TLR7 induces anergy in human CD4 + T cells

TLR7 induces nergy in humn CD T cells Mrgrit Dominguez-Villr 1, Anne-Sophie Gutron 1, Mrine de Mrcken 1, Mrl J Keller & Dvid A Hfler 1 The recognition of microil ptterns y Toll-like receptors (TLRs) is criticl for ctivtion of the innte immune system. Although TLRs re expressed y humn CD T cells, their function is not well understood. Here we found tht enggement of TLR7 in CD T cells induced intrcellulr clcium flux with ctivtion of n nergic gene-expression progrm dependent on the trnscription fctor NFATc, s well s unresponsiveness of T cells. As chronic infection with RNA viruses such s humn immunodeficiency virus type 1 (HIV-1) induces profound dysfunction of CD T cells, we investigted the role of TLR7-induced nergy in HIV-1 infection. Silencing of TLR7 mrkedly decresed the frequency of HIV-1-infected CD T cells nd restored the responsiveness of those HIV-1 CD T cells. Our results elucidte previously unknown function for microil pttern recognition receptors in the downregultion of immune responses. Toll-like receptors (TLRs) represent the mjor pthwy y which microorgnisms interct with host cells. They re fmily of highly conserved pttern-recognition receptors tht recognize distinct pthogen-ssocited moleculr ptterns tht re conserved in specific clsses of microorgnisms 1. The humn TLR fmily consists of t lest ten memers tht cn e clssified into two different groups on the sis of their cellulr loction. Intrcellulr TLRs (TLR3, TLR7, TLR8 nd TLR9) recognize nucleic cids; TLR7 nd TLR8 recognize singlestrnded RNA,3, wheres TLR3 nd TLR9 re receptors for doulestrnded RNA nd doule-strnded DNA, respectively. In contrst, cell surfce TLRs (TLR1, TLR, TLR, TLR nd TLR6) recognize vrious components of cteri 1. In mice, lthough TLR7 nd TLR8 re expressed t low levels in CD T cells, there re species-specific differences in the recognition of lignds 3 s well s in their functionlity. Specificlly, mouse TLR7 nd humn TLR8 medite species-specific recognition of GU-rich single-strnded RNA. It hs een suggested tht in contrst to its humn TLR counterprt, mouse TLR8 is not functionl nd TLR7 is the only TLR tht recognizes single-strnded RNA. The expression nd signling pthwys triggered y stimultion of TLRs hve een descried in ntigen-presenting cells (APCs) in process tht leds to the ctivtion of APCs with the secretion of inflmmtory nd ntivirl cytokines 1,. Although TLR expression hs een studied minly in APCs, severl reports hve descried the expression of TLRs on lymphocytes 6, nd specificlly on CD T cells. As with APCs, such studies indicte tht the enggement of TLRs cts s positive costimultory signl tht increses the secretion of proinflmmtory cytokines, prolifertion nd cell survivl 7,8. While TLRs re centrl to the erly host immune response to cute virl infection, more-chronic infectious diseses re chrcterized y the inility of the host immune system to mount strong, long-lsting response to the infectious gent. In prticulr, it hs een shown tht during infection with RNA viruses such s heptitis C virus nd humn immunodeficiency virus type 1 (HIV-1), the immune responses medited y CD helper T cells nd CD8 cytotoxic T cells determine the outcome of the infection, with chronic infections eing correlted with lte, trnsient or nrrowly focused responses y CD or CD8 T cells 9 11. Severl studies hve demonstrted impirment in the ctivtion nd/or function of T cells during infection with HIV-1. Specificlly, CD T cells from ptients chroniclly infected with HIV-1 disply n nergic phenotype with defects in prolifertion nd the secretion of interleukin (IL-) nd interferon-γ (IFN γ). The mechnisms y which RNA viruses impir T cell function re not well understood. Here we descrie previously unrecognized pthwy of TLRmedited negtive regultion of oth the ctivtion nd the cytokine production of CD T cells. Engging TLR7 expressed on CD T cells resulted in complete nergy y inducing intrcellulr clcium flux, with ctivtion of n nergic gene-expression progrm dependent on the trnscription fctor NFATc nd with susequent T cell unresponsiveness tht ws reversed y knockdown of TLR7 with short hirpin RNA (shrna). In studies of the potentil physiologicl relevnce of these findings, we found tht knockdown of TLR7 vi shrna decresed the frequency of HIV-1-infected CD T cells in vitro nd restored the responsiveness of those HIV-1 CD T cells in vitro. Our results elucidte previously unknown function for microil pttern recognition receptors in the downregultion of immune responses, inducing nergy y incresing intrcellulr clcium concentrtions nd interfering with secondry costimultion signls in the presence of signling vi TLRs 7. RESULTS Inhiition of the ctivtion of CD T cells y TLR7 While investigting potentil costimultory role for TLRs in CD T cells, we oserved tht the entry of CD T cells into the cell cycle fter crosslinking of the T cell ntigen receptor (TCR) with ntiody 1 Deprtment of Neurology nd Deprtment of Immunoiology, Yle School of Medicine, New Hven, Connecticut, USA. Alert Einstein College of Medicine nd Montefiore Medicl Center, Bronx, New York, USA. Correspondence should e ddressed to M.D.-V. (mrgrit.dominguez-villr@yle.edu) or D.A.H. (dvid.hfler@yle.edu). Received 18 June; ccepted Octoer; pulished online 17 Novemer 1; doi:1.138/ni.336 118 VOLUME 16 NUMBER 1 JANUARY nture immunology

Figure 1 TLR7 signling inhiits the prolifertion nd cytokine secretion of CD T cells. () Prolifertion of CD T cells leled with the cytosolic dye CFSE nd stimulted for 3 d with vrious concentrtions (ove plots) of imiquimod (). Numers ove rcketed lines indicte percent vile proliferting CD T cells. () Frequency of vile proliferting CD T cells stimulted for 3 d with vrious concentrtions (horizontl xis) of imiquimod. (c) ELISA of cytokine secretion y CD T cells stimulted for 3 d with vrious concentrtions of imiquimod. (d) Intrcellulr stining of IFN-γ nd IL- (top) nd of IL-17 nd IL- (ottom) in CD T cells stimulted for d with vrious concentrtions of imiquimod nd then stimulted for with h PMA nd ionomycin. Numers in qudrnts indicte percent cells in ech throughout. (e) Frequency of cytokine-producing CD T cells (s in d). (f) Expression of CD (left), CD69 (middle) nd CD137 (right) on CD T cells stimulted with nti-cd3 nd nti-cd8 in the presence (α-cd3 nd α-cd8 ) or sence (α-cd3 