Abstract
Toll-like receptors (TLRs) control activation of adaptive immune responses by antigen-presenting cells (APCs). However, initiation of adaptive immune responses is also controlled by regulatory T cells (TR cells), which act to prevent activation of autoreactive T cells. Here we describe a second mechanism of immune induction by TLRs, which is independent of effects on costimulation. Microbial induction of the Toll pathway blocked the suppressive effect of CD4+CD25+ TR cells, allowing activation of pathogen-specific adaptive immune responses. This block of suppressor activity was dependent in part on interleukin-6, which was induced by TLRs upon recognition of microbial products.
TLRs play an essential role in innate host defense as well as in the control of adaptive immune responses (1, 2). These receptors evolved to detect the presence of infection through recognition of conserved pathogen-associated molecular patterns (PAMPs) (3). Because PAMPs are produced by pathogens, but not by the host cells, their recognition by TLRs allows for self-nonself discrimination (4). TLRs control activation of the adaptive immune responses by inducing dendritic cell (DC) maturation (1,5). Maturation of DCs is characterized by the up-regulation of major histocompatibility complex (MHC) class II and costimulatory molecules (CD80/CD86), which provide the two requisite signals for naı̈ve T cell activation (6).
Induction of CD80/CD86 expression on APCs is not the only mechanism that controls T cell activation. T cell responses are also regulated by CD4+CD25+ suppressor or regulatory T cells (TR cells) (7–10). These cells are critical for the maintenance of peripheral T cell tolerance, because their depletion leads to organ-specific autoimmune diseases (11, 12). Although the molecular mechanism of TR cell–mediated suppression is currently unknown, it has been shown to be cell-contact dependent and to result in the inhibition of interleukin-2 (IL-2) transcription in responder T cells (13, 14). The peripheral T cell repertoire consists of both self-reactive T cells and a majority of T cells with potentially useful specificities for pathogen-derived antigens. Although inhibition of the former by TR cells is clearly beneficial, suppression of T cells specific for pathogens could be detrimental to effective protective immunity to microbial infection. Because microbial infection is first detected and subsequently regulated by TLRs (1, 2), we hypothesized that the suppressor activity of TR cells may also be regulated by TLRs themselves.
Ligation of TLRs on DCs overcomes CD4+CD25+T cell–mediated suppression.
To test the above hypothesis, we first established that CD4+CD25+ TR cells could effectively suppress CD4+CD25– T cell activation when freshly isolated splenic DCs (15) were used as APCs (fig. S1). These cells express low levels of CD80 and CD86, which is characteristic of immature DCs (16), and stimulation with TLR ligands such as lipopolysaccharide (LPS) or CpG leads to their maturation, characterized by up-regulation of cell-surface MHC class II and the costimulatory molecules CD80 and CD86, as well as in the secretion of cytokines, chemokines, and other soluble factors (6). Stimulation of DCs with TLR4 and TLR9 ligands [LPS (17, 18) and CpG (19), respectively] reversed the TRcell–mediated suppression, restoring T cell proliferation to near normal levels (15) (Fig. 1A). This effect was observed even at a high (1:1) ratio of TR cells to responder T cells (Fig. 1B). Further, when DCs from TLR4-deficient mice were used as APCs, suppression was blocked in the presence of CpG but not LPS (16). These results suggest that microbial induction of DC maturation by TLRs is important for abrogating the suppressive effects of TR cells.
CD25+ T cell–mediated suppression of CD4+CD25– T cells is blocked by activation of TLRs on DCs. (A and B) T cell proliferation assays were set up as described (15) in the presence of the indicated numbers of CD4+CD25+ TRcells, with or without LPS (100 ng/ml) (left) or CpG (1 μM) (right). T cell proliferative responses are expressed as the mean ± SE of triplicate cultures (A) or as the percentage of total counts (B), with 100% representing counts in the absence of TR cells separately for each condition. Standard errors were less than 10%.
Block of TR cell–mediated suppression is independent of costimulation.
