stimulated with 0, 1 or 10 µg/ml of wall-bound anti-human CD3{epsilon} antibody (MAB100, R&D systems, Minneapolis, MN) in a 96 U-well culture plate (Nunclon Surface, Nunc, Roskilde, Denmark) for short-term (48 h) or long-term (96 h). For anti-CD28-induced co-stimulation, T-cells were exposed for 96 h with or without 1 µg/ml of anti-CD28 (37407.111, R&D Systems).

The number of CD8+ and CD4+ T-cells present in a fixed volume (100 µl) were sampled, stained, washed and resuspended in 400 µl of 0.5% formaldehyde in PBS. The number of cells counted per minute in the flow cytometer was then used to estimate the expansion of CD8+ and CD4+ T-cells in combined cultures of CD4+ and CD8+ T-cells.

For selected cultures, 1 µg/ml of anti-TGF-ß1,2,3 (ID11, R&D Systems) was added in the absence of exogenous TGF-ß1 and 1µg/ml of anti-IL-10 (25209, R&D Systems) was added to parallel cultures stimulated in the presence of 10 ng/ml TGF-ß1. Similarly, 20 WHO units/ml of IL-2 ( 202-IL, R&D Systems) was added daily to selected cultures during {alpha}CD3 (1 µg/ml or 10 µg/ml) stimulation, with or without anti-CD28 (1µg/ml) co-stimulation in the presence or absence of TGF-ß1 (10 ng/ml). All culture conditions were performed in triplicate wells. The proliferation of T-cells was analyzed by adding 2.5 µCi/ml of tritiated [3H]thymidine (TRK 296, Amersham Biosciences, Piscataway, NJ) into 96-well culture plates containing 200 µl of 2 x 105 cell/well 16 h before harvesting. Radioactivity was analysed with a liquid scintillation analyser.

Assessment of apoptosis/vitality
Negatively selected T-cells were cultured (106/ml) in a serum free medium Aim-V (12055-091, Gibco BRL, Paisley, UK) with or without 10 ng/ml of human recombinant TGF-ß1 (240-B-002, R&D Systems). The cells were stimulated with 0, 1 or 10 µg/ml of wall-bound anti-human CD3{epsilon} antibody (MAB100, R&D Systems) in a 96 U-well culture plate (Nunclon Surface) for short-term (48 h) or long-term (96 h). The viability and apoptosis was analyzed by harvesting 100 µl from selected culture conditions and staining with AnnexinV–FITC and propidium iodide (PI) (TA5532, R&D Systems) as described by the producer's manual. The percentage of apoptotic (AnnexinV+ and PI–) cells was determined using flow cytometric analysis.

Assessment of cytokine secretion
Cell culture supernatants were collected at the end of selected culture conditions. The Human Th1/Th2 Cytokine Cytometric Bead Array assay (550749, Beckton Dickinson) was used to evaluate cytokine secretion. The secreted amount of the cytokines interleukin-2 (IL-2), IL-4, IL-5, IL-10, tumour necrosis factor {alpha} (TNF{alpha}) and interferon {gamma} (IFN{gamma}) was analysed by flow cytometry as instructed by the producer's manual. The range of the assay was 5–10 000 pg/ml.

Asessment of TGF-ß1 dose effect
Cord blood mononuclear cells (1 x 106/ml) were cultured in a serum free medium with 0, 0.1, 1, 10 and 100 ng/ml of acid-activated human recombinant TGF-ß1 (240-B-002, R&D Systems). The cells were stimulated with 10 µg/ml of wall-bound anti-human CD3{epsilon} antibody (MAB100, R&D Systems) in a 96 U-well culture plate (Nunclon Surface) for long-term (96 h). For anti-CD28-induced co-stimulation, T-cells were exposed to 1 µg/ml of anti-CD28 (37407.111, R&D Systems) for 96 h.