nd α-cd8) of imiquimod, nd of cells stined with isotype-mtched control ntiody (Isotype). MFI, men fluorescence intensity. P <., P <. nd P <. (pired t-test). Dt re representtive of eight ( d) or six (e,f) independent experiments with one donor in ech (men nd s.e.m. in,c,e,f). to the invrint signling protein CD3 (nti-cd3) nd crosslinking of the coreceptor CD8 with nti-cd8 ws locked y coenggement of TLR7 (Fig. 1, nd Supplementry Fig. 1,). Tretment with the synthetic TLR7 gonist imiquimod resulted in considerly less prolifertion of CD T cells thn tht of vehicle-treted control cells, s well s less secretion of IFN-γ nd IL-17, in dose-dependent fshion (Fig. 1c e nd Supplementry Fig. 1c,d). We oserved this inhiitory effect s soon s 1 h fter ctivtion, with much less induction of the expression of IL, IFNG nd IL fter tretment with imiquimod (Supplementry Fig. 1e). We ssessed the effect of concentrtions of up to µg/ml of imiquimod ut found no effect on cell viility (dt not shown). The diminished prolifertion correlted with less secretion of the cytokines IFN-γ, IL-17, IL- nd IL-, s mesured y enzyme-linked immunosorent ssy (ELISA), t dy 3 fter stimultion (Fig. 1c). We confirmed the diminished cytokine secretion t the single-cell level, s the frequency of cytokine-producing cells ws lso diminished in dose-dependent mnner with incresing doses of imiquimod in culture (Fig. 1d,e). Furthermore, stimultion of CD T cells in the presence of imiquimod inhiited the expression of ctivtion mrkers such s CD, CD69 nd CD137, mesured 8 h nture immunology VOLUME 16 NUMBER 1 JANUARY 119

c IL- (U/ml) CFSE GDQ Lox CL6 7.9 8.3 9.1.3 1 1 3 1 1 IFN-γ (ng/ml) 3 1 IL- (pg/ml) 1, 7 fter ctivtion (Fig. 1f). Of note, the effect of imiquimod ws not relted to the conversion of CD T cells into popultion of regultory T cells (T reg cells), s neither expression of the trnscription fctor Foxp3 nor secretion of IL-1 ws incresed in the presence of imiquimod (dt not shown). The unresponsive phenotype oserved ws not due to n indirect effect of TLR7 on T reg cells, s we otined the sme results with CD T cell popultion sorted s CD3 CD or CD CD lo CD17 cells nd depleted of T reg cells (dt not shown). Only stimultion of TLR9 with the synthetic lignd oligodeoxynucleotide ODN6 lso decresed the frequency of proliferting CD T cells, ut it did so to lesser extent thn did stimultion of TLR7 (Supplementry Fig. 1,). The effect oserved ws not due to n indirect effect of the triggering of TLR7 on contminnt APCs in the culture, s imiquimod exerted the sme effect on T cell clones grown from single donor (Supplementry Fig. ). To confirm the specificity of our results, we stimulted CD T cells in the presence of other synthetic lignds of TLR7, such s loxoriine, CL6 or grdiquimod (Fig. ). The three lignds tested induced significntly less CD T cell prolifertion thn did vehicle control, with grdiquimod nd loxoriine inhiiting prolifertion to degree similr to tht chieved y imiquimod tretment (Fig.,). The secretion of IL-, IFN-γ, IL- nd IL-17 ws lso inhiited fter stimultion in the presence of ech of the three lignds, s mesured y ELISA fter 3 d in culture (Fig. c). The specificity of the phenotype oserved ws further demonstrted y silencing of TLR7 in CD T cells (Fig. d). While control CD T cells trnsduced with nontrgeting shrna responded to tretment with imiquimod y decresing their secretion of IFN-γ nd IL-, CD T cells in which TLR7 ws IL-17 (ng/ml) Figure The inhiitory effect of imiquimod is TLR7 specific. () Prolifertion of CFSEleled CD T cells stimulted for 3 d with nti-cd3 nd nti-cd8 in the presence of vehicle () or the TLR7 gonists grdiquimod (GDQ), loxoriine (Lox) or CL6 (numers ove rcketed lines s in Fig. 1). () Frequency of vile proliferting. 1.. GDQ Lox CL6 Proliferting CD d TLR7 mrna (AU) 8 6. 1.. Isotype 1 1 3 1 1 TLR7 GDQ Lox CL6 e IFN-γ (ng/ml) 3 1 Isotype g IL- (pg/ml) IL- (pg/ml) 3 1, 8 6 IL- (U/ml).1.. 1. ssrna (µg/ml).1.. 1. ssrna (µg/ml) Proliferting CD.1.. 1. ssrna (µg/ml) silenced tht were stimulted with imiquimod produced concentrtions of IFN-γ nd IL- similr to those produced fter tretment with vehicle (Fig. e). Both TLR7 nd TLR8 re expressed in humn CD T cell supopultions 6,8 ; however, lthough reports suggest tht oth TLR7 nd TLR8 recognize single-strnded RNA s their nturl lignd in APCs,3, we oserved no such effect when we used the TLR8 lignd ssrna ( single-strnded RNA) in the trnsfection regent LyoVec in these experiments in prllel with TLR7 lignds (Fig. f,g). Insted, stimultion of T cells in the presence of ssrna significntly incresed the production of IFN-γ nd inhiited the secretion of IL- y CD T cells (Fig. g), with no effect on prolifertion (Fig. f). icle lone (LyoVec) did not hve ny effect on cytokine secretion or prolifertion under these experimentl conditions (dt not shown). We otined the sme results with CL7, nother lignd predominntly of TLR8 (dt not shown). These dt were in greement with pulished report 8 nd suggested tht despite shring lignds, the signling pthwys tht TLR7 nd TLR8 triggered on CD T cells led to different phenotypic outcomes. Ligtion of TLR7 induces the ctivtion nd mturtion of APCs, with upregultion of ctivtion mrkers nd secretion of proinflmmtory cytokines 1 1. In greement with pulished dt, CD1 monocytes isolted from the sme donors studied ove (Figs. 1 nd ) nd stimulted in the presence or sence of imiquimod showed n ctivted phenotype, with upregultion of the expression of the humn leukocyte ntigen HLA-DR, the costimultory molecule CD8 nd the cytokine receptor CD nd downregultion of the expression of the costimultory molecule CD86 (Supplementry Fig. 3). Upon eing stimulted with imiquimod, CD1 monocytes lso secreted the f IFN-γ (ng/ml) IL-17 (ng/ml) 8 6 8 6 3 1.1.. 1. ssrna (µg/ml).1.. 1. ssrna (µg/ml) CD T cells s in. (c) ELISA of cytokine secretion y CD T cells s in. (d) Expression of TLR7 mrna (top) nd TLR7 protein (ottom) in CD T cells stimulted with nti-cd3 nd nti-cd8 in the presence of shrna specific for TLR7 (construct designtion, TRCN6973; ) or nontrgeting shrna (), nd lso contining sequence encoding green fluorescent protein, then sorted t dy on the sis of the expression of green fluorescent protein; mrna results re presented in ritrry units (AU). Isotype (ottom), isotype-mtched control ntiody. (e) ELISA of IFN-γ nd IL- secreted y CD T cells trnsduced with shrna s in d (horizontl xis) nd stimulted for 3 d with nti-cd3 nd nti-cd8 in the presence of vehicle or imiquimod (key). (f) Prolifertion of CFSE-leled CD T cells stimulted with nti-cd3 nd nti-cd8 in the presence of vrious doses of ssrna (horizontl xis). (g) ELISA of IL-, IFN-γ, IL- nd IL-17 secreted y CD T cells t dy 3 fter ctivtion with nti-cd3 nd nti-cd8 in the presence of vrious doses of ssrna. P <., P <. nd P <. (one-wy nlysis of vrince (ANOVA) with Tukey s post-test (,c) or pired t-test (d,e,g)). Dt re representtive of seven independent experiments (,,d,e; men nd s.e.m. in d,e), five experiments (c; men nd s.e.m.) or four independent experiments (f,g; men ± s.e.m. of n = donors). 1 VOLUME 16 NUMBER 1 JANUARY nture immunology

