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Keywords:

  • T lymphocyte;
  • Tolerance/suppression/anergy;
  • IL-2 receptor;
  • Cytokines;
  • Costimulation

Abstract

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods

The in vivo differentiation/survival of CD4+CD25+ T suppressor cells is dependent on IL-2 and CD28-mediated costimulatory signals. To determine the cytokine and costimulatory requirements for CD25+ T cells in vitro, we established a two-stage culture system where CD25+ T cells were activated in a primary culture. In the subsequent culture, activated CD4+CD25+ cells were then mixed with responders in order to assess their suppressor function. Pre-culture of CD25+ T cells with anti-CD3 alone resulted in poor survival and minimal induction of suppressor activity. Pre-culture in the presence of anti-CD3 and IL-2 or IL-4, but not IL-6, IL-7, IL-9, IL-10 or IL-15, resulted in proliferation of the CD25+ cells and induction of potent suppressor function. Inhibition of the interaction of CD28 or cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) with CD80/CD86 in the pre-culture of CD4+CD25+ cells did not prevent the induction of suppressor function. Furthermore, the inhibition of costimulatory signals did not inhibit the ability of fresh CD25+ T cells to inhibit CD8+ responders under conditions where activation of the responders was independent of CD80/CD86. These studies support the view that activation of CD25+ T cells requires IL-2/IL-4 for their survival/differentiation into effector cells, but is independent of CD28/CTLA-4-mediated costimulation.

Abbreviations:
IBD:

Inflammatory bowel disease

AIG:

Autoimmune gastritis

Tg:

Transgenic

WT:

Wild type

CTLA-4:

Cytotoxic T lymphocyte-associated antigen-4

HA:

Hemagglutinin

1 Introduction

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods

CD4+CD25+ T cells are potent immunoregulatory cells that not only suppress T cell proliferation in vitro, but also have the capacity to suppress immune responses to auto- and alloantigens, tumor antigens, and infectious agents in vivo. However, the mechanism by which these cells mediate suppression remains elusive. In vitro studies of CD4+CD25+ T cells 1, 2 have demonstrated that CD4+CD25+ T cells require stimulation through their TCR in order to be suppressive. Suppression is cell contact-dependent, cytokine-independent, and results in inhibition of IL-2 production by the CD4+CD25 responders. In contrast, CD4+CD25+T cells exert their effects in vivo by both cytokine-independent and cytokine-dependent mechanisms 35.

Activation of naive CD4+CD25 T cells requires two signals: one through the TCR by interaction with MHC/peptide and a second through costimulation delivered by APC 6, 7. The primary costimulatory signal is mediated through CD28 upon binding to CD80 or CD86 expressed on APC. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152), a homologue of CD28 that also binds both CD80/CD86, is induced upon T cell activation and negatively regulates T cell activation. CD4+CD25+ cells require activation through their TCR in order to acquire suppressive activity, but once activated, their suppressor effector function is completely nonspecific and does not require re-engagement of their TCR 1, 8. The nature of the costimulatory signals required for activation of CD4+CD25+ suppressor effector function is unknown. Surprisingly, CD4+CD25+ are the only cells in the naive animal that express detectable levels of CTLA-4 in the absence of activation 911. This finding raised the question of whether the expression of CTLA-4 on CD4+CD25+ T cells is of functional importance.

Takahashi et al. 10 have reported that the inhibition of proliferation seen when CD4+CD25 T cells are co-cultured with CD4+CD25+ cells could be abrogated by anti-CTLA-4 mAb or by its Fab fragment. In addition, autoimmune gastritis (AIG) could be induced in normal BALB/c animals by treatment with anti-CTLA-4 in the presence or absence of a depleting anti-CD25 mAb, and Read et al. 11 have demonstrated that the CD4+CD25+ T cell-mediated inhibition of inflammatory bowel disease (IBD) induced by transfer of CD45RBhi T cells to severe combined immunodeficiency (SCID) recipients can be abrogated by administration of anti-CTLA-4 mAb. Both of these studies concluded that engagement of CTLA-4 on CD4+CD25+ T cells by its target ligands plays a critical role in the activation of the suppressive function of CD4+CD25+ T cells. However, in both studies, it was difficult to rule out the possibility that anti-CTLA-4 acted, in part, upon the CD25 responder cells to decrease their threshold for activation rendering them more resistant to CD25-mediated suppression. In addition, these findings are quite contrary to the widely accepted concept that engagement of CTLA-4 is coupled to a biochemical pathway that induces a negative signal for T cell activation 6, 7.