We next determined whether the TLR-induced block of suppression was dependent on induction of costimulatory molecules or secreted cytokines. TLR4 signals through at least two distinct pathways: a MyD88-dependent pathway, which is essential for production of inflammatory cytokines, and a MyD88-independent pathway, which is sufficient for the up-regulation of MHC class II and costimulatory molecules in DCs (20). Thus, when MyD88-deficient DCs (15) are stimulated with LPS, they up-regulate normal levels of costimulatory molecules. When these DCs were used as APCs, neither LPS nor CpG treatment was able to interfere with TR cell–mediated suppression (Fig. 2A) despite normal up-regulation of CD80, CD86, and CD40 in MyD88-deficient DCs by LPS (fig. S2). This result demonstrates that the block of TRcell–mediated suppression was independent of expression of costimulatory molecules on DCs.
Block of suppression is independent of costimulation but dependent on cytokine(s) secreted by DCs in response to TLR ligation. (A to C) TR cell suppression assays were done with either WT (A and B) or MyD88–/– splenic DCs (A to C) as APCs, with or without LPS or CpG (A). LPS DC CM from either WT (B and C) or the indicated TLR4–/– mutant mice (C) was added at the beginning of cultures. T cell responses are shown as the mean ± SE of triplicate cultures (A) or as the percentage total counts, with 100% representing the counts in the absence of TR cells separately for each condition (B and C). Standard errors were less than 10%.
Cytokines(s) secreted by DCs are required for overcoming suppression.
To explore whether secreted cytokines might be responsible for the observed effects of LPS and CpG, we added conditioned medium (CM) from DCs treated with LPS (LPS CM) (Fig. 2B) or CpG (16) to the TR cell assays. Whether we used wild-type (WT) DCs or MyD88-deficient DCs as APCs, the addition of WT LPS CM led to a block of TR cell–mediated suppression (Fig. 2B). In contrast, CM from MyD88-deficient or TLR4-deficient DCs stimulated with LPS had no effect on block of suppression (Fig. 2C). CM from WT DCs not treated with TLR ligands had no effect on suppression (16). Stimulation of the TLR/MyD88 pathway in DCs therefore leads to induction of a secreted factor(s) that is responsible for the block of suppression, which is independent of the costimulatory pathway of T cell activation.
TLRs are also expressed in many cell types other than DCs, where they can induce expression of distinct subsets of genes (1). We therefore sought to determine whether the ability of TLRs to induce factor(s) responsible for blocking of suppression was restricted to professional APCs. In assays with CM from LPS-stimulated DCs, macrophages, B cells, and fibroblasts, block of TRcell–mediated suppression was observed only in LPS CM from DCs and macrophages (fig. S3A). CM from B cells and fibroblasts stimulated with LPS failed to block the suppressive activity of TR cells (fig. S3B).
We next tested whether the soluble factor(s) present within LPS and CpG CM acted directly on TR cells to turn them off or rendered responder T cells refractory to suppression by TRcells. TR cells were incubated for 36 hours with antibodies to CD3 (anti-CD3) and MyD88–/– DCs in the presence or absence of CM from LPS-treated WT DCs and then used in a suppression assay. TR cells preincubated with LPS CM were capable of suppressing responder T cell activation as efficiently as those that were not exposed to DC CM and freshly isolated TR cells (Fig. 3A). These data suggest that a cytokine(s) present in LPS-stimulated DC CM acts on responder T cells and makes them refractory to TR cell–mediated suppression.