d Indo-1 AM (/7) 1, CASP3 mrna (reltive 1 3 ) IKZF1 mrna (reltive 1 3 ) RGS mrna (reltive 1 3 ) 8 6 1 3 1 3 1 ( µg/ml) Ionomycin 6 Time (s) CD98 mrna (expression 1 3 ) KMD6B mrna (reltive 1 3 ) SOCS mrna (reltive 1 3 ) (1 µg/ml) 3 1 1 9 6 3 3 1 ssrna FASL mrna (reltive 1 3 ) LDHA mrna (reltive 1 3 ) TNFRSF9 mrna (reltive 1 3 ) 6 6 1 Indo-1 AM (/7) IRS Ctrl IRS 661 6 8 Time (s) GRG mrna (reltive 1 3 ) RAB1 mrna (reltive 1 3 ) TRAF mrna (reltive 1 3 ) 8 6 1. 7... 6 c NFATc/p-NFATc (AU).. 1.. NFATc e IFN-γ (ng/ml) β-ctin CBLB mrna (reltive 1 3 ) EGR3 mrna (reltive 1 3 ) 6 8 6 3 1 9 NFATc DGKA mrna (reltive 1 3 ) ITCH mrna (reltive 1 3 ) IL- (pg/ml) 3 1 8 6 p-nfatc (AU) 1..8.6.. p-nfatc β-ctin EGR mrna (reltive 1 3 ) SIRT1 mrna (reltive 1 3 ) f IFN-γ (ng/ml) 1 3 8 6 3 1 3 1 IL- (pg/ml) 18 13 9 IRS 661 97 kd IRS 661 Figure 3 Mechnism of TLR7-induced nergy. () Clcium flux over time in CD T cells stimulted (downwrd rrows) with µg/ml or 1 µg/ml of imiquimod (top row), ionomycin (ottom left) or ssrna in LyoVec (ottom right), ssessed s the rtio of Indo-1 AM fluorescence t nm to tht t 7 nm (/7). () Clcium flux in CD T cells preincuted for 1 h with control sequence (IRS Ctrl ) or with the TLR7-specific inhiitory sequence IRS 661 (1 µm; IRS 661 ) nd stimulted with imiquimod ( µg/ml), presented s in. (c) Immunolot nlysis of totl NFATc (left) or phosphorylted (p-) NFATc (right) nd β-ctin (loding control) in CD T cells t, nd 9 min (left) or, 1,, 3 nd min (right) fter tretment with imiquimod (ottom), nd rtio of dephosphorylted NFATc to phosphorylted NFATc (top left) or intensity of phosphorylted NFATc, normlized to results otined for β-ctin (top right), ssessed fter stimultion s in lots elow. (d) Expression of nergy-relted genes y CD T cells stimulted for h with vehicle, imiquimod or IRS 661 plus imiquimod (key). (e) ELISA of IL- nd IFN-γ secreted y CD T cells trnsduced with nontrgeting or NFATC-specific shrna nd stimulted for 3 d with nti-cd3 nd nti-cd8 in the presence or sence of imiquimod. (f) ELISA of IL- nd IFN-γ secreted y CD T cells incuted for h with vehicle or imiquimod in the presence or sence or IRS 661, then wshed nd llowed to rest for 1 h, then stimulted for d with nti-cd3 nd nti-cd8. P <., P <. nd P <. (pired t-test (c,e) or one-wy ANOVA with Tukey s post-test (d,f)). Dt re representtive of six independent experiments (,), five experiments (c,d), three experiments (e) or four experiments (f) with one donor in ech (men nd s.e.m. in c f). proinflmmtory cytokines IL-6, tumor-necrosis fctor nd IL-1β nd decresed their secretion of IL-1 (Supplementry Fig. 3), wheres we oserved no effect on monocyte prolifertion (dt not shown); this suggested tht stimultion of CD T cells nd CD1 monocytes with imiquimod led to completely different outcomes. To confirm the specificity of the unresponsive phenotype driven y TLR7 signling, we stimulted CD T cells with nti-cd3 nd nti- CD8 in the presence of shrna specific for TLR7 or nontrgeting shrna, s control. After d of culture, we confirmed tht the efficiency with which protein nd RNA ws silenced ws >8% (Fig. d). We stimulted shrna-treted resting CD T cells with with nti-cd3 nd nti-cd8 in the presence or sence of imiquimod nd mesured the secretion of IL- nd IFN-γ. While CD T cells trnsduced with nontrgeting shrna showed decrese in the production of IL- nd IFN-γ secretion fter tretment with imiquimod, this effect ws olished in cells expressing TLR7-specific shrna (Fig. e), which confirmed tht the unresponsive stte of T cells oserved in the presence of imiquimod ws TLR7 specific. Induction of clcium-nfatc driven CD T cell nergy y TLR7 The inhiition of the prolifertion, cytokine secretion nd ctivtion of CD T cells upon stimultion in the presence of TLR7 lignds nture immunology VOLUME 16 NUMBER 1 JANUARY 11

c 1 1 3 1 1 p-irak 1 1 3 1 1 p-jnk p-jnk (MFI) p-irak (MFI) 3 6 9 1 IRS 661 IRS 661 3 6 9 1 d 1 1 3 1 1 p-nf-κb 1 1 3 1 1 p-p38 p-p38 (MFI) p-nf-κb (MFI) 3 IRS 661 3 6 9 1 IRS 661 3 6 9 1 Figure Imiquimod inhiits the phosphoryltion of Jnk. Phosphorylted IRAK (), NF-κB (p6) (), Jnk (c) nd p38 (d) in CD T cells stimulted for 3 min with vehicle or imiquimod (left) or for 1 min with vehicle lone or with imiquimod in the presence or sence of IRS 661 (right). P <., P <. nd P <. (pired t-test). Dt re representtive of eight independent experiments with one donor ech (men ± s.e.m. t right). resemled the unresponsive phenotype tht chrcterizes clonl nergy. Vrious model systems hve een used to induce clonl nergy, including tretment with the clcium ionophore ionomycin 16,17, nd the ctivtion of clcium signling in the sence of ctivting signls in costimultory signling pthwys is common in ll these models. Thus, the min chrcteristic of n stimulus tht elicits nergy is its ility to induce n unopposed increse in intrcellulr clcium concentrtions 17. To test the hypothesis tht TLR7 signling on CD T cells ws inducing clonl nergy, we stined sorted CD T cells with the rtiometric clcium indictor Indo-1 AM nd treted them with vrious doses of imiquimod (Fig. 3). Imiquimod induced significnt nd sustined (mintined for t lest min) increse in intrcellulr clcium concentrtion in dose-dependent mnner tht ws TLR7 specific, s locking TLR7 with IRS 661, specific inhiitory oligonucleotide sequence 18, impired the increse in clcium concentrtion upon stimultion with imiquimod (Fig. 3 nd Supplementry Fig. ). We did not oserve tht increse in intrcellulr clcium concentrtion in cells stimulted with the TLR8 gonist ssrna or with lignds of other intrcellulr TLRs, such s the synthetic RNA