ARTICLES TLR7 induces anergy in human CD4+ T cells Margarita Dominguez-Villar1, Anne-Sophie Gautron1, Marine de Marcken1, Marla J Keller2 & David A Hafler1 The recognition of microbial patterns by Toll-like receptors (TLRs) is critical for activation of the innate immune system. Although TLRs are expressed by human CD4+ T cells, their function is not well understood. Here we found that engagement of TLR7 in CD4+ T cells induced intracellular calcium flux with activation of an anergic gene-expression program dependent on the transcription factor NFATc2, as well as unresponsiveness of T cells. As chronic infection with RNA viruses such as human immunodeficiency virus type 1 (HIV-1) induces profound dysfunction of CD4+ T cells, we investigated the role of TLR7-induced anergy in HIV-1 infection. Silencing of TLR7 markedly decreased the frequency of HIV-1-infected CD4+ T cells and restored the responsiveness of those HIV-1+ CD4+ T cells. Our results elucidate a previously unknown function for microbial pattern– recognition receptors in the downregulation of immune responses. Toll-like receptors (TLRs) represent the major pathway by which immunodeficiency virus type 1 (HIV-1), the immune responses medi- microorganisms interact with host cells. They are a family of highly ated by CD4+ helper T cells and CD8+ cytotoxic T cells determine the out- conserved pattern-recognition receptors that recognize distinct come of the infection, with chronic infections being correlated with late, pathogen-associated molecular patterns that are conserved in specific transient or narrowly focused responses by CD4+ or CD8+ T cells9–11. classes of microorganisms1. The human TLR family consists of at least Several studies have demonstrated impairment in the activation and/or ten members that can be classified into two different groups on the basis function of T cells during infection with HIV-1. Specifically, CD4+ of their cellular location. Intracellular TLRs (TLR3, TLR7, TLR8 and T cells from patients chronically infected with HIV-1 display an anergic TLR9) recognize nucleic acids; TLR7 and TLR8 recognize single- phenotype with defects in proliferation and the secretion of interleukin stranded RNA2,3, whereas TLR3 and TLR9 are receptors for double- 2 (IL-2) and interferon-γ (IFN–γ). The mechanisms by which RNA stranded RNA and double-stranded DNA, respectively. In contrast, viruses impair T cell function are not well understood. Nature America, Inc. All rights reserved. America, Inc. Nature cell surface TLRs (TLR1, TLR2, TLR4, TLR5 and TLR6) recognize Here we describe a previously unrecognized pathway of TLR- 5 various components of bacteria1. In mice, although TLR7 and TLR8 are mediated negative regulation of both the activation and the cytokine expressed at low levels in CD4+ T cells, there are species-specific dif- production of CD4+ T cells. Engaging TLR7 expressed on CD4+ © 201 ferences in the recognition of ligands3 as well as in their functionality. T cells resulted in complete anergy by inducing intracellular calcium Specifically, mouse TLR7 and human TLR8 mediate species-specific flux, with activation of an anergic gene-expression program depend- recognition of GU-rich single-stranded RNA. It has been suggested ent on the transcription factor NFATc2 and with subsequent T cell npg that in contrast to its human TLR counterpart, mouse TLR8 is not unresponsiveness that was reversed by knockdown of TLR7 with functional and TLR7 is the only TLR that recognizes single-stranded short hairpin RNA (shRNA). In studies of the potential physiological RNA4. The expression and signaling pathways triggered by stimula- relevance of these findings, we found that knockdown of TLR7 via tion of TLRs have been described in antigen-presenting cells (APCs) shRNA decreased the frequency of HIV-1-infected CD4+ T cells in a process that leads to the activation of APCs with the secretion in vitro and restored the responsiveness of those HIV-1+ CD4+ of inflammatory and antiviral cytokines1,5. Although TLR expression T cells in vitro. Our results elucidate a previously unknown function has been studied mainly in APCs, several reports have described the for microbial pattern–recognition receptors in the downregulation expression of TLRs on lymphocytes6, and specifically on CD4+ T cells. of immune responses, inducing anergy by increasing intracellular As with APCs, such studies indicate that the engagement of TLRs acts calcium concentrations and interfering with secondary costimulation as a positive costimulatory signal that increases the secretion of proin- signals in the presence of signaling via TLRs7. flammatory cytokines, proliferation and cell survival7,8. While TLRs are central to the