Cs and Cytokines Cooperate for the Induction of Tregs

6.1 Interleukin-10 Modulates DCs for Tolerance Induction 98

6.2 IL-10-Dependent Feedback Mechanisms Between Treg and DCs . 99

6.3 TNFa and "Semi-mature" DCs 100

6.4 Suppressive Effects Mediated by TGF-P 101

6.5 Pharmaceuticals Interfere with DC Maturation 103

References 103

Abstract. Regulatory T cells (Treg) in broader terms consist of different subsets of T cells that are characterized by their ability to suppress proliferation of conventional effector T cells by various means. To date, three main groups of Treg can de distinguished, mainly by their functional properties (for review see Jonuleit and Schmitt 2003) Briefly, T regulatory (Tr)-1 cells as well as T helper (Th)-3 T cells express common T cell markers such as CD4 and are characterized by secretion of IL-10 and TGF-P, which provides a means by which proliferation of conventional CD4+ cells is blocked. In contrast, genuine Treg that are characterized by their expression of CD25 block T cell proliferation by an unknown cell-to-cell contact-dependent mechanism. However, there are many overlapping features shared by the different subtypes of regulatory T cell and the common denominator is the production of regulatory cytokines such as IL-10 and TGF-p.

6.1 Interleukin-10 Modulates DCs for Tolerance Induction

IL-10 was originally described as cytokine-synthesis-inhibiting factor (CSIF) with regard to its effects exerted on IFNy production of TH1 T cells. Meanwhile, it has been found to exert suppressive effects on a wide range of different populations of lymphocytes. When human or murine DCs are exposed to IL-10 in vitro culture systems, the cells display reduced surface expression of MHC class I and MHC class II molecules and reduced expression of T cell costimulatory molecules of the B7 family. In addition, the release of pro-inflammatory cytokines, i.e., IL-1a, IL-6, TNFa and most markedly IL-12, is abolished after IL-10 treatment (Griffen et al. 2001; ten Dijke and Hill 2004). However, all of these effects could only be recorded when immature DCs were exposed to IL-10. In contrast, mature DCs are insensitive to IL-10 and display a stable phenotype in the presence of IL-10 once they have matured (Gorelik et al. 2002; Skapenko et al. 2004).

According to their reduced MHC and B7 expression, the IL-10 treated DCs are inferior in T cell stimulation as opposed to their fully activated counterparts, but IL-10 does not merely keep DCs in an immature state; instead there is evidence that IL-10 modulates DC maturation, enabling DCs to induce T cells with regulatory properties. For example, freshly isolated Langerhans cells inhibit proliferation of TH1 cells after exposure to IL-10 but had no effect on TH2 cells (Szabo et al; 2002). Moreover, it has been shown that IL-10 modulated DCs from peripheral blood induce alloantigen specific anergy or anergy in melanoma-specific CD4+ and CD8+ T cells (Foussat et al; 2003; Cottrez et al; 2000). Further analysis of these anergic T cells revealed reduced IL-2 and IFN-y production and in contrast to genuine Treg, reduced expression of the IL-2 receptor a-chain CD25. However, in addition to these anergic T cells, some authors have also observed the emergence of genuine Treg after injection of IL-10, as indicated by CD25+ upregulation and cell-to-cell contact requirement for their suppressive activity (Wakkach et al. 2003).

The therapeutic use of these IL-10 modulated DCs is under investigation since injection of in vitro-generated, IL-10-modified DCs is able to prevent autoimmunity in a murine model of multiple sclerosis (EAE) and prolonged graft survival significantly in an murine GVHD model (Muller et al. 2002; Sato et al. 2003). Although most of these protocols involved in vitro exposure of DCs to IL-10, there is recent evidence that IL-10-driven DC modulation may also play a role in generation of regulatory T cells in vivo. For instance, Wakkach et al. were able to not only confirm previous in vitro results showing that addition of IL-10 to in vitro cultures differentiated DCs to a CD45hlgh tolerogenic phenotype, but they also demonstrated that this tolerogenic phenotype, along with regulatory Tr1 cells, is significantly enriched in spleens of IL-10 transgenic mice (Wakkach et al. 2003). Thus these data show that IL-10 plays an important role in rendering DCs not merely immature but also modifies their ability to induce regulatory T cells in vivo.