duplex poly(i:c) (for TLR3) or ODN6 (for TLR9) (Fig. 3 nd Supplementry Fig. ). As positive control, we treted cells with the clcium ionophore ionomycin, which hs een used s n nergy-inducing gent in in vitro experiments 17,19, nd otined concentrtions of intrcellulr clcium similr to those induced y 1 µg/ml of imiquimod (Fig. 3 nd Supplementry Fig. ). An immedite consequence of n increse in the concentrtion of intrcellulr clcium is the ctivtion of NFATc, trnscription fctor tht is highly phosphorylted in resting cells nd ecomes dephosphorylted y the clcium-clmodulin dependent phosphtse clcineurin when the concentrtion of intrcellulr clcium is incresed,1. To determine whether stimultion with imiquimod induced dephosphoryltion of NFATc, we stimulted CD T cells with imiquimod nd purified totl protein extrcts, nd 9 min lter. Immunolot nlysis with nti-nfatc showed tht tretment with imiquimod resulted in dephosphoryltion of NFATc; we confirmed these results y nlysis of totl extrcts of CD T cells stimulted for min with imiquimod nd proed with phosphoryltion-specific ntiody to NFATc (Fig. 3c). After eing dephosphorylted, NFATc trnsloctes to the nucleus, where it ecomes trnscriptionlly ctive. We used nucler nd cytoplsmic protein extrcts to further confirm trnsloction of NFATc from the cytoplsm to the nucleus upon stimultion with imiquimod (dt not shown). This trnsloction of NFATc to the nucleus in the sence of concomitnt costimultory signl leds to the trnscription of set of NFATc-dependent, nergy-relted genes tht re different from those upregulted with full ctivtion in the presence of costimultory signl 17. To investigte whether stimultion of TLR7 nd susequent dephosphoryltion of NFATc induce the expression of nergy-relted genes 17, we incuted CD T cells for 16 h with imiquimod in the presence or sence of the TLR7-inhiitory sequence IRS 661 nd isolted RNA for nlysis. As control for the expression of nergy-relted genes, we incuted CD T cells with either ionomycin plus the phorol ester PMA ( non-nergic stimulus) or ionomycin lone (n nergic stimulus) 17 (Supplementry Fig. ). Ten of the twelve nergy-relted genes exmined were significntly upregulted in cells stimulted with imiquimod reltive to their expression in the control cells stimulted with vehicle (Fig. 3d nd Supplementry Fig. ). The effect oserved in the regultion of these genes ws TLR7 specific, s preincution of CD T cells with IRS 661 efore tretment with imiquimod rogted the increse in the expression of nergy-relted genes (Fig. 3d). Furthermore, we lso ssessed the expression of other genes encoding molecules tht hve een functionlly relted to estlishment nd mintennce of the nergic phenotype nd re trgets of NFAT, such s SIRT1 (ref. 3), ITCH, CBLB,, DGKA 6, EGR nd EGR3 (ref. 7), nd found tht ll of these ut EGR showed significnt upregultion upon tretment with imiquimod (Fig. 3d). Our dt suggested tht imiquimod induced clonl nergy in CD T cells vi n increse in intrcellulr clcium concentrtion nd ctivtion of n NFATc-dependent nergy-relted gene-expression progrm. To further investigte the role of NFATc in TLR7-induced T cell nergy, we used two shrnas to silence NFATC nd used imiquimod to induce nergy in resting CD T cells fter knockdown of NFATc. After 3 d 1 VOLUME 16 NUMBER 1 JANUARY nture immunology

Figure Imiquimod inhiits the ctivtion of Jnk nd Jun fter full stimultion of CD T cells through signling vi the TCR nd costimultion. () Phosphorylted NF-κB (p6), Jnk, p38 nd Jun in CD T cells stimulted for 6 min (left of ech pir) or 1 min (right of ech pir) with vehicle (left) or with PMA nd ionomycin (iono) in the presence or sence of imiquimod (right). () Expression of FOS nd JUN mrna in CD T cells stimulted for 1 96 h with nti-cd3 nd nti-cd8 of in the presence or sence of imiquimod. P <., P <. nd P <. (pired t-test). Dt re representtive of eight independent experiments with one donor ech () or four independent experiments (; men ± s.e.m. of n = donors). in culture, cells trnsduced with nontrgeting shrna nd stimulted with imiquimod significntly reduced their production of IL- nd IFN-γ, while cells trnsduced with NFATC-specific shrna were not ffected y tretment with imiquimod (Fig. 3e nd Supplementry Fig. 6); this suggested tht NFATc ws necessry for the nergic phenotype driven y TLR7 signling in CD 3 T cells. Tretment with imiquimod did not hve n effect on the expression of NFATC (Supplementry Fig. 6). In greement with those dt nd pulished oservtions 17, tretment with imiquimod filed to upregulte the expression of nergy-relted genes (KMD6B, IKZF1, GRG nd RAB1) in CD T cells in which NFATc ws knocked down reltive to their expression in cells trnsduced with nontrgeting shrna (Supplementry Fig. 6), which confirmed tht this gene-expression progrm driven y tretment of CD T cells with imiquimod ws lrgely NFATc dependent. As reported in pulished studies of n ionomycin-induced nergy model 17, we hypothesized tht pretretment of CD T cells with imiquimod would e sufficient to diminish their susequent cytokine secretion nd prolifertive response to stimultion with oth crosslinking of the TCR nd costimultory signling through CD8. We pretreted memory CD T cells for h with imiquimod in the presence or sence of the inhiitory sequence IRS 661 nd llowed them to rest for 1 h fter wshout to remove ll trces of imiquimod nd then stimulted the cells for 3 d with nti-cd3 nd nti- CD8. pretreted with imiquimod showed significntly lower production of oth IL- nd IFN-γ thn tht of cells pretreted with vehicle (Fig. 3f). We oserved no significnt chnge in cell viility upon pretretment wtih imiquimod (dt not shown). Agin, the pronergic effect of imiquimod ws prevented y pretretment with the TLR7 ntgonist IRS 661 (Fig. 3f). These dt suggested tht imiquimod induced clonl nergy in CD T cells y inducing clciumdependent ctivtion of NFATc nd susequent expression of nergyrelted genes. Effect of TLR7 signling on costimultory signls TLR7 signling hs een studied minly in APCs, in which it is linked to ctivtion of the trnscription fctors IRF7 nd NF-κB nd the kinse Jnk through pthwys dependent on or independent of the dptor MyD88 nd the kinse IRAK (ref. 1). To ssess the consequences of TLR7 signling on CD T cells reltive to the consequences of conventionl TLR signling, we isolted CD T cells from helthy donors nd stimulted the