The cytokine(s) that is responsible for block of suppression acts on responder T cells and does not signal through the common gamma chain. (A) CD4+CD25+ T cells were cultured in the presence of MyD88–/– DCs for 36 hours with anti-CD3 (0.1 μg/ml) alone or with anti-CD3 and LPS-treated WT DC CM. After 36 hours, the indicated number of freshly isolated TR cells or cells as treated above were used in a TR cell assay with MyD88–/– DCs as APCs. T cell responses are shown as the mean ± SE of triplicate cultures. (B) TRcell suppression assay was set up with MyD88–/– splenic DCs as APCs. CD4+CD25– T cells were first stained with a mixture of anti-γc antibodies (10 μg/ml) for 2 hours wherever indicated. The anti-γc antibodies were present in the culture throughout the entire period of the assay. LPS-treated WT DC CM was added at the beginning of the cultures. T cell responses are shown as the mean ± SE of triplicate cultures.
To demonstrate that LPS CM recovers proliferation of responder T cells rather than induces proliferation of TR cells, we performed suppression assays with 5- (and 6-) carboxyfluorescein diacetate succininyl ester (CFSE)–labeled responders or suppressors. LPS CM had no effect on TR cell proliferation but recovered proliferation of responder T cells (fig. S4).
IL-6 is critical for overcoming suppression.
We next attempted to identify the cytokine(s) responsible for block of suppression. Exogenous IL-2 has been shown to block suppression mediated by TR cells (13,14). LPS- or CpG-treated CM from IL-2–/– DCs blocked suppression mediated by TR cells at similar levels to WT CM (16), demonstrating that IL-2 plays no role in the TLR-mediated block of suppression. Moreover, release of suppression was not abrogated by blocking antibodies to common γ chain (γc), ruling out involvement of all the cytokines that signal through this receptor, including IL-7 and IL-15 (21) (Fig. 3B and table S1). Several additional candidate cytokines were tested, and their involvement in the blockade of suppression was ruled out by various methods (see table S1 for a complete listing of the cytokines tested).
We next sought to identify the cytokine(s) responsible for block of suppression, using size exclusion chromatography to fractionate LPS-treated DC CM (15). When these fractions were tested in TR cell suppression assays, the activity was found in fractions 10 through 14 (Fig. 4A). DCs secrete proinflammatory cytokines such as IL-12, IL-6, and TNF-α (tumor necrosis factor), as well as a variety of chemokines, when stimulated by TLR ligands. To determine whether the activity in the fractions correlated with the presence of any of the cytokines known to be induced by TLRs in DCs, we assayed all the fractions for these cytokines by ELISA (enzyme-linked immunosorbent assay). We found that all the active fractions contained IL-6 (Fig. 4A), whereas TNF-α was present in inactive fractions and IL-12 was found in some, but not all, active fractions (16). We used antibodies against each of these cytokines individually to neutralize their activity in LPS-treated WT DC CM. We found that neutralization of IL-12 or TNF-α had no effect (16), but neutralization of IL-6 almost completely abrogated the ability of DC CM to block suppression (Fig. 4B). Moreover, LPS- or CpG-treated DC CM from IL-12 and TNF-α–deficient DCs blocked suppression mediated by TR cells comparably to that mediated by WT DC CM (16) (table S1). By contrast, CM from LPS-treated IL-6–/– DCs did not lead to block of suppression (Fig. 4C). Recombinant IL-6 alone or together with MyD88–/– DC CM did not lead to block of suppression (16), although the addition of recombinant IL-6 restored the ability of DC CM from IL-6–/– DCs to block suppression (Fig. 4D). This result suggests that IL-6 needs to act synergistically with another TLR-induced cytokine(s) for efficient blockade of TR cell function. These data, combined with the presence of IL-6 in all the active fractions, indicated that IL-6 has a major role in the TLR-mediated block of suppression. This conclusion was further confirmed when suppression assays were performed with DCs from WT, MyD88–/–, and IL-6–/– mice as APCs in the presence or absence of LPS (Fig. 4E).
