Activated CD4+ CD25+ T cells suppress antigen-specific CD4+ and CD8+ T cells but induce a suppressive phenotype only in CD4+ T cells

Authors


Dr G. Schuler, Department of Dermatology, University Hospital of Erlangen-Nuremberg, Hartmannstrasse 14, 91052 Erlangen, Germany.
Email: Detlef.Dieckmann@derma.imed.uni-erlangen.de
Senior author: Detlef Dieckmann, email: Gerold.Schuler@derma.imed.uni-erlangen.de

Summary

CD4+ CD25+ regulatory T cells are increasingly recognized as central players in the regulation of immune responses. In vitro studies have mostly employed allogeneic or polyclonal responses to monitor suppression. Little is known about the ability of CD4+ CD25+ regulatory T cells to suppress antigen-specific immune responses in humans. It has been previously shown that CD4+ CD25+ regulatory T cells anergize CD4+ T cells and turn them into suppressor T cells. In the present study we demonstrate for the first time in humans that CD4+ CD25+ T cells are able to inhibit the proliferation and cytokine production of antigen specific CD4+ and CD8+ T cells. This suppression only occurs when CD4+ CD25+ T cells are preactivated. Furthermore, we could demonstrate that CD4+ T-cell clones stop secreting interferon-γ (IFN-γ), start to produce interleukin-10 and transforming growth factor-β after coculture with preactivated CD4+ CD25+ T cells and become suppressive themselves. Surprisingly preactivated CD4+ CD25+ T cells affect CD8+ T cells differently, leading to reduced proliferation and reduced production of IFN-γ. This effect is sustained and cannot be reverted by exogenous interleukin-2. Yet CD8+ T cells, unlike CD4+ T cells do not start to produce immunoregulatory cytokines and do not become suppressive after coculture with CD4+ CD25+ T cells.

Abbreviations:
T reg

regulatory T cells

DC

dendritic cells

Introduction

There is now compelling evidence that CD4+ T cells specialized for the suppression of immune responses play a critical role in immune regulation.1 Since their first description in 1995, it has been convincingly demonstrated in rodents that cells with this function are enriched within the CD4+ CD25+ subset.2,3 Recent studies demonstrate that CD4+ CD25+ T cells play a similar role as an immune regulator in humans.4–7 It was shown that CD4+ CD25+ T cells, similar to their rodent counterpart, constitute a small fraction of CD4+ T cells (average 6%). They are naturally anergic to mitogenic stimuli, inhibit the proliferation of CD4+ and CD8+ T cells after stimulation via their T-cell receptor (TCR) and do so in a cytokine-independent yet cell contact-dependent manner.4–7

Progress has been made in elucidating the mechanisms by which CD4+ CD25+ T cells exert their regulatory function. It has been suggested that CD4+ CD25+ T cells bind transforming growth factor-β (TGF-β) on their cell surface and thereby mediate contact dependent suppression of other T cells.8 Two groups have described the increased expression of glucocorticoid-induced tumour necrosis factor (TNF) receptor (GITR) on CD4+ CD25+ T cells compared to resting CD4+ CD25 T cells. Furthermore, they show that anti-GITR antibodies abrogate CD4+ CD25+ mediated suppression.9,10 Cytotoxic T-lymphocyte antigen-4 (CTLA-4) is constitutively expressed on CD4+ CD25+ regulatory T cells (Treg). As a known immunomodulatory receptor it was always speculated to play an important role in the suppressive capacity of CD4+ CD25+ Treg. So far reports are conflicting, as some studies could not demonstrate a role of CTLA-4,5,7 while others found it to be important, especially in vivo in mice.11,12 A recent report showed, that CD4+ CD25+ Treg can induce the immunosuppressive tryptophan catabolism in dendritic cells (DC) in a CTLA-4-dependent fashion.13 Lately the important role of the transcription factor Foxp3 has been demonstrated in mice. Transfection of CD4+ CD25 T cells with Foxp3 leads to the induction of CD25 expressing regulatory T cells.14–16 Presumably Foxp3 also plays an important role in humans, as the so called IPEX syndrome (immune dysregulation, polyendocrinopathy, enteropathy and X-linked inheritance) is caused by a mutation in the Foxp3 gene17 and has a similar phenotype as the scurfy mouse which shows equivalent genetic alterations.18

