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Abstract

  1. Top of page
  2. Abstract
  3. Foreword
  4. Introduction
  5. Generating suppressors by exogenous antigens
  6. Lifestyle of suppressor T cells
  7. Dynamics of in vivo suppression
  8. IL-2 dependence of suppressor cells
  9. Conclusion
  10. References

Recent years have witnessed the revival of suppressor T cells that control immunity by interfering with the generation of effector T-cell function in vivo. The discovery that CD4 T cells with the CD25 surface marker were enriched in suppressor activity enabled further phenotypic and functional analysis of the so-called natural suppressor cells. In vitro characterization showed that these cells were anergic, i.e. did not respond to antigenic stimulation with proliferation and, instead they suppressed other cells through direct cell contact resulting in inhibition of interleukin-2 gene transcription. We have analysed the generation and function of suppressor T cells in T-cell receptor (TCR) transgenic mice. The results showed that such cells can be generated intrathymically when agonist TCR ligands are expressed on thymic epithelial cells. Thus generated cells constitute a lineage of cells committed to suppression only with the ability to survive for prolonged periods of time in the absence of the inducing ligand. Because of appropriate homing receptors such cells can accumulate and proliferate in antigen draining lymphnodes after antigenic stimulation and suppress proliferation and cytokine secretion of CD4 and CD8 T cells as well as CD8 T-cell-mediated cytotoxicity. We also attempted to generate such cells from naïve T cells in secondary lymphoid tissue under conditions where expansion of already preformed suppressor T cells could be excluded. The results showed that subimmunogenic peptide delivery by osmotic minipumps or by peptide containing DEC 205 antibodies yielded CD25+ suppressor cells that were phenotypically and functionally indistinguishable from intrathymically generated suppressor cells. The experiments with DEC205 antibodies revealed (i) dose-dependent proliferation of naïve T cells and (ii) conversion into suppressor T cells of only those T cells that underwent a limited number of cell divisions. These results are compatible with other studies that were, however, less rigorous in excluding expansion of existing cells as opposed to de novo generation of suppressor cells from naïve T cells. The fact that natural suppressor cells have an essential role in preventing autoimmunity and that they can be specifically induced by TCR agonist ligands opens new perspectives in preventing autoimmunity, transplant rejection and allergy.


Foreword

  1. Top of page
  2. Abstract
  3. Foreword
  4. Introduction
  5. Generating suppressors by exogenous antigens
  6. Lifestyle of suppressor T cells
  7. Dynamics of in vivo suppression
  8. IL-2 dependence of suppressor cells
  9. Conclusion
  10. References

Having been a long-term member of the Basel Institute for Immunology from 1973 until 1996, I had ample opportunity to meet and interact with Ivan Lefkovits. In fact, I did not: Ivan was always the friendly fellow who had important things to discuss with Niels Jerne, the director and with Charley Steinberg, the guru. I must confess that initially I was a bit intimidated by this intellectual elite and as expected from Thomas Bernhard's description of the ‘Alte Meister’, some of the ‘Ehrfurcht’ diminished upon continued contact with and closer inspection of the triumvirate but it never went completely. Then, when Fritz Melchers took over, Ivan and I teamed up on occasions, because we both had an innate dislike of the ‘Obrigkeit’ and I still remember dragging Ivan physically into the meeting of the Institute's advisors in spite of the fact that this was supposed to be a secret affair that could only be witnessed by highly selected individuals.

Years later, when I was just about leaving Basel, I learned about Ivan's ‘Geschichte’ which has been so ‘beeindruckend’ described in a little book by his mother. As a consequence, we got to know each other better, and we intensified our contacts in spite of my departure from Basel in 1996. Actually, Ivan and Hana have spent some time in my laboratory in Paris, and I am still ashamed by the terrible accommodation I provided them with: I had not even inspected the ‘guest apartment’ of the University of Descartes which turned out to be unlivable and unbelievable, to such an extent that Ivan and Hana moved to various locations in Paris (and also to Fontainbleau) and in this way learned more about this fascinating city. In spite of this little mishap, we have remained friends and have met on several occasions over the years, one of which was Ivan's farewell symposium at the International Conference on Immunology in Montreal, which by many criteria was the most original symposium of the entire conference. The symposium was followed the next day by an even more original dinner with much remembering of the good old days in Basel.