We have recently developed a two-step culture system in which the CD4+CD25+ cells are pre-activated in the absence of CD4+CD25 T cells 8. Here, we use this system to examine the contribution of both cytokines and the CD28-CTLA-4/CD80-CD86 pathway in the activation of CD4+CD25+ T cells. We demonstrate that IL-2 or IL-4, but not other cytokines, are needed together with the TCR stimulus to generate potent suppressor activity. Furthermore, we demonstrate that activation of CD4+CD25+ T cells to exert potent suppressor function is completely independent of the engagement of CD28/CTLA-4 with CD80/CD86.

2 Results

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods

2.1 Temporal requirements for activation of CD25-mediated suppressor effector function

CD4+CD25+ T cells, purified from wild-type (WT) mice, were activated with soluble anti-CD3 and APC in the presence of IL-2 for 3 days and then expanded in media containing IL-2 for an additional 4 days 8. These 7-day-activated CD4+CD25+ cells were harvested and co-cultured with CD4+CD25 T cells from mice expressing a transgenic (Tg) TCR in the presence of APC and the appropriate peptide. In order to determine how quickly the suppressive capabilities of CD4+CD25+ cells are acquired, CD4+CD25+ cells from BALB/c mice were pre-activated for either 1, 2, or 3 days before assaying their suppressive activity on the proliferative responses of CD4+CD25 T cells from mice expressing a Tg TCR specific for hemagglutinin (HA). In the presence of IL-2 alone, the recovery of CD4+CD25+ cells was 75% of the input at day 3 (Fig. 1A). However, these cells were not suppressive (data not shown).

The CD4+CD25+ T cells were also cultured with soluble anti-CD3 and APC or with plate-bound anti-CD3 in the presence or absence of IL-2. In the absence of IL-2, recovery was very poor and decreased with time. The recovered cells were small and appeared pre-apoptotic. When these few remaining cells were assayed for their suppressive function, they were minimally suppressive at the highest number of cells tested (data not shown). When CD4+CD25+ cells were activated in the presence of IL-2, either with soluble or plate-bound anti-CD3, the cells appeared healthy and blast formation was observed as early as day 1 of culture. When freshly isolated CD4+CD25+ cells were co-cultured with HA TCR Tg CD4+ cells stimulated with peptide, no suppressive activity was detected, similar to our previously published results with responder cells from DO11.10 mice 8. However, CD4+CD25+ T cells, activated in the presence of IL-2, acquired their full suppressive capacity between 2 and 3 days (Fig. 1B, C). These cells were extremely potent suppressors as 85–95% suppression was seen at suppressor-to-responder ratios of 1:8. More detailed studies of the activation requirements for CD25-mediated suppressor function were therefore performed by pre-culturing the CD4+CD25+ T cells under various conditions for 3 days prior to mixing them with CD4+CD25 T cells.

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Figure 1. Time course for induction of CD4+CD25+ suppressor effector cells. (A) CD4+CD25+ cells were stimulated with soluble (sol) anti-CD3 and APC or plate-bound (pb) anti-CD3 (5 μg/ml) in the absence or presence of IL-2 (100 U/ml). Cells were harvested on the indicated days. Results are expressed as percent recovery of CD4+CD25+ cells compared to input. (B, C) Harvested cells, stimulated in the first culture with soluble (sol) anti-CD3, APC and IL-2 for the indicated times (B) or plate-bound (pb) anti-CD3 and IL-2 for the indicated times (C), were co-cultured with HA TCR Tg CD4+ cells stimulated with APC and HA peptide. Results are expressed as percent suppression based on counts for CD4+ T cells without CD4+CD25+ cells for that time point.

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2.2 Induction of CD25-mediated suppressor function requires relatively high concentrations of IL-2

As shown in Fig. 1, IL-2 is required for the proliferation of CD4+CD25+ cells as well as the induction of suppressor function. However, the concentration of IL-2 used in the pre-activation step was most likely maximal. In order to further examine the role of IL-2 in the induction of suppressor effector function versus its role in proliferation, we compared the concentration of IL-2 needed for induction of proliferation of CD4+CD25+ T cells with the concentration of IL-2 needed for induction of suppressor activity.

In the standard proliferation assay, CD4+CD25+ cells were non-responsive when stimulated with soluble anti-CD3 and APC in the absence of IL-2. The maximum proliferative response was typically seen at 12.5 U/ml (Fig. 2A). CD25-mediated suppression of proliferation could be abrogated when relatively low concentrations (6.25 U/ml) of IL-2 were added to co-cultures of freshly isolated CD25+ and CD25 T cells (Fig. 2B). However, in the pre-activation step of the two-stage assay, maximum recovery typically required higher concentrations (25 U/ml) of IL-2 (Fig. 2C). Higher concentrations (50–100 U/ ml) were also required to generate cells with potent suppressive function when mixed with HA TCR Tg responders in the second culture (Fig. 2D). With most IL-2 concentrations, potent suppressor function was noted at the higher ratios of CD4+CD25+ cells to HA TCR Tg CD4+ cells, but significant differences in the potency of suppression were clearly observed at ratios of 1:32. These results suggest that IL-2 may play distinct roles in stimulating the proliferation of CD25+ cells and in generating their suppressor function.

