Naturally occurring and antigen-induced CD4+ CD25+ Treg cells have been extensively studied in mice and humans. Depletion of the naturally occurring subset of CD4+ CD25+ Treg cells results in various types of autoimmune disease.11,12,27,41 CD4+ CD25+ Treg cells inhibit a wide range of autoimmune and inflammatory manifestations such asgastritis, oophoritis, orchitis, thyroiditis, inflammatory bowel disease and spontaneous autoimmune diabetes.11,12,27,41,51 Although CD4+ CD25+ Treg cells play a critical role in the regulation of immunity, we know little about the regulation of CD4+ CD25+ Treg cells. Interestingly, new evidence suggests that TLR signalling may directly or indirectly regulate the immunosuppressive function of CD4+ CD25+ Treg cells in immune responses.16,17
To date, differential TLR expression on CD4+ T cells and CD4+ CD25+ Treg cells has been found, suggesting that TLRs may be directly involved in adaptive immune responses, but how TLRs directly regulate adaptive immune responses is still not fully understood.52,53 Recently, a close relationship between TLRs and autoimmune diseases has been reported in mouse models. In a mouse model of systemic lupus erythematosus (SLE), when TLR9 was absent, SLE was exacerbated with increased activation of lymphocytes and plasmacytoid DCs as well as enhanced levels of immunoglobulin G (IgG) and interferon-α (IFN-α) in sera. In contrast, TLR7-deficient mice had ameliorated SLE with decreased lymphocyte activation and serum IgG levels.54 These data reveal opposing inflammatory and regulatory roles for TLR7 and TLR9 in SLE. Importantly, it is interesting that, in in vitro CD4+ CD25+ Treg cell suppression assays, engagement of TLR4 or TLR9 on freshly isolated mouse splenic DCs could significantly abrogate the immunosuppressive function of CD4+ CD25+ Treg cells, by rendering effector T cells resistant to CD4+ CD25+ Treg cell-mediated suppression through DC expression of interleukin-6 (IL-6), but not the effects of costimulation.39 Thus, the regulation of TLRs on CD4+ CD25+ Treg cells may directly or indirectly play a role in the occurrence of autoimmune diseases.
There is accumulating evidence of significant alterations in TLR expression on T cells in patients who have infectious diseases, although it is unclear whether altered TLR expression on T cells contributes directly to pathophysiology or immune defence in these patients.55,56 Furthermore, cross-talk between TLRs and CD4+ CD25+ Treg cells in tumours has also been noted.5,21 Elucidation of TLR signalling in adaptive immune responses in terms of both direct and indirect regulation of the immunosuppressive function of CD4+ CD25+ Treg cells may therefore have therapeutic benefits in inflammation, cancers and autoimmune diseases (Table 3).
The direct regulatory effects of TLRs on CD4+ CD25+ Treg cells
TLR2–/– mice, unlike TLR4–/– mice, contain significantly fewer CD4+ CD25+ Treg cells than control mice.21 Administration of TLR2 ligands to wild-type mice results in significantly increased CD4+ CD25+ Treg cell numbers.5,21 In the presence of a TLR2 agonist, such as the synthetic bacterial lipoprotein Pam3Cys-SK4, CD4+ CD25+ Treg cells expand markedly but their immunosuppressive function is temporarily abrogated.5 However, studies have shown that hsp60 could act as a costimulator of CD4+ CD25+ Treg cells.21,46,57 Treatment of CD4+ CD25+ Treg cells with hsp60 or its peptide p277 before anti-CD3 mAb-induced activation significantly enhanced the ability of the CD4+ CD25+ Treg cells to down-regulate the function of CD4+CD25– or CD8+ target T cells, detected through the inhibition of target T-cell proliferation and IFN-γ and tumour necrosis factor (TNF)-α secretion.58 The enhanced costimulatory effects of hsp60 on CD4+ CD25+ Treg cells involved innate signalling via TLR2, which led to activation of protein kinase C, phosphatidyl-inositol-3-kinase, and p38.58 hsp60-treated CD4+ CD25+ Treg cells suppressed target T cells both by cell contact mechanisms and by secretion of cytokine transforming growth factor (TGF)-β and IL-10.58 Thus, hsp60, a self-molecule, can down-regulate adaptive immune responses by up-regulating CD4+ CD25+ Treg cells innately through TLR2 signalling and not through APCs. Thus, the effect of TLR2 on CD4+ CD25+ Treg cells is somewhat controversial and further investigation is required.
