Toll-Like Receptor 8-Mediated Reversal of CD4+ Regulatory T Cell Function

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Science  26 Aug 2005:
Vol. 309, Issue 5739, pp. 1380-1384
DOI: 10.1126/science.1113401


CD4+ regulatory T (Treg) cells have a profound ability to suppress host immune responses, yet little is understood about how these cells are regulated. We describe a mechanism linking Toll-like receptor (TLR) 8 signaling to the control of Treg cell function, in which synthetic and natural ligands for human TLR8 can reverse Treg cell function. This effect was independent of dendritic cells but required functional TLR8-MyD88-IRAK4 signaling in Treg cells. Adoptive transfer of TLR8 ligand-stimulated Treg cells into tumor-bearing mice enhanced anti-tumor immunity. These results suggest that TLR8 signaling could play a critical role in controlling immune responses to cancer and other diseases.

Regulatory T (Treg) cells actively suppress host immune responses and, in consequence, play an important role in preventing autoimmunity, as well as in the general regulation of immune responses to tumors and infectious diseases (14). Naturally occurring CD4+ CD25+ Treg cells represent a subset of Treg cells that exist without specific antigen stimulation and suppress immune responses through a cell-cell contact mechanism (4). In comparison, antigen-induced Treg cells mediate immune suppression either through cell-cell contact or through secretion of soluble factors such as interleukin (IL)–10 and transforming growth factor–β (2, 4, 5). It is possible that both types of Treg cells may have detrimental effects on cancer immunotherapy, because of their potent ability to suppress immune responses (4, 5). Consistent with this hypothesis, increased proportions of CD4+ CD25+ Treg cells have been observed in patients with different types of cancers (6, 7). Furthermore, we recently demonstrated that antigen-specific CD4+ Treg cells present in tumor-infiltrating lymphocyte (TIL) lines suppressed the proliferation of naïve CD4+ T cells through a cell contact–dependent mechanism (5, 8).

Toll-like receptors (TLRs) recognize a set of conserved molecular structures, so called pathogen-associated molecular patterns, that allow them to sense and initiate innate and adaptive immune responses (9, 10). Such responses are generally thought to be produced through the induction of dendritic cell (DC) maturation, which enables DCs to activate naïve T cells and to render effector cells refractory to Treg cells (1114). Whether such TLR signals directly regulate the suppressive function of CD4+ Treg cells without the participation of DCs remains unknown.

We established two CD4+ Treg cell lines (Treg102 and Treg164) by pooling a panel of Treg cell clones (5, 8) and tested whether their suppressive function could be reversed. A previous study has shown that stimulation of mouse DCs with TLR ligands, such as lipopolysaccharide (LPS, a TLR4 ligand) and CpG motif–containing oligonucleotides (a TLR9 ligand), induces DCs to secrete cytokines, including IL-6, that render CD4+ effector cells refractory to Treg cell–mediated suppression (14). In light of these results, we tested whether DCs might have the potential to influence the suppressive effects of human Treg cells on naïve CD4+ T cell proliferation in the presence of either CpG-A [also called type-D CpG oligonucleotides (1517)], LPS, IL-6, or tumor necrosis factor (TNF)–α. Although CpG-A could reverse Treg cell–mediated suppression and restored the proliferation of naïve CD4+ T cells to ∼60% of normal levels, LPS, IL-6, and interferon (IFN)–α lacked this ability (Fig. 1A). In control experiments, neither CpG-A nor the cytokines used could appreciably affect the baseline proliferation of any of these cell populations (Fig. 1A and fig. S1). Proliferation experiments performed with plates coated with antibody to CD3 revealed that CpG-A reversed Treg cell–mediated suppression more strongly in the absence of DCs (Fig. 1B). Again, this contrasted with the failure of another class of CpG oligonucleotide, CpG-B (also called type K CpG), as well as of LPS, TNF-α, IL-6, and IFN-α, to block the suppressive activity of CD4+ Treg cells (Fig. 1B). Furthermore, cultured CD4+ Treg cells at 100% purity (5, 8) that had been pretreated with CpG-A for 3 days became nonsuppressive (Fig. 1C). These results demonstrated that CpG-A can directly mediate reversal effect on Treg cell function in the absence of DCs.

Fig. 1.

Reversal of the suppressive function of CD4+ Treg cells by CpG-A. (A) Restoration of Treg cell–suppressed proliferation of naïve CD4+ T cells by CpG-A. The purified naïve CD4+ T cells and DCs were mixed with CD4+ Treg cells at an 1:10 ratio of Treg cells to naïve T cells, in medium containing soluble antibody to CD3 with or without CpG-A, LPS, IL-6, or INF-α. Naïve T cells plus DCs, DCs plus Treg cells, Treg cells alone, DCs alone, and naïve T cells alone, in the presence or absence of CpG-A, LPS, IL-6, or INF-α, served as controls. CPM, counts per minute. (B) Requirement for DCs in reversing the suppressive effect of Treg cells on naïve T cell proliferation. The freshly prepared CD4+ (responding) T cells weremixed with CD4+ Treg cells in plates coated with antibody to CD3, in medium containing CpG-A, CpG-B, TNF-α, LPS, IL-6, or INF-α but without DCs. (C) Reversal of Treg cell suppressive function by pretreatment with CpG-A or non-CpG-A. CD4+ Treg cell clones were pretreated with CpG-A or CpG-B for 3 days before we evaluated their ability to suppress naïve CD4+ T cell proliferation.

Because both CpG-A and CpG-B contain the CpG motif sequences responsible for binding to TLR9, we next tested whether the CpG motif itself conferred the ability to reverse Treg suppressive function. CD4+ Treg cell lines that had been pretreated for 3 days with CpG-A, non-CpG-A (in which the CG sequence was changed to GC), CpG-B, or non-CpG-B (CG changed to GC) (18) were used in proliferation assays. Pretreatment with CpG-A and non-CpG-A reversed the suppressive activities of CD4+ Treg cells equally well (Fig. 2A), indicating that the reversal effect of CpG-A did not depend on the CpG motif. To test whether the Poly-G tail (containing five guanosine nucleotides), which is present in CpG-A but absent in CpG-B, was responsible for the observed effect, oligonucleotides were generated in which Poly-G tails had been replaced with Poly-A (CpG-NG). Other oligonucleotides were also tested, in which the CpG motif had been deleted but 10 guanosine nucleosides were retained with the phosphorothioate backbone (Poly-G10) (18). The ability to reverse suppression was entirely lost in CpG-NG but retained and enhanced in the CpG-deleted Poly-G10 oligonucleotides, indicating that Poly-G oligonucleotides were necessary and sufficient to reverse Treg cell function, most likely through a receptor distinct from TLR9 (Fig. 2B and fig. S2).

Fig. 2.

Identification of sequence elements in CpG-A responsible for the direct reversal of Treg cell suppressive function. (A) Reversal of Treg cell function by CpG-A and non-CpG-A. CD4+ Treg cells were pretreated with CpG-A, non-CpG-A, CpG-B, or non-CpG-B for 3 days before we evaluated their ability to suppress naïve CD4+ T cell proliferation. (B) Poly-G, but not the CpG motif, is responsible for the observed reversal effect. Naïve CD4+ T cells were mixed with CD4+ Treg cells in the presence of CpG-NG, CpG-A, or Poly-G10. Poly-A10, Poly-T10, and Poly-C10 served as controls for Poly-G10 oligonucleotides. (C) Poly-G2 restores the proliferation of naïve T cells suppressed by Treg102 and naturally occurring CD4+ CD25+ Treg cells. Naïve CD4+ T cells or Treg cells were labeled with carboxyfluorescein diacetate succinimidyl ester (CFSE). Cell culture conditions are indicated. After 3 days of culture, cell division was analyzed by FACS gated on the CFSE-labeled cells without OKT3 (antibody to CD3) stimulation.

Using CD4+ CD25+ and CD4+ CD25 T cell populations purified from fresh peripheral blood lymphocytes by fluorescence-activated cell sorting (FACS) (18), we again found that the suppressive activity of naturally occurring CD4+ CD25+ Treg cells could be reversed by Poly-G10 oligonucleotides, but not by a control oligonucleotide (Poly-T10) (fig. S3). In culture, proliferation was restricted to naïve CD4+ T cells, whereas Treg cells were non-responsive, regardless of the presence or absence of Poly-G2 oligonucleotides (Fig. 2C). This T cell receptor–induced proliferation of naïve CD4+ T cells was completely suppressed by both naturally occurring CD4+ CD25+ Treg cells and Treg 102 cells and was reversed by treatment with Poly-G2 oligonucleotides (Fig. 2C). From these experiments, we conclude that ligands capable of reversing Treg cell function can influence both antigen-specific as well as naturally occurring CD4+ CD25+ Treg cells.

Because TLRs have been implicated in the regulation of innate and adaptive immunity and might play a role in controlling Treg cell function (10, 12), we assumed that Poly-G oligonucleotides might mediate reversal of Treg cell function through one of the TLRs. Because MyD88, along with IRAK4, is essential for signaling in TLRs (10), we sought to test whether these two adaptors were involved in mediating Poly-G oligonucleotide response. We approached this through specific knockdown using enhanced green fluorescent protein (GFP)–expressing, lentivirus-based RNA interference (18, 19). Inhibition of expression of IRAK4, MyD88, and three of the TLRs by the corresponding small interfering RNAs (siRNAs) was evident in HEK293 cells transfected with target genes (fig. S4). For IRAK4 and MyD88, specific inhibition was also apparent in FACS-sorted GFP+ (transduced) Treg cells versus untransduced (GFP) Treg cells (Fig. 3A). The suppressive ability of these transduced antigen-specific Treg cells, as well as transduced naturally occurring CD4+ CD25+ Treg cells, could no longer be reversed by Poly-G10 oligonucleotides (Fig. 3B and fig. S5), suggesting that direct reversal of the suppressive activity of Treg cells by Poly-G oligonucleotides depends directly on the activity of both adaptors.

Fig. 3.

The TLR8-MyD88-IRAK4 pathway is required to reverse the suppressive function of Treg cells. (A) Knockdown of IRAK4, MyD88, and TLR8 in naturally occurring CD4+ CD25+ Treg cells by RNA interference. Specific knockdown of target genes was observed with real-time PCR analysis, and expression of an irrelevant gene was essentially unchanged. (B) Evaluation of the reversibility of IRAK4 siRNA, MyD88 siRNA–, or control siRNA–transduced (GFP+) and untransduced (GFP) Treg102 and CD4+ CD25+ Treg cells by Poly-G10 oligonucleotides. Uninfected parental Treg cells and Poly-T10 oligonucleotides served as controls. (C) The expression pattern of TLR7, TLR8, and TLR9 in Treg cells as determined by RT-PCR with gene-specific primers. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) amplification was used as an internal control. PBMCs, peripheral blood mononuclear cells; mDC, mature dendritic cell; LCL, EBV-transformed B lymphoblastoid cell line; 293, HEK293 cells. (D) The loss of reversible suppressive function by Treg102 and CD4+ CD25+ Treg cells transduced with TLR8 siRNA. Treg cells infected with TLR8 siRNA or TLR9 siRNA served as controls. Treg cells were sorted into transduced (GFP+) and untransduced (GFP) cell populations for testing of their suppressive function in the presence of Poly-G10 or Poly-T10.

Because TLR7, TLR8, and TLR9 have been identified as specific receptors for nucleic acid ligands (10, 2025), we tested whether they may serve as receptors for Poly-G oligonucleotides. Reverse transcription polymerase chain reaction (RT-PCR) (Fig. 3C), as well as quantitative PCR (fig. S6), revealed TLR8 as the only receptor consistently expressed by naturally occurring CD4+ CD25+ Treg cells and antigen-specific Treg cell lines. Consistent with this, the suppressive function of TLR8 siRNA–transduced Treg102 and naturally occurring CD4+ CD25+ Treg cells could not be reversed by Poly-G10, whereas TLR7 or TLR9 siRNA–transduced Treg cells retained their reversible suppressive function (Fig. 3D). Taken together, these results indicate that the TRL8-MyD88 signaling pathway controls the suppressive function of both antigen-specific Treg and naturally occurring CD4+ CD25+ Treg cells.

These results suggest that the TLR8 signaling pathway may be necessary and sufficient for direct reversal of the suppressive function of Treg cells, meaning that it should be possible to reproduce the effect with natural ligands for human TLR8. We found that two such ligands, ssRNA40 and ssRNA33 in complexes with cationic lipid (23), completely reversed the suppressive function of antigen-specific Treg102 cells and naturally occurring CD4+ CD25+ Treg cells (Fig. 4A). In contrast, ligands for other TLRs failed to do so (Fig. 4A). Consistent with this result, we found that none of the TLR8 ligands could reverse the suppressive function of Treg102 cells when TLR8 expression was specifically inhibited by siRNA (Fig. 4B).

Fig. 4.

Evaluation of Poly-G and natural TLR ligands for their ability to reverse Treg cell function and their effects on antitumor immunity. (A) Naïve CD4+ T cells were mixed with Treg102 and naturally occurring CD4+ CD25+ Treg cells in the presence of different TLR ligands. (B) The reversibility of TLR8 siRNA–transduced (GFP+) and untransduced (GFP) Treg cells in response to Poly-G10, ssRNA40, and CpG-A oligonucleotides. Uninfected parental and control siRNA-transduced Treg102 cells and Poly-T10 served as controls. (C) Poly-G10–induced reversal of Treg cell function enhances antitumor immunity in vivo. Rag1-deficient mice were injected with human 586mel tumor cells on day 0 and then treated with autologous tumor-specific CD8+ TIL586 cells, either alone or plus Treg102 cells with or without Poly-G10 or Poly-T10 on day 3. Treg cells were pre-activated with OKT3 and washed before adoptive transfer. Tumor volumes were measured and presented as means ± SD (n = 6 mice per group). (D) Experimental procedures, tumor cells, and T cells were as in (C), except that Rag2-γC-deficient mice were used. P values in (B) and (C) were determined by the Wilcoxon rank-sum test.

We next evaluated the ability of Poly-G10 to reverse Treg cell function in a mouse tumor model (18, 26). When injected into B and T cell–deficient Rag1–/– mice, the human 586mel tumor cell line showed progressive growth but were inhibited when co-injected with autologous, tumor-specific CD8+ TIL586 cells (26) (Fig. 4C). When CD8+ TIL586 and Treg102 cells treated with or without Poly-T10 (a control oligonucleotide) were adoptively transferred into tumor-bearing mice, tumor cells again grew progressively and more rapidly than in mice receiving 586mel cells alone (P = 0.04), suggesting that the Treg102 cells had inhibited tumor-specific immune responses. By contrast, adoptive transfer of TIL586 cells and Poly-G10–treated Treg102 cells restored, and in some cases enhanced, tumor growth inhibition, compared with that in mice treated with TIL586 cells alone (Fig. 4C) (P = 0.004).

To exclude a potential role of the host natural killer cells in controlling tumor growth in vivo, we further investigated the effect of Poly-G treatment on Treg cell-mediated suppression of antitumor immunity in Rag2-γC–deficient mice that lacked B, T, and natural killer cells. The tumor growth rate in Rag2-γC-deficient mice that received TIL586 cells plus Treg cells treated with control oligonucleotides did not differ significantly from that in mice receiving tumor cells without treatment (Fig. 4D) (P = 0.07). In contrast, adoptive transfer of TIL586 along with Poly-G10–treated Treg cells into tumor-bearing mice completely restored, but did not enhance, antitumor activity mediated by TIL586 cells (P = 0.10). This further supports the conclusion that antitumor immunity mediated by TIL586 cells can be directly controlled by the functional (suppressive versus nonsuppressive) status of Treg cells and that Poly-G oligonucleotide stimulation of Treg cells could reverse this immune suppression.

This study identifies a set of guanosine-containing DNA oligonucleotides and natural TLR ligands that can trigger the TLR8-MyD88-IRAK4 signaling pathway and reverse the suppressive function of different Treg populations. Because most TLRs use the MyD88-IRAK4 pathway, it is not entirely clear why only TLR8 ligands can reverse the suppressive function of Treg cells. In part, this may be explained by the expression pattern of TLRs in Treg cells, because human Treg cells express a relatively high level of TLR8 but little or no TLR7 or TLR9 (Fig. 3C). However, it is also possible that TLR8-MyD88-IRAK4 complexes might recruit a unique downstream signaling pathway of MyD88 required for the control of Treg cell function. Although the findings presented here suggest a mechanism linking TLR8 signaling to the functional regulation of Treg cells, it will be important to establish the physiological relevance of this activity for normal immune responses in vivo. Nevertheless, the results presented here suggest that, by shifting the functional balance between Treg and effector T cells through TLR8 signaling, Poly-G oligonucleotides or similar ligands might be useful in clinical settings to enhance the efficacy of immunotherapy directed toward cancer and infectious diseases.

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Materials and Methods

Figs. S1 to S6

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