Activator protein 1 suppresses antitumor T-cellfunction via the induction of programmed death 1Gang Xiao, Anqi Deng, Haifeng Liu, Gaoxiang Ge, and Xiaolong Liu1

State Key Laboratory of Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences,Shanghai 200031, China

Edited by Michael Karin, University of California at San Diego School of Medicine, La Jolla, CA, and approved August 13, 2012 (received for review April16, 2012)

T cells play a critical role in tumor immunosurveillance by eliminatingnewly transformed somatic cells. However, tumor cell variants canescape from immunological control after immunoediting, leading totumor progression. Whether and how T cells respond to tumorgrowth remain unclear. Here,we found that tumor-infiltrating T cellsexhibited persistently up-regulated expression of the activator pro-tein 1 (AP-1) subunit c-Fos during tumor progression. The ectopicexpressionof c-Fos in T cells exacerbated tumor growth,whereas theT-cell–specific deletion of c-Fos reduced tumor malignancy. This un-expected immunosuppressive effect of c-Fos was mediated throughthe induced expression of immune inhibitory receptor programmeddeath 1 (PD-1) via the direct binding of c-Fos to the AP-1–binding sitein the Pdcd1 (gene encoding PD-1) promoter. A knock-in mutation ofthis binding site abrogated PD-1 induction, augmented antitumor T-cell function and repressed tumor growth. Taken together, thesefindings indicate that T-cell c-Fos subsequently induces PD-1 expres-sion in response to tumor progression and that disrupting such in-duction is essential for repression of tumor growth.

antitumor immunity | T cell response | transactivation

In a process known as tumor immunosurveillance, effector Tcells, in cooperation with other immune cells, can recognize

and destroy premalignant somatic cells in the initial stages oftumor development (1). Transformed cells may not be com-pletely eliminated, leading to the establishment of a state ofequilibrium in which the immune system is still able to controlthe net tumor growth (2). However, the continual interactionbetween immune cells and tumor cells in such a state can resultin the selection of tumor cell variants with the ability to evadeimmunological control (3). Tumor cell variants escape immuneelimination by attenuating antigen processing and presentationor by becoming insensitive to the effects of antitumor cytokinesthrough a process termed immunoediting (4). Whether T cellscan respond to and still have an impact on tumor growth remainto be determined.Activator protein 1 (AP-1) is a sequence-specific transcrip-

tional activator that is composed of protein heterodimers of Fosand Jun family members (5). As a multifunctional transcriptionfactor that is encoded by a protooncogene, the AP-1 subunit c-Fospromotes cell proliferation and transformation (6). In T cells, c-Fos functions as a molecular counter to integrate intermittent T-cell receptor (TCR) stimulation and the expression of c-Fosreflects T-cell activation even after a weak or intermittent stim-ulus (7). c-Fos is immediately activated and dimerizes with Junproteins to elicit the transcription of AP-1–responsive genes thatare essential for T-cell activation and function following TCRstimulation (8). Some AP-1 targets, such as Fas ligand and IFN-γ(9), are important for antitumor activities of T cells.To address whether T cells respond to tumor growth, we in-

oculated mice with immunoedited cancer cell lines that had beenshown previously to be able to grow rapidly as solid tumors inimplanted mice (10, 11). We found that tumor-infiltrating T cellspersistently elevated the expression of c-Fos during tumor pro-gression. Unexpectedly, the up-regulation of c-Fos expression intumor-infiltrating T cells was shown to promote rather than represstumor growth because of the direct induction of the expression

of the immunoinhibitory receptor programmed death 1 (PD-1).Importantly, the disruption of c-Fos (AP-1)–mediated PD-1expression restored antitumor T-cell function. Altogether,these findings indicate that, although tumor-infiltrating T cellsare able to respond to tumor progression, they eventually fail torepress tumor growth because of c-Fos (AP-1)–mediated in-duction of PD-1 expression.