Statistical analysis
The Sigma Stat program was used to analyse the data. Data fulfilling the criteria for normal distribution were analysed with a paired t-test. Data with a non-parametric distribution were analysed with the Wilcoxon signed rank test. P < 0.05 was considered significant.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The naïve phenotype of selected cord blood T-cells
The phenotype of selected cells was consistent with a highly purified naïve cord blood T-cells. Thus, purified T-cells were consistently <1% CD45RO positive. Furthermore, the expression of the activation marker CD69 was consistently <1%. Interestingly, 9% of CD4+ T-cells expressed CD25 while <1% of CD8+ T-cells expressed that marker (P < 0.001, data not shown). However, the CD4+ T-cells expressing CD25 were CTLA-4 negative (<1%). Overall, CD4+ T-cells were more L-selectin positive (>93%) than CD8+ T-cells (59%; P = 0.031, data not shown). Thus, the above data demonstrate that the study population consisted of highly purified T-cells expressing a phenotype consistent with uncommitted naïve umbilical cord blood T-cells.

TGF-ß1 dose effect
The dose of TGF-ß1 causing the greatest anti-proliferative effect upon CBMCs was found to range between 1 and 10 ng/ml, depending on the presence of co-stimulation. Ten nanogram per milliliter had a maximal effect when stimulated with a high dose of anti-CD3 only, but 1 ng/ml had a slightly stronger effect than 10 ng/ml when optimally stimulated with both anti-CD3 and anti-CD28 (data not shown). Therefore 10 ng/ml of TGF-ß1 were used throughout the study. None of the examined doses of TGF-ß1 (0, 0.1, 1, 10 and 100 ng/ml) induced proliferation.

The effect of TGF-ß1 on T-cells during sub-optimal stimulation with {alpha}CD3
We first evaluated if the dose and duration of TCR-directed T-cell stimulation would influence their responsiveness towards TGF-ß1. Therefore, naïve human umbilical cord blood T-cells were exposed to short (48 h) and long-term (96 h) stimulation with low (0.1 and 1 µg/ml) and high dose (10 µg/ml) solid phase anti-CD3 mAb. Since 0.1 µg/ml of anti-CD3 mAb did not give any significant stimulation of naïve T-cells under these conditions, all data reported below on low dose stimulation are based upon 1 µg/ml of anti-CD3. In addition, the cells were exposed to various concentrations of TGF-ß1 (0, 0.1, 1, 10 and 100 ng/ml). During short-term stimulation, CD8+ T-cell expansion was inhibited by 31% (Fig. 1B; P = 0.049) in the presence of TGF-ß1 while CD4+ T-cells were not significantly affected. In contrast, during long-term stimulation, both CD8+ and CD4+ T-cell expansion were affected following low dose stimulation (Fig. 1C and D) (% inhibition; CD4 = 44%, P = 0.011 vs CD8 = 69%, P = 0.031), whereas only CD8+ T-cells were affected during high dose stimulation (41% inhibition, P = 0.007). The effect of TGF-ß1 upon T-cell expansion was also reflected when we evaluated the proliferation during the above combined naïve CD4+ and CD8+ T-cell culture condition. Thus, as shown in Fig. 2, the greatest relative inhibitory effect of TGF-ß1 on suboptimally stimulated cells was observed during long-term low dose stimulatory conditions (% inhibition = 84%, P = 0.013). Thus, if TGF-ß1 is present at the time of suboptimal TCR-driven stimulatory condition, its main effect upon T-cell expansion is inhibitory, particularly if the avidity of such stimulation is low.



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Fig. 1. TGF-b1 selectively inhibits the expansion of CD8+ T-cells. Negatively selected naïve human cord blood T-cells were stimulated during short-term (A and B, n = 4) or long-term (C and D, n = 9) with no, low (1 mg/ml) or high (10 mg/ml) dose of immobilized anti-CD3 with or without TGF-b1 (10 ng/ml). After stimulation, 100 ml of cell culture were sampled, analysed and counted on time by flow cytometric analysis. Results are expressed as mean cell number ± 1 SD. *P < 0.05, **P < 0.01.

 


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Fig. 2. The anti-proliferative effect of TGF-ß1 on naïve human T-cells is decreased by CD28 co-stimulation. Negatively selected naïve cord blood T-cells were stimulated (2 x 105/well) with increasing doses of immobilized anti-CD3 for 96 h with or without anti-CD28 co-stimulation (1 µg/ml) and with or without TGF-ß1 (10 ng/ml). Cells were pulsed with radioactive thymidine 16 h before harvesting. Results presented are means of triplicate experiments performed on three individual samples ± 1 SD. *P < 0.05, **P < 0.01, ***P < 0.001.