To date, very little is known about the requirements for the development and physiological regulation of CD4+ CD25+ T-cell function. Survival and/or expansion of CD4+ CD25+ T cells in the periphery seems to be dependent on interleukin-2 (IL-2) and costimulatory molecules, as mice lacking these components show major deficiencies in CD4+ CD25+ T cells.19–21 It is difficult to understand how CD4+ CD25+ T cells exert their suppressive function in vivo, considering that they constitute only up to 6% of CD4+ T cells and need direct cell contact and activation via their TCR to suppress other T cells. We and others could show that coculture of CD4+ T cells with CD4+ CD25+ Treg leads to anergic cells producing high levels of IL-10 and TGF-β. By means of these cytokines they are able to suppress proliferation of other T cells.22,23 These two studies also offer an explanation for conflicting results concerning cytokine dependency of immune suppression. On the one hand, it was convincingly demonstrated that CD4+ CD25+ Treg act via direct cell contact and not via secreted cytokines in vitro.24 On the other hand, it could be demonstrated that in particular, IL-10 is necessary to achieve regulation in vivo.25 Comparatively little is known about the effects of CD4+ CD25+ Treg on CD8+ T cells. We could demonstrate that the allospecific CD8+ T-cell proliferation was reduced during coculture with CD4+ CD25+ T cells.5 Other studies demonstrating that interferon-γ (IFN-γ) production was reduced and that proliferation could not be restored by IL-2, were conducted in the murine system.26 One recent report addressed the effects of human CD4+ CD25+ T cells on polyclonal and allogeneic stimulation of CD8+ T cells27 and found effects similar to those described for the mouse studies. Apart from this limited knowledge on the effects of CD4+ CD25+ Treg on polyclonal and allospecific stimulation no information is available regarding the Influence of CD4+ CD25+ Treg on antigen-specific responses in humans.28

In the present report we demonstrate that coculture of antigen-specific CD4+ or CD8+ T-cell clones with CD4+ CD25+ Treg results in suppressed proliferation of the CD4+ and CD8+ T-cell clones with specificity for nominal antigens. These effects can only be seen if CD4+ CD25+ T cells are preactivated before coculture. Furthermore the anergized CD4+ T-cell clones switch their cytokine production from IFN-γ towards IL-10 and TGF-β and thereby become suppressive and keep their antigen specificity. In sharp contrast to CD4+ T cells, however, CD8+ T cells do not start to produce suppressive cytokines and do not become suppressors after coculture.

Our findings further elucidate the mechanisms of action of CD4+ CD25+ Treg and might also be of clinical relevance, as anergized CD4+ T-cell clones might be interesting tools in the treatment of autoimmune disorders and transplantation.

Materials and methods

Culture medium

RPMI-1640 (BioWhittaker, Rockland, ME) supplemented with 1% heat-inactivated autologous plasma, 20 µg/ml gentamicin (Merck, Darmstadt, Germany) and 2 mm glutamine (BioWhittaker) was used for the generation of DC, X-VIVO-20 (BioWhittaker) supplemented with 1% heat-inactivated single donor human serum, 20 µg/ml gentamicin (Merck) and 2 mm glutamine (BioWhittaker) for T-cell culture.

Cytokines

All cytokines used in this study were recombinant human proteins. Final concentrations were: granulocyte–macrophage colony-stimulating factor (GM-CSF 1000 U/ml; LeukomaxTM; Novartis, Basel, Switzerland), IL-4 800 U/ml (Novartis), IL-2 10 U/ml (Proleukin; Chiron Behring, Marburg, Germany) and IL-7 (PeproTech, Frankfurt/Main, Germany) 10 ng/ml; for DC maturation we used a cocktail consisting of IL-1β 2 ng/ml (Sigma, St Louis, MO); IL-6 1000 U/ml (Novartis); TNF-α 10 ng/ml (Bender, Vienna, Austria), and prostaglandin E2 (PGE2 1 µg/ml; Sigma).

Antibodies

For immunostaining phycoerythrin (PE)- and fluoroscein isothiocyanate-conjugated antibodies (all from BD Pharmingen) against CD3 (UCHT 1), CD4 (RPA-T4), CD8 (RPA-T8), CD14 (M5E2), CD19 (HIB 19), CD25 (M-A251), CD45 RO (UCHL 1), CD56 (B159), CD80 (L307.4), CD83 HB15e), CD86 (FUN-1), HLA-A0201 (BB7.2) and respective mouse isotype controls were employed. Unconjugated anti-IL-10 (JES3-19F1) (Pharmingen, San Diego, CA) and anti-TGF-β (R & D Systems, Minneapolis, MN) were used for neutralization experiments; anti-CD3 (UCHT1) and anti-CD28 (CD28.2) were used for polyclonal activation of T cells.

Cytokine measurement

The cytokines IL-10, IFN-γ and TGF-β were measured by enzyme-linked immunosorbent assay (ELISA) according to the manufacturers instructions; IL-10 and IFN-γ (Biosource-Diagnostics, Nivelles, Belgium), TGF-β (BD Pharmingen).

