in vitro priming of adoptively transferred t cells ... · small-molecule rorg agonist have enhanced...

Tumor Biology and Immunology

In Vitro Priming of Adoptively Transferred T Cellswith a RORg Agonist Confers DurableMemory andStemness In VivoXiao Hu1, Kinga Majchrzak2, Xikui Liu1, Megan M.Wyatt2, Chauncey J. Spooner1,Jacques Moisan1,Weiping Zou3, Laura L. Carter1, and Chrystal M. Paulos2

Abstract

Adoptive T-cell transfer therapy is an FDA-approved treatment for leukemia that relies on theex vivo expansion and reinfusion of a patient'simmune cells, which can be engineered with a chi-meric antigen receptor (CAR) for more efficienttumor recognition. Type 17 T cells, controlled tran-scriptionally by RORg , have been reported to medi-ate potent antitumor effects superior to thoseobserved with conventionally expanded T cells.Here, we demonstrate that addition of a synthetic,small-molecule RORg agonist during ex vivo expan-sion potentiates the antitumor activity of humanTh17 and Tc17 cells redirected with a CAR. Likewise,ex vivo use of this agonist bolstered the antitumorproperties of murine tumor-specific CD4þ andCD8þ T cells. Expansion in the presence of the RORgagonist enhanced IL17A production withoutcompromising IFNg secretion in vitro. In vivo, cyto-kine neutralization studies revealed that IFNg andIL17A were required to regress murine melanomatumors. The enhanced antitumor effect of RORgagonist treatment was associated with recovery ofmore donor T cells in the tumor and spleen; thesecells produced elevated levels of cytokines monthsafter infusion and expressed markers of long-livedstem and central memory cells such as Tcf7and CD62L. Conversely, untreated cells mainlyexhibited effector phenotypes in the tumor. Curedmice previously treated with agonist-primed T cells were protected from tumor rechallenge. Collectively, our work reveals that in vitrotreatment with a RORg agonist generates potent antitumor Type 17 effector cells that persist as long-lived memory cells in vivo.

Significance: RORg agonists can be used in vitro during T-cell expansion to enhance the efficacy of adoptive cell therapy(e.g., CAR-T) and to provide long-term protection against tumors.

Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/14/3888/F1.large.jpg. Cancer Res; 78(14); 3888–98. �2018 AACR.

© 2018 American Association for Cancer Research

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1Lycera Corp, Ann Arbor, Michigan. 2Medical University of South Carolina,Hollings Cancer Center, Charleston, South Carolina. 3University of Michigan,School of Medicine, Ann Arbor, Michigan.

Note: Supplementary data for this article are available at Cancer ResearchOnline (http://cancerres.aacrjournals.org/).

Corresponding Authors: Chrystal M. Paulos, Medical University of South Car-olina, 86 Jonathan Lucas Street, Charleston, SC 29425. Phone: 202-557-1868;

Fax: 843-327-9885; E-mail: [email protected]; Xiao Hu, Lycera Corp. Phone:734-233-3059; E-mail: [email protected]; and Laura L. Carter, Lycera Corp. Phone:734-233-3058; E-mail: [email protected]

doi: 10.1158/0008-5472.CAN-17-3973

�2018 American Association for Cancer Research.

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IntroductionThe nuclear receptor RORgt is a master transcription factor

that controls the development of CD4þ and CD8þ lymphocytesthat secrete IL17A, called T helper 17 (Th17) and T cytotoxic 17cells (Tc17), respectively (1). RORgt also plays a role in thedifferentiation of IL17A-producing innate immune cells, suchas innate lymphoid cells, NK cells and gd T cells (2, 3).Although defined by IL17 secretion, Type 17 cells are poly-functional effectors that can co-secrete IL22 and IFNg upontumor recall responses and possess durable memory propertiesin vivo (4, 5). The involvement of Th17 and Tc17 cells inautoimmunity, tumor immunity, and mucosal defense hasbeen well established (6, 7). Importantly, a recent case reportof a patient with colon cancer treated with the checkpointinhibitor pembrolizumab revealed that IL17 blockade withsecukinumab provided dramatic relief of immune-mediatedskin toxic effects but was associated with a subsequent loss ofthe antitumor efficacy, suggesting that the IL17/Th17 axis playsa role in the antitumor effects of immunotherapy (8).