Even though I was not listed as a speaker in the symposium's program (my abstract was lost by nobody's fault), I was included last minute so to speak, which serves as a convenient excuse for the somewhat sloppy scientific text that follows and has little to do with Ivan's preoccupation with real science, which is appropriately ‘gewuerdigt’ by his other friends who collaborated with him scientifically.

[The words ‘Ehrfrucht’, ‘Obrigkeit’, ‘beeindruckend’ and ‘gewuerdigt’ used about do have quite adequate English equivalents (see webster), but for the cognoscenti the German version sounds incommensurably better]

Introduction

  1. Top of page
  2. Abstract
  3. Foreword
  4. Introduction
  5. Generating suppressors by exogenous antigens
  6. Lifestyle of suppressor T cells
  7. Dynamics of in vivo suppression
  8. IL-2 dependence of suppressor cells
  9. Conclusion
  10. References

Recent years have witnessed the revival of suppressor T cells that control immunity by interfering with the generation of effector T-cell function in vivo. A major discovery was the identification of suppressor cells that expressed the α chain of the interleukin-2 (IL-2) receptor (CD25) on their surface and the fact that CD25+4+ T cells were highly enriched in suppressive activity [1]. This enabled the phenotypic and functional analysis of so-called natural suppressor cells. With regard to gene expression, it was found that such cells had upregulated several activation markers and downregulated proteins characteristic of naïve T cells [2–5]. Of great significance was the observation that these cells express high levels of the transcription factor Foxp3 that, when defective, leads to an early-onset of fatal autoimmune disease [6–9]. Experiments concerned with the genesis of such cells conducted in T-cell receptor (TCR) transgenic mice revealed that coexpression of a class II major histocompatibility complex (MHC)-restricted TCR and its ligand within the same organism drove the generation of such cells [10–12] and that expression on thymic stroma was an effective [11–13] but not exclusive [13] means of generating such cells. Here, we are concerned with the generation of CD25+ suppressor cells by exogenous antigens in the fully mature immune system as well as with their in vivo stability and function. The novel evidence suggests that the CD25+ suppressors represent a suitable tool to induce antigen-specific immunological tolerance in the fully mature immune system in the absence of general immune suppression that often has undesired side-effects ranging from increased risk of infection to development of life-threatening lymphoma.

Generating suppressors by exogenous antigens

  1. Top of page
  2. Abstract
  3. Foreword
  4. Introduction
  5. Generating suppressors by exogenous antigens
  6. Lifestyle of suppressor T cells
  7. Dynamics of in vivo suppression
  8. IL-2 dependence of suppressor cells
  9. Conclusion
  10. References

There has been a long history of attempts to induce specific in vivo immune suppression by exogenous antigens by delivering such entities via the oral route, intranasally, intravenously or subcutaneously [14–16] in attempts to either induce and/or expand [17–21] suppressor cells. There is no doubt that some of these experiments have worked but since the readout of most experiments was immunosuppression rather than the generation of a well-defined class of Foxp3-expressing suppressor cells, there was no emerging consensus on what might represent the most useful approach to specifically prevent unwanted immunity. It turned out that ‘chronic’ antigenic stimulation could result in CD4+ T cells that produced mostly IL-10 [22–24]. However, in some instances, these cells were only able to suppress certain immune reactions and had in fact a phenotype that differed from the later described CD25+ suppressors [24]. Before the recognition of Foxp3 expression as marker for suppressor T cells, findings were reported that the subimmunogenic presentation of proteins or peptides could result in the generation of CD25+ T cells that at least in some assays qualified as suppressor cells [14, 25–27]. Based on our own limited and initial studies in this direction [13], we pursued a larger study of developing suitable protocols of extrathymic generation of CD25+ suppressors and a comparative analysis of such peripherally generated cells with intrathymically generated CD25+ suppressor T cells.