The presence of IL-6 in DC CM is critical for block of TR cell–mediated suppression. (A) Fractions from size exclusion chromatography were tested for their activity to block suppression. MyD88–/– splenic DCs were used as APCs to set up TR cell suppression assays. T cell proliferative responses in the presence of TR cells are shown. T cell proliferative responses in the absence of TRcells were ∼235,000 counts per minute (cpm) for fractions 10 to 14 and for unfractionated DC CM, and ∼180,000 cpm for all the other fractions. Fractions were analyzed for the presence of various cytokines. Data are presented as relative units of IL-6 found in these fractions. The apparent discrepancy between activity of fractions and distribution of IL-6 amounts in the fractions is due to differences in sensitivities of the two assays. (B to D) TR cell suppression assays were set up as described, and MyD88–/– mice were used as APCs. LPS-treated DC CM from WT (B, C, and D), MyD88–/– (C), or IL-6–/–mice (C and D) was added at the beginning of the cultures. To neutralize IL-6, DC CM was preincubated with anti–IL-6 neutralizing antibody (10 μg/ml) (B). Recombinant mouse IL-6 (20 ng/ml) was used to replenish DC CM from IL-6–deficient mice (D). T cell responses are shown as the percentage of total counts, with 100% representing the counts in the absence of TR cells separately for each condition. Standard errors were ∼5%. (E) A T cell proliferation assay was set up in the presence or absence of TR cells with splenic DCs from indicated mice. LPS was used at 100 ng/ml. T cell responses are shown as the mean ± SE of triplicate cultures.
To further test the contribution of IL-6 to T cell activation in the presence of TR cells, we set up in vitro priming assays using total CD4 T cells from WT mice with titrating doses of anti-CD3. DCs from WT or IL-6–deficient mice were used as APCs either in the presence or absence of LPS. Although LPS significantly increased the ability of WT DCs to prime naı̈ve T cells, it had minimal effect on the ability of IL-6–deficient DCs to prime T cells in assays that contain physiological ratios of responder and TR cells (Fig. 5A). However, in the absence of TR cells, IL-6–/– DCs induce T cell activation similar to that induced by WT DCs (Fig. 5B). Collectively, these results demonstrate that IL-6 plays a critical role in T cell activation by overcoming TR cell–mediated suppression.
Production of IL-6 by DCs stimulated with TLR ligands is important for in vitro T cell priming. (A) Total CD4 T cells from WT mice were stimulated with titrating doses of anti-CD3, with DCs from either WT or IL-6–deficient mice as APCs. LPS was used at 100 ng/ml. T cell proliferative responses are shown as the mean ± SE of triplicate cultures. (B) CD4+CD25– T cells from WT mice were stimulated with titrating doses of anti-CD3, using either WT or IL-6–deficient DCs. LPS (100 ng/ml) was used to stimulate DCs. T cell proliferative responses are shown as the mean ± SE of triplicate cultures.
IL-6 is required for T cell activation in vivo.
If IL-6 induced during infection allows effector T cells to overcome suppression by TR cells, then IL-6–deficient mice should have a defect in T cell activation in vivo. To test this hypothesis, we immunized WT and IL-6–deficient mice with ovalbumin, using LPS as an adjuvant (15). We found that IL-6–deficient mice were severely compromised in their induction of T cell responses, as measured by proliferation (Fig. 6A) and secretion of IL-2 (Fig. 6B). IL-6–/– DCs matured and produced cytokines, in response to LPS, similar to WT DCs (fig. S5). To determine whether T cells can be primed in the absence of TR cells in IL-6–/– mice, we set up monoclonal antibody (mAb)–based in vivo depletion of CD25+ T cells, using a previously published protocol (22) (fig. S6). Depletion of TR cells followed by immunization led to T cell priming in IL-6–/– mice (Fig. 6C), suggesting that the defect in T cell activation in IL-6–/– mice is due to the inability to overcome TR-mediated suppression. The difference in T cell responses between WT mice and TR-depleted IL-6–/– mice is probably due to the fact that in vivo depletion is transient (22) (fig. S6B) and newly generated TR cells may not allow the same level of T cell responses in IL-6–/–mice. These results do demonstrate, however, that induction of IL-6 by microbial products is required to induce T cell activation in the presence of TR cells. When TR cells are depleted, induction of costimulation seems to be sufficient to induce T cell priming. Depletion of TR cells in WT mice during priming results in enhanced T cell responses compared with control mice (Fig. 6D). This result demonstrates that even T cells specific for foreign antigens are subject to TR cell–mediated suppression in vivo. Taken together, these data demonstrate that IL-6 plays a major role in T cell activation both in vitro and in vivo because of its ability to overcome suppression mediated by TR cells.