To quantitate antigen-specific, IFN-γ-releasing, peptide-specific effector T cells, an enzyme-linked immunospot (ELISPOT) assay was used as described.29

For analysis of intracellular cytokine production T cells were either stimulated with phorbol 12-myristate 13-acetate (PMA) 20 ng/ml and Ca2+ ionophore A23187 500 µg/ml (both from Sigma) for 6 hr or with platebound anti-CD3 and soluble anti-CD28 antibody for 6 hr. Monensin, 2 µm (Sigma) was added for the last 5 hr of culture. Cells were collected, washed, fixed and saponin-permeabilized (Fix/perm solution, BD Pharmingen) and stained with cytokine-specific antibody or isotype. The antibody used for intracellular cytokine staining was PE-conjugated anti IL-10 (JES3-19F1) from BD Pharmingen.

DC generation and establishment of T cell clones

DC were generated from whole blood or leukapheresis products (obtained from the Department of Transfusion medicine. University of Erlangen, from healthy donors after Informed consent was given) as described.29 In brief, peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll density gradient centrifugation. Monocytes were isolated by plastic adherence and cultured in RPMI medium, supplemented with IL-4 and GM-CSF. At day 6 a maturation cocktail (IL-1β, IL-6, PGE2 and TNF-α) was added. At day 7–8 non-adherent cells were harvested and constituted mature DC, which were >90% double positive for costimulatory molecules (CD80, CD86) and CD83 and negative for lineage marker (CD3, CD4, CD8, CD14, CD19, CD56). Tetanus toxoid (TT; Chiron Behring) or keyhole limpet haemocyanin (KLH; Calbiochem, San Diego, CA), both at 10 µg/ml, were added at day 6 before maturation as indicated.

CD4+ T cells were isolated from PBMC with a negative CD4+ T-cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). CD4+ CD25+ T cells were isolated from the pure, untouched CD4+ T cells using CD25 Microbeads (Miltenyi Biotech). Purity was assessed by fluorescence-activated cell sorting (FACS) and was generally > 90%.

For induction of specific T cells, CD4+ T cells were isolated by positive selection with magnetic beads (Miltenyi Biotech). T cells (2 × 105) were cocultured with autologous DC loaded with TT or KLH at a 1 : 20 ratio in 96 flat-bottomed wells. IL-2 (10 U/ml) and IL-7 (10 ng/ml) were added every other day. After the second stimulation T cells were tested for their reactivity towards the respective antigen. Consequently 104 T cells were cocultured with syngeneic antigen-loaded DC or control DC at a 1 : 20 ratio in 96 round bottom wells for 16 hr. The supernatant was subsequently harvested and analysed by ELISA for IFN-γ production. Positive populations (at least 3× background IFN-γ production) were cloned and restimulated with antigen loaded syngeneic DC as described above.

CD8+ T cells of HLA-A*0201 donors were isolated by positive selection with magnetic beads (Miltenyi Biotech). T cells (2 × 106) were cocultured at a 1 : 20 ratio with autologous DC loaded with the Influenza matrix peptide (IMP.A2): GILGFVFTL30 or Melan-A.A2 ana: ELAGIGILTV31 (Clinalfa, Darmstadt, Germany; 10 µm each for 1 hr). IL-2 (20 U/ml) was added every other day and T cells were restimulated weekly. After two stimulations T cells were stained with IMP.A2 or Melan-A.A2 specific Tetramers (Proimmune, Oxford, UK). Positive populations were sorted on a FACS VantageTM (Becton Dickinson, San Jose, CA). Cells were cloned and further restimulated with antigen loaded syngeneic DC and an allogeneic irradiated B cell line.

Flow cytometric analysis

For immunofluorescence staining, cells were washed and stained for 20 min at 4° with optimal dilution of each antibody or for 15 min at 37° with optimal dilution of each tetramer. Cells were washed again and analysed by flow cytometry (FACS ScanTM and CELLQuestTM software; Becton Dickinson).

Induction of anergized T cells

To induce anergized antigen-specific T cells, CD8+ CTL or CD4+ T-cell clones were cocultured with CD4+ CD25+ T cells for 5–7 days at indicated ratios. CD4+ CD25+ T cells were isolated as described above and either used ex vivo, or preactivated with plate-bound anti-CD3 (10 µg/ml) and soluble anti-CD28 (10 µg/ml) for 24 hr. The CD4+ CD25+ T cells were either syngeneic to the clone cells used or allogeneic as depicted. In some experiments CD4+ CD25 + Treg were HLA-A*0201+ and the respective clone cells HLA-A*0201 negative. After coculture cells were labelled with the HLA-A*0201-specific AB BB7.2 and sorted with anti mouse microbeads (Miltenyi, Germany). Pure, anergized cloned T cells were then used for further experiments.

