PerspectiveImmunology

T Cell Activation by TLRs: A Role for TLRs in the Adaptive Immune Response

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Science's STKE  04 Sep 2007:
Vol. 2007, Issue 402, pp. pe48
DOI: 10.1126/stke.4022007pe48

Abstract

Toll-like receptor (TLR) activation is primarily thought to affect antigen-presenting cells (APCs) by inducing an innate immune response that can subsequently activate the adaptive immune system. However, there are increasing data that demonstrate expression and activation of TLRs on T cells, thus providing evidence for a direct role for TLRs in the activation of an adaptive immune response. A study recently demonstrated that Pam3CSK {N-palmitoyl-S-[2,3-bis(palmitoloxy)-(2RS)-propyl]-Cys-Ser-Lys4}, a TLR2 agonist lipopeptide, activates T helper 1 (TH1) cells and induces interferon-γ (IFN-γ) production, even in the absence of TLR1, which differs from its mechanism of activation of APCs. Moreover, whereas Pam3CSK-stimulated IFN-γ production by TH1 cells is ablated in the absence of both myeloid differentiation marker 88 (MyD88), an adaptor protein in the TLR pathway, and interleukin-1 receptor (IL-1R)–associated kinase–4 (IRAK4), the mitogen-activated protein kinases p38 and c-Jun N-terminal kinase (JNK) are still phosphorylated. These data suggest that TLR2 activation of TH1 cells occurs through a mechanism different from that described for APCs and provides further evidence of direct TLR activation of the adaptive immune system.

Toll-like receptors (TLRs) are immune recognition receptors that respond to a plethora of microbial components, such as lipopolysaccharide (LPS), lipoproteins, porins, peptidoglycan, flagellin, single- and double-stranded RNA, and unmethylated CpG oligonucleotides (16). Cellular activation by TLR agonists induces signal transduction events that result in the transcription of many immune and inflammatory genes. Antigen-presenting cells (APCs) have been the primary focus of TLR investigation, although TLRs are also expressed on cells of the adaptive immune system, such as T cells. A study by Imanishi et al. recently demonstrated that murine T helper 1 (TH1) effector T cells, but not TH2 cells, are responsive to TLR2 agonists but not to other TLR ligands (7). The authors demonstrated that the bacterial lipopeptides Pam3CSK {N-palmitoyl-S-[2,3-bis(palmitoloxy)-(2RS)-propyl]-Cys-Ser-Lys4} and MALP2 (macrophage-activating lipopeptide), both TLR2 agonists, stimulate interferon-γ (IFN-γ) production and proliferation and survival of TH1 cells in the absence of T cell receptor (TCR) signaling and that this effect is enhanced by the addition of either interleukin-2 (IL-2) or IL-12. Thus, TH1 effector cell function can be induced by two methods, either through direct interactions between T cells and APCs or through direct activation with microbial products in the absence of APCs.

Stimulation of CD4+ T cells results in their differentiation into TH1, TH2, or the more recently described TH17 cells. These cells are distinguishable by their transcription factor requirements and cytokine profiles. TH1 cells require the transcription factor T-bet (T box expressed in T cells) and produce IFN-γ; TH2 cells require GATA3 and secrete the effector cytokines IL-4, IL-5, and IL-13, whereas cells of the TH17 lineage require the transcription factor retinoic acid receptor–related orphan receptor γ-T (RORγt) to produce IL-17 (8, 9). TLRs influence the differentiation of TH1 and TH2 cells through the activation of non-T cells, resulting in the generation of lineage-specific cytokines. IL-12, IL-23, and IL-27 promote TH1 differentiation of naïve T cells and are often produced by dendritic cells and macrophages that have been activated by TLR ligation (9). Thus, TLR agonists can induce a TH1-mediated immune response, which is important in fighting many microbial infections. However, there is evidence that TLR activation of APCs can also direct differentiation of naïve CD4+ T cells into TH2 cells, which are involved in antibody production, important in fighting parasitic infections, and involved in the pathologies of allergy and asthma. In a mouse model of allergic asthma generated by ovalbumin and the TLR4 agonist LPS, the symptoms and pathology induced are mediated by TH2 cells. The response is dependent, however, on the dose of LPS administered, because a low dose of LPS generates a TH2 response, whereas a high dose of LPS induces a TH1 response (10). In this situation, the effect of TLR stimulation is mostly on APCs, resulting in the induction of cytokines that can direct differentiation of naïve T cells to either TH1 or TH2 lineages, as opposed to a direct effect of TLR stimulation on T cells.