Initially, we attempted to mimic a situation that may occur in vivo when cell-specific proteins are expressed at low levels, broken down by proteases and peptides are presented by steady state (mostly non-activated) dendritic cells. To this end, we constantly infused relatively small doses of peptides with the aid of osmotic mini-pumps that were transplanted subcutaneously and monitored the appearance of cells with different phenotypes over time. We first conducted these studies in TCR-HA transgenic mice on the RAG-2–/– background that expressed a TCR specific for the peptide 107–119 of influenza hemagglutinin presented by Ed MHC molecules in naïve T cells. In order to exclude any influence of the thymus, these mice were thymectomized prior to implantation of peptide-delivering osmotic pumps. The observation was that the continuous supply of peptides at a wide range of doses (103−10 μg per day), resulted in downregulation (endocytosis) of the TCR on many T cells as well as the appearance of CD25+ T cells with suppressive activity at day 14 of peptide-infusion in the apparent absence of immunological priming.

The experiments were then repeated by transferring selected CD25-naïve TCR-HA expressing T cells into nu/nµ recipient mice and implanting peptide-delivering pumps into these. The same result was obtained, arguing that it was possible to de novo generate CD25+ cells with suppressive activity from CD25 naïve T cells in the absence of any ‘tutoring’ by other intrathymically generated T cells, the latter representing a phenomenon implicit in so-called ‘infectious tolerance’[28].

Finally, a similar approach was used to generate suppressors from small numbers of naïve CD4+ T cells in normal mice, emphasizing that the protocol was suitable to generate suppressors from a small number of naïve T cells in a normal environment. In transgenic as well as non-transgenic systems, the thus induced CD25+ cells prevented the development of antigen-specific effector T-cell responses in various in vivo readouts [29].

The transfer of naïve T cells from RAG–/–, TCR-HA transgenic mice was then used to analyze whether constant infusion provided an advantage over single-dose injection of peptides, and whether targeting of peptide to steady state dendritic cells was a suitable mode to induce such cells. This approach was carried out because previous experiments using the DEC-205 antibody to target peptides to dendritic cells had led to somewhat different outcomes in different laboratories: While in some studies this mode of peptide-delivery resulted in moderate cellular expansion followed by deletion and/or anergy [30, 31], it had in at least one experimental setting also resulted in the generation of CD25+ cells with suppressive activity but it was not clear whether preexisting CD25+ cells were expanded, because naïve T cells with the transgenic TCR were not on the RAG–/– background. Also, Foxp3 expression was not analyzed in these experiments [32]. Our results using RAG–/–, TCR-HA-expressing CD25 T cells showed (i) that DEC-205 delivered peptide-induced dose-dependent proliferation; (ii) that the cells which proliferated most extensively did not acquire constitutive CD25 expression and (iii) that cells which went through only a few divisions converted most efficiently into CD25+ suppressors during 14 days after i.p. delivery of the antibody-peptide fusion protein. Thus, there was an inverse relationship of cell division and CD25 conversion but not in the sense as previously reported that cells which did not divide at all converted best [14]. Instead, cells that were weakly activated and proliferated to some extent showed the best conversion rate which was better than that of cells that had not divided at all and probably had not encountered any antigen.

Thus, there is no doubt that in addition to intrathymic generation of CD25+ suppressor cells by ‘self’ agonist ligands of the TCR, the recently obtained information can be exploited to generate in the fully mature immune system in the absence of a functioning thymus suppressor cells that are specific for exogenous antigens [29]. This obviously opens new avenues of inducing specific immune tolerance, an old dream of immunologists that has been experimentally addressed decades ago but has resulted in erratic results, most likely due to the fact that protocols aiming to induce specific immunosuppression could not be optimized because retrospectively, knowledge on the nature of suppressor cells was not available at the time. Also, the literature on this topic tends to suggest that peptide delivery under subimmunogenic conditions results in the generation of suppressor cells when distinct antigens and different TCR transgenic mice were used [14, 32].