Proliferation assays

In order to assess proliferation of the differently cultured CD4+ and CD8+ T cells, 105 sorted T cells were incubated in X-VIVO-20 with 5 × 103 DC in 96-well U-bottomed plates or 10 µg/ml of plate-bound anti-CD3+ 10 µg/ml soluble anti-CD28 in 96-well flat-bottomed plates. For assessment of regulatory properties 105 resting T cells were cultured with 5 × 103 DC in 96-well U-bottomed plates. Purified CD4+ CD25+, CD4+ CD25- T cells and anergized cloned T cells were added at a 1 : 1 ratio unless otherwise indicated. The optimal time point for addition of [3H]Tdr was evaluated for each clone individually. Usually an interval of 2–3 days was found optimal. Therefore after 2–3 days of culture [3H]Tdr (37 kBq/well) was added for an additional 16 hr. Proliferation was measured using a liquid scintillation counter.

Transwell experiments

Transwell experiments were performed in 24-well plates. CD4+ T-cell clones (106) were stimulated with 5 × 104 antigen loaded m-DC. In addition, 106 anergized CD4+ T cells were either added directly to the culture or were placed in transwell chambers and activated with antigen loaded DC (Millicell, 0·4 µm; Millipore). After 5 days of coculture, T cells were transferred to 96-well plates (105 cells/well) in triplicates. Proliferation was measured after 16 hr pulse with [3H]Tdr using a liquid scintillation counter.

Results

CD4+ CD25+ Treg inhibit proliferation of antigen-specific T cells after preactivation

Most studies demonstrating the regulatory potency of CD4+ CD25+ Treg used allo-responses or polyclonal stimuli. Our aim was to determine the effects of CD4+ CD25+ Treg on antigen specific CD4+ and CD8+ clones. These consist of 100% of antigen-specific T cells, which are strongly stimulated by their cognate antigen. If ex vivo isolated CD4+ CD25+ Treg were used, no marked inhibitory effect could be seen even if CD4+ CD25+ Treg and T cells were used at high ratios of 10 : 1 (data not shown). This was true for autologous and allogeneic CD4+ CD25+ Treg alike, indicating that it is not just related to a lack of TCR stimulation with syngeneic DC (Fig. 1 and data not shown). If CD4+ CD25+ Treg were preactivated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hr they showed strong regulatory potency on CD4+ and CD8+ T-cell clones (Fig. 1). This effect was slightly increased when preactivated allo-CD4+ CD25+ Treg were used (Fig. 1). As previously reported, inhibition of CD4+ CD25+ Treg on CD4+ and CD8+ T-cell clones required direct cell contact (data not shown).5,27 Preactivated CD4+ CD25 T cells on the other hand had no inhibitory effects. The experiments shown in Fig. 1 were performed with TT- and IMP-specific clones. Similar results were obtained with KLH- and Melan-A specific clones (data not shown).

Figure 1.

Coculture of antigen specific CD4+ and CD8+ T cell clones with preactivated CD4+ CD25+ T cells results in reduced antigen-specific proliferation. Antigen-specific T cell clones (CD4+ T cells: TT-specific; CD8+ T cell: Influenza matrix peptide A2 (GILGFVFTL) specific) were cocultured with MACS-sorted CD4+ CD25+ and CD4+ CD25 T cells at a 1 : 1 ratio. CD4+ CD25+ T cells were either freshly isolated autologous, or autologous and allogeneic cells preactivated with plate-bound anti-CD3 soluble anti-CD28 (10 µg/ml each) for 24 hr. CD4+ CD25 T cells were used preactivated for control purposes. Cocultures of antigen-specific T cells and CD4+ CD25+ T cells were stimulated with antigen-loaded DC syngeneic to the antigen specific T cells at a 1 : 20 ratio for 2–3 days. [3HT]Tdr (37 kBq/well) was added for an additional 16 hr. One out of five independent experiments with comparable results is shown.

The inhibitory effect of preactivated CD4+ CD25+ Treg is dose dependent

We further titrated the ratio of CD4+ CD25+ Treg: clone cells. Inhibition of CD4+ and CD8+ T-cell clone proliferation was concentration dependent in both cases (Fig. 2). At a 1 : 1 ratio 75% inhibition of CD4+ T-cell proliferation was achieved, whilst around 60% inhibition of CD8+ T-cell proliferation occurred.

Figure 2.