Resultsc-Fos Expression in T Cells Is Elevated During Tumor Progression. Toexamine whether T cells respond to tumor growth, a pilot exper-iment was conducted to assess the expression levels of members ofthe AP-1 family. Whereas c-Fos mRNA levels were significantlyup-regulated in tumor-infiltrating T cells after the i.v. inoculationof Lewis lung carcinoma (LLC) cells, the mRNA levels of theother Fos and Jun subunits that constitute the AP-1 familyremained largely unchanged (Fig. 1A). To further monitor c-Fosexpression during tumor progression, we used the T-cell–specificc-Fos EGFP reporter mouse model (12) (Cd4Cre+c-Fos+/fl), the Tcells of which express EGFP when c-Fos expression is induced(Fig. S1A). Following the inoculation of the LLC cells into thereporter mice, an EGFP signal was detected subsequent to tumordevelopment; additionally, EGFP levels were continuously ele-vated in lung T cells throughout tumor progression (Fig. 1B andFig. S1B). To examine whether the elevated EGFP expressionresulted from increased gene transcription, we assessed c-FosmRNA levels and found that c-FosmRNA levels were significantlyhigher in EGFP+ T cells than in EGFP− T cells (Fig. 1C).The surface expression of CD44 was elevated following T-cell

activation in the lymph nodes (13) and at the tumor sites (14).After LLC inoculation, all of the EGFP+ T cells were CD44hi, andthe number of EGFP+ CD44hi T cells markedly increased in thelungs and was moderately enhanced in the mediastinal lymphnodes (MLNs) but remained unchanged in the spleen (SPL) (Fig.1D). Thus, tumor-infiltrating T cells can respond to tumor pro-gression and exhibit persistently increased c-Fos transcription.

Ectopic Expression of c-Fos in T Cells Promotes Tumor Growth. Toexamine the role of c-Fos in T cells during tumor progression, weinoculated LLC cells into c-FosTg (FosTg) mice that specificallyoverexpressed c-Fos in their T lineage cells (15). FosTg miceexhibited higher mortality rates compared with control micefollowing the LLC inoculation (Fig. 2A). Consistent with thisfinding, the number of lung tumors significantly increased in theFosTg mice (Fig. 2B). The lungs of the FosTg mice also showedsignificantly more tumor growth than littermate controls at both3 and 4 wk post-LLC inoculation (Fig. 2C). Histological exami-nations confirmed these findings (Fig. 2D). Although tumor cellproliferation was not affected (Fig. 2E), the number of apoptotic

Author contributions: G.X., G.G., and X.L. designed research; G.X., A.D., and H.L. per-formed research; G.X., G.G., and X.L. analyzed data; and G.X. and X.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: liux@sibs.ac.cn.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1206370109/-/DCSupplemental.

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tumor cells significantly decreased when c-Fos was ectopicallyexpressed in the T cells (Fig. 2F). These results suggest that theectopic expression of c-Fos in T cells promotes tumor growthrather than exerting antitumor functions.

Deletion of c-Fos in T Cells Reduces Tumor Growth. To verify theunexpected protumor activity of c-Fos in T cells, Cd4Cre+c-Fosfl/fl

(Foscko) mice were inoculated with LLC cells. Foscko mice havea T-cell–specific deletion of c-Fos that is mediated throughCd4Cre expression (Fig. S2A). These mice exhibit similar numbersof peripheral T cells, although they have fewer CD4 single-positivethymocytes than their littermate controls (Fig. S2B). Consistentwith the results obtained using FosTg mice, a survival advantagewas observed in the LLC-inoculated Foscko mice compared withthe control mice (Fig. 3A). Similarly, tumor numbers were signif-icantly reduced (Fig. 3B) and tumor growth was notably repressed(Fig. 3C) in the Foscko mice. Histological analyses of tissue sec-tions also showed repressed tumor growth in the Fosckomice (Fig.3D). In the absence of c-Fos in T cells, tumor cell proliferationdecreased (Fig. 3E), whereas tumor cell apoptosis increased (Fig.3F). Notably, c-Fos expression in other tumor-infiltrating immunecells [e.g., dendritic cells (DCs), macrophages (Macs), and naturalkiller (NK) cells] was not altered (Fig. S2C). Similar results wereobtained when FosTg and Foscko mice were inoculated with B16-F10 melanoma cells (Fig. S2 D–G). These findings suggest thatT-cell c-Fos expression promotes tumor growth following in-oculation with tumor cells.