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Figure 2. IL-2 exerts multiple effects on CD4+CD25+ T cells. (A) CD4+CD25 cells or CD4+CD25+ cells were stimulated with anti-CD3 and APC in the presence of the indicated IL-2 concentrations. (B) CD4+CD25 cells co-cultured without or with freshly isolated CD4+CD25+ cells were stimulated with anti-CD3 and APC in the presence of the indicated IL-2 concentrations. (C) CD4+CD25+ cells were stimulated with anti-CD3 and APC in the presence of the indicated IL-2 concentrations. Recovery of cells after 3 days is indicated as a percentage of cells initially plated. (D) Cells harvested in (C) were co-cultured with HA TCR Tg CD4+ cells stimulated with APC and HA peptide.

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2.3 Suppressor function can be generated with cytokines other than IL-2

We next determined whether other cytokines had the ability to promote the proliferation of CD4+CD25+ cells and the generation of suppressor function. In a standard proliferation assay, CD4+CD25+ cells proliferated when stimulated with soluble anti-CD3 in the presence of IL-4 and to a small degree in the presence of IL-7 (Fig. 3A). Both IL-4 and IL-7 were able to abrogate suppression when added to co-cultures of freshly isolated CD25+ and CD25 T cells (Fig. 3B).

To determine whether the proliferation of CD4+CD25+ cells correlated with the induction of suppressor function, we used our two-stage assay system. CD4+CD25+ cells from normal BALB/c mice were stimulated for 3 days with anti-CD3 in the presence of cytokines, harvested and co-cultured with TCR Tg CD4+ cells. Only IL-2 and IL-4 promoted the generation of potent suppressors (Fig. 3C). In some experiments, culture of the CD25+ T cells in the presence of anti-CD3 and IL-6, IL-7, IL-15 and IL-9 resulted in the generation of modest suppressor activity when higher numbers of the activated suppressors were added to the responders. In multiple studies, only the addition of IL-7 consistently resulted in the generation of suppressors and these were never as potent as those generated with IL-2 or IL-4. The capacity of IL-10 and TFG-β to generate suppressor function was also tested; however, viable cells were not recovered.

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Figure 3. IL-2 and IL-4 are the most efficient inducers of proliferation and suppressor effector function. (A) CD4+CD25– cells or CD4+CD25+ cells were stimulated with anti-CD3 and APC in the presence of the indicated cytokines. (B) IL-2 and IL-4 reverse CD4+CD25+ T cell-mediated suppression. CD4+CD25 cells or CD4+CD25 cells co-cultured with freshly isolated CD4+CD25+ cells were stimulated with anti-CD3 and APC in the presence of the indicated cytokines. (C) CD4+CD25+ cells were activated with anti-CD3 and APC in the presence of the indicated cytokines for 3 days. Cells were harvested and co-cultured with HA TCR Tg CD4+ cells stimulated with APC and HA peptide.

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2.4 Costimulatory requirements for the induction of CD4+CD25+ suppressor function

We previously demonstrated that addition of anti-CTLA-4 (10 μg/ml) to co-cultures of CD25+ and CD25 T cells failed to abrogate suppression 1. However, Takahashi et al. subsequently demonstrated that addition of higher concentrations (100 μg/ml) of anti-CTLA-4 or very high concentrations (300 μg/ml) of its Fab fragment significantly diminished suppression 10. They hypothesized that the use of lower concentrations of anti-CTLA-4, rather than blocking the interaction of CTLA-4 with its ligands, facilitated cross-linking of the antibody bound to the CD25+ T cells by FcR-bearing APC and resulted in actual enhancement of suppressor function. We therefore repeated these studies using commercial preparations of two anti-CTLA-4 mAb (4F10 and 9H10), as well as our own preparation of 4F10 purified on a Protein A column from ascites.