TLRs are expressed on CD4+ T cell subsets, and these cells can be directly activated by TLR agonists. Although naïve T cells are generally unresponsive to TLR ligands, TCR activation up-regulates TLR expression, rendering these T cells responsive to TLR agonists, which function in a costimulatory capacity (1113). Memory T cells are also responsive to TLR agonists, especially those for TLR2, TLR5, and TLR8, and this stimulation also requires TCR activation or the presence of cytokines such as IL-2 or IL-15 (11, 13). These data suggest that TLR stimulation of T cells can function not only in a costimulatory capacity during the initiation of an adaptive immune response but also as an aid in the survival of memory T cells and the rapid induction of a memory response.

Regulatory T (Treg) cells, CD4+ CD25+ T cells that express the transcription factor Foxp3, perform a critical function by controlling the immune response and aiding in the prevention of autoimmunity (14). Treg cells are also sensitive to TLR activation, which results in a reversal of their suppressive activity (1518). This is a transient effect, however, and Treg cells regain their suppressive function after TLR agonists have been removed (17).

Stimulation of TLRs occurs through dimerization (mainly homodimerization) of the TLR upon ligand binding. However, previous studies performed with APCs demonstrated that TLR1, TLR2, and TLR6 are unusual because TLR2 forms heterodimers with both TLR1 and TLR6 (1921). This may in part explain why TLR2 is able to recognize such a large number of structurally unrelated ligands. However, Imanishi et al. used TLR1-deficient mice to demonstrate for the first time that stimulation of TH1 cells by Pam3CSK can occur in the absence of TLR1. This suggests that activation of TH1 cells may occur solely through TLR2 dimers or through the involvement of an unidentified coreceptor. Moreover, TLR2 is expressed on both TH1 and TH2 cells, but only TH1 cells increase their expression of activation markers and produce cytokines in response to treatment with TLR2 agonists. It is unclear whether the lack of stimulation of TH2 cells by Pam3CSK is due to the absence of TLR1, because the expression of TLR1 on TH2 cells is unknown, or whether the unresponsiveness of these cells is due to another mechanism. MALP2, another TLR2 agonist, requires TLR6 for the cellular activation of APCs (22). It would be useful to determine whether MALP2 can stimulate TH1 cells in the absence of TLR6. Overall, these data indicate that TLRs can stimulate TH1 cells in a manner different from that by which they stimulate APCs.

Accessory molecules are also involved in TLR signaling. Recognition of LPS requires the accessory molecules MD2 (a small secreted protein that binds to the extracellular domain of TLR4) and CD14 in addition to TLR4 (2327). CD14, although not required for TLR2 signaling, has also been implicated in enhancing TLR2-induced activation of the transcription factor nuclear factor κB (NF-κB) through an undefined mechanism (2831). It is unclear at this point whether CD14 or other accessory molecules are involved in the stimulation of TH1 cells by TLR2 ligands, but they provide another possible mechanism by which TH1 cells could be activated by Pam3CSK in the absence of TLR1.

TLRs are composed of two regions, the extracellular ligand-recognition portion, which contains leucine-rich repeats, and the intracellular signaling TIR (Toll-IL-1 receptor) domain, which interacts with the TIR domains of the appropriate adaptor molecules. Two pathways are induced after TLR4 stimulation: (i) the myeloid differentiation marker 88 (MyD88)–dependent pathway, which involves MyD88 and TIRAP [TIR domain–containing adaptor protein, also known as Mal (MyD88 adaptor-like)], and (ii) the MyD88-independent pathway, which involves the adaptors TRIF [TIR domain–containing adaptor inducing IFN-β, also known as TICAM-1 (TIR domain–containing adaptor molecule 1)] and TRAM [TRIF-related adaptor molecule, also known as TICAM-2] (32). TLR3 signaling requires TRIF but not MyD88, whereas MyD88 is absolutely necessary for downstream signaling by TLR5 and TLR7 to TLR9. Similar to TLR4 signaling, which is MyD88-dependent, signaling from heterodimers of TLR2 and either TLR1 or TLR6 requires the adaptor molecules MyD88 and TIRAP.