PD-1 Is a Downstream Target of c-Fos (AP-1) in Tumor-Infiltrating TCells. Tumor cells use peripheral tolerance mechanisms to avoidtumor-specific T cells to exert antitumor functions (16). Onemechanism of peripheral tolerance is the attenuation of TCRsignaling through immune inhibitory receptors. We examinedinhibitory receptor expression in tumor-infiltrating T cells fromFosTg mice. Among the inhibitory receptors that were examined,PD-1 mRNA levels were shown to significantly increase in bothCD44hi CD4 and CD44hi CD8 T cells from FosTg mice (Fig. 4A).All of the EGFP+ tumor-infiltrating T cells were CD44hi in theCd4Cre+c-Fos+/fl mice; thus, the expression of inhibitory recep-tors in the EGFP+ CD4 and EGFP+ CD8 tumor-infiltrating T

cells was examined in Foscko mice. In accordance with the resultsobtained using the FosTg mice, PD-1 mRNA levels decreasedwhen c-Fos was depleted (Fig. 4B).PD-1 is expressed in tumor-specific T cells and is responsible

for T-cell functional impairment and exhaustion (17). Therefore,it is possible that c-Fos (AP-1) may transactivate PD-1 expressionin response to tumor progression. Indeed, whereas PD-1 expres-sion was significantly augmented in tumor-infiltrating CD44hiT cells from FosTg mice (Fig. 4C), it was reduced in tumor-infiltrating EGFP+ T cells from Foscko mice (Fig. 4D).

c-Fos (AP-1) Transactivates the Pdcd1 Proximal Promoter. Ourin vitro experiments suggested that PD-1 was a downstreamtarget of c-Fos (AP-1) because its expression was elevated in thepresence of ectopically expressed c-Fos and was attenuated inthe absence of c-Fos (Fig. S3). PD-1 expression is controlled byseveral cis elements that are located in the distal conserved re-gion (CR-C) of the Pdcd1 (gene encoding PD-1) promoter (18,19). There is also a proximal conserved region (CR-B) that lacksFig. 1. Tumor-infiltrating T cells exhibit increased c-Fos expression during

tumor progression. (A) Lung-infiltrating T cells were FACS-purified from LLC-inoculated or noninoculated C57BL/6 mice at day 21 postinoculation formRNA quantification (mean ± SD; n = 3; **P < 0.005 by Student t test). (B)EGFP expression in lung-infiltrating T cells from treated Cd4Cre+c-Fos+/fl mice(mean ± SEM; n = 5; *P < 0.05 and **P < 0.005 by Student t test). (C) EGFP+ Tcells from LLC-inoculated reporter mice at day 21 postinoculation were pu-rified with their EGFP− counterparts for c-Fos mRNA quantification (mean ±SD; n = 3; ***P < 0.001 and **P < 0.005 by Student t test). (D) Flow-cyto-metric analysis of CD44 and EGFP expression in T cells from the lung, MLN,and SPL of reporter mice at day 21 post-LLC inoculation. Statistical analysis isshown in Table S1.