 
TGF-ß1 inhibits the proliferation of highly purified CD4+ and CD8+ naïve human cord blood T-cells
We next wanted to examine if the observed difference between CD8+ and CD4+ T-cells was caused by a real difference in sensitivity or by possible interaction between the phenotypes within the above culture conditions. In addition, it has been suggested that positively selected naïve murine CD4+ and CD8+ T-cells may have different sensitivity to the regulatory effects of TGF-ß1 (19). Therefore, we next analyzed the effect of TGF-ß1 on isolated cultures of positively selected CD4+ and CD8+ T-cells. As shown in Fig. 3(A and B), during long-term low and high dose suboptimal stimulatory conditions, the proliferation of both CD4+ and CD8+ T-cells was significantly inhibited in the presence of TGF-ß1. Interestingly, during low dose stimulation, proliferation of CD8+ T-cells was almost blocked in the presence of TGF-ß1 while CD4+ T-cells were less affected (% inhibition; CD4+ = 57% (P = 0.02), vs CD8+ = 93% (P ≤ 0.001); Fig. 3A and B). However, TGF-ß1 inhibited the proliferation of both phenotypes equally during high dose stimulation (% inhibition CD4+ = 56% (P ≤ 0.001) vs CD8+ = 52% (P = 0.002); Fig. 3A and B). In summary, the strongest inhibitory effect of TGF-ß1 during suboptimal stimulatory conditions on both phenotypes was observed during low dose {alpha}CD3 stimulation.



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Fig. 3. The anti-proliferative effect of TGF-ß1 on purified CD4+ (A) and CD8+ (B) T-cells is decreased by CD28 co-stimulation. Positively selected (1 x 105/well) naïve cord blood CD4+ and CD8+ T-cells were stimulated with increasing doses of immobilized anti-CD3 for 96 h with or without anti-CD28 co-stimulation (1 µg/ml) and with or without TGF-ß1 (10 ng/ml). Cells were pulsed with [3H]thymidine 16 h before harvesting. Results presented are means of triplicate experiments performed on three individual samples ± 1 SD. *P < 0.05, **P < 0.01, ***P < 0.001.

 
Viability and necrosis of negatively selected T-cells
It has been suggested that TGF-ß1 may induce apoptosis of T-lymphocytes (23). Therefore, we evaluated if the inhibitory effect of TGF-ß1 on the expansion and proliferation of naïve human cord blood T-cells could be explained by TGF-ß1-induced apoptosis. As shown in Fig. 4(A and B), no increase in apoptotic T-cells was observed during any of the suboptimal stimulatory conditions tested in the presence of TGF-ß1, thus indicating that the observed suppression of expansion and proliferation is due to an anti-proliferative effect rather than a TGF-ß1-induced apoptosis.



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Fig. 4. TGF-ß1 does not affect apoptosis of naïve human T-cells. Negatively selected naïve human T-cells (106/ml) from three cord blood samples were stimulated for 48 (A) or 96 h (B) with no, low (1 mg/ml) or high (10 mg/ml) dose of immobilized anti-CD3 with or without TGF-ß1 (10 ng/ml). Results are presented as mean % apoptosis (Annexin V positive and PI negative). No significant difference was observed.

 
Cytokine secretion of negatively selected T-cells
Secretion of IL-2.
Since IL-2 is one of the key regulators of T-cell proliferation, its secretion by naïve T-cells was evaluated. IL-2 was only measurable following high dose stimulation and, as expected, IL-2 secretion increased proportionally with the strength and duration of the T-cell stimulation (Table 1). The above effect of TGF-ß1 on T-cell expansion was accompanied by its pronounced effects on IL-2 secretion. As shown in Table 1, the inhibitory effect of TGF-ß1 upon IL-2 secretion was much more pronounced during high dose long-term stimulation compared to high dose short-term stimulation (34-fold inhibition vs 2-fold inhibition, Table 1). It is therefore conceivable that TGF-ß1 is primarily inhibiting naïve CD8+ T-cell expansion via its effect upon IL-2 secretion. However, when we added IL-2 exogenously to the cultures of purified both CD4+ and CD8+ T-cells (20 IU/ml daily) the anti-proliferative effects of TGF-ß1 was not prevented (data not shown).