cells ex vivo with PMA iono PMA iono 1 1 3 1 1 p-nf-κb 1 1 3 1 1 p-p38 JUN mrna (reltive 1 3 ) p-nf-κb (MFI) p-p38 (MFI) 3 3 6 9 1 3 3 6 9 1 1 36 8 7 96 Time (h) FOS mrna (reltive 1 3 ) 6 1 1 3 1 1 p-jnk 1 1 3 1 1 p-jun 1 36 8 7 96 Time (h) PMA iono PMA iono 3 6 9 1 α-cd3 α-cd8 α-cd3 α-cd8 imiquimod, then ssessed the phosphoryltion sttus of IRAK, Jnk, the p6 suunit of NF-κB nd the mitogen-ctivted protein kinse p38 y flow cytometry. In greement with pulished reports of other cell types,3,, stimultion of TLR7 with imiquimod induced phosphoryltion of IRAK, NF-κB nd p38 t different time points thn did tretment with vehicle (Fig. ). Preincution with IRS 661 efore stimultion with imiquimod inhiited the phosphoryltion of these molecules (Fig. ), which suggested tht the effect oserved ws TLR7 specific. Incution with IRS 661 y itself did not hve ny effect on protein phosphoryltion (dt not shown). Enggement of TLR7 decresed the sl levels of phosphorylted Jnk (Fig. ). One of the trgets phosphorylted y ctivted Jnk is Jun, component of AP-1, which is n essentil trnscription fctor involved in costimultory signl trnsduction 17. We hypothesized tht inhiition of Jnk ctivity y imiquimod might explin, t lest in prt, the nergic phenotype oserved in CD T cells fter stimultion with imiquimod in the presence of full stimultion of the TCR nd costimultory signling (Figs. 1 nd ). To test this hypothesis, we stimulted CD T cells with PMA plus ionomycin in the presence or sence of imiquimod nd ssessed the phosphoryltion sttus of Jnk nd Jun t vrious time points. We used NF-κB nd p38 s positive controls. Although tretment with imiquimod did not produce n dditive effect on the phosphoryltion of NF-κB with stimultion, imiquimod further incresed the phosphoryltion of p38 (Fig. ). Of note, upon stimultion with PMA plus ionomycin, the phosphoryltion of Jnk ws inhiited y imiquimod, nd this decrese in Jnk ctivity ws ccompnied y decrese in the phosphoryltion of Jun (Fig. ). Moreover, imiquimod decresed the ctivity of oth Jnk nd Jun, s mesured y phosphoryltion fter ctivtion (Fig. ), nd it decresed JUN expression (Fig. ). These dt supported the hypothesis tht imiquimod tretment oth induced nergy in CD T cells nd interfered with costimultory signling during T cell stimultion. The decrese in the expression of CD69 nd CD137 (oth trnscriptionl trgets of AP-1 (refs. 8,9)) oserved fter stimultion in the presence of imiquimod (Fig. 1f) further supported this hypothesis. p-jnk (MFI) p-jun (MFI) 3 6 3 6 9 1 nture immunology VOLUME 16 NUMBER 1 JANUARY 13

Inhiition of HIV-1 in vitro vi TLR7-C signling lockde To investigte the potentil iologicl nd clinicl relevnce of the oservtions noted ove in ex vivo nd in vitro model systems, we determined whether enggement of TLR7 y single-strnded RNA virus,3 could medite CD T cell nergy. The responses of CD T cells from ptients chroniclly infected with HIV-1 re impired nd re insufficient to cler the virus, while the cells disply fetures of nergy 3 33. Severl virl proteins hve een shown to d CD g CASP3 mrna (reltive 1 3 ) IKZF1 mrna (reltive 1 3 ) RGS mrna (reltive 1 3 ) Viility 1 3 1, 1, 3 TLR7() 1.1.6 3.1. 1 1 IRS 661 (µm) CD98 mrna (reltive 1 3 ) e IFN-γ 1 1 3 1 1 1 CD 3 1 TLR7() 1 3 6 7 8 9 1 11 h i j 1 1 3 1 1 1 1 1 3 1 1 1 1 1 3 1 1 1 13. 1 1 1 1 3 1 Viility 1 1 1 1 3 1 Viility 1.6 11.3 1 1 1 1 3 1 Viility c IRS 661 (µm) 1 Quin- AM NFATc(1) VIVIT..6 1. Viility TLR7() Mock 6. 8.3.9.8 3.8 1.9 3. 6. 1 1 IL- (IRS Ctrl) HIV-1NL-D (IRS 661) 1, 1, FASL mrna (reltive 1 3 ) 3 KMD6B mrna (reltive 1 3 ) SOCS mrna (reltive 1 3 ) 3 3 LDHA mrna (reltive 1 3 ) TNFRSF9 mrna (reltive 1 3 ) 1, 1, 3.7 1 1 3 1 GRG mrna (reltive 1 3 ) RAB1 mrna (reltive 1 3 ) TRAF mrna (reltive 1 3 ) 1, 8 6 3 19. 9. 1.3 f IL- cells (%) NFATc().8.3.3 8 6 Mock. TLR7() CD CD CD 1 1 Quin- AM 7 9 11 NFATc(1) NFATc() 1 7 9 11 VIVIT IFN-γ cells (%) 1 7 9 11 Mock TLR7() Figure 6 Knockdown of TLR7 or NFAT olishes infection with HIV-1. () Viility of CD T cells trnsduced with nontrgeting shrna or TLR7-specific shrna (clone 3 or ) (ove plots), then infected with d lter, nd ssessed 7 d fter infection. Numers djcent to outlined res indicte percent vile HIV-1 cells. () Kinetics of the infection shrna-trnsduced CD T cells (s in ) with. (c) Viility of CD T cells stimulted for d with nti-cd3 nd nti-cd8 in the presence of vrious doses of IRS 661 (ove plots), then infected with nd ssessed t dy 7 fter infection (numers in plots s in ). (d) Frequency of cells mong CD T cells incuted with vrious doses of IRS 661 (horizontl xis) nd infected with. (e) Expression of IFN-γ nd IL- y CD T cells stimulted for d with nti-cd3 nd nti-cd8 in the presence of shrna s in, then mock infected (Mock) or infected with nd then, 7 d lter, stimulted for h with PMA nd ionomycin. (f) Frequency of IL-- or IFN-γproducing CD T cells mong cells treted s in e. (g) Expression of nergy-relted genes y CD T cells left uninfected with no further tretment ( ) or infected in vitro with in the presence of IRS 661 or control sequence, nd sorted (s (uninfected) or (infected)) t dy 7 fter infection. (h) Viility of CD T cells preincuted with vehicle or Quin- AM (ove plots) nd then infected with, ssessed t dy 7 fter infection (left) or on dys 11 fter infection (right). (i) Viility of CD T cells stimulted in the presence of nontrgeting shrna or NFATC-specific shrna (clone 1 or ) nd infected with d lter, ssessed t dy 7 fter infection (left) or on dys 11 fter infection (right). (j) Viility of CD T cells preincuted with vehicle or VIVIT peptide nd infected with, ssessed t dy 7 fter infection (left) or on dys 11 fter infection (right). Numers in plots (left, h j) s in. P <., P <. nd P <. (one-wy ANOVA with Tukey s post-test (,f,g,i,j) or pired t-test (d,h)). Dt re from one experiment representtive of five (,c) or four (e) independent experiments with one donor ech (,c,e) or re representtive of five independent experiments (,d; men ± s.e.m. of n = donors), four independent experiments (f; men ± s.e.m. of n = donors), six independent experiments (g; men nd s.e.m. of n = 6 donors) or six independent experiments with one donor ech (h j; men ± s.e.m. t right). 1 VOLUME 16 NUMBER 1 JANUARY nture immunology