While IL17þT cells are abundant in the mucosal tissues andsupport gut-related homeostasis (9), few such cells exist in theblood of healthy individuals. However, many Th17 and Tc17cells infiltrate tumors, especially compared with the density ofthese cells in the nontumor tissue of patients (10). This height-ened presence of Type 17 cells in tumor tissue holds true formany types of malignancies, implying that tumors themselvesproduce factors that promote RORgt expression. Approximately15% of human CD4þ T cells in tumors express RORgt and thistranscription factor is induced by cytokines TGFb and IL6, bothproduced at high levels in inflamed tissues and transformedcells (11). We reported that RORg activation with a novel smallmolecule agonist potentiates the function of antitumor Th17cells to a greater extent than the endogenous agonist desmos-terol (11, 12). This activation is associated with enhancedcytokine production and CTL activity as well as reduced Tregformation in vitro and effective antitumor immunity in synge-neic models (11).

In the context of cellular therapy for cancer, several reportshave shown that Type 17 T cells eradicate large humanand murine tumors to a far greater extent than bulk CD4T cells, Th1 or Th2 cells (4, 5, 13–15). Additional investigationrevealed that tumor-specific RORgtþ Th17 cells persisted longerthan T-bet expressing IL2-expanded Th0 and Th1 cells due totheir stem-like memory properties. Thus, we were interested inhow the addition of a synthetic RORg agonist to the ex vivoexpansion of TCR or CAR T-cell cultures would affect theirfunction, memory phenotype, persistence, and antitumor activ-ity when infused into mice with large murine or human tumors.To address this question, murine pmel-1 TCR transgenic CD8,TRP-1 TCR transgenic CD4 and mesothelin human CAR T-cellmodels were used (4, 16, 17).

Herein, we report that the RORg agonist LYC-54143 potenti-ates the antitumor activity of Th17 and Tc17 cells when addedto ex vivo cell expansion cultures of both CAR-expressinghuman T cells and tumor-specific CD4 and CD8 T cells.LYC-54143 treatment generates cells that produce elevatedeffector cytokines and increased markers associated withstem-like memory T cells (18, 19). When lymphocytes frommice receiving LYC-54143–treated Th17 plus Tc17 cells wereanalyzed after eradicating or controlling tumors, we found thatthe donor cells were more prevalent and were composed of

diverse memory phenotypes compared with mice unfused withuntreated cells. In addition, the agonist-primed cells expressedheightened Tcf7, a transcription factor downstream of thecanonical Wnt pathway essential for stemness (20) and long-lasting immunity to cancer (19). Importantly, mice cured withLYC-54143–primed cells were protected from repeated tumorchallenges several months later. Collectively, our work revealsthat antitumor Type 17 cells generated in the presence of asmall-molecule RORg agonist have enhanced antitumor activ-ity and persist as long-lived memory cells in vivo. These datasupport the utility of including these drugs as part of cellmanufacturing and expansion protocols.

Materials and MethodsMice and tumor lines

C57BL/6J (B6), TRP-1 TCR transgenic mice, pmel-1 TCRtransgenic mice, and NOD/scid/g chain knock out (NSG) micewere purchased from The Jackson Laboratory, housed, and bredin the Medical University of South Carolina Hollings CancerCenter (MUSC, Charleston, SC). NSG mice were housed underspecific pathogen-free conditions in micro-isolator cages andgiven autoclaved food and acidified water. Housing and experi-ments were conducted with Institutional Animal Care andUse Committee's approval at Medical University of SouthCarolina. B16F10 (H-2b) melanoma was maintained in culturemedia (RPMI 1640 w/L-glutamine, 10% FBS, 1% penicillin–streptomycin, NEAA, and Na Pyruvate, and 0.1% BME andHepes). M108 xenograft tumors were cultured and engrafted asdescribed previously (15).

T-cell culturesTRP-1 cells. TRP-1 splenocytes (which contain MHC-II–restrictedCD4þ T cells expressing TRP-1-recognizing transgenic TCR Vb14)were activated using 10 Gy-irradiated B6 456 splenocytes (feedercells) pulsed with 1 mmol/L TRP-1 peptide and polarized to aTh17 phenotype at 2 � 106 cells/2 mL of cell media in one wellof a 24-well plate with the following cocktail: 100 ng/mL rhIL6(NIH repository), 100 ng/mL rhIL21 (Shenandoah), 30 ng/mLrhTGFb1 (Biolegend), 10 ng/mL rhIL1b (NIH Repository),10 mg/mL each of anti-mIFNg clone XMG1.2, anti-mIL4clone 11B11, and anti-mIL2 clone JES6-1A12 (Bio X Cell).Th0 polarization occurred under peptide activation with irradi-ated feeder cells with the following cocktail: 100 IU/mLrhIL2 (NIH repository). Cultured cells were supplemented withnew media containing 100 IU/mL rhIL2 (NIH repository)throughout expansion. In experiments where indicated, RORgagonist LYC-54143 (synthesized at Lycera) was added to thecultures during peptide activation and then again 2 days later(10 mmol/L). For further description of RORg agonist and theiruse in immunotherapy of cancer, see, for example, internationalpatent application publication WO 2015/131035.