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Table 1. Secretion of IL-2a

 
Secretion of IFN{gamma} and TNF{alpha}.
Similarly, we evaluated the effect of TGF-ß1 on the secretion of the Th1/Tc1 cytokines IFN{gamma} and TNF{alpha}. Interestingly, although the secretion of both cytokines increased with increasing dose and duration of {alpha}CD3 stimulation, their secretion was differently regulated by TGF-ß1. While TGF-ß1 caused a significant reduction of IFN{gamma} secretion during long-term, high dose (10 µg/ml) stimulation (P = 0.009), it tended to enhance the secretion of TNF{alpha} under the same conditions (P = 0.068) (Table 2).


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Table 2. Secretion of IFN{gamma} and TNF{alpha}a

 
Secretion of IL-4, IL-5 and IL-10.
It has been suggested that naïve human umbilical cord T-cells are skewed towards Th-2 cytokine secretion. However, of the Th/Tc2 cytokines tested (IL-4, IL-5 and IL-10), only IL-10 was secreted in detectable (>20 pg/ml) amounts in our system (Table 3). After short-term, high dose {alpha}CD3 stimulation, IL-10 was only secreted in detectable amounts in the presence of TGF-ß1, and TGF-ß1 significantly increased the secretion of IL-10 by T-cells that were stimulated with a high dose of {alpha}CD3 for 96 h (Table 3).


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Table 3. The effect of TGF-ß1 on the secretion of IL-10a

 
We next wanted to evaluate if endogenous TGF-ß secretion was a significant factor in our system. As shown in Fig. 5(A and B), the administration of TGF-ß1,2,3 mAb into the culture system did not have any effect. However, anti IL-10 mAb administration tended to reduce the anti-proliferative effect of TGF-ß1 on CD4+ T-cells during high dose {alpha}CD3 stimulation (Fig. 5A) (% TGF-ß1-induced inhibition on proliferation without anti-IL-10: 45% vs with anti-IL-10: 25%). In contrast, this was not observed among CD8+ T-cells. Taken together, the above data demonstrate that changes in the intensity and duration of stimulatory signal directed through the TCR on naïve cord blood T-cells have a quantitative but not qualitative influence on TGF-ß1-mediated cytokine regulation. Furthermore, the effect of TGF-ß1 on the cytokine secretion of human naïve cord blood T-cell is independent of the Th/Tc0, Th/Tc1, and Th/Tc2 or Th/Tc3 cytokine pattern phenotypes as currently defined. Finally, it is possible that the anti-proliferative effects of TGF-ß1 on CD4+ T-cells are partially driven through IL-10.



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Fig. 5. Neutralization of TGF-ß and IL-10 activity indicates that TGF-ß1 inhibits T-cell proliferation independently. Isolated CD4+ (A) and CD8+ (B) T-cells (5 x 105) were stimulated in separate culture systems for 96 h with 10 µg/ml of immobilized anti-CD3. Neutralizing antibodies (10 µg/ml) against TGF-ß1, 2 and 3 were added to cultures in the absence of TGF-ß1, while neutralizing antibodies against IL-10 (10 µg/ml) were added to cultures in the presence of TGF-ß1 (10 ng/ml). Cells were pulsed with [3H]thymidine 16 h before harvesting. Results presented are means of triplicate experiments performed on two individual samples ± 1 SD. **P < 0.01, ***P < 0.001.