(µg/ml) 1 1 1 8.3 17.8 1. 1 3 1 1 1 1 1 1 1 3 1 Viility Figure 7 Clcium-induced nergy fvors HIV-1 repliction. () Viility of CD T cells stimulted for d with nti-cd3 nd nti-cd8 in the presence of vrious doses of imiquimod (ove plots) nd infected with, ssessed t dy 7 fter infection. () Frequency of CD T cells mong cells treted s in. (c) Viility of CD T cells preincuted for 1 h with vehicle or ionomycin.1. 1 1 induce the stte of T cell unresponsiveness tht precedes the loss of CD T cells in HIV-1-infected ptients 3. We hypothesized tht virus CD T cell interction through TLR7 would e responsile, t lest in prt, for the nergic phenotype oserved in HIV-1-infected CD T cells from ptients. First, we isolted CD T cells from four helthy donors, infected the cells in vitro with physiologicl concentrtions (multiplicity of infection,.1) of repliction-competent strin of HIV-1 derived from the prototype HIV-1 NL3 virus nd tgged with the red fluorescent reporter DsRed ( ) 3,3 nd ssessed their ility to produce IL- nd IFN-γ 7 d fter infection. In greement with pulished reports 3 33, infection with mrkedly diminished the ility of vile CD T cells to produce IL- nd IFN-γ fter h of stimultion with PMA nd ionomycin (Supplementry Fig. 7). We did not oserve this decrese in cytokine secretion only in the ulk T cell popultion infected with the virus ut specificlly in -infected cells, which suggested tht direct interction of the virus with infected CD T cells rendered the CD T cells unresponsive. To test the hypothesis tht TLR7 signling in CD T cells ccounted in prt for the nergic phenotype fter infection with HIV-1, we isolted CD T cells from helthy donors nd treted the cells ex vivo with either of two shrnas specific for TLR7 (clone 3 or ), to silence TLR7, or with nontrgeting shrna, s control (Fig. 6). After d, we infected the shrna-treted CD T cells with concentrtions of within the physiologicl rnge 3 (multiplicity of infection,.1) nd mesured the frequency of infected cells s well s their ility to produce proinflmmtory cytokines every 8 h for totl of 11 d. Although CD T cells trnsduced with nontrgeting shrna were infected with to n extent similr to tht of untrnsduced cells, cell popultions trnsduced with TLR7-specific shrna hd much lower frequency of -infected CD T cells t ll time points exmined (Fig. 6,). This lower frequency of HIV-1 cells ws not due to specific increse in the deth of cells in which TLR7 ws silenced, s there ws no difference etween cell popultions trnsduced with TLR7-specific shrna nd control cell popultions trnsduced with nontrgeting shrna in terms of the frequency CD e 1 1 3 1 1 1 3 1 1 1 1 1 3 1 Viility (µg/ml) α-cd3 1.3.1 c 1 1 3 1 1 1 f CD Iono 1.1 1 1 1 1 3 1 Viility 3 1 α-cd3 3 7 9 11 CD 3 7 9 11 of erly or lte poptotic CD T cells fter infection (Supplementry Fig. 8). We further confirmed the role of TLR7 in decresing the infection rte y lockde of TLR7 on CD T cells from helthy donors with vrious doses of IRS 661 efore in vitro infection (Fig. 6c,d). Moreover, the secretion of cytokines y -infected cells ws significntly different for cells trnsduced with nontrgeting shrna nd their counterprts trnsduced with TLR7-specific shrna (Fig. 6e,f). Although -infected cells in cultures of CD T cells trnsduced with nontrgeting shrna produced less IL- nd IFN-γ protein thn did mock-infected CD T cells, the low frequency of -infected CD T cells trnsduced with TLR7-specific shrna secreted significntly more IL- nd IFN-γ thn did their counterprts trnsduced with nontrgeting shrna (Fig. 6e,f), which suggested tht the nergic phenotype did not develop fter infection in cells trnsduced with TLR7-specific shrna. To confirm tht oservtion, we ssessed the expression of nergy-relted genes in CD T cells otined from helthy donors, infected with in vitro in the presence of IRS 661 (to inhiit TLR7) or control sequence nd ssessed t dy 7 fter infection. Sorted CD T cells showed higher expression of eight of the twelve nergy-relted genes exmined thn did CD T cells, while HIV-1 CD T cells sorted from IRS 661 treted cultures did not upregulte ny of these genes t the time point nlyzed (Fig. 6g). The expression of other genes encoding molecules tht hve een functionlly linked to nergy, such s ITCH, DGKA, CBLB nd SIRT1, ws lso upregulted in CD T cells ut not in IRS 661 treted T cells (dt not shown). These dt suggested tht n interction of with TLR7 ws responsile, t lest in prt, for the nergic phenotype oserved in - infected CD T cells. We investigted the role of the intrcellulr clcium nd NFATc- ctivted gene-expression signling events we oserved y enggement of TLR7 with imiquimod, fter infection with in vitro. We hypothesized tht lockde of intrcellulr clcium nd silencing of NFATC would led to decrese in the frequency of HIV-1 T cells even in the presence of functionl TLR7. To induce d CD 3 1 1 Iono CsA 7 9 11 (ove plots), then infected with nd ssessed t dy 9 fter infection. (d) Frequency of CD T cells mong cells treted s in c, ssessed on dys 3 11 fter infection. (e) Viility of CD T cells preincuted for 1 h with vehicle or nti-cd3 (ove plots), then infected with nd ssessed t dy 9 fter infection. (f) Frequency of CD T cells mong cells treted s in e, ssessed on dys 3 11 fter infection. (g) Frequency of CD T cells mong cells stimulted for d with nti-cd3 nd nti-cd8, with vehicle or cyclosporine A (CsA) dded to the cultures 6 h efore infection with, ssessed on dys 11. Numers in plots (,c,e) s in Figure 6. P <., P <. nd P <. (pired t-test). Dt re from one experiment representtive of three () or six (c,e) independent experiments with one donor ech (,c,e) or re representtive of three independent experiments with one donor ech (; men ± s.e.m.) or six independent experiments (d,f,g; men ± s.e.m. of n = 6 donors). g nture immunology VOLUME 16 NUMBER 1 JANUARY