The inhibitory effect of preactivated CD4+ CD25+ T cells on antigen-specific T cells is dose dependent and more pronounced on CD4+ antigen-specific T cells. Antigen specific CD4+ and CD8+ T cells were cocultured with increasing doses of preactivated CD4+ CD25+ T cells and stimulated with syngeneic antigen loaded DC (5 × 103). After 2–3 days proliferation was measured by [3HT]Tdr incorporation. Results were similar in five independent experiments.

CD4+ CD25+ Treg induce a cytokine switch from IFN-γ to IL-10 and TGF-β in CD4+ T-cell clones

In order to investigate the effects of the coculture of preactivated CD4+ CD25+ Treg with antigen specific CD4+ and CD8+ T-cell clones we measured the cytokine levels of IFN-γ, IL-10 and TGF-β after 72 hr of coculture. For this purpose CD4+ CD25+ Treg were activated with anti-CD3 and anti-CD28 for 24 hr. Cells were subsequently harvested and cocultured with the respective clone cells at a 1 : 1 ratio. Culture supernatant was harvested at 72 hr. Usually supernatant was harvested from cultures containing clone cells and CD4+ CD25+ Treg. In some experiments cells were sorted after 72 hr of coculture according to their human leucocyte antigen (HLA) haplotype or their expression of CD4 in order to prove that cytokine production was attributable to the anergized clone cells. CD4+ CD25+ Treg were from HLA-A*0201+ donors, labelled with the specific antibody BB7.2 and sorted with magnetic beads. Less then 1% CD4+ CD25+ T cells were left in culture after this procedure (data not shown). Remaining cells were left to rest for 3–4 days, then restimulated and supernatant analysed for cytokines by ELISA. This effort revealed that changes in cytokine profile were related to the anergized clone cells. Because in the coculture of CD8 + clones and CD4+ CD25+ T cells some IL-10 was always detectable, additional intracellular FACS stainings for IL-10 were performed. Therefore at least 5 days after onset of coculture cells were stimulated with PMA and A24187 or plate-bound anti-CD3 soluble anti CD28 with the addition of monensin. After a surface staining with the anti-CD4 antibody, cells were fixed and permeabilized and stained for IL-10. As shown in Fig. 3 IL-10 could only be detected in the CD4+ CD25+ T cells and not in the CD8 + clone cells. As depicted in Fig. 3, coculture of CD4+ TT-specific T-cell clones resulted in an almost complete shutdown of IFN-γ production with a concurrent upregulation of IL-10 and to a lesser extent of TGF-β production.

Figure 3.

Coculture of CD4+ but not CD8+ antigen-specific T cells with CD4+ CD25+ T cells led to increased secretion of IL-10 and TGF-β. Antigen-specific T-cell clones were either cultured alone or cocultured with preactivated CD4+ CD25+ T cells (TT clone (a)* = anergized TT-specific T cell clone; IMP clone (a)** = anergized IMP.A2-specific CD8+ T-cell clone) or cocultured with preactivated CD4+ CD25 T cells (CD25) and stimulated with antigen-loaded DC. After 72 hr of coculture supernatant was harvested and analysed for IFN-γ, IL-10 and TGF-β by ELISA. One out of five individual experiments is shown. In order to ascertain that IL-10 production in the coculture of CD4+ CD25+ and CD8+ IMP specific cell lines was attributable to the CD4+ T cells, intracellular staining for IL-10 plus surface staining for CD4+ was performed (FACS graph insert).

When coculture of CD4+ CD25+ Treg with CD8+ IMP.A2-specific clones was analysed no induction of IL-10 or TGF-β production was apparent. A greater than 60% reduction of IFN-γ production could be observed (Fig. 3). Coculture of clone cells with preactivated CD4+ CD25 T cells did not induce changes in cytokine pattern (Fig. 3). Similar results were obtained with KLH- and Melan-A-specific clones (data not shown).

IL-10 Production of anergized CD4+ T-cell clones is stable over a long period of time and can not be reverted by restimulation with mature DC

To analyse weather anergized CD4+ clone cells produce IL-10 only transiently we analysed the IL-10 production in the supernatant of cocultures after different time points (1–7 days). These experiments revealed, that IL-10 production peaked after 4 days and then remained essentially stable (Fig. 4a). For proof that anergized CD4+ clone cells are converted into long-time IL-10-producing cells we restimulated them with highly immunogenic mature DC several times. As depicted in Figs 4(b) and 3, restimulations after the primary anergizing coculture with preactivated CD4+ CD25+ T cells did not significantly diminish the IL-10 production. To prove that IL-10 production is related to anergized CD4+ CD25 T cells, in some experiments CD4+ CD25+ and CD4+ CD25 were separated based on their expression of HLA-A*0201 as described for Fig. 3. These results yielded essentially the same results as the unfractionated cells (data not shown).

Figure 4.