 
Phenotypic changes following TGF-ß1 exposure
As various co-stimulatory molecules and cytokine receptors have been associated with an inhibitory function, we next evaluated the expansion of negatively selected CD25, CTLA-4 and CD28 positive T-cells before and after the various above stimulatory conditions. Surprisingly, we did not detect any significant changes or phenotype alterations in the above surface molecule expression pattern following stimulation in the presence of TGF-ß1 (data not shown). The frequency of CD25 positive cells was increased within both the CD4+ and CD8+ T-cell population with increasing stimulatory intensity (Fig. 6). Finally, the expression of the TGF-ßRII was highly variable and did not differ significantly between the CD4+ and CD8+ T-cells during all experimental conditions tested. Initially, CD4+ and CD8+ naïve T-cells are nearly identical, both towards their ability to respond to TGF-ß1 and their known regulatory phenotypic surface molecule expression patterns. Such phenotypic difference could not be found accountable for their different sensitivity towards TGF-ß1 in our study.



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Fig. 6. The percentage of T-cells expressing CD25+ is not affected by TGF-ß1. T-cells (1 x 106/ml) were stimulated with increasing amount of immobilized anti-CD3 with or without TGF-ß1 for short-term (48 h) and long-term (96 h). After staining, the cells were analysed by flow cytometry. Results are expressed as mean percentage of CD25+/CD4+ T-cells (A, CD4+, n = 4) or CD25+/CD8+ T-cells (B,CD8+, n = 3) ± 1 SD.

 
The effect of TGF-ß1 on T-cells during optimal stimulation with {alpha}CD3 and {alpha}CD28
In the next set of experiments we assessed whether the {alpha}CD28-driven co-stimulation would influence the responsiveness of naïve human cord blood T-cells to TGF-ß1.

We evaluated if co-stimulation with {alpha}CD28 altered the T-cell proliferative response pattern towards TGF-ß1. The anti-proliferative effect of TGF-ß1 is partially inhibited during optimal stimulation with anti-CD28 in addition to anti-CD3. Thus, as shown in Fig. 2, the anti-proliferative effect of TGF-ß1 upon naïve T-cells was reduced if T-cells were co-stimulated with {alpha}CD28. During low dose {alpha}CD3 stimulation the percentage inhibition caused by TGF-ß1 decreased from 91% to 54% in the presence of {alpha}CD28 co-stimulation and became non-significant during high dose {alpha}CD3 stimulation (Fig. 2).

We next evaluated the above co-stimulatory effects in a pure CD4+ and CD8+ culture system. As shown in Fig. 3(A and B), the proliferation pattern of isolated CD4+ and CD8+ T-cells is very similar to that of a combined culture system. In all cases the additional stimulation with anti-CD28 reduced the inhibitory effect of TGF-ß1 most significantly during high dose {alpha}CD3 stimulation.

Thus, taken together the above data suggest that TGF-ß1 has predominantly inhibitory effect on TCR-induced stimulation of naïve T-cells, particularly under suboptimal stimulatory conditions.

Cytokine secretion of negatively selected T-cells
Since {alpha}CD28 was significantly affecting the sensitivity of T-cells towards TGF-ß1, we evaluated if this could be reflected in cytokine pattern alterations. As shown in Fig. 7, even under optimal primary stimulatory conditions, the cytokine pattern of naïve umbilical cord blood T-cells is skewed towards a Th-1/Tc-1 phenotype. Furthermore, the anti-CD28 co-stimulation caused a much higher relative increase in cytokine secretion during low dose anti-CD3 stimulation than during high dose stimulation. Thus, during high dose {alpha}CD3-stimulation with anti-CD28 it induced the IL-2 secretion ~8-fold and TNF{alpha} ~4-fold. However, during low dose stimulation, {alpha}CD28 co-stimulation induced a marked increase of nearly all the cytokines tested (IL-2 68-fold, IFN{gamma} 11-fold, TNF{alpha} 11-fold, IL-10 2-fold and IL-5 from 0 to 13 pg/ml) (Fig. 7). Finally, even under optimal primary stimulatory conditions, we observed only marginal IL-4 secretion from naïve T-cells.



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Fig. 7. Co-stimulation with anti-CD28 alters the effect of TGF-ß1 on cytokine secretion. 106/ml T-cells were stimulated with increasing doses of immobilized anti-CD3 with or without 1 µg/ml of soluble anti-CD28 co-stimulation and with or without TGF-ß1 (10 ng/ml) for 96 h. Cell culture supernatants were collected and analysed with a cytokine bead array system in flow cytometry. The analysis detection range was 5–10 000 pg/ml. Results are expressed as mean ± 1 SD of three individual experiments.