p (ng/ml) 1.. IRS Ctrl IRS 661 p (ng/ml). 1.. IRS Ctrl IRS 661 c p (ng/ml) 1.. TLR7() d p (ng/ml).. 1.. TLR7() e DNA provirl lod (%) 8 6 IRS Ctrl IRS 661 f DNA provirl lod (%) 8 6 TLR7() 7 9 11 1 7 9 11 1 Figure 8 Inhiition of TLR7 decreses infection in HIV-1 ptients. () Concentrtion of p in CD T cells isolted from n HIV-1-infected ptient nd stimulted for 1 d (horizontl xis) in vitro with nti-cd3 nd nti-cd8 in the presence of control sequence (IRS Ctrl) or IRS 661 (key). () Concentrtion of p in CD T cells treted s in, ssessed on dy 1. (c) Concentrtion of p in CD T cells isolted from n HIV-1-infected ptient nd stimulted for 1 d (horizontl xis) in vitro with nti-cd3 nd nti-cd8 in the presence of nontrgeting shrna or TLR7-specific shrna (clone 3 or ). (d) Concentrtion of p in CD T cells treted s in c, ssessed on dy 1. (e) DNA provirl lod in CD T cells otined from HIV-1-infected ptients nd stimulted for 11 d in vitro s in. (f) DNA provirl lod in CD T cells from five HIV-1-infected ptients nd stimulted for 11 d in vitro in the presence shrna s in d. P <. nd P <. (pired t-test (,e) or one-wy ANOVA with Tukey s post-test (d,f)). Dt re from one experiment representtive of seven independent experiments with nine donors (,c) or re representtive of seven independent experiments (,d f; men nd s.e.m. of n = 9 donors). clcium lockde, we preincuted CD T cells with the cheltion gent Quin- AM 36 efore infection with. Clcium cheltion significntly decresed the frequency of vile cells, even t the lowest concentrtion of the chelting gent (Fig. 6h), perhps due to the essentil role of clcium in mny cellulr processes. Nevertheless, there ws lower frequency of T cells mong the remining vile CD T cells in cultures treted with Quin- AM thn mong CD T cells treted with vehicle lone (Fig. 6h), in greement with pulished investigtions suggesting role for clcium in the HIV-1 life cycle 37. We then silenced NFATC with either of two different shrnas or locked NFATc with VIVIT peptide, which interferes with the clcineurin-nfatc interction nd inhiits dephosphoryltion of NFAT 38. In oth cses, the sence of functionl NFATc led to significntly lower frequency of -infected T cells thn mong CD T cells left untreted or treted with vehicle lone (Fig. 6i,j), which indicted role for NFATc in HIV-1 infection, s previously suggested 39. These dt suggested tht clcium-dependent ctivtion of NFATc upon triggering of TLR7 ws involved in infection in vitro. Clcium-induced nergy fvors HIV-1 repliction Given the decresed infection rte of cells in which TLR7 ws silenced nd the non-nergic phenotype of infected CD T cells in which TLR7 ws silenced, we hypothesized tht the nergic stte induced y stimultion vi TLR7 during infection with HIV-1 would e necessry step for HIV-1 to replicte in the host. In the sence of signling vi TLR7, the virus would not e le to render the infecting cell nergic nd long-term infection would not occur. To test this hypothesis, we otined CD T cells from helthy donors, induced nergy in the cells vi the TLR7 pthwy with vrious doses of imiquimod nd susequently infected them with nd nlyzed them 7 d lter. Of note, the frequency of -infected CD T cells directly correlted with the concentrtion of imiquimod used nd the increse in intrcellulr clcium (Fig. 7,), nd thus with the degree of nergy in the culture, which suggested tht clcium-induced nergy fvored infection with HIV-1. Moreover, the induction of nergy vi other well-estlished in vitro methods, such s tretment with ionomycin (Fig. 7c,d) or stimultion with nti-cd3 without costimultory signls (Fig. 7e,f), efore infection with lso incresed the frequency of -infected cells. These dt supported the hypothesis tht HIV-1-induced nergy vi ligtion of TLR7 nd n increse in intrcellulr clcium concentrtion were prerequisites for productive infection with HIV-1. To further investigte whether inhiiting TLR7-induced nergy would ffect the frequency of HIV-1-infected cells, we dded either the clcineurin inhiitor cyclosporine A or concentrtions of IL- tht hve een shown to reverse nergy in severl in vitro settings 17 efore in vitro infection of cells with. Although the ddition of IL- 6 h efore infection with did not ffect the frequency of -infected cells (dt not shown), perhps due to response kinetics, the rod spectrum of IL- functions or the inility of IL- to rescue CD T cells from TLR7-induced nergy, locking nergy with cyclosporine A significntly decresed the frequency of T cells (Fig. 7g). This further supported the hypothesis tht HIV-1-induced nergy ws necessry for productive infection with HIV-1. Inhiition of TLR7 diminishes HIV-1 in T cells ex vivo We next sought to directly investigte the role of TLR7 in CD T cells from HIV-1-infected ptients. Specificlly, on the sis of the in vitro model system with, we hypothesized tht inhiition of the TLR7 pthwy in CD T cells from HIV-1-infected ptients would decrese the infection rte. We isolted CD T cells from HIV-1 infected ptients (Supplementry Tle 1) nd stimulted the cells in the presence of IRS 661 or trnsduced them with either of two TLR7-specific shrnas, then collected superntnts every 3 d for totl of 1 d to mesure virus concentrtion y ELISA of the HIV-1 core ntigen p. The inhiition of TLR7 y IRS 661 sustntilly decresed the concentrtion of p in culture (Fig. 8,), nd we otined similr results for cells trnsduced with TLR7- specific shrna (Fig. 8c,d). We ssyed CD T cells from helthy donors in prllel s negtive control ut found no detectle p t ny time point (dt not shown). Furthermore, we mesured the lod of provirl integrted DNA t dy 7 fter stimultion with nti- CD3 nd nti-cd8 in the presence of IRS 661 or control sequence nd fter trnsduction with TLR7-specific or nontrgeting shrna to monitor the cellulr virl reservoir. Inhiition of TLR7 y either IRS 661 (Fig. 8e) or knockdown of TLR7 (Fig. 8f) resulted in significntly lower provirl DNA lod thn tht in control cells. Together these results showed tht inhiition of TLR7 signling ws le to diminish the HIV-1 lod in infected CD T cells derived from infected ptients. 16 VOLUME 16 NUMBER 1 JANUARY nture immunology