Although all of these preparations could block the binding of PE-conjugated anti-CTLA-4 to CTLA-4-expressing target cells, none was capable of reversing suppression at any concentration tested. However, two independently purified preparations of 4F10 purified on protein G columns partially reversed CD4+CD25+ T cell-mediated suppression (Fig. 4A) in a standard proliferation assay. Curiously, when co-cultures were stimulated with a low concentration (0.5 μg/ml) of anti-CD3, only modest abrogation of suppression was noted at the highest concentration of anti-CTLA-4 tested. However, when the cells were stimulated with a high concentration (10 μg/ml) of anti-CD3, more marked abrogation of suppression was observed even though the magnitude of stimulation and the extent of suppression were similar at both concentrations of anti-CD3 used (Fig. 4B). As Takahashi et al. observed more significant reversal of suppression with Fab fragments of anti-CTLA-4, we prepared Fab fragments of our active preparation. Addition of very low concentrations of these Fab fragments resulted in marked abrogation of suppression at both concentrations of anti-CD3 (Fig. 4C, D). However, interpretation of these results is complicated as addition of the Fab fragments also significantly augmented the proliferative responses of the responder CD4+CD25 T cells cultured alone.

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Figure 4. Does anti-CTLA-4 mAb reverse suppression? (A, B) BALB/c CD4+CD25 cells (squares), CD4+CD25+ cells (diamonds) or CD4+CD25 cells co-cultured with CD4+CD25+ cells (circles) were stimulated with 0.5 μg/ml (A) or 10 μg/ml (B) anti-CD3 and APC in the presence of the indicated amounts of anti-CTLA-4 (4F10). (C, D) BALB/c CD4+CD25 cells (squares), CD4+CD25+ cells (diamonds) or CD4+CD25 cells co-cultured with CD4+CD25+ cells (circles) were stimulated with 0.5 μg/ml (C) or 10 μg/ml (D) anti-CD3 and APC in the presence of the indicated amounts of anti-CTLA-4 Fab fragments.

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2.5 Costimulatory requirements for the induction of CD4+CD25+ suppressor effector function

The previous experiments indicate that results of studies in which anti-CTLA-4 or its fragments are added to co-cultures of CD25+ and CD25 T cells are difficult to interpret as they are complicated by the potential effects of the antibody on the suppressors, the responders, or both populations. As an alternative approach to determining the role of CTLA-4 in the activation of CD4+CD25+ suppressor function, we used our two-stage assay system and added anti-CTLA-4 or other reagents that inhibit the CD28-CTLA-4/CD80-CD86 interactions to the pre-activation cultures of BALB/c CD4+CD25+ T cells that were activated by anti-CD3, APC, and IL-2 (100 U/ml). The activated suppressors were then harvested, washed, and cultured in the second step with fresh TCR Tg CD4+CD25 responders that were stimulated with fresh APC and peptide in the absence of inhibitors.

In the absence of costimulation blockade, activated CD4+CD25+ T cells suppressed the responses of the HA-specific responders by >95% (Fig. 5A). None of the inhibitory reagents prevented the induction of suppressor effector function, even when lower numbers of activated suppressors were added to the second culture (data not shown). Furthermore, suppressors generated with lower concentrations (25 U/ml and 10 U/ml) of IL-2 in the presence of anti-CD80/CD86 were as effective as suppressors generated in the absence of costimulatory blockade (Fig. 5B). In contrast to the in vivo requirement for CD28 12, under these in vitro conditions, no role for CD80/CD86-CD28/CTLA-4 interactions for the induction of suppressor activation could be demonstrated.

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Figure 5. Anti-CTLA-4 mAb does not inhibit the generation of CD4+CD25+ suppressor effector cells in the two-step culture system. (A) BALB/c CD4+CD25+ cells were stimulated with anti-CD3, APC and IL-2 (100 U/ml) for 3 days in the presence of the indicated reagents. In the second culture, pre-activated cells from the first culture were co-cultured with HA TCR Tg CD4+CD25 cells, APC and HA peptide. (B) BALB/c CD4+CD25+ cells were stimulated with anti-CD3, APC and various doses of IL-2 for 3 days in the presence of anti-CD80/CD86. In the second culture, pre-activated cells from the first culture at the indicated number were co-cultured with HA TCR Tg CD4+CD25 cells, APC and HA peptide.

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2.6 Activation of CD4+CD25+ cells is independent of CD80/CD86 costimulation

The nature of the costimulatory signal for T cell activation delivered by the interaction of CD80/CD86 with CD28 is complex 13. In some studies, increasing the strength of the TCR signal can overcome a lack of costimulation, while in others the addition of exogenous IL-2 can readily replace costimulation. We were still concerned that the presence of IL-2 in our assay, which is required for the proliferation and survival of CD4+CD25+ cells, could remove the need for costimulation. It was therefore of interest to directly compare the costimulatory requirements for induction of proliferation of CD25+ and CD25 T cells in our culture system that uses a relatively low concentration of anti-CD3. In the presence of CD80/CD86–/– APC, the addition of a high concentration of IL-2 failed to promote proliferation of CD25 responders, while the response of CD25+ T cells was identical either in the presence of WT or CD80/CD86–/– APC (Fig. 6). Thus, IL-2, in our system, does not substitute for costimulation of CD4+CD25 cells. These studies demonstrate that the requirements for the induction of T cell proliferation, even in the presence of IL-2, are distinct for CD25+ and CD25 T cells.

