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. 2006 May 2;103(18):7048-53.
doi: 10.1073/pnas.0601554103. Epub 2006 Apr 21.

Toll-like receptor 2 signaling modulates the functions of CD4+ CD25+ regulatory T cells

Haiying Liu  1 , Mousa Komai-Koma, Damo Xu, Foo Y Liew
Affiliations

Toll-like receptor 2 signaling modulates the functions of CD4+ CD25+ regulatory T cells

Haiying Liu et al. Proc Natl Acad Sci U S A. .

Abstract

Toll-like receptors (TLRs) are primary sensors of both innate and adaptive immune systems and play a pivotal role in response against structurally conserved components of pathogens. Synthetic bacterial lipoprotein (BLP) Pam3Cys-SK4 is a TLR2 agonist that is capable of modulating T cell immune responses. We show here that BLP, together with anti-CD3 antibody [T cell receptor (TcR) activation], induced proliferation of both CD4+ CD25+ regulatory T cells (Tregs) and CD4+ CD25- (effector) T cells in the absence of antigen-presenting cells. The expanded Tregs showed a transient loss of suppressive activity. Moreover, BLP rendered effectors resistant to the suppression of Tregs by increasing IL-2 secretion. BLP also transiently suppressed the induction of Foxp3 (X-linked forkhead/winged helix transcription factor) mRNA in Tregs at the first 8-15 h after T cell receptor activation. Consistent with this observation, BLP-stimulated Tregs regained their inhibitory activity and prevented spontaneous colitis induced by effectors in severe combined immunodeficient mice. Our results demonstrate a previously unrecognized pathway by which TLR expressed on T cells may directly modulate the immune response. Thus, during an acute bacterial infection, BLP may rapidly increase the host's adaptive immunity by expanding effectors and also by attenuating the suppressive activity of Tregs. In the process, BLP also expands the Tregs, which recover their suppressive activity when the infection has subsided, in time to limit potential autoimmunity that might result from the overactivated effectors.