Fig. 2. T-cell c-Fos overexpression promotes tumor growth. (A) Survival ofLLC-inoculated FosTg mice compared with littermate controls (FosTg, n = 11;control, n = 10; P < 0.005 by log-rank test). (B) Number of lung tumors at day28 post-LLC inoculation (FosTg, n = 8; control, n = 8; ***P < 0.001 by Studentt test). (C) Lungs at days 21 and 28 post-LLC inoculation. (Scale bars, 5 mm.)The results are representative of four independent experiments. (D) Hema-toxylin and eosin (HE)-stained lung sections at day 28 post-LLC inoculation(magnification, 40× and 400×). (Scale bars, 200 μm.) (E) Ki-67 staining of lungsections. Proliferating tumor cells of each tumor were quantified as de-scribed in Materials and Methods; more than 50 HPFs were counted fromeach group (magnification, 400×; mean ± SEM; FosTg, n = 15 from 3 mice;control, n = 8 from 3 mice). (Scale bars, 200 μm.) (F) Cleaved caspase-3staining and apoptotic tumor cell quantification (magnification, 400×;mean ± SEM; FosTg, n = 14 from 3 mice; control, n = 7 from 3 mice; *P < 0.05by Student t test). (Scale bars, 200 μm.)

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any defined cis elements but is indispensable for PD-1 expression(18). Interestingly, the proximal promoter (the 1 kb upstream ofPdcd1, including CR-B) was transactivated following phorbol 12-myristate 13-acetate (PMA) and ionomycin (P+I) stimulation,which induce c-Fos expression (Fig. 5A and Fig. S4A). Fourpotential AP-1–binding sites (A through D) were predicted inthe proximal promoter (Fig. S4B). Notably, all of the three Junproteins can activate the proximal promoter when coexpressedwith c-Fos, but JunB/c-Fos composition showed the most markedactivating activity (Fig. 5B), and JunB is expressed at a higherlevel than cJun and JunD in tumor-infiltrating T cells (Fig. S4C).The dose-dependent activation of the proximal promoter wasobserved when different concentrations of c-Fos and JunB werecoexpressed (Fig. 5C). Out of the four potential AP-1–bindingsites, only site D, which is located in CR-B, effectively bound c-Fos (AP-1) in P+I-stimulated T cells (Fig. S4D). Similar resultswere observed in tumor-infiltrating T cells (Fig. 5D). Mutationanalysis confirmed that the AP-1–binding site D was responsiblefor the c-Fos (AP-1)–mediated transcriptional activity of CR-B(Fig. 5E).

Knock-In Mutation of the AP-1–Binding Site Represses Tumor Growth.To determine whether c-Fos (AP-1)–induced PD-1 up-regulationcontributed to the attenuated antitumor activities of the tumor-infiltrating T cells, we generated mice with mutations in the AP-1–binding site D in the CR-B (Fig. S5 A–D). The proportions ofCD4 and CD8 T cells and TCR Vβ use were comparable betweenthe homozygous mutant mice (Pdcd1KI/KI) and littermate controls(Pdcd1+/KI) (Fig. S5 E–H). T cells from the Pdcd1KI/KI mice butnot those from Pdcd1+/KI mice failed to up-regulate surface PD-1expression compared with their counterparts from Pdcd1+/+ micefollowing TCR stimulation (Fig. S6A). The decreased PD-1 sur-face expression could result from impaired Pdcd1 transcription(Fig. S6B). The induction of c-Fos mRNA transcription was notaltered (Fig. S6C); however, c-Fos failed to bind to the mutantAP-1–binding site D in Pdcd1KI/KI T cells (Fig. S4E).Following inoculation with LLC cells, the Pdcd1KI/KI mice

showed substantially improved survival rates (Fig. 6A), signifi-cantly decreased tumor numbers (Fig. 6B), and barely detectabletumor nodules (Fig. 6 C and D), suggesting that tumor growthwas better controlled in the Pdcd1KI/KI mice compared with thelittermate control mice. Importantly, tumor cell proliferationdecreased (Fig. 6E), and tumor cell apoptosis significantly in-creased in the Pdcd1KI/KI mice (Fig. 6F). Similar results wereobserved after inoculation with B16-F10 melanoma cells (Fig. S7A and B). Intriguingly, mutation of the AP-1–binding site Dsubstantially attenuates the protumor effects of the ectopicallyexpressed c-Fos in FosTg mice; tumor numbers were notablyreduced and tumor growth was effectively repressed in thePdcd1KI/KI FosTg mice compared with the control mice (Fig. S7C).PD-1 expression was significantly impaired in tumor-infiltrating