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Figure 6. Activation of CD4+CD25+ cells is independent of CD80/CD86 costimulation. BALB/c CD4+CD25 cells or CD4+CD25+ cells were stimulated with anti-CD3 in the presence of IL-2 (100 U/ml). APC were either from WT BALB/c mice or from CD80/CD86–/– mice.

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2.7 CD4+CD25+ T cell-mediated suppression of CD8+ T cells does not require CTLA-4

Although these studies strongly suggest that activation of suppressor effector function is completely independent of the CTLA-4/CD28 pathway, it is still possible that the addition of IL-2, which is required for generation of activated suppressors (Fig. 2), in some manner substitutes for a CTLA-4- or CD28-derived costimulatory signal for activation of suppressor activity. CD4+CD25+ T cells suppress the proliferation and the production of IFN-γ by CD8+ T cells 14. As the activation of CD8+ T cells is less dependent on CD28-derived costimulatory signals than the activation of CD4+ T cells, we examined the effect of completely blocking the CD28/CTLA-4 pathway in CD4+CD25+ T cell-mediated suppression of CD8+ T cell proliferation.

When CD8+ T cells were activated in the presence of CD80/CD86–/– APC, the proliferative response was ∼ 50% of that seen in the presence of WT APC (Fig. 7A); nevertheless, both responses were markedly inhibited by the addition of CD4+CD25+ T cells. The addition of anti-CTLA-4 Fab alone enhanced the proliferative response of CD8+ responders in the absence of CD25+ suppressors (Fig. 7B), but this enhanced response was suppressed by the addition of CD4+CD25+ T cells to the same extent as that seen in the absence of the anti-CTLA-4 Fab. When anti-CD80/CD86 was combined with anti-CTLA-4, the response of the CD8+ T cells was inhibited by 50–60% (Fig. 7C). However, the residual response in the presence of costimulatory blockade was still substantial and was readily inhibited by CD4+CD25+ T cells. These results demonstrate that engagement of CTLA-4 by its target ligands expressed on APC or on activated T cells is not required for activation of CD4+CD25+ T cell-mediated suppression of CD8 responses.

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Figure 7. CD4+CD25+ T cell-mediated suppression of the proliferative response of CD8+ cells is independent of costimulation through CD28 or CTLA-4. (A) C57BL/6 CD8+ cells were stimulated with anti-CD3 (1.0 μg/ml) and either WT or CD80/CD86–/– APC and were co-cultured with titrated numbers of CD4+CD25+ cells. (B) C57BL/6 CD8+ cells were stimulated with anti-CD3 (1.0 μg/ml) and APC (5×104) and were co-cultured with titrated numbers of CD4+CD25+ cells in the presence of control antibody or anti-CTLA-4 Fab fragment. (C) C57BL/6 CD8+ cells were stimulated with anti-CD3 (1.0 μg/ml) and APC and were co-cultured with titrated numbers of CD4+CD25+ cells in the presence of control antibody or anti-CTLA-4 Fab fragment plus anti-CD80/CD86 mAb.

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3 Discussion

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods

CD4+CD25+ T cells have emerged as one of the major populations of T regulatory cells that exhibit potent suppressor activity in vivo and in vitro. CD4+CD25+ T cells fail to proliferate when stimulated via their TCR even in the presence of a costimulatory signal delivered by anti-CD28. The basis for this non-responsive state is the failure of CD25+ T cells to transcribe their IL-2 gene. However, CD4+CD25+ T cells will readily proliferate when stimulated via the TCR in the presence of exogenous IL-2 and this property has facilitated functional studies of CD25+ T cells that have been expanded in vitro. In vitro activated CD4+CD25+ suppressor effector T cells remain non-responsive to TCR stimulation, have enhanced suppressor activity, and do not require re-engagement of their TCR to manifest suppressor activity.