Researchers observed that exposure of CD4+ CD25+ Treg cells to the TLR4 ligand lipopolysaccharide (LPS) induced up-regulation of several activation markers and enhanced their survival or proliferation.49 The proliferative response does not require APCs and is augmented by TCR triggering and IL-2 stimulation.49 Most importantly, LPS treatment increases the immunosuppressive ability of CD4+ CD25+ Treg cells by 10-fold.49 Moreover, LPS-activated CD4+ CD25+ Treg cells can efficiently control the occurrence of naïve CD4+ effector T cell-mediated diseases.49 These findings provide the first evidence that CD4+ CD25+ Treg cells respond directly to pro-inflammatory bacterial products, a mechanism that is likely to contribute to the control of inflammatory responses. However, others failed to observe effects of LPS on CD4+ CD25+ Treg cells.41,45,59 Thus, LPS-induced signalling on CD4+ CD25+ Treg cells is still controversial.
TLR5 ligand flagellin has important effects in regulating mucosal immune responses.41,60,61 Both human CD4+ CD25+ Treg cells and CD4+CD25– T cells express TLR5 at levels comparable to those on monocytes and DCs. Costimulation of CD4+ effector T cells with anti-CD3 mAb and flagellin resulted in enhanced proliferation and production of IL-2 at levels equivalent to those achieved by costimulation with CD28. In contrast, costimulation with flagellin did not break the hyporesponsiveness of CD4+ CD25+ Treg cells, but rather potently increased their immunosuppressive capacity and enhanced expression of Forkhead box protein 3 (Foxp3).41,60 However, in a tumour mouse model, administration of flagellin, which is specifically recognized by TLR5, at 8–10 days after tumour implantation produced significant inhibition of the growth of the antigenic tumour, which was associated with an increased IFN-γ:IL-4 ratio and a decreased frequency of CD4+ CD25+ Treg cells.62 In contrast, flagellin administered at the time of tumour implantation led to accelerated tumour growth, which was associated with a decreased IFN-γ:IL-4 ratio and an increased CD4+ CD25+ Treg cell frequency.62 Whether the contrasting effects of activation of TLR5 by flagellin on tumour growth are attributable to the different impacts on effector T cells and CD4+ CD25+ Treg cells and/or the different effects on naïve and activated/memory CD4+ CD25+ Treg cells has not yet been determined.
In addition, TLR8 could directly reverse the immunosuppressive function of CD4+ CD25+ Treg cells.21,52 It has been reported that CpG-A and poly (G10) oligonucleotides could directly reverse the immunosuppressive function of CD4+ CD25+ Treg cells in the absence of DCs, but the exact functional ingredients were not identified in that study.49 Further experiments indicated that short poly (G) oligonucleotides [poly (G2), poly (G3) and poly (G4) with phosphorothioate linkages] had a more potent ability to reverse CD4+ CD25+ Treg cell function than longer oligonucleotides [poly (G5), poly (G7) and poly (G10)].52 Interestingly, when TLR8 and myeloid differentiation primary response protein 88 (MyD88) were knocked down using an RNA interference method, the response ability of CD4+ CD25+ Treg cells to poly (G) oligonucleotides was abolished. Consistent with these results, TLR8 was consistently expressed by naturally occurring as well as inducing CD4+ CD25+ Treg cells.52,63,64 A recent study more clearly demonstrated that the immunosuppression of CD4+ CD25+ Treg cells is abrogated by TLR8 triggering directly on the CD4+ CD25+ Treg cells, not on the CD4+ effector T cells.52 These results consistently support the hypothesis that the TLR8–MyD88 signalling pathway directly controls the immunosuppressive function of CD4+ CD25+ Treg cells without the involvement of APCs.
The TLR9 ligand CpG oligodeoxynucleotide synergizes with anti-CD3 mAb to induce proliferation of both rat CD4+ CD25– and CD4+ CD25+ Treg cells.65 Surprisingly, TLR9 ligand partially abrogates the suppressive activity mediated by CD4+ CD25+ Treg cells, which is partially attributable to the direct effect of TLR9 ligand on effector T cells which are rendered more resistant to the regulation exerted by CD4+ CD25+ Treg cells.65 Thus, TLR9 ligand may rapidly increase the host's adaptive immunity by expanding effector T cells and also by attenuating the suppressive activity mediated by CD4+ CD25+ Treg cells.
Taking these results together, it is obvious that CD4+ CD25+ Treg cells express certain TLRs. In general, TLR2(?), TLR8 or TLR9 ligation abrogates or reverses the immunosuppressive function of CD4+ CD25+ Treg cells, whereas TLR2(?), TLR4 or TLR5 ligation enhances CD4+ CD25+ Treg cell suppressive capacity (Table 4).