Fig. 3. T-cell c-Fos depletion reduces tumor growth. (A) Survival of LLC-in-oculated Foscko mice compared with littermate controls (Foscko, n = 10;control, n = 18; P < 0.001 by log-rank test). (B) Number of lung tumors at day28 post-LLC inoculation (Foscko, n= 7; control, n= 12; **P< 0.005 by Student ttest). (C) Lungs at days 21 and 28 post-LLC inoculation. (Scale bars, 5 mm.) Theresults are representative of nine independent experiments. (D) HE-stainedlung sections at day 28 post-LLC inoculation (magnification, 40× and 400×.)(Scale bars, 200 μm.) (E) Ki-67 staining of lung sections. Proliferating tumorcells were quantified (magnification, 400×; mean ± SEM; Foscko, n = 9 from 3mice; control, n = 10 from 3 mice; **P < 0.005 by Student t test). (Scale bars,200 μm.) (F) Cleaved caspase-3 staining and apoptotic tumor cell quantifica-tion (magnification, 400×; mean ± SEM; Foscko, n = 14 from 3 mice; control,n = 13 from 3 mice; ***P < 0.001 by Student t test). (Scale bars, 200 μm.)

Fig. 4. PD-1 is a potential downstream target of c-Fos in tumor-infiltratingT cells. (A) CD44hi tumor-infiltrating T cells were purified from FosTg andcontrol mice at day 21 post-LLC inoculation for mRNA quantification (mean ±SD; n = 3; **P < 0.005 by Student t test). (B) EGFP+ tumor-infiltrating T cellswere purified from Foscko and control mice at day 21 post-LLC inoculationfor mRNA quantification (mean ± SD; n = 3; **P < 0.005 by Student t test).(C and D) Flow-cytometric analysis of PD-1 expression in CD44hi tumor-infiltrating T cells from FosTg mice (C), EGFP+ tumor-infiltrating T cells fromFoscko mice (D), and controls at day 28 post-LLC inoculation (solid line, anti-PD-1; shaded in gray, isotype control). The results are representative of threeindependent experiments.

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T cells from Pdcd1KI/KI FosTg mice (Fig. S7D), suggesting thatthe c-Fos–mediated immune suppressive effect is largely due tothe induction of PD-1 expression.

Disruption of c-Fos (AP-1)–Induced PD-1 Expression Enhances AntitumorT-Cell Function. The repressed growth of the immunoedited tumorcells in the Pdcd1KI/KI mice may have resulted from the en-

hanced functioning of the T cells. As expected, the Pdcd1KI/KI

mice possessed more CD44hi lung-infiltrating T cells comparedwith the littermate controls following but not before LLC inocu-lation (Fig. 6G and Fig. S8A); furthermore, these T cells failed toup-regulate PD-1 expression (Fig. 6H). Importantly, lung-in-filtrating CD44hi T cells from the LLC-inoculated Pdcd1KI/KI

mice, but not from the untreated Pdcd1KI/KI mice, produced more

Fig. 5. c-Fos (AP-1) transactivates PD-1 expression by directly binding to the AP-1–binding site D in the Pdcd1 promoter. (A) Jurkat cells were electroporatedwith thepGL3 vector or Pdcd1-pGL3 (as shown in Fig. S4B) for reporter assay (mean ± SD; n = 3; **P < 0.005 by Student t test). (B) c-Fos plasmids combined with Jun plasmidswere cotransfected with the pGL3 vector or the Pdcd1-pGL3 into Jurkat cells for reporter assay (mean ± SD; n = 3; **P < 0.005 and *P < 0.05 by Student t test). (C)Increasing concentrations (μg) of the AP-1 (c-Fos and JunB) constructs were cotransfected with the pGL3 vector or the Pdcd1-pGL3 into Jurkat cells for reporter assay(mean ± SD; n = 3; ***P < 0.001 and **P < 0.005 by Student t test). (D) Tumor-infiltrating T cells were purified from C57BL/6 mice at day 21 post-LLC inoculation andlysed for ChIP assay (mean± SD; n = 3; *P < 0.05 by Student t test). (E) The reporter activity of themutant formDM-pGL3 and Pdcd1-pGL3 was measured (mean± SD;n = 3; ***P < 0.001 by Student t test).