Pmel-1 T cells. pmel-1 splenocytes (which containMHC-I-restrict-ed CD8þ T cells expressing gp100-recognizing transgenic TCRVb13) were activated using 1 mmol/L hgp100 peptide þ 100 IUrhIL2/mL and primed on day 1 with type 17-polarizing cytokineswith our without RORg agonist (as above for TRP-1 cells).Cells were supplemented with culture media containing 100 IUrhIL2/mL and expanded as indicated. CD8þ T-cell cultures

RORg Agonist-Treated T Cells Have Durable Antitumor Memory

www.aacrjournals.org Cancer Res; 78(14) July 15, 2018 3889




were in vitro activated with feeder cells and peptide 12 hoursbefore transfer.

Human normal donor peripheral Th17/Tc17 cellsTo generate mesothelin-specific T cells, sorted pan T (CD4þ or

CD8þ) cells were activated with CD3/CD28-coated beads andprogrammed to a Th17 or Tc17 phenotype and then transducedwith a chimeric anti-mesothelin single-chain variable fragment(scFv) fusion protein containing the T-cell receptor z (TCRz)signaling domain and 4-1BB that was generated as describedpreviously (13). CD4 T cells or CD8þ T cells were polarized toTh17 or Tc17 phenotype as follows: 10 ng/mL rhIL1b, 10 ng/mLrhIL6, 20 ng/mL rhIL23, 10 mg/mL anti-hIL4 clone 11B11, andanti-hIFNg clone H22 (eBioscience). Cells were either primedwith RORg agonist or not at 10 mmol/L on day 0 and 2 postbeadactivation. Experiments were conducted with fetal calf serumcontaining endogenous sources of TGFb. Cell cultures weremain-tained with 100 IU/mL of rhIL2 and cells were expanded for up totwo weeks.

Adoptive cell therapyB6mice were given 4.5� 105 B16F10 cells subcutaneously and

tumors were allowed to establish between 7 and 10 days beforeACT. One day before therapy, mice received nonmyeloablative5 Gy total body irradiation. T cells were infused via tail vein. NSGmice were given 5 � 106 M108 suspended in Matrigel subcuta-neously. Tumors were allowed to establish for 35 days prior toadoptive therapy. In all experiments, mice were randomized totreatment groups and tumor burden was monitored in blindedfashion using perpendicular caliper measurements and reportedas tumor area (mm2).

Tissue distribution assaysSpleens from treated mice were harvested and mechanically

disrupted using the tip of a syringe plunger. Cells were filteredthrough a wire mesh, red blood cells lysed with RBC lysis buffer(Biolegend), and then resuspended in cell media for analysis.Tumors were sectioned, then incubated in 1 mg/mL collagenasetype II (Life Technologies) at 37�C for 1 hour. Digested tissuewas filtered, resuspended in cell media, and plated for assay.Before probing with antibodies, FC block (Biolegend) wasapplied to cells at 1 mg/100 mL. TRP-1 donor T cells wereidentified as CD4þVb14þ cells while pmel-1 donor T cells asCD8þ Vb13þ cells.

Flow cytometry and ELISAFlowcytometrywasperformedwith aBDFACSverse 500 instru-

ment. Intracellular staining of cytokines were conducted usingIC fixation and permeabilization system (Thermo Fisher Scien-tific). Staining of transcription factors was conducted usingFOXP3 fixation and permeabilization system (Thermo FisherScientific) per manufacturer's instructions. Antibodies used wereanti-mCD3-efluor450 clone 17A2, IL17A, and IFNg , anti-h/mRORgt-PE clone AFKJS-501 9, anti-h/mCD44-PerCPCy5.5clone IM7, anti-hCD4-APCH7 clone RPA-T4, anti-mCD4-APC/PE clone RM4-5, and anti-mCD62L- APC clone MEL-14(eBioscience). Anti-human CD45, anti-mouse Vb13, anti-mouseTCF7 were from Biolegend, and anti-mouse Vb14 was purchasedfrom BD Biosciences. ELISA for IL17A, IL22, and IFNg wasperformed using DuoSet ELISA kits (R&D Systems) per themanufacturer's instructions.