T cell priming is defective in vivo in IL-6–/– mice. (A and B) Purified CD4 T cells from draining lymph nodes of WT or IL-6–deficient mice, immunized with ovalbumin and LPS emulsified in incomplete Freund's adjuvant (IFA), were restimulated with titrating doses of ovalbumin. Irradiated (2000 rads) B cells from WT mice were used as APCs. Data are shown as proliferative responses (A) and IL-2 production (B) by responding T cells. (C and D) Anti-CD25 (Clone PC61) or control rat immunoglobulin G was injected intravenously into mice on day 0. Depletion of CD25+ T cells was confirmed by staining the peripheral blood mononuclear cells (PBMCs) on day 3 (fig. S6). Mice were then immunized on day 3 with ovalbumin and LPS emulsified in IFA. Purified CD4 T cells from draining lymph nodes of WT or IL-6–deficient mice were restimulated on day 7 of immunization with titrating doses of ovalbumin. Irradiated (2000 rads) B cells from WT mice were used as APCs. Data are shown as proliferative responses and are representative of three independent experiments.
Conclusions.
The results presented in this study suggest that induction of T cell responses is controlled by TLRs at least at two different levels. TLR-mediated recognition of PAMPs and induction of costimulation on DCs is required to direct T cell responses against pathogen-derived antigens in a cognate T cell–APC interaction. However, induction of costimulation alone does not seem to be sufficient to induce T cell activation in vivo. Indeed we find that production of IL-6 by DCs in response to TLR ligation during infection is critical for T cell activation, because it allows pathogen-specific T cells to overcome the suppressive effect of CD4+CD25+ TRcells. Removal of TR cells, however, allows T cell activation even in the absence of IL-6 (in IL-6–deficient mice), suggesting that induction of costimulation on DCs is sufficient for T cell activation in the absence of TR cells.
Although IL-6 can act systemically, production of high levels of IL-6 during infection will not normally result in a nonspecific block of suppression, because activation of antigen-specific T cells still requires the costimulatory signals provided in a cognate T cell–APC interaction. However, it is tempting to speculate that during chronic infections, conditions may arise that lead to IL-6–mediated release of suppression of self-reactive T cells, which may explain the link between infection and some autoimmune diseases (23). IL-6–deficient mice, on the other hand, are resistant to autoimmune diseases such as experimental autoimmune encephalitis and rheumatoid arthritis (24–27). In addition, administration of IL-6R mAb to mice with severe combined immunodeficiency (SCID), after transfer of CD45 RBhigh T cells, confers protection from colitis (28), and IL-6–deficient mice have been shown to be less susceptible to colitis (29). These reports support our findings, and our results seem to provide a mechanistic explanation for these observations.
IL-6 has multiple well-characterized functions, particularly in the context of the acute phase response and B cell differentiation. Our study suggests that the failure to overcome TR-mediated suppression contributes to the phenotype of IL-6–/– mice, including their susceptibility to infection and resistance to autoimmunity.
In conclusion, the present study demonstrates that innate immune recognition by TLRs controls the activation of adaptive immune responses by at least two distinct mechanisms: the induction of costimulatory molecules on DCs and the production of IL-6, which renders pathogen-specific T cells refractory to the suppressive activity of CD4+CD25+ TRcells.
Supporting Online Material
www.sciencemag.org/cgi/content/full/1078231/DC1
Materials and Methods
Figs. S1 to S6
Table S1
References
↵* To whom correspondence should be addressed. E-mail: ruslan.medzhitov{at}yale.edu