We have analyzed in some detail the requirements for growth, expansion, and differentiation of CD4+CD25+ T cells to the suppressor effector stage in vitro. Although CD25+ T cells proliferate when triggered via their TCR in the presence of IL-2, about tenfold higher concentrations of IL-2 are required to generate a level of proliferation equivalent to that seen with CD4+CD25 T cells. One explanation for this relative non-responsiveness is the selective induction of high levels of suppressor of cytokine signaling (SOCS) gene expression in the CD25+ population upon activation 15, 16. Furthermore, the concentration of IL-2 needed for induction of suppressor effector function was two- to threefold higher than that needed for the generation of a maximum proliferative response. This result suggests that IL-2 may also function as a differentiation factor for the induction of T suppressor function in vitro.

IL-4 and, to a much lesser extent, IL-7 were capable of inducing proliferation of CD25+ T cells. Although the receptors for IL-2, IL-4, IL-7, IL-9, and IL-15 all use the γ common chain in their receptor complex 17, only IL-4 was consistently similar to IL-2 in its effects on CD4+CD25+ cells. IL-6, although being an effective survival factor for T cells 18, 19, had no effect on CD4+CD25+ T cells. Notably, both IL-10 and TGF-β have been reported to induce suppressor T cells 20, 21. However, using purified CD4+CD25+ cells, we were unable to elicit a proliferative response or generate suppressors in the presence of IL-10 or TGF-β. In fact, cells cultured in the presence of TGF-β were not viable after 3 days and could not be assayed for suppressive function. CD4+CD25+ T cells also appear to differ in their growth requirements in vitro from human T regulatory type 1 clones that preferentially respond to IL-15 in vitro22.

When we compared the suppressive activity of cells grown in the presence of different cytokines, only cells grown in the presence of IL-2 and IL-4 were potent suppressor effector cells. Modestsuppressor activity was seen when cells were expanded in the presence of IL-7, but only at the highest cell concentrations tested. Although cells grown with the other cytokines or with anti-CD3 alone manifested some suppressor activity at the highest concentrations tested, recovery of the activated cells was very low under these conditions; it also remains possible that small amounts of IL-2 were produced in these cultures by contaminating CD25 T cells or CD25+ effector cells. The requirement for IL-2 or IL-4 for development of suppressor activity in the two-stage culture system is somewhat surprising in view of the ability of fresh CD25+ T cells to suppress proliferation of CD25 T cells in co-culture studies. Although CD25+ T cells suppress the transcription of the IL-2 gene in the co-cultures, under most conditions suppression is not complete. An intriguing possibility is that a small amount of IL-2 (or IL-4) must first be produced bythe CD25 T cells and is required for induction of CD25-mediated suppression. In vivo during an ongoing inflammatory response, the required cytokines would be produced by the activated effectors.

The second related question addressed in this study was the role of the CD28-CTLA-4/CD80-CD86 pathway in the regulation of CD4+CD25+ T suppressor function in vitro. IL-2 is critical for the differentiation and or maintenance of CD4+CD25+ T cells in vivo as mice with deficiencies in IL-2 production, IL-2 responsiveness, or costimulatory molecules have been reported to have both quantitative and qualitative deficiencies in CD4+CD25+ T cells 9, 2325. Furthermore, in contrast to CD4+CD25+ T cells from WT mice, CD4+CD25+ T cells from IL-2R β-chain–/– mice did not expand or prevent autoimmune disease when transferred into IL-2–/– recipients 26. More recent studies have suggested that a strong TCR stimulus together with a maximal CD28-derived costimulatory signal are required for the development of CD25+ T cells in the thymus and perhaps for their maintenance in the periphery 27. IL-2 cannot be substituted for this CD28 signal. Although signaling through CD28 appears to be required for the development of CD4+CD25+ cells, their activation requirements, once in the periphery, have not been examined. It is therefore interesting that we can readily expand and induce the differentiation of potent suppressor T cells in vitro in the absence of CD28 signaling as blocking costimulation with anti-CD80/CD86, CTLA-4-Ig, or the use of CD80/CD86–/– APC had no effect on the generation of suppressor activity in vitro.

Takahashi et al. 10 and Read et al. 11 have proposed that CTLA-4 functions as a costimulatory molecule on CD4+CD25+ T cells for induction of suppressor activity. We have previously been unable to reverse suppression in vitro by the addition of anti-CTLA-4 to co-cultures of CD4+CD25+ and CD4+CD25 T cells in response to both anti-CD3 and peptide antigens (1, and data not shown). In our current studies, significant variation in the capacity of anti-CTLA-4 to inhibitsuppression was seen with different preparations of antibody. With intact antibody preparations that reversed suppression, we reproducibly observed that suppression was only abrogated in the presence of both high concentrations of anti-CD3 and anti-CTLA-4. This result is surprising, as one might have predicted that suppression would be more easily reversed at low concentrations of the stimulatory mAb. As reported by Takahashi et al. 10, reversal of suppression was readily observed when Fab fragments were used. However, CD4+CD25 cells stimulated in the presence of anti-CTLA-4, and more dramatically in the presence of its Fab fragments, exhibited a higher proliferative response (Fig. 4). Thus, in the co-culture, it is possible that anti-CTLA-4 blocks the interaction of CD80/CD86 with CTLA-4 at the level of the responder CD25 T cells and thereby raises the threshold required for CD4+CD25+ T cells to mediate suppression.