Fig. 6. Knock-inmutation of the AP-1–binding site Dleads to repressed tumor growth and enhanced T-cellantitumor functions. (A) Survival of LLC-inoculatedPdcd1KI/KI mice compared with littermate controls(Pdcd1KI/KI, n = 17; control, n = 16; P < 0.001 by log-rank test). (B) Lung tumor numbers were counted atday 35 post-LLC inoculation (Pdcd1KI/KI, n = 9; control,n = 7; **P < 0.005 by Student t test). (C) Images oflungs at days 28 and 35 post-LLC inoculation. (Scalebars, 5 mm.) The results are representative of nineindependent experiments. (D) Lung sections with HEstaining at day 28 post-LLC inoculation (magnifica-tion, 40× and 400×). Ki-67 (E) and cleaved caspase-3(F) staining of lung sections were performed. (Scalebars, 200 μm.) Quantification was not performed be-cause of the small tumor sizes observed in Pdcd1KI/KI

mice. (G) CD44 expression in tumor-infiltrating T cellsat day 35 post-LLC inoculation (statistically analyzed inTable S1). (H) Flow-cytometric analysis of PD-1 ex-pression in CD44hi tumor-infiltrating T cells at day 35post-LLC inoculation (solid line, anti-PD-1; shaded ingray, isotype control). The results are representativeof three independent experiments. (I) Tumor-in-filtrating lymphocytes were harvested at day 35 post-LLC inoculation. Cytokine expression was assessed. (J)Surface CD25 and intracellular Foxp3 expression intumor-infiltratingCD4T cells. Productionof IL-17a (K),IL-4 (L), and IFN-γ (M) from tumor-infiltrating CD4 Tcells was analyzed. Granzyme B (N) and perforin (O)expression in tumor-infiltrating CD8 T cells assessed.Results from (I) to (O) were statistically analyzed asshown in Table S1.

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antitumor cytokines, such as IL-2, IFN-γ, and TNF-α, comparedwith those from the littermate controls (Fig. 6I and Fig. S8B).In addition to cytokines, regulatory T cells (Tregs) also play

key roles in antitumor immunity. Tregs are recruited to tumorsites to suppress T-cell function (20). Fewer Tregs were observedin tumors from Pdcd1KI/KI mice compared with the littermatecontrols (Fig. 6J). In contrast to Tregs, the proportion of Th1cells, but not of Th17 or Th2 cells, increased in the tumor-in-filtrating T cells from Pdcd1KI/KI mice (Fig. 6 K–M), indicatingthat the enhanced antitumor T helper cell function in thePdcd1KI/KI mice may have been attributable to an enhanced Th1response. Granzyme B and perforin are important effectormolecules that are used by CD8 T cells to kill target cells. Al-though the number of granzyme B–expressing CD8 T cells wasfound to be somewhat decreased in the Pdcd1KI/KI mice, thenumber of perforin-expressing CD8 T cells increased in thePdcd1KI/KI mice (Fig. 6 N and O). Taken together, the enhancedcytokine production and effector function of T cells may pro-mote antitumor immunity when c-Fos (AP-1) fails to up-regulatePD-1 during tumor progression.