 
When we examined the effect of {alpha}CD28 on TGF-ß1 mediated cytokine secretion, we observed that it modified the effect of TGF-ß1 on IL-2 and IFN{gamma} secretion in a similar manner as it did on proliferation. During low dose stimulation ({alpha}CD3 = 1 µg/ml), the TGF-ß1-mediated suppression of IL-2 (100% inhibition) and IFN{gamma} (79% inhibition) was not reversed by {alpha}CD28 co-stimulation (Fig. 7A and B). In contrast, during high dose stimulation ({alpha}CD3 = 10 µg/ml), the inhibitory effect of TGF-ß1 on the secretion of IL-2 and IFN{gamma} was largely reversed in the presence of {alpha}CD28, mirroring the effect of TGF-ß1 on proliferation. IL-5 continued to be equally down regulated by TGF-ß1 during optimal stimulatory conditions (Fig. 7E). Finally, and to our surprise, TNF{alpha} secretion peaked when naïve T-cells were stimulated under maximal stimulatory conditions in the presence of TGF-ß1, however this was not observed for IL-10 secretion (Fig. 7C and D). Conversely, co-stimulation with anti-CD28 did not alter the cytokine pattern for IL-10 secretion during high dose anti-CD3 stimulation.

However, during low dose anti-CD3, co-stimulation with anti-CD28, TGF-ß1 induced the secretion of IL-10 4-fold, while TNF{alpha} secretion was unaffected by its presence.


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
The main objective of this study was to evaluate whether the phenotype and stimulatory conditions influence the responsiveness of naïve human umbilical cord blood T-cells towards TGF-ß1.

In this paper we show that all of the examined factors affect the responsiveness of naïve human cord blood T-cells towards TGF-ß1. Not only do we demonstrate that the two examined naïve human CD4+ and CD8+ cord blood T-cell phenotypes respond differently to TGF-ß1, but also that the stimulatory mode, signaling intensity and duration markedly alters their sensitivity to this pleiotropic cytokine.

Our main observation is that CD8+ T-cells are more sensitive than CD4+ T-cells towards the inhibitory effect of TGF-ß1. A closer examination of individual T-cell subsets shows that this is true for both combined and separate cultures of CD4+ and CD8+ T-cells. To our knowledge, this is the first study demonstrating increased sensitivity of human naïve CD8+ T-cells towards TGF-ß1. Our results are, however, in agreement with other in vitro studies where murine CD8+ T-cells are shown to be more sensitive towards TGF-ß1 than CD4+ T-cells (19). Yet, in contrast to our findings, the murine CD8+ T-cells were shown to be selectively co-stimulated in the presence of TGF-ß1 while the CD4+ T-cells were not (19). The different effect of TGF-ß1 observed in these studies may be explained by the difference in experimental conditions. In addition to the species difference, our T-cells were cultured in a serum free medium whereas the murine T-cells were not. Therefore, the presence of other cytokines affecting the murine T-cells cannot be excluded.

TGF-ß1 is relatively most inhibitive during long-term (96 h) low dose (1 µg/ml) anti-CD3 stimulation in both combined and pure cultures of CD4+ and CD8+ T-cells. During short-term stimulation TGF-ß1 only inhibits the expansion of CD8+ T-cells following low dose stimulation. Yet, during long-term high dose stimulation, purified CD4+ and CD8+ T-cells are almost equally affected by TGF-ß1. Thus, it is clear from this data that both the avidity and duration of stimulation alters the TGF-ß1 responsiveness of the T-cells.

Our results on cytokine secretion show that TGF-ß1 down-regulates the secretion of both Th1/Tc1 and Th2/Tc2 cytokines. This is not surprising considering that TGF-ß1 down-regulates the transcription factors T-bet (24) and GATA-3 (25), which regulate the development of Th1 and Th2 cells, respectively. The cytokines mostly affected by TGF-ß1 are IL-2 and IFN{gamma}. Interestingly, TGF-ß1 has a positive effect on the secretion of both IL-10 and TNF{alpha}. Although the changes observed in secreted IL-2 levels in response to TGF-ß1 harmonize well with changes in proliferation, daily addition of exogenous IL-2 did not inhibit the anti-proliferative effect of TGF-ß1. Thus, in our system, the mechanism of TGF-ß1-mediated anti-proliferative effect is not only driven through its inhibitory effect upon IL-2 secretion. This is in concert with previous findings in IL-2 –/– mice where {alpha}CD3-driven TGF-ß1-mediated T-cell suppression of autoimmunity was not driven through IL-2 (8,26).