6.2 IL-10-Dependent Feedback Mechanisms Between Treg and DCs

Many results support the concept that DCs are inducers of Treg under certain circumstances. However, recent results imply that Treg, on the other hand, also affect DC functions (Serra et al. 2003). For example, Misra et al. have shown that DC cocultured with Treg remain in an immature state as judged by surface marker expression (Misra et al. 2004). These "Treg-exposed" DCs were inferior in induction of T cell proliferation and produced significant amounts of IL-10. In another murine cardiac transplantation model, increased numbers of splenic CD4+/CD25+ Treg and immature DCs were observed after treatment of the recipients with 15-deoxyspergualin, a commonly used antirejection drug (Min et al; 2003). As expected, these immature DCs purified from tolerant recipients induced the generation of CD4+/CD25+ Treg when incubated with naive T cells. Surprisingly, when these Treg isolated from tolerant recipients were incubated with DC progenitors, generation of DCs with tolerogenic properties, i.e., inferior T cell stimulatory capacity and IL-10 production, was observed. In conclusion, these results support the notion that IL-10 is a critical factor in a self-maintaining feed back loop, i.e., IL-10 derived from regulatory T cells has been shown to play a role in locking immature DCs in a tolerogenic state, which in turn induces further regulatory T cells that may contribute to IL-10 production (Misra et al. 2004). However, this positive feed back loop can ensure prolonged immunosuppression and does not only rely on the cell-to-cell contact required by genuine Treg.

6.3 TNFa and "Semi-mature" DCs

The term "immature" is not accurately defined in many aspects, and according to a long-standing definition, "real" immature DCs are only found in peripheral tissues; whereas the impetus to migrate toward regional lymph nodes requires at least some activation. Indeed there are reports showing that lung-derived "migratory" DCs (and hence "partly" activated DCs) account for the induction of regulatory T cells (Akbari et al. 2001). Therefore, tolerogenic DCs found in the lymph node may be differentially activated or "semi-mature".

In this regard, TNFa may play a role, since it has been shown that injection of DCs cultivated in presence of TNFa acted in a tolerogenic fashion (Menges et al. 2002). In these experiments, DCs were able to block autoimmunity in a murine model of multiple sclerosis (EAE). This suppressive effect was mediated by the induction of IL-10-producing regulatory T cells. The subsequent phenotypic analysis revealed that the DCs expressed regular amounts of MHC class II, and T cell costim-ulatory molecules, i.e., according to the authors, these DCs displayed a mature phenotype as judged by their surface marker expression. In contrast, these DCs failed to secrete IL1p, IL-6, TNFa, and in particular IL-12. The importance of IL-12 production for full maturation of DCs and acquisition of an immunstimulatory phenotype is further substantiated by results showing that IL-10 as well as cAMP are potent agonists of IL-12p70 secretion. And in fact, DCs treated with these agents are resistant to terminal maturation and induce T cell unrespon-siveness in vitro (Griffin et al. 2001). In conclusion, maturity of DCs may not merely be judged by their surface marker expression, instead cytokine expression also has to be taken into account and only upregu-lation of several different indicators warrant a fully activated phenotype of DCs.

6.4 Suppressive Effects Mediated by TGF-0

Transforming growth factor (TGF)-P comprises different members of a larger family of similar molecules and can be divided into three closely related members: TGF-^1, TGF-^2, TGF-^3. All three members have an important role in regulation of an immune response. Most of the effects of these different TGFs are indistinguishable in in vitro cultures, because of their similar signaling pathway: i.e., all TGF-^s engage the same receptor complex that eventually activates transcription factors of the Smad family (ten Dijke and Hill 2004). Many studies have shown that TGF-P has the ability to block proliferation of effector T cells by limiting IL-2 production and upregulation of cell cycle inhibitors. Moreover, TGF-P can block differentiation of TH1 and TH2 subsets (Gorelik et al. 2002) by blocking IFN-P and IL-4 production, respectively. The latter is mediated by blocking the transcription factors T-bet (IFN-y) and GATA-3 (IL-4) (Skapenko et al. 2004; Szabo et al. 2002).

These immunosuppressive qualities make TGF-P an ideal candidate molecule to convey suppression mediated by regulatory T cell. Despite its immunosuppressive activities, the direct involvement of TGF-P in effects induced by naturally occurring CD25+CD4+ Treg is still being debated since the main immunosuppressive mechanism seems to rely on cell-to-cell contact and is independent of soluble factors such as TGF-p.