Knock-In Mutation of the AP-1–Binding Site Suppresses SpontaneousTumor Growth. Finally, we examined whether the disruption of c-Fos (AP-1)–induced PD-1 expression can also suppress sponta-neous tumor growth. The mammary epithelium–specific trans-genic mice expressing the polyomavirus middle T antigen(PyMT) under the transcriptional control of the mouse mam-mary tumor virus long terminal repeat (21), which developspontaneous mammary tumors in multiple mammary glands,were crossed with Pdcd1KI/KI mice. By blockage of c-Fos (AP-1)–induced PD-1 expression (Fig. 7A), the time of tumor onset inPdcd1KI/KI PyMT mice was significantly delayed (Fig. 7B).Consistent with the prolonged tumor latency, mammary tumor

growth was also decelerated in these mice (Fig. 7C). Histologicalexamination of tumors showed invasive tumor growth in controlmice, whereas tumor development was notably repressed inPdcd1KI/KI PyMT mice (Fig. 7D). Although tumor cell pro-liferation was not notably affected, tumor cell apoptosis signifi-cantly increased in the Pdcd1KI/KI PyMT mice (Fig. 7 E and F).Consistent with the observations in allograft studies, tumor-

infiltrating CD44hi T cells from the Pdcd1KI/KI PyMT mice pro-duced more antitumor cytokines than those from the controlmice (Fig. 7G). Less Tregs, Th17, and Th2 cells, but more Th1cells, were observed in tumors from Pdcd1KI/KI PyMT mice (Fig.7 H–K). In addition, the number of granzyme B–expressing CD8T cells decreased, whereas the number of perforin-expressingCD8 T cells increased in the Pdcd1KI/KI PyMT mice (Fig. 7 L andM). Altogether, the disruption of c-Fos (AP-1)–induced PD-1expression enhances antitumor T-cell function and, thus, sup-presses spontaneous tumor growth.

DiscussionIt has been reported that c-Fos inhibits the inflammatory re-sponse of myeloid-derived immune cells (22, 23). In this study,we demonstrated that c-Fos (AP-1) also suppresses antitumorT-cell function. The expression of c-Fos is persistently up-regu-lated during tumor progression, which likely occurs through TCRsignaling alone or in combination with stimulation from tumormicroenvironment–derived cytokines and chemokines (24, 25). In-triguingly, up-regulated c-Fos sequentially promotes the expressionof PD-1, an effective inhibitor of T-cell activation. In the tumormicroenvironment, multiple cell types, including tumor cells,endothelial cells, and immune cells, express the PD-1 ligandsthat can negatively signal to T cells (26). Thus, tumor-infiltratingT cells eventually fail to repress tumor growth, although up-

Fig. 7. Mutation of the AP-1–binding site relievesspontaneous tumor growth and enhanced T-cellantitumor function in Pdcd1KI/KI PyMT mice. (A)Flow-cytometric analysis of PD-1 expression inCD44hi tumor-infiltrating T cells from mice at day130 after birth (solid line, anti-PD-1; shaded in gray,isotype control). (B) Tumor onset was monitored fortumor-free survival analysis (Pdcd1KI/KI PyMT, n = 9;control, n = 20; P < 0.001 by log-rank test). (C)Mouse mammary tumor growth was monitoredstarting from 6 wk after birth, up to 15 wk (mean ±SEM, Pdcd1KI/KI PyMT, n = 9; control, n = 18; *P <0.05 by Student t test with Welch’s correction). (D)Mouse tumor sections with HE staining at day 130after birth (magnification, 40× and 400×). (Scalebars, 200 μm.) Ki-67 (E) and cleaved caspase-3 (F)staining of tumor sections from (D) were per-formed, more than 150 HPFs were counted for eachgroup (magnification, 400×; mean ± SEM; Pdcd1KI/KI

PyMT, n = 10 from 3 mice for Ki-67; n = 11 from 3mice for cleaved caspase-3; control, n = 11 from 3mice for Ki-67; n = 10 from 3 mice for cleaved cas-pase-3; *P < 0.05 by Student t test). (Scale bars, 200μm.) (G) Cytokine expression was assessed. (H) Sur-face CD25 and intracellular Foxp3 expression in tu-mor-infiltrating CD4 T cells. Production of IL-17a (I),IL-4 (J), and IFN-γ (K) from tumor-infiltrating CD4 Tcells was analyzed as in G. Granzyme B (L) andperforin (M) expression in tumor-infiltrating CD8 Tcells from mice in A was assessed. Results from G–Mwere statistically analyzed as shown in Table S1.