Lifestyle of suppressor T cells

  1. Top of page
  2. Abstract
  3. Foreword
  4. Introduction
  5. Generating suppressors by exogenous antigens
  6. Lifestyle of suppressor T cells
  7. Dynamics of in vivo suppression
  8. IL-2 dependence of suppressor cells
  9. Conclusion
  10. References

Naturally occurring suppressor T cells have been extensively characterized in vitro and the conclusion was (i) that these cells were intrinsically anergic, i.e. did not respond to antigenic stimulation with proliferation; (ii) that these cells suppressed other cells through a specific T–T cell contact resulting in inhibition of IL-2 gene transcription and (iii) that production of IL-10 was not required for the suppressive effect whereas a contribution of transforming growth factor-β (TGF-β) is still being debated [33].

These experiments could not provide information on the question whether CD25+CD4+Foxp3 expressing T cells represented a lineage of irreversibly committed cells or an effector T-cell population that had acquired a suppressor phenotype when antigenically stimulated. This question was therefore addressed with the TCR-HA TCR transgenic system by transferring CD4+25+ T cells with a TCR of known antigen specificity into an antigen-free environment and following the fate of either such cells or induced CD4+25+ cells of 14 day-peptide-infused mice over prolonged time periods [34]. The conclusion from such experiments was that after their induction by cognate antigen CD4+25+ suppressors could survive for long periods of time in a normal lymphoid environment without any requirement of antigenic stimulation by TCR agonist ligands. Some cells had an intermitotic lifespan that in murine models lasted for months [34], an observation well compatible with the notion that some CD4+25+ cells in normal mice had an intermitotic lifespan of at least 70 days [35]. Whenever tested during the observation period, the activation of these cells by cognate antigen resulted in potent suppressor activity in vitro and CD4+25+ cells did not lose their anergic in vitro phenotype when maintained for long time periods in an antigen-free environment. Thus, by these criteria CD4+25+ suppressor T cells represent a long-lived, stable lineage of T cells committed to immunosuppression only.

Dynamics of in vivo suppression

  1. Top of page
  2. Abstract
  3. Foreword
  4. Introduction
  5. Generating suppressors by exogenous antigens
  6. Lifestyle of suppressor T cells
  7. Dynamics of in vivo suppression
  8. IL-2 dependence of suppressor cells
  9. Conclusion
  10. References

Contrary to in vitro readouts, the effect of suppression by CD4+25+ cells on the immune response of CD4+ or CD8+ cells in vivo is only observed at later stages especially under conditions where responses begin with a relatively low frequency of suppressor T cells and other T cells responding to antigenic challenge. In vivo suppressor T cells are not anergic and respond to antigenic challenge with strong proliferation [34–38] as naïve or memory T cells do. Thus both suppressor T cells and naïve CD4+ T cells will initially expand in a similar fashion in antigen draining lymph nodes to which both suppressor T cells and CD4 T cells home with similar efficacy. The specific homing to and expansion in antigen-draining lymph nodes appears an extremely important function of suppressors that enables optimal contact of antigen-specific suppressors with T cells that are specific for antigen which is presented in the same draining lymph node. Later during the response, the proliferation of CD4+ T cells is selectively diminished, and their cytokine secretion is suppressed while the activated suppressor cells produce predominantly IL-10 [34]. IL-10 is, however, not always essential for the observed suppression of CD4 T cells and the molecular effector mechanism of the in vivo suppression is at present unknown. It is also worth mentioning that under these in vivo conditions, the commitment of CD4 T cells to produce IL-2 or (-interferon is not prevented, i.e. these cytokines are produced in increased amounts when the suppressed CD4 T cells are separated from the suppressors and antigenically stimulated [10, 39]. Thus, in vivo CD4 T cells can be suppressed after they have proliferated and had become effector T cells.