early host immune response to acute RESULTS viral infection, more-chronic infectious diseases are characterized by Inhibition of the activation of CD4+ T cells by TLR7 the inability of the host immune system to mount a strong, long-lasting While investigating a potential costimulatory role for TLRs in CD4+ response to the infectious agent. In particular, it has been shown that T cells, we observed that the entry of CD4+ T cells into the cell cycle during infection with RNA viruses such as hepatitis C virus and human after crosslinking of the T cell antigen receptor (TCR) with antibody 1Department of Neurology and Department of Immunobiology, Yale School of Medicine, New Haven, Connecticut, USA. 2Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, New York, USA. Correspondence should be addressed to M.D.-V. ([email protected]) or D.A.H. ([email protected]). Received 18 June; accepted 20 October; published online 17 November 2014; doi:10.1038/ni.3036 118 VOLUME 16 NUMBER 1 JANUARY 2015 NATURE IMMUNOLOGY ARTICLES Nature America, Inc. All rights reserved. America, Inc. Nature 5 © 201 npg Figure 1 TLR7 signaling inhibits the proliferation and cytokine secretion of CD4+ T cells. (a) Proliferation of CD4+ T cells labeled with the cytosolic dye CFSE and stimulated for 3 d with various concentrations (above plots) of imiquimod (IMQ). Numbers above bracketed lines indicate percent viable proliferating CD4+ T cells. (b) Frequency of viable proliferating CD4+ T cells stimulated for 3 d with various concentrations (horizontal axis) of imiquimod. (c) ELISA of cytokine secretion by CD4+ T cells stimulated for 3 d with various concentrations of imiquimod. (d) Intracellular staining of IFN-γ and IL-2 (top) and of IL-17 and IL-4 (bottom) in CD4+ T cells stimulated for 4 d with various concentrations of imiquimod and then stimulated for with 4 h PMA and ionomycin. Numbers in quadrants indicate percent cells in each throughout. (e) Frequency of cytokine-producing CD4+ T cells (as in d). (f) Expression of CD25 (left), CD69 (middle) and CD137 (right) on CD4+ T cells stimulated with anti-CD3 and anti-CD28 in the presence (α-CD3 and α-CD28 + IMQ) or absence (α-CD3 and α-CD28) of imiquimod, and of cells stained with isotype-matched control antibody (Isotype). MFI, mean fluorescence intensity. *P < 0.05, **P < 0.005 and ***P < 0.0005 (paired t-test). Data are representative of eight (a–d) or six (e,f) independent experiments with one donor in each (mean and s.e.m. in b,c,e,f). to the invariant signaling protein CD3 (anti-CD3) and crosslinking of of up to 15 µg/ml of imiquimod but found no effect on cell viability the coreceptor CD28 with anti-CD28 was blocked by coengagement (data not shown). The diminished proliferation correlated with less of TLR7 (Fig. 1a,b and Supplementary Fig. 1a,b). Treatment with the secretion of the cytokines IFN-γ, IL-17, IL-2 and IL-4, as measured by synthetic TLR7 agonist imiquimod resulted in considerably less pro- enzyme-linked immunosorbent assay (ELISA), at day 3 after stimu- liferation of CD4+ T cells than that of vehicle-treated control cells, as lation (Fig. 1c). We confirmed the diminished cytokine secretion at well as less secretion of IFN-γ and IL-17, in a dose-dependent fashion the single-cell level, as the frequency of cytokine-producing cells was (Fig. 1c–e and Supplementary Fig. 1c,d). We observed this inhibitory also diminished in a dose-dependent manner with increasing doses of effect as soon as 12 h after activation, with much less induction of imiquimod in culture (Fig. 1d,e). Furthermore, stimulation of CD4+ the expression of IL2, IFNG and IL4 after treatment with imiquimod T cells in the presence of imiquimod inhibited the expression of (Supplementary Fig. 1e). We assessed the effect of concentrations activation markers such as CD25, CD69 and CD137, measured 48 h NATURE IMMUNOLOGY VOLUME 16 NUMBER 1 JANUARY 2015 119 ARTICLES a b Veh e Veh f GDQ IMQ 100 Veh GDQ Lox CL264 80 Lox 4 150 + 80 150 + CL264 60 ** ) 3 60 100 ml 100 / ng 40 70.9 8.3 9.1 55.3 40 2 γ ( 50 T cells (%) 50 20 IL-2 (U/ml) T cells (%) *** Proliferating CD4 Cells *** IFN- 2 3 4 5 20 1 0 10 10 10 10 *** *** Proliferating CD4 0 0 0.1 0.25 0.5 1 2.5 CFSE 0 0 0 NT TLR7(3) NT TLR7(3) ssRNA40 (µg/ml) Veh 150 4 1,000 2.0 400 8 c GDQ d NT g TLR7(3) * Lox 2.0 3 750 1.5 300 ) 6 ) CL264 ml 100 / * ml / 1.5 200 ng 4 ng 2 500 1.0 γ ( ** γ ( IL-2 (pg/ml) 100 IFN- 2 IL-2 (U/ml) 1.0 50 IL-4 (pg/ml) IL-17 (ng/ml) IFN- * *** 1 ** 250 0.5 mRNA (AU) *** * 0 0 *** * * * 0.5 0 0.1 0.25 0.5 1 2.5 0 0.1 0.25 0.5 1 2.5 * * TLR7 0 0 0 0 *** ssRNA40 (µg/ml) ssRNA40 (µg/ml) 0 1,000 4 NT TLR7(3) 800 Figure 2 The inhibitory effect of imiquimod 3 150 Isotype Isotype * * is TLR7 specific.