However, so-called Tr1 cells display a unique cytokine profile since they neither express typical TH1 (IFN-P) nor TH2 (IL-4) cytokines, but produce sizable amounts of IL-10 and TGF-p. These cytokines are responsible for the suppressive effects exerted by Tr1 cells. Accordingly, via the secretion of TGF-P (and IL-10), Tr1 cells are able to suppress TH1- as well as TH2-mediated pathologies (Fousat et al. 2003; Cottrez et al; 2000). The differentiation of Tr1 cells is supposedly dependent on TGF-P too. It is thought that in a first step, naive CD4+ T cells can be activated by antigen-presenting cells (APCs) in the presence of IL-10 and/or TGF-P, resulting in an anergic state that is already able to suppress effector T cells in a cell contact-dependent manner. However, in a second differentiation step, limited in vivo proliferation of these otherwise anergic T cells occurs and "fully differentiated" Tr1 cells will develop that produce IL-10 and TGF-P, but still remain unable to produce IL-2 and IL-4 (Levings et al. 2001).

Even more recently, a model has been proposed in which the production of TGF-P is a key factor leading to renewal of "Treg" in a self-sustained cascade of events. In this model, Zheng et al. (Zheng et al. 2004) showed that CD4+/CD25- T cells develop into CD4+/CD25+ Treg after proliferation in presence of TGF-p. Thereafter these cells acquire immunosuppressive capacity along with the ability to secrete TGF-P and IL-10. As outlined above, these two cytokines are able to further generate "fresh" CD4+/CD25+ Tregs from a pool of naive CD4+/CD25- T cells, which via secretion of TGF-P can then further propel the generation of third-, fourth-, fifth-, etc. generation Tregs.

Further evidence that TGF-P has potent effects on sustaining Treg-mediated suppression derives from the investigations of Jonuleit et al. (Jonuleit et al. 2002), which demonstrate the importance of TGF-|3 in so-called infectious tolerance. Here the authors have shown that CD4+/CD25+ Treg can "educate" naive CD4+/CD25- T cells to acquire regulatory capabilities. Briefly, naive CD4+ T cells were incubated with naturally occurring CD25+ Treg, then recovered from these cocul-tures and finally coincubated with CD4+ effector T cells. In stimulation assays, these precultured T cells were now able to suppress proliferation of the freshly isolated effector T cells. Thus, these data show that contact to naturally occurring CD25+ Treg "infected" naive T cells to gain regulatory capacity. However, these second-generation regulatory T cells do not express the marker CD25 and the suppressive activity is cell-to-cell contact-independent. In fact, it has been shown that TGF-|3 is critically involved in mediating suppression of infectious tolerance. In summary, these data show that TGF-P plays a major role in maintenance of regulatory T cell function.

As outlined above, the effects of TGF-P on T cells is well documented; however, TGF-P also has immunosuppressive activity on APCs. To this end, it has been shown that Trl-derived TGF-P is able to reduce the antigen presenting capacity of DCs in vitro (Geissmann et al. 1999) and Trl clones are able to limit production of immunoglobulin by B cells (Roes et al. 2003). Therefore, in addition to the direct effects on T cells, TGF-P can further augment immunosuppression in vivo by downregulating APC functions and thus further limiting the activation of effector T cells in vivo.

6.5 Pharmaceuticals Interfere with DC Maturation

In accordance with the concept that immature DCs induce Treg rather than effector T cells, several pharmaceuticals have been tested for their ability to induce Treg by affecting the maturation status of DCs. Among them are the vitamin D3 methobolite 1a,25-(OH)2D3, n-acethyl-l-cysteine and common immunosuppressive drugs such as corticosteroids, cyclosporin A, rapamycin, and aspirin (Greissman et al. 1999; Roes et al. 2003; Piemonti et al. 1999,2000; Hackstein et al. 2001); Matyszak et al; 2000; Verhasselt et al. 1999). All of them have been shown to suppress DC maturation and as a consequence, anergy and/or regulatory T cells were induced. The effects are numerous and in the following examples are depicted only.

Direct induction of Treg in vitro by pharmacologically treated DCs has been observed after exposure of DCs to n-acetyl-l-cysteine and injection of DCs exposed to a mixture of vitamin D3 and mycophenolate mofetil, which induced full tolerance in an murine allograft model (Gre-gori et al. 2001). Interestingly, adoptive transfer of T cells from such tolerant mice into previously untreated mice prevented the rejection of respective allografts, thus indicating that probably regulatory T cells had been induced by vitamin D3-treated DCs in vivo. Furthermore, administration of rapamycin in clinically relevant doses prevented the full maturation of DCs and downregulated their IL-12 secretion and their capacity to induce T cell proliferation in vitro. Upon adoptive transfer of these rapamycin-treated DCs, an allo-antigen-specific T cell hypore-sponsiveness could be observed in the recipients (Hackstein et al. 2003). In sum, there is abundant evidence showing that drugs affecting DC maturation by means of preventing DC maturation are also most likely inducers of Treg in vivo.


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