DISCUSSION Microil pttern recognition receptors re criticl for the erly sensing of diverse cteril nd virl infections for ctivtion of the innte immune system. Here we hve descried previously unknown function of these pttern-recognition receptors in shutting off the immune responses of humn CD T cells. Enggement of TLR7 y its lignds in CD T cells prevented entry into the cell cycle nd the secretion of proinflmmtory cytokines fter stimultion. Mechnisticlly, stimultion of TLR7 incresed intrcellulr clcium concentrtions, which led to dephosphoryltion of NFATc nd its trnsloction to the cell nucleus; this ctivted n nergic gene-expression progrm. Furthermore, TLR7 signling interfered with costimultory signls, s ctivtion of Jnk nd Jun ws inhiited in the presence of TLR7 lignds nd full stimultion through the TCR nd costimultion vi CD8. Our results hve potentil clinicl implictions, s silencing of TLR7 inhiited the frequency of cells infected in vitro with, nd those infected cells did not disply n nergic phenotype, which suggested tht the clcium-induced TLR7-dependent nergic stte of -infected cells might hve role in HIV-1 persistence. TLR signling hs een studied predominntly in APCs, in which enggement of the lignd induces the secretion of proinflmmtory cytokines nd upregultion of ctivtion molecules 1 1. Although TLR signling in CD T cells hs not een studied in depth, the few reports pulished hve demonstrted positive costimultory role for TLR signling 7,8,. Our results hve demonstrted previously undescried TLR signling pthwy with n inhiitory effect on T cell prolifertion nd cytokine secretion. The differences etween the phenotypes of monocytes nd those of CD T cells upon TLR7 stimultion could e due to the intrinsic differences in ctivtion requirements for ech cell popultion. Although CD T cells need enggement of the TCR nd costimultory signl to enter cell cycle, CD1 cells require one signl. Thus, enggement of TLR7 y its lignd is sufficient to induce ctivtion nd secretion of proinflmmtory cytokines in monocytes, s expected for cell of the innte immune system. In contrst, stimultion of TLR7 in CD T cells leds to significnt increse in intrcellulr clcium tht in the sence of costimultory signl triggers the TLR7-driven nergic progrm 17. In this context, our results lso showed decrese in CD T cell prolifertion upon stimultion of TLR9, with no effect on IFN-γ secretion nd trend towrd incresed IL-17 secretion, lthough this result ws not significnt. Furthermore, there ws no increse in the intrcellulr clcium concentrtion when CD T cells were stimulted with CpG B, which suggested tht the mechnism y which CpG B modifies CD T cell functionlity is not common to ligtion of TLR7. We note tht ll the lignds we used here were phrmcologicl gonists of TLR7. We hve not een le to find ny immunostimultory RNA specific for humn TLR7, which would e more physiologicl lignd, s these immunostimultory RNA sequences re specific for mouse TLR7 ut recognize humn TLR8 insted. We hve overcome this issue through the use of HIV-1 s potentil nturl lignd of TLR7. The physiologicl relevnce of our dt is suggested y the effect of TLR7 signling on HIV-1-infected cells. Severl reports hve highlighted the role of innte sensors in infection with HIV-1, focusing on the function of these molecules in CD T cells tht hd not een productively infected 1,. Our results dd new lyer of complexity to the understnding of the HIV virl life cycle, which needs further investigtion, nd re in contrst with pulished dt descriing the cytoplsmic sensors of virl DNA tht trigger cell deth y pyroptosis of unproductively infected CD T cells 1,. Moreover, the oservtions reported here suggest involvement of TLR7 oth in the induction of the nergic phenotype oserved in HIV-1-infected cells nd in increses in the degree of infection of CD T cells, which suggests tht TLR7-induced upregultion of clcium is involved in the virl life cycle. In this context, TLR8-dependent ctivtion of NF-κB hs een shown to e criticl for HIV-1 repliction in dendritic cells 3. Although we did not investigte the role of TLR8 in the infection of CD T cells y HIV-1, it is possile tht contriutions of oth TLR7-driven ctivtion of NF-κB nd TLR7-induced nergy hve role in the incresed virl lod. The oservtion tht nergy fvored HIV-1 infection is somewht prdoxicl given existing literture showing tht HIV-1 infection is fvored in ctivted CD T cells 11. We speculte tht HIV-1 might trigger TLR7 to induce n increse in intrcellulr clcium concentrtions, which hs een suggested to e importnt for the virl life cycle 37. Whether the induction of nergy in host cells y HIV-1 infection vi TLR7 is ystnder consequence of the increse in intrcellulr clcium driven y TLR7, nd whether this sttus is eneficil for the host or not, re currently unknown. However, these oservtions open up new field of investigtion relted to the mechnism, kinetics nd consequences of the interction of HIV-1 with the host cell through TLR7. Although infections re generlly regrded s illnesses, mmmls re colonized with cteri nd viruses. The prime exmple of this is cteri dopted for digestion, lthough humns re lso colonized with common DNA nd RNA viruses, including endogenous retroviruses. It hs een suggested tht these endogenous retroviruses provide n doptive selective dvntge in generting genetic diversity. On the sis of our dt here, we speculte tht TLR7 might hve een co-opted in humn CD T cells to co-evolve so tht the cells do not enter into the cell cycle in response to endogenous retroviruses, with potentil consequences such s leukemi (HTLV-1) nd utoimmune diseses. There re exmples in the literture showing tht certin endogenous retrovirus sequences re upregulted in humn utoimmune diseses. Although it will e of interest in future investigtions to elucidte precisely when TLR7 is engged in the life cycle of HIV-1 during infection, these dt demonstrte mechnism y which HIV-1 my void elimintion y co-opting NFAT-dependent TLR7-induced T cell nergy. In summry, we hve demonstrted previously unknown role for TLR7 in CD T cell function tht is in direct opposition to its role in cells of the innte immune system. Moreover, TLR7 lignds my e used s mens of inducing tolernce on CD T cells in humn utoimmune diseses. Finlly, our dt hve demonstrted novel function for microil pttern recognition receptors in the inhiition of immune responses. Methods Methods nd ny ssocited references re ville in the online version of the pper. Note: Any Supplementry Informtion nd Source Dt files re ville in the online version of the pper. Acknowledgments We thnk L. Devine nd Z. Wng for technicl ssistnce; Y. Tsunetsugu-Yokot (Tokyo University of Technology) for provirl DNA; D. Bruce, H. Zpt nd B.C. Herold nd the lortory of B.C. Herold for the recruitment of ptients; nd R. Medzhitov, A. Iwski nd memers of the Hfler lortory for comments nd suggestions. Supported y the Ntionl MS Society (CA161- A-18), the US Ntionl Institutes of Helth (P1 AI77, U19 AI613, U19 AI73 nd P1 AI39671 to D.A.H., nd R1 AI639 to M.J.K.), the Pentes Foundtion (D.A.H.), the Nncy Tylor Foundtion for Chronic Diseses (D.A.H.) nd the Rce to Erse MS Foundtion (M.D.-V.). nture immunology VOLUME 16 NUMBER 1 JANUARY 17