Anergized CD4+ clone cells produce IL-10 for a long period of time and their phenotype is not changed after multiple restimulations with mature DC. (a) CD4+ clone cells were cocultured with preactivated CD4+ CD25+ T cells. Supernatant was harvested at the indicated time points and analysed by ELISA for secretion of IL-10. One of five individual experiments with similar results is shown. (b) Anergized CD4+ clone cells were restimulated with mature DC at a 1 : 20 ratio three times. This procedure did not change the IL-10 secretion. One representative out of five independent experiments is shown. Similar results were also obtained with KLH- and Melan-A-specific clones.

CD8+ T-cell clones are stably anergized and impaired in IFN-γ production after coculture with preactivated CD4+ CD25+ T cells

To analyse the anergic state of the antigen-specific CD8+ T cells they were cocultured with preactivated CD4+ CD25+ or CD4+ CD25 T cells. After 3 days cells were separated by magnetic-activated cell sorting (MACS) based on their expression of CD4. CD8+ T cells were left to rest for another 4 days. After that they were partly used for analysis by ELISPOT and proliferation assay (both stimulated with syngeneic DC) or restimulated with antigen loaded DC. This was repeated at weekly intervals three times. The anergic state of antigen specific CD8+ T cells acquired after one 3 day co culture with preactivated CD4+ CD25+ was stable over at least 3 weekly stimulations with mature DC. The same holds true for the impairment of IFN-γ production (Fig. 5).

Figure 5.

CD8+ clone cells remain anergic and defective in IFN-γ production after coculture with CD4+ CD25+ T cells even after multiple restimulations with mature DC. IMP-specific CD8+ clone cells were cocultivated with preactivated CD4+ CD25+ and CD4+ CD25 T cells. After 3 days cells were MACS separated, based on their expression of CD4+. CD8+ T cells were left to rest for 4 days and then analysed. (a) CD8+ T cells (105) were cocultivated with 5 × 103 antigen-loaded DC. Proliferation was assessed 2–3 days after stimulation by [3HT]Tdr incorporation. To analyse IFN-γ production 105 CD8+ T cells were cocultured with 5 × 103 antigen loaded DC in an ELISPOT assay (b). The mean proliferation/IFN-γ production of the same clone not cocultivated with any CD4+ T cell served as a control.

CD4+ anergized clone cells suppress proliferation of other T cells after antigen-specific activation

As it has been reported that coculture of CD4+ CD25+ T cells with other CD4+ T cells confers suppressive potency to the anergized T cells, we analysed our anergized CD4+ and CD8+ T cells for their suppressive capacity. Anergized CD4+ TT-specific T-cell clones showed strong inhibition of other T cells when stimulated with their cognate antigen. This inhibition was not restricted to T cells of the same specificity, but also applied to T cells recognizing a different antigen, such as KLH-specific T-cell clones. When CD4+ clone cells were cocultured with preactivated CD4+ CD25 T cells they did not show any inhibitory capacity (Fig. 6a). Anergized CD8+ T cells did not show any suppressive capacity (Fig. 6b) but retained their anergic phenotype even after multiple restimulations with their cognate antigen + IL-2 (data not shown). Similar results were obtained when Melan-A-specific CD8+ CTL or KLH-specific CD4+ T cells were used (data not shown).

Figure 6.

Anergized CD4+ and CD8+ antigen-specific T cells suppress proliferation of other T cells after antigen specific activation. (a) A TT-specific CD4+ T-cell clone was cocultured with the anergized TT-specific T-cell clone (TT clone (a), e.g. after coculture with CD4+ CD25+ T cells) at a 1 : 1 ratio and activated with syngeneic DC loaded with TT. As a control KLH-specific CD4+ T cells were cocultured with anergized TT-specific T cells and activated with KLH-loaded DC. Furthermore, both CD4+ T-cell clones (TT and KLH) were cultured alone and activated with syngeneic DC loaded with the respective antigen. Additionally, KLH specific T cells were cocultured with anergized TT-specific T cells and activated with DC loaded separately with TT and KLH. For control purposes TT clone cells were also cocultured with preactivated CD4+ CD25T cells. After 2–3 days of culture proliferation was measured by [3HT]Tdr incorporation. One of four independent experiments is shown. (b) Anergized IMP.A2-specific T cells (IMP clone (a)) were cocultured with Melan-A.A2- or IMP.A2-specific T-cell clones each at a 1 : 1 ratio and activated with the respective antigen loaded on syngeneic DC. Additionally, anergized IMP.A2-specific T cells were cocultured with Melan-A.A2-specific T cells and activated with DC loaded separately with Melan-A.A2 and IMP-A2. For control purposes both CTL clones were cultured alone and activated with their cognate antigen loaded on syngeneic DC. [3HT]Tdr incorporation was measured after 2–3 days of culture. One out of four independent experiments with similar results is shown.