Furthermore, a recent study on the cross-talk between IL-2 and TGF-ß in CD8+ T-cells has revealed that TGF-ß selectively inhibits IL-2-induced proliferation and not apoptosis by inhibiting genes involved in proliferation (c-myc, cyclin D2 and cyclin E) rather than disrupting the two major IL-2R signaling pathways Shc and STAT5 (27). In contrast, it has been demonstrated that the anti-proliferative effect of TGF-ß1 is driven through Smad3 signaling but not cyclin inhibitors (28). Thus, the exact molecular mechanisms of TGF-ß1-mediated anti-proliferative effect remain to be further investigated. Our second main observation is that co-stimulation through CD28 reduces the inhibitory effect of TGF-ß1 on both combined and pure cultures of CD4+ and CD8+ T-cells. Our results indicate that signaling through CD28 modifies the responsiveness of the responding T-cell towards TGF-ß1. However, this effect depends on the avidity of the signal received simultaneously through CD3, since only T-cells receiving high dose anti-CD3 stimulation become less responsive towards TGF-ß1 in the presence of {alpha}CD28 stimulation. The modifying effect of anti-CD28 co-stimulation also affects the secretion of both IL-2 and IFN{gamma} in a similar way. Thus, the inhibition of IL-2 and IFN{gamma} by TGF-ß1 is greatly reduced with anti-CD28 during high dose anti-CD3 stimulation, and unaffected during low dose stimulation. The inhibitory effect observed by TGF-ß1 on IL-5 was, however, not changed by the presence of anti-CD28 co-stimulation, irrespectively of anti-CD3 stimulation dose. Recently, Sung et al. (29) reported a similar but a more radical change in responsiveness of naïve (CD62LHI) murine CD4+ T-cells towards TGF-ß1 following anti-CD28 co-stimulation. TGF-ß1 inhibited proliferation and IL-2 secretion of the T-cells during anti-CD3 stimulation alone, whereas, addition of anti-CD28 modified the effect of TGF-ß1, which enhanced proliferation while still down regulating IL-2 secretion. In contrast to the study of Sung et al., our results suggest that the addition of anti-CD28 modifies the effect of TGF-ß1 on both IL-2 secretion and proliferation similarly and reduces the inhibitory effect of TGF-ß1 on both factors. In contrast, our results concur with those of Tzachanis et al. (30). They have recently demonstrated how the inhibitory effect of TGF-ß1 on IL-2 secretion may be modified by stimulatory conditions of the responding T-cell. They reported that Tob, a negative regulator of T-cell activation, is down regulated during optimal stimulatory conditions, but not when stimulated suboptimally with anti-CD3 or anti-CD28 alone (30). Tob acts a co-repressor with the Smad2–Smad4 complex, which can be formed in response to TGF-ß signaling, and aids the binding of the complex to the IL-2 promoter, thereby down regulating IL-2 transcription. This mechanism, however, only explains the observed change in the TGF-ß1 inhibition on IL-2 secretion, which does not fully explain how TGF-ß1 inhibits proliferation or secretion of other cytokines. Tob shares many of the inhibitory characteristics of TGF-ß1, thus raising the question if some of the other observed effects of TGF-ß1 can result from a cross-talk between TGF-ß and Tob. Another point of interest is that Tzachanis et al. (30) observed that CD8+ peripheral T-cells expressed less Tob than CD4+ T-cells and were less responsive towards the modifying effect produced by co-stimulation. In our study, however, both phenotypes respond similarly to the presence of the co-stimulation. Therefore our results may indicate a similar expression of Tob in both phenotypes of naïve cord blood T-cells. Although TGF-ß1 induces the secretion of both IL-10 and TNF{alpha}, these cytokines respond differently towards the {alpha}CD28 co-stimulation. The TNF{alpha} secretion is highly induced by {alpha}CD28 while IL-10 secretion is not. IL-10 plays a vital inhibitory role in vivo whereas TNF{alpha} does not. Thus, the presence of a co-stimulatory signal favours the release of TNF{alpha} and not IL-10, indicating that the secretion of these cytokines is regulated by TGF-ß1 through different molecular mechanisms. Although TGF-ß1 has previously been shown to induce the secretion of both TNF{alpha} and IL-10, the molecular pathway for this regulation is complex and only partially understood (31,32). The anti-proliferative effect of IL-10 is well known, also the fact that IL-10 secretion is induced by TGF-ß1 (31). Our data indicate that increased IL-10 secretion may have a role in the anti-proliferative effect of TGF-ß1 upon CD4+ but not CD8+ T-cells. However, this remains to be further elucidated.