Statistical analysisKaplan–Meier survival curves were assessed for significance

using a log-rank test between treatment groups. A P value of<0.05 was considered significant. Comparisons between twogroups were analyzed using Student t tests with Welch's cor-rection for parametric distribution or Mann–Whitney signedrank tests for nonparametric distribution. A P value of <0.05was considered significant. For comparisons between multiplegroups, a one-way ANOVA was performed followed by multi-ple comparisons. A P value of <0.05 was considered statisticallysignificant.

ResultsRORg agonist augments the function of CAR human Th17 andTc17 cells

We reported that a series of RORg agonists could augmentthe effector function ofmurine Type 17 T cells in vitro and improvetheir antitumor activity when administered as an oral therapy inmice bearing syngeneic tumors (11). To extend these results andevaluate how RORg agonists would affect human chimeric anti-gen receptor (CAR) T cells, human total T (CD4þ and CD8þ) cellswere enriched from the peripheral blood of healthy individuals,programmed using Type 17 polarizing conditions (IL1b, IL6,IL23), activated with anti-CD3/CD28 beads and 1 day afteractivation, transduced with a lentiviral vector that encodes a CARrecognizing mesothelin (Fig. 1A). The redirected T cells werefurther expanded in the presence of IL2 and IL23. The resultsshowed that 10 days after expansion, a representative agonist(LYC-54143) increased RORgt by approximately 20% comparedwith untreated Type 17 cells with similar numbers of total cells inboth conditions (Fig. 1B). Consistentwith thefindings inmouse Tcells (11), this in vitro agonist treatment induced the cells toproduce 6-fold more IL17A without compromising their abilityto produce IFNg (Fig. 1C and D). Moreover, CAR transductionefficiencywas not compromised in T cells treatedwith this agonist(Supplementary Fig. S1). Previously, we reported that Type 17CAR T cells primed in the presence of an agonist lyse humantumors better than untreated cells (11). We next sought to testif the function of CAR Type 17 cells was heightened whenreactivated against a battery of different tumors expressingmesothelin, including leukemia (K562-meso), mesothelioma(M108), ovarian cancer (Ov79), and pancreatic cancer (Panc1;ref. 21). We found that agonist-treated CAR Type 17 cells secretedmore IL17A when cocultured with mesothelin-positive tumorscompared with untreated CAR T cells (Fig. 1E). As an importantcontrol, IL17A production was nominal when CAR T cells wereincubated withmesothelin-negative tumor lines (such as K562 orK562 lines overexpressing CD19), regardless of if they wereprimed with an agonist. Collectively, our data indicate thatthis RORg agonist augments the functional capacity of humanType 17 cells.

Transfer of human CAR Type 17 cells regresses mesotheliomain vivo when primed in vitro with a RORg agonist

T cells redirected with CAR constructs containing the ICOScytoplasmic tail induce RORg and IL17 expression and regresshuman tumors more effectively than those activated throughCD28 (10). Moreover, activator beads coated with anti-CD3/ICOS augment the function and antitumor activity of humanCAR Th17 cells more effectively than those stimulated with

Hu et al.

Cancer Res; 78(14) July 15, 2018 Cancer Research3890




anti-CD3/CD28 beads (13). Consequently, we posited that asmall molecule that activates RORgt in human CAR Type 17cells stimulated with anti-CD3/CD28 beads would regresstumors in vivo more effectively than untreated cohorts. To testthis idea, we co-infused Th17/Tc17 cells that had been redir-ected with a mesothelin CAR, primed with the RORg agonistLYC-54143 and activated with CD3/CD28 beads into NSGmice bearing a human mesothelioma. As a control, an equalnumber of untreated CAR Th17/Tc17 cells were co-infused intomesothelioma-bearing mice. As in Fig. 2A, RORg agonist–treated cells mediated the best antitumor activity in mice. Whilethe infusion of untreated cells was initially effective, theresponse was less durable than that observed with infused cellstreated in vitro with the RORg agonist. Importantly, these cellsmust be redirected with a CAR to mediate tumor regression, asmock transduced Th17/Tc17 cells (untransduced) were onlyslightly (not significantly) different from untreated animals. We

next asked if extended duration of antitumor activity andregression observed with agonist-primed CAR Th17/Tc17 cellsmight be the result of better persistence of these cells in vivocompared with untreated CAR cells. Indeed, 30 days afterinfusion, more than twice as many agonist-treated cells weredetected in tumor than untreated control cells (Fig. 2B). Col-lectively, our data suggest that in vitro priming of human