Inhibitory capacity of anergized CD4+ antigen-specific T cells is not related to residual activity of CD4+ CD25+ T cells in the culture

In order to prove that the observed effects are not related to residual CD4+ CD25+ T cells in the cultures, HLA-A*0201 positive CD4+ CD25+ Treg cells were cocultured with HLA-A*0201 negative TT specific clone cells. After coculture cells were separated based on their expression of HLA-A*0201 and pure anergized clone cells (less then 1% CD4+ CD25+ T cells left; data not shown) were cocultured with fresh TT-specific clone cells. The inhibitory capacity of anergized TT-specific clone cells could still be observed, while coculture of TT specific clones with CD4+ CD25 T cells did not lead to the induction of suppressive T cells (Fig. 7). To exclude that activation induced cell death was responsible for the effects seen, Propidium iodide staining was performed. No significant increase in apoptosis could be detected.

Figure 7.

Inhibitory capacity of anergized TT clone T cells is not related to residual CD4+ CD25+ T cells. CD4+ CD25+ and CD4+ CD25 T cells of a HLA.A0201 positive donor were preactivated and mixed at a 1 : 1 ratio with TT-specific clone cells in order to prove that the regulatory potential seen after coculture of TT-specific clone cells with preactivated CD4+ CD25+ T cells is caused by the anergized clone cells. After 5 days of coculture CD4+ CD25+ T cells were depleted from the culture by MACS sorting with the HLA.A*0201-specific antibody BB7.2 (<1% HLA.A*0201+ cells left). After a 1 day rest these anergized TT-specific clone cells were mixed with normal TT specific clone cells at a 1 : 1 ratio and activated with syngeneic TT loaded DC. After 2–3 days of coculture proliferation was measured by [3HT]Tdr incorporation. One of four independent experiments is shown. Depletion is indicated by X.

Inhibition of T-cell proliferation by anergized CD4+ T-cell clones is dependent on IL-10 and TGF-β

We performed transwell and blocking experiments with neutralizing antibodies for IL-10 and TGF-β in order to analyse which components contribute to the inhibitory action of anergized CD4+ T cells. If anergized T-cell clones and responder CD4+ T cells were separated by a semipermeable membrane, allowing free passage of soluble mediators but no direct cell contact, no reduction in inhibition was observed, suggesting that direct cell contact is not required. Blocking antibodies towards IL-10, on the other hand, showed marked reduction of inhibition. When anti-TGF-β antibodies were applied the effect was less pronounced. Combination of both antibodies abrogated inhibition almost completely, suggesting that these two cytokines predominantly mediate suppression of anergized T-cell clones (Fig. 8).

Figure 8.

The suppressive capacity of anergized clone cells is dependent on IL-10 and TGF-β. To further elucidate the mechanisms by which regulation of anergized TT clone cells is mediated, TT clone cells were cocultured with anergized TT clone cells [TT(a)] and activated with TT-loaded DC, in the presence or absence of neutralizing anti IL-10 antibodies, anti TGF-β antibodies or a mixture of both (10 µg/ml each). In a parallel transwell experiment the anergized TT clone cells and the normal TT clone cells were separated by a semipermeable membrane (indicated by ⊔) and activated with TT-loaded DC from the same batch separately. After 2–3 days proliferation was measured by [3HT]Tdr incorporation. One representative out of four independent experiments is shown. X denotes the presence of the indicated component.

Discussion

The present data demonstrate that antigen-specific CD4+ and CD8+ clone cells can be suppressed by preactivated CD4+ CD25+ Treg cells in vitro. Anergized CD4+ T-cell clones undergo a switch in cytokine production, from the predominant secretion of IFN-γ to the preferential secretion of IL-10 and to a lesser extend TGF-β. Through this cytokine pattern they are able to suppress other T-cell proliferation. This mechanism quite surprisingly does not apply to CD8+ T cells. Anergized CD8+ T cells reduce their IFN-γ production, without the induction of IL-10 or TGF-β secretion and are not able to inhibit other T-cell functions, but keep their anergic phenotype despite restimulation with their cognate antigen and IL-2.