The administration of TGF-ß1 into the culture in our study could be inducing its own secretion as has been shown for murine (33) and human (34) T-cells. In addition, it is conceivable that under our culture conditions, TGF-ß1 might be inducing the differentiation of T-regulatory cells as others have reported (35,36). Finally, since some groups have shown that T-regulatory cells (CD4/CD25+) secrete high levels of TGF-ß1 under certain stimulatory conditions (37), these regulatory cells could also be mediating their regulatory effects in our system. However, we did not observe an increased expansion of either CD4+/CD25+ or CD8+/CD25+ potential regulatory T-cells in the presence of TGF-ß1. In fact >80% of responding T-cells acquired this phenotype after stimulation, regardless of the presence of TGF-ß1. Furthermore, our results suggest that there is no significant endogenous secretion of TGF-ß1, as the addition of anti-TGF-ß1, 2 and 3 did not change the proliferation of CD4+ and CD8+ T-cells during anti-CD3 stimulation.

It has been suggested that cytokine secretion of T-cells in neonates is biased towards Th2 responses (38). However, in our system, the key Th/Tc2 cytokines IL-4 and IL-5 were hardly detectable, even under optimal stimulation conditions, whereas the key Th/Tc1 cytokines IFN{gamma} and TNF{alpha} were secreted in highly significant amounts. The impaired capacity of T-cells in young children to develop a Th/Tc1 response may thus be attributed to the antigen-presenting cells that differ from their counterparts in adults in the ability to induce a Th/Tc1 polarization (39).

The sensitivity of naïve T-cells towards the modifying effect of TGF-ß1 on cytokine secretion may be cell cycle dependent, particularly as we observe the strongest inhibitory effect during high dose TCR-driven stimulation. This is supported by the observation that cytokine secretion of T-cells is cell cycle dependent (40). In that study, the secretion of IL-2 and IFN{gamma} appeared to be somewhat cell cycle independent whereas IL-4 expression required three cell divisions. This is mirrored by our observations where TGF-ß1 had a significant effect on the secretion of IL-2 and IFN{gamma} but had only a minor effect on IL-4.

It is not surprising that there exists a regulatory system where anti-CD28 can counteract the TGF-ß1 suppression, bearing in mind the role that TGF-ß1 has in T-cell homeostasis. One can speculate that TGF-ß1-mediated suppression is most needed when T-cells are presented with weak or non-specific stimuli (false alarm). However, when a T-cell is stimulated simultaneously through the TCR and through the CD28 co-receptor, it is most likely that it has met the antigen which it is designed to respond to. Therefore, at this standpoint the TGF-ß1 regulation is not desirable. The molecular background for these co-stimulatory linked processes is thus of great importance and remains to be investigated.


    Acknowledgements
 
The Landspitali, University Hospital science fund and the research fund of the University of Iceland supported this work. We would also like to thank Inga Skaftadóttir for her technical assistance and Helgi Valdimarsson for his helpful comments on the manuscript.


    Abbreviations
 
NK   natural killer
Tc1   T cytotoxic 1
Tc2   T cytotoxic 2

    Notes
 
Transmitting editor: W. Strober

Received 16 March 2004, accepted 8 October 2004.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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