The mechanisms by which TLRs modulate the immunosuppressive ability of CD4+ CD25+ Treg cells are not clear. One explanation is that the up-regulation or down-regulation of Foxp3 expression following stimulation by different TLRs may be related to the functional alteration of CD4+ CD25+ Treg cells. However, how TLR signalling affects Foxp3 expression needs to be addressed. Another possibility to explain the abrogated immunosuppressive function but enhanced proliferative capacity of CD4+ CD25+ Treg cells after TLR stimulation is suggested by reports indicating that CD4+ CD25+ Treg cells rapidly lose their ability to inhibit proliferation after receiving strong activation signals. Experimental data support the idea that TLR2 on murine CD4+ CD25+ Treg cells might function as a strong costimulatory trigger.5,37 We may speculate that some TLR-mediated signals force CD4+ CD25+ Treg cells into the proliferative pathway, which might be paralleled by the reversal of their immunosuppressive capabilities. However, TLR4 may induce CD4+ CD25+ Treg cell proliferation while enhancing the immunosuppressive ability of CD4+ CD25+ Treg cells, which does not support this speculation.
The indirect regulatory effects of TLRs on CD4+ CD25+ Treg cells
A central function of TLRs is to directly activate acute antimicrobial defence systems. TLRs are known to induce the production of antimicrobial proteins and peptides by various cell types, including cells of myeloid origin in the gut epithelium.4,41,66 Activation of resident macrophages through TLRs also leads to production of various cytokines (IL-1, IL-6, TNF-α, etc.) and chemokines (monocyte chemotactic protein 1, etc.), which collectively orchestrate the acute inflammatory response to infections.53,67
DCs are pivotally positioned at the interface of innate and adaptive immunity. Pathogen recognition is mediated by TLRs which are expressed at high levels on the surface of DCs. Analysis of MyD88-deficient mice demonstrated the critical role of TLRs in DC maturation and induction of adaptive immune responses.3,68,69 MyD88-deficient mouse DCs could not prime antigen-specific naïve T cells in vitro.70 While antigen mixed with complete Freund's adjuvant (CFA), which contains several ligands for TLRs, leads to a robust immune response in wild-type mice, it fails to trigger T-cell proliferation and IFN-γ production in MyD88-deficient mice.71,72 These data suggest that engagement of TLRs on DCs in vivo is required for DC maturation, secretion of cytokines and chemokines and antigen-presenting ability, which subsequently control the choice of the effector T cells of adaptive immune responses. TLR signalling in macrophages and DCs leads to secretion of IL-12 which skews the resultant CD4+ effector T-cell response towards the T helper type 1 (Th1) phenotype.23 Moreover, TLR triggering in APCs contributes to the adaptive immune response by indirectly controlling the immunosuppression of CD4+ CD25+ Treg cells.53
Some researchers found that the immunosuppressive function of CD4+ CD25+ Treg cells is critically dependent on immature DCs and is readily reversed by TLR-induced activation of DCs.59 The potential responsiveness of CD4+ CD25+ Treg cells to IL-2 was increased by the co-operative effects of IL-6 and IL-1, both of which are produced by TLR-activated mature DCs.5,58,73 The pro-inflammatory cytokines produced by TLR-activated mature DCs are required for reversal of CD4+ CD25+ Treg cell anergy, but whether they are required to overcome the immunosuppression of CD4+ CD25+ Treg cells is a matter of controversy.39,59 However, cytokines secreted by APCs in response to TLR ligands are also important for CD4+ effector T cells to overcome the immunosuppressive effects of CD4+ CD25+ Treg cells.18,23,74
Some studies showed that decreased immunosuppression of CD4+ CD25+ Treg cells on effector T cells was mediated by soluble factors produced by DCs in response to the TLR4 ligand LPS or the TLR9 ligand CpG oligonucleotide.5,19,23 The activity of LPS-treated DC supernatants was specifically abrogated by neutralizing anti-IL-6 mAbs, thus indicating the significance of IL-6 in the control of CD4+ CD25+ Treg cells on CD4+ CD25– effector T cells.19 These conclusions were further supported by the observation that the supernatant of LPS-stimulated DCs from IL-6–/– knock-out mice was unable to reverse suppression of CD4+ CD25+ Treg cells.75 However, recombinant IL-6 did not substitute for DC supernatant. This suggests that, in addition to IL-6, some factors, as yet undefined, are essential in this process. However, preincubation of CD4+ CD25+ Treg cells with the supernatant of LPS-activated DCs did not block the immunosuppressive capacity of CD4+ CD25+ Treg cells,76 suggesting that the supernatant of LPS-activated DCs may render CD4+ CD25– effector T cells refractory to suppression rather than blocking the immunosuppressive capacity of CD4+ CD25+ Treg cells.23,59,76,77 This regulating network may allow CD4+ CD25+ Treg cells to maintain their function and prevent self-reactive T-cell activation during an ongoing immune response to infections and other damage stimuli.
Although some TLR signalling on DCs by CpG or LPS rendered CD4+ effector T cells refractory to CD4+ CD25+ Treg cell-mediated suppression, TLR-induced mature DCs are also capable of stimulating the proliferation of CD4+ CD25+ Treg cells,78,79 which may be beneficial for the efficient down-regulation of the immune response as soon as the pathogens have been cleared.