It has been convincingly demonstrated that Treg play a dominant role in the maintenance of peripheral tolerance in mice.32 The best characterized amongst Treg cells are CD4+ CD25+ T cells, so-called naturally occurring or constitutional Treg cells.33 Lately their prominent role in establishing peripheral tolerance has been also shown in humans. Functional properties seem to be very similar in the two species.33

It is known that CD4+ CD25+ Treg suppress CD4+ and CD8+ T cells in polyclonal or allospecific assays.5 In contrast, suppression of antigen-specific clones by CD4+ CD25+ T cells has been assumed, based on mouse studies,34,35 but never been formally demonstrated in humans. No inhibition could be observed if syngeneic CD4+ CD25+ Treg, isolated ex vivo were added to cultures of antigen specific clonal CD4+ and CD8+ T cells. This data is not surprising as T-cell clones consist to 100% of antigen-specific T cells, that all become immediately activated by their cognate antigen and syngeneic ex vivo isolated Treg will not be activated strongly enough to mediate suppression. This is especially true if DC are loaded with a HLA class I restricted peptide, but also if mature DC are loaded with protein (TT), that will be presented via HLA class II. As there is a concentration dependent effect of CD4+ CD25+ T cell suppression and only few CD4+ CD25+ Treg isolated ex vivo will be activated by the presented antigen, the ratio of suppressor: target might simply be too low. Upon activation of CD4+ CD25+ Treg by a polyclonal stimulus prior to the inhibition assays, a strong reduction in CD4+ T-cell clone proliferation could be observed. This inhibition was concentration dependent as reported for polyclonal/allospecific readout systems.5,7 If allogeneic CD4+ CD25+ Treg were used, the inhibitory effect was slightly increased probably because additional activation of CD4+ CD25+ Treg occurred via allostimulation.

Cytokine analysis revealed that CD4+ and CD8+ T cells unexpectedly behave differently after contact with CD4+ CD25+ Treg. CD8+ T-cell IFN-γ secretion is highly impaired, with a prolonged anergic phenotype. CD4+ T cells, on the other hand, shut down their IFN-γ production completely whilst up-regulating IL-10 production strongly and TGF-β production slightly. These changes in cytokine production of CD4+ T cells are permanent, as they are not reversible by restimulation with allomature DC. IL-10 secretion starts at about 3 days of coculture, peaks at 4 days and then remains stable. These findings are in line with prior observations obtained in polyclonally or allospecifically activated T-cell assays.22,23

We could also show that CD4+ T cells become suppressor cells after contact with CD4+ CD25+ Treg, which is not true for CD8+ T cells. More importantly, we could demonstrate that anergized CD4+ T-cell clones retain their antigen specificity despite their functional changes. If these anergized CD4+ T-cell clones were activated with their cognate antigen they suppressed proliferation of other T cells. In this respect action of anergized CD4+ T-cell clones is antigen specific. The suppression however, applies to any T cell regardless of their specificity, as demonstrated by the suppression of KLH-specific ‘bystander’ CD4+ T cells in our assay. The inhibitory function of anergized CD4+ T cells is dependent on secreted cytokines, predominantly IL-10 and to a lesser extend TGF-β, as demonstrated by blocking and transwell experiments. This is also in line with prior findings which were however, not obtained with T cells specific for nominal antigens.22,23

In conclusion we showed, that CD4+ CD25+ Treg inhibit clonal T-cell proliferation only after preactivation caused by numeric effects. Drastic changes in cytokine production can only be observed in CD4+ T cells. This is in line with the finding that only CD4+ T cells, after coculture with CD4+ CD25+ Treg, become suppressor cells. These suppressive CD4+ T cells retained their antigen specificity as they secreted IL-10 and TGF-β and inhibited T-cell function only after stimulation with their cognate antigen. These findings suggest a predominant effect of CD4+ CD25+ Treg on antigen-specific CD4+ T cells. It will be interesting to witness, whether this holds also true in vivo in humans, as current efforts try to employ the depletion of CD4+ CD25+ Tregs in humans to enhance vaccine efficacy in cancer, primarily by using HLA class I-restricted peptides aiming at the induction of antigen-specific CD8+ T cells.

This study extends the knowledge on CD4+ CD25+ Treg function in several ways. First, we now show that activated CD4+ CD25+ Treg can suppress both CD4+ and CD8+ T cells specific for nominal antigens by direct cell contact. Second, we demonstrate that antigen-specific CD4+, but not CD8+ T cells anergized by activated CD4+ CD25+ Treg, switch from IFN-γ to IL-10 and TGF-β production and acquire suppressive activity, which does not require cell–cell contact and is mediated by the combined action of IL-10 and TGF-β. Third, we suggest that our findings may not only be relevant for immunobiology and immunopathology, but might also be important in clinical terms for the use of CD4+ CD25+ Treg to treat autoimmune diseases. Now it might be possible to generate specific CD4+ T-cell lines, anergize them with preactivated CD4+ CD25 + Treg in vitro and adoptively transfer them to the patient.

Acknowledgement

This study was supported by the German science foundation (grant No. DI 895/1-1 to D.D.).

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