The binding of ligand to TLR2 on APCs results in the recruitment of MyD88 and TIRAP and the subsequent activation of IL-1R–associated kinase (IRAK). TRAF6 [tumor necrosis factor receptor (TNFR)–associated factor 6] is then activated and associates with a complex containing the mitogen-activated protein kinase (MAPK) kinase kinase TAK1 [transforming growth factor (TGF)–β–activated kinase] and the adaptor proteins TAB1 (TAK-binding protein 1) and TAB2, which results in the phosphorylation of TAB2 and TAK1 by TRAF6. Subsequent downstream signaling includes the activation of the MAPKs—extracellular signal–regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun N-terminal kinase (JNK)—and the successive phosphorylation and polyubiquitination of IκBα (inhibitor of NF-κB α), resulting in translocation of NF-κB to the nucleus, where it can initiate transcription (33).

APCs treated with TLR2 agonists require the recruitment of MyD88 and TIRAP and the activation of IRAK4 to induce an inflammatory response (3436). However, Imanishi and colleagues demonstrated that TLR2 stimulation of TH1 cells from either MyD88- or IRAK4-deficient mice still results in p38 and JNK phosphorylation but does not induce ERK phosphorylation or NF-κB nuclear translocation (Fig. 1). Although p38 and JNK are activated by TLR2 agonists in the absence of MyD88 or IRAK4, the production of IFN-γ is greatly reduced when compared with that of wild-type cells, suggesting that the signaling events induced by TLR2 ligands in TH1 cells differ from those induced in APCs. It is possible that another adaptor molecule, such as TIRAP, IRAK1, or IRAK2, may be able to compensate for the loss of MyD88 or IRAK4 in TH1 cells. The use of additional adaptors may also address the lack of a requirement for TLR1 for stimulation.

Fig. 1.

Signaling events involved in TLR2-induced activation of TH1 cells. Stimulation of TH1 cells (expressing either TLR2 as a homodimer or as a heterodimer with a coreceptor) with the synthetic lipopeptide Pam3CSK results in the activation of ERK1/2, p38, JNK, and NF-κB, and the production of IFN-γ. Both MyD88 and IRAK4 are necessary for the activation of ERK and NF-κB but are only partially required for the activation of p38 and JNK. The addition of either IL-2 or IL-12 results in enhanced p38 and JNK phosphorylation (symobolized by the blue arrows) together with increased IFN-γ production, which is somewhat dependent on p38 and JNK. IKK, IκB kinase.

Although TH1 cells are activated by TLR2 ligands, the production of IFN-γ is enhanced in the presence of either IL-2 or IL-12, indicating a synergistic effect of TLR stimulation with cytokine responsiveness. This has also been demonstrated in memory T cells stimulated with TLR agonists in the presence of IL-2 or an antibody against CD2, which mimics the binding of a T cell to an APC, resulting in costimulation of the T cell (11). The enhanced production of IFN-γ is partly due to the increased activation of p38 and JNK, because specific inhibitors of these pathways reduce the secretion of IFN-γ. Interestingly, Imanishi et al. also showed that there is no effect on IFN-γ secretion when p38 is inhibited in TH1 cells stimulated with an antibody against CD3, indicating that different signaling pathways are involved in IFN-γ production by T cells depending on the stimulus.

The ability of TLR agonists to directly activate both the innate and adaptive arms of the immune system has important implications for how we think about the ways in which the body fights infections. In the context of the work performed by Imanishi and colleagues, TLR stimulation of APCs results in the production of IL-12, which can then act synergistically on TLR2-stimulated TH1 cells to increase IFN-γ secretion, which is necessary for the activation of macrophages. However, the signaling events involved in TLR2-induced activation of TH1 cells differ from those in APCs. Further work should focus on the differences in these signaling mechanisms and on how they affect the immune response to pathogens.

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