The suppression of CD8+ T cells in vivo does initially not affect their proliferation at all when injected together with suppressors at a 1 : 1 ratio such that they accumulate at very similar frequencies whether or not suppressors are present [40, 41]. However, by about day 7 of a primary CD8 immune response in the presence of suppressors, one can observe specific suppression of cytolytic activity of the expanded CD8+ T cells [41]. It is of special interest that in some instances control of CD8 T-cell responses by suppressor cells was found to be entirely dependent on TGF-β signalling in the CD8+ T cells as the cytolytic activity of CD8+ T cells from mice expressing a dominant negative TGF-β receptor were resistant to suppression by CD4+25+ suppressor cells [41], and CD8 T cells responsible for autoimmunity in a particular model of diabetes could not be suppressed by CD4+25+ cells when they expressed the same dominant negative TGF-β receptor [42]. Thus, in this particular scenario, essential molecular pathways are emerging of how suppressors interact with CD8+ T cells but it is not clear whether under these circumstances TGF-β has to be produced by the suppressors themselves or is provided by other cells.

IL-2 dependence of suppressor cells

  1. Top of page
  2. Abstract
  3. Foreword
  4. Introduction
  5. Generating suppressors by exogenous antigens
  6. Lifestyle of suppressor T cells
  7. Dynamics of in vivo suppression
  8. IL-2 dependence of suppressor cells
  9. Conclusion
  10. References

It has become clear that IL-2 or IL-2 receptor-deficient mice lack CD4+25+ suppressor cells in peripheral lymphoid tissue [43] and are therefore highly susceptible to autoimmune disease which in IL-2 receptor-deficient mice can be cured by providing the mice with IL-2 receptor-positive CD4+25+ suppressor T cells [44]. Thus, these experiments challenge earlier notions that the autoimmune disease in IL-2 and IL-2 receptor-deficient mice is due to a deficiency in activation-induced cell death [45, 46]. When analyzed in some more detail, it became clear that the antigen-induced proliferation of suppressors in vivo is not dependent on IL-2 (IL-4 can substitute for IL-2 in vitro[47]) but that suppressors that are stimulated by antigen in the absence of IL-2 lose their expression of CD25 and become less potent suppressors (unpublished results), a scenario likely to cause autoimmunity in IL-2 or IL-2 receptor-deficient mice that do generate CD4+25+ cells in the thymus [44].

Conclusion

  1. Top of page
  2. Abstract
  3. Foreword
  4. Introduction
  5. Generating suppressors by exogenous antigens
  6. Lifestyle of suppressor T cells
  7. Dynamics of in vivo suppression
  8. IL-2 dependence of suppressor cells
  9. Conclusion
  10. References

The fact that CD4+25+ Foxp3-expressing suppressor cells have an essential role in preventing an early-onset of autoimmune disease in mammals, and the fact that these cells can be artificially induced through subimmunogenic presentation of TCR agonist ligands opens new possibilities to exploit these cells to induce specific tolerance in the fully mature immune system. This may become a powerful tool in the prevention of allergies and transplant rejection. Inducing such cells by organ-specific antigens such as insulin or myelin basic protein peptides may become an effective means to prevent autoimmunity in patients at risk of developing such disease. Such attempts have been reported to have success in animal models of disease [21, 48] but have failed in clinical trials [49]. There is hope, however, that our acquisition of knowledge on the ‘lifestyle’ of suppressor T cells will help to develop more suitable and reliable procedures that may be exploited for the induction of specific immunologic tolerance in the clinic without the use of dangerous general immunosuppression.

References

  1. Top of page
  2. Abstract
  3. Foreword
  4. Introduction
  5. Generating suppressors by exogenous antigens
  6. Lifestyle of suppressor T cells
  7. Dynamics of in vivo suppression
  8. IL-2 dependence of suppressor cells
  9. Conclusion
  10. References