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regulated c-Fos may also initially activate the expression of genesthat are essential for T-cell function.PD-1 plays a critical role in maintaining the homeostasis of

T-cell–mediated adaptive immunity, and PD-1 deficiency leadsto autoimmune diseases (27, 28). Although PD-1 was initiallydiscovered to be a negative regulator that leads to T-cell ex-haustion during chronic viral infection (29), it was subsequentlyfound to be responsible for the functional impairment of tumor-infiltrating T cells (17, 30). Importantly, blocking the PD-1pathway by anti–PD-1 antibody activates antitumor immunity(31). Several cis elements have been identified to regulate PD-1expression (18, 19, 32). However, the mechanism of transcrip-tional regulation of PD-1 in T cells during tumor progressionremains largely unknown. TCR signaling is responsible for PD-1induction (26), and cytokines have also been shown to reinforcePD-1 expression (19). As a common target for both TCR andcytokine signaling, c-Fos (AP-1) could be a primary factor that isresponsible for the activation of PD-1 transcription during tumorprogression, which was indeed confirmed by analysis of the AP-1–binding site mutant mouse. c-Fos (AP-1)–mediated PD-1expression suppresses antitumor T-cell function; however, thistranscriptional regulation may be an important factor in theavoidance of autoimmune diseases given that the rapid up-regulation of PD-1 is essential for T-cell tolerance (33).Tumor-infiltrating T cells produce more antitumor cytokines

when c-Fos (AP-1)–induced PD-1 expression was disrupted. Suchdisruption also modulates the composition of the effector T-cellsubsets and results in optimal antitumor effects. Specifically, theproportion of Tregs was decreased in the tumor microenviron-ment of Pdcd1KI/KI mice. This observation is consistent withprevious report that PD-1 signaling regulates the developmentand maintenance of induced Tregs (34). Importantly, similareffects of c-Fos (AP-1)–induced PD-1 expression were observed

when a spontaneous mammary tumor model that mimics humanbreast cancer was used. It would be interesting to examinewhether the c-Fos expression is also up-regulated in tumor-in-filtrating T cells from patients.Helper function, but not the early activation of T cells, has

been shown to be inhibited during the Treg-mediated suppres-sion of the IgE response (35). Interestingly, a similar dissectionbetween the activation and functionality of T cells may also beinduced by tumor cells. In addition to passively escaping T-cell–mediated immune recognition, tumor cells can actively suppressthe antitumor T-cell function through the induction of PD-1expression, resulting in tumor immune escape and subsequentaggravated tumor growth. Disruption of c-Fos (AP-1)–mediatedPD-1 induction, however, can effectively restore the antitumoractivities of tumor-infiltrating T cells, as evidenced by the knock-in mouse model. Thus, the early blockade of c-Fos (AP-1)–in-duced PD-1 up-regulation may offer new opportunities for drugdevelopment for the prevention of many diseases, includingcancer and chronic infection.

Materials and MethodsFull methods are provided in the SI Materials and Methods. Briefly, individualmice were i.v.-injected with 2 × 106 LLC cells or 2 × 105 B16-F10 cells. Mousemammary tumors were examined by visual inspection and palpation tomonitor tumor onset. For the knock-in mutation, the targeted embryonicstem cell clone was microinjected into C57BL/6 blastocysts to generate chi-meric mice.

ACKNOWLEDGMENTS.We thank Dr. X. Wu (Fudan University) for providingthe PgkCre transgenic mice and our colleague Dr. L. Hui for providing the c-Fosfl/fl mice. This work was funded through National Basic Research Pro-gram of China Grant 2012CB518700, National Natural Science Foundationof China Grant 30925031, and Shanghai Municipal Government Grant12XD1405800.

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