Takahashi et al. 10 attempted to determine the cellular site of action of anti-CTLA-4 using responder CD4+CD25 T cells from CLTA-4–/– mice and CD4+CD25+ T cells from WT mice. Reversal of suppression was seen in these co-cultures, but only in the presence of very high concentration of the anti-CTLA-4 Fab fragment. However, they also reported that CD25+ T cells from CTLA-4–/– mice did exhibit significant suppressor activity. We have not used either suppressors or responders from CTLA-4–/– miceas they develop autoimmune disease at a very early age and it is difficult to use CD25 as marker for regulatory T cells under these conditions. As CD4+CD25+ T cells can readily suppress the activation of CD8+ responders and activation of CD8+ T cells is much less dependent on CD28-derived costimulatory signals, we assayed the ability of CD4+CD25+ T cells to suppress the responses of CD8+ T cells under conditions of costimulatory blockade. The proliferative responses of CD8+ T cells were reduced by 50% when they were cultured in the presence of CD80/CD86–/– APC, CLTA-4-Ig, or anti-CD80/CD86. However, the residual proliferative responses were still highly significant and were readily inhibited by CD4+CD25+ T cells.

The effects of anti-CTLA-4 in reversing suppression in vivo were not addressed in our studies although we have not observed reversal of CD25-mediated suppression of AIG using anti-CTLA-4 using a protocol similar to that used by Read et al. 11 to reverse suppression of IBD. The pathogenesis of AIG and IBD are quite distinct as bacteria play no role in the former and CD4+CD25+ T cells from IL-10-deficient mice protect from disease 3, 28, while the induction of IBD requires the presence of intestinal flora and CD4+CD25+ T cells from IL-10-deficient mice do not protect 3, 4, 29. Thus, it remains possible that engagement of CTLA-4by CD80/CD86 may be required, not for the initial activation of the CD4+CD25+ suppressors, but at some later time point during the generation of IL-10-producing T cells in the IBDmodel. All of the studies on the reversal of suppression in vivo are difficult to interpret because the effects of the anti-CTLA-4 may be mediated on the CD25 effector cells. Although some studies did not observe enhanced disease when recipients of effector cells alone were treated with anti-CTLA-4 11, other studies have reported significant disease enhancement 30.

Taken together, we conclude from our results on pre-activation of CD4+CD25+ T cells in the absence of responder T cells, as well as our studies using fresh CD25+ T cells in co-cultures, that CTLA-4 is not required for effector function, nor is the activation of suppressor effector function in vitro dependent on costimulation mediated by CD28/CTLA-4 interactions with CD80/CD86. We cannot exclude that some of the effects of anti-CTLA-4 or its Fab fragments were actually mediated by interaction with CTLA-4 on the surface of the CD25+ T cells. However, it is not clear that such effects are secondary to blockade of the interaction of CTLA-4 with its ligands CD80/CD86 on APC. Anti-CTLA-4 and even its Fab fragments may inhibit or disrupt the interaction of CTLA-4 with the CD3 complex in formation of the immunological synapse 31 in regulatory T cells. Indeed, reversal of suppression was more pronounced at higher concentration of anti-CD3 and accumulation of CTLA-4 in the synapse has been reported to be proportional to the strength of the TCR stimulus. We cannot exclude the intriguing possibility that other costimulatory pathways play a role in the activation of this unique population of cells. Our results raise the issue of the significance of the selective expression of CTLA-4 on freshly explanted CD4+CD25+ T cells.

Is expression of this membrane marker indicative of a unique functional role or does expression merely indicate that the CD4+CD25+ T cells have undergone a complex activationevent during intra-thymic development as we have previously hypothesized 32, or repeated activation in the periphery? We would favor the view that CTLA-4 expression is not critical to the function of these cells and merely reflects their chronic state of activation in vivo secondary to continual stimulation by autoantigens. Although CD28-derived costimulatory signals are needed for the generation, maintenance and survival of CD4+CD25+ T cells, it remains possible that activation with peptide/MHC alone may facilitate the induction of T cell suppression in sites of inflammation where the number of professional APC may be limiting.

4 Materials and methods

  1. Top of page
  2. Abstract
  3. 1 Introduction
  4. 2 Results
  5. 3 Discussion
  6. 4 Materials and methods

4.1 Mice and cell lines

Female BALB/c mice and C57BL/6 mice were obtained from the National Cancer Institute, (Frederick, MD). Mice expressing a Tg TCR specific for HA110–119/I-Ed33 were maintained at Taconic (Germantown, NY) under NIAID contract. CD80/CD86–/– mice on the BALB/c background 34 were kindly provided by R. Hodes (NCI, NIH).

4.2 Media, reagents and antibodies

Cells were grown as previously reported 1. Biotin-anti-CD25 (7D4), PE-streptavidin, purified anti-CD3 (2C11), anti-CD80 and anti-CD86 were purchased from BD PharMingen (San Diego, CA). Anti-CTLA-4 (4F10 and 9H10) was purchased from BD PharMingen. The 4F10 cell line was grown in Celline 350 flasks, and antibody purified on a protein G column. Anti-CTLA-4 Fab fragments were made using the ImmunoPure Fab preparation kit (Pierce, Rockford, IL). Protein G-purified 4F10 and Fab fragments were prepared by the Antibody Production Facility (NIAID, NIH). Tricolor-anti-CD4 was purchased from Caltag (Burlingame, CA). CD152 (CTLA-4) (mouse) Ig fusion protein was purchased from Alexis Biochemicals (San Diego, CA). Anti-CD90 (Thy1.2), anti-CD8, anti-CD4 and anti-PE magnetic beads were purchased from Miltenyi Biotec, Inc. (Auburn, CA). Human rIL-2 and hIL-15 were purchased from Peprotech (Rocky Hill, NJ). Mouse (m) IL-4, mIL-6 and mIL-9 were purchased from R & D Systems. mIL-10 was purchased from Chemicon. HA110–119 peptide was synthesized and purified by HPLC by the Peptide Synthesis Laboratory (NIAID, NIH) and used at 5 μM final concentration. Flow cytometry analysis to assess cell purity was performed using CellQuest® software.

4.3 Cell purification

CD4+CD25+ cells were purified as previously reported 1 with the following modifications: CD8+ T cells were depleted from enriched T cells with anti-CD8 beads using an autoMACS (Miltenyi). CD4+ T cells were incubated with biotin-anti-CD25 (15 μg/108 cells) followed by PE-conjugated streptavidin (7.5 μg/108 cells) followed by anti-PE beads and purified on an autoMACS. Purity ranged 95–98%. For some experiments, CD4+CD25 and CD4+CD25+ cells were purified by flow cytometry on a FACStar Cell Sorter (Becton Dickinson). LN cells were labeled with biotin-anti-CD25/PE-streptavidin and tricolor-anti-CD4. The purity of cells was >99%. T-depleted spleen cells were used as APC and were prepared by lysing erythrocytes with ACK lysis buffer, and depleting of CD90+ cells on an autoMACS. APC were irradiated at 3,000 rad.

4.4 Proliferation assays

WT BALB/c or HA TCR Tg CD4+CD25 cells (5×104), CD4+CD25+ cells (5×104) or CD4+CD25 cells (5×104) co-cultured with CD4+CD25+ cells (2.5×104) were cultured in 96-well plates (0.2 ml) with APC (5×104) and 0.5 μg/ml anti-CD3 or HA peptide, respectively, for 72 h at 37°C/7% CO2. Cultures were pulsed with [3H]thymidine for the last 6 h of culture. All results are expressed as the mean cpm of triplicate cultures. The standard deviation was always <10% of the mean.

Two-stage proliferation assays were set up as follows: CD4+CD25+ cells (5×105) were cultured with an equivalent number of APC and 0.5 μg/ml anti-CD3 for 3 days. When indicated, the cells were cultured in the presence of 100 U/ml IL-2, 20 ng/ml IL-15, 20 ng/ml IL-4, 10 ng/ml IL-10, 10 ng/ml IL-6, 10 ng/ml IL-7, 10 ng/ml IL-9, 100 μg/ml hamster IgG, 10 μg/ml each of anti-CD80 and anti-CD86, 20 μg/ml mCTLA-4-Ig fusion protein, 200 μg/ml anti-CTLA-4 or 20 μg/ml anti-CTLA-4 Fab. After 3 days, the cells were harvested and washed. The 3-day-activated CD4+CD25+ cells (2.5×104) were then co-cultured with HA TCR Tg CD4+CD25 cells (5×104), APC (5×104) and peptide in 96-well plates (0.2 ml) for 72 h at 37°C/7% CO2. Cultures were pulsed with [3H]thymidine for the last 6 h of culture.

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