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Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
CD4+CD25− (effector) and CD4+CD25+ (regulatory) T cells functionally expressed TLR2. (A) FACS analysis shows the purity of Tregs, which were Foxp3+. The same degree of purity was achieved for effectors (data not shown), which were Foxp3−. −ve, isotype control. (B) Cells were either freshly isolated or activated for 72 h with plate-bound αCD3. Fluorescence density was measured by gating CD4+ and CD25+ cells. Control was isotype-matched IgG. T cells from TLR2−/− mice stained negative (data not shown). (C) Tregs were labeled with 5-(-6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE) and cultured with APC+ αCD3 and BLP (5 μg/ml) for 3 days. (D) Tregs and effectors were cultured with αCD3 and BLP for 3 days, and cellular proliferation was determined by [3H]thymidine incorporation. (E) Tregs and effectors from WT or TLR2−/− mice were cultured as in D, and cell proliferation was determined. IL-2 in the supernatant was measured by using ELISA. Data are mean ± SD (n = 3) and are representative of three experiments. ∗∗, P < 0.01 compared with control culture without BLP.
Fig. 2.
Fig. 2.
BLP abrogated the suppressive functions of Treg cells. (A) Tregs and effectors from C57BL/6 WT or TLR2−/− mice were cultured alone or together (1:1 ratio) with sαCD3 and APC from TLR2−/− mice, with or without BLP, for 3 days. Cellular proliferation and IFN-γ synthesis were determined. (B) Tregs and effectors from C57BL/6 WT or TLR2−/− mice were cultured as in A in various combinations as indicated. BLP partially reversed the suppressive effect of Tregs, even when either cell type was nonresponsive to BLP (TLR−/−). Data are mean ± SD (n = 3) and are representative of at least three experiments. ∗∗, P < 0.01 compared with culture without BLP.
Fig. 3.
Fig. 3.
IL-2 produced by effectors played an important role in the BLP-mediated abrogation of the suppressive effect of Tregs. Effectors and Tregs were cultured separately or together with sαCD3 and APC for 3 days. (A) Only effectors produced appreciable amounts of IL-2 in response to BLP. (B) Tregs and effectors were cultured together with or without BLP and αIL-2 or normal IgG. (C) The proliferation of effectors could be completely suppressed by formalin-fixed Tregs. This suppression was reversed by the addition of IL-2. (D) Supernatant was collected from cultures of effectors stimulated for 3 days with BLP. Supernatant from WT, but not TLR2−/−, mice reversed the suppressive effect of Tregs. The reversal was neutralized by αIL-2. (E) Effectors were cocultured with Tregs in the presence of the supernatant (1 or 1/5 dilution) of effectors stimulated with or without (con) BLP. (F) Tregs were cultured with supernatants from effectors (of WT or IL-2−/− mice) stimulated with or without (con) BLP. Tregs proliferated significantly when cultured with BLP alone or with the supernatant of effectors from WT, but not IL-2−/−, mice. Data are mean ± SD (n = 3–4) and are representative of at least three experiments. ∗∗, P < 0.01 compared with controls.
Fig. 4.
Fig. 4.
BLP treatment transiently abrogated the suppressive activity and reduced the expression of Foxp3 of Tregs. (A) Effector and Tregs from BALB/c mice were cultured alone or together for 3 days with sαCD3 and APC. In the cocultures, Tregs were preactivated with αCD3 and BLP for 15 h or for 3 days and rested for another 3 days before being cocultured with freshly isolated effectors (7 days). BLP-treated Tregs regained their suppressive activity after the rest period. Data are mean ± SD (n = 3) and are representative of three experiments. ∗∗, P < 0.01 compared with effectors alone. (B and C) Tregs and effectors from BALB/c (B) or TLR2−/− and WT C57BL/6 (C) mice were activated by αCD3 with or without BLP. Cells were analyzed for Foxp3 mRNA expression by real-time PCR. TcR triggering markedly elevated Foxp3 expression at 8–15 h. BLP significantly suppressed Foxp3 expression in the Tregs from WT, but not TLR2−/−, mice. Results are presented as percentage of hypoxanthine phosphoribosyltransferase expression and are representative of at least three experiments. ∗∗, P < 0.01 compared with nonactivated cells. Attempts to demonstrate the effects of BLP on Foxp3 expression by using intracellular staining were not successful, probably because of the lower sensitivity of flow cytometry (data not shown).
Fig. 5.
Fig. 5.
BLP-stimulated Tregs recovered their suppressive functions in vivo. SCID mice were adoptively transferred i.p. with 5 × 105 effectors and infected with L. major in the footpad. The diseased mice were injected i.p. (on day 21) with PBS (CD25−/PBS), 5 × 105 Tregs (CD25−/CD25+), or BLP-activated Tregs for 15 h (αCD3 plus BLP). (A and B) Mice were monitored for body weight (A) and histology of the colon (B) at the end of experiment (8 weeks). Mesenteric lymph node cells were collected and cultured in vitro with αCD3. (C) Cytokine productions were determined by using ELISA. (D) Leishmania infection was also determined as the difference in the thickness between the infected right footpad and the uninfected left footpad. The footpad lesions parallel the parasite load in the footpad (ref. and data not shown). Data are mean ± SD (n = 6) and are representative of two experiments.
Fig. 6.
Fig. 6.
Schematic representation of the role of BLP in T cell homeostasis. Mammalian hosts could use BLP produced by pathogens to enhance the proliferation of, and IL-2 production by, CD4+CD25− effector (TEff) cells to combat the pathogens. BLP also transiently switches off Tregs (probably by down-regulating Foxp3) to boost the effectors. IL-2 produced by TEff could also transiently switch off the Treg function. Both BLP and IL-2 could also expand Tregs. When the level of BLP is reduced after elimination of the pathogens, the expanded Tregs could regain their suppressive function to prevent potential autoimmunity that might result from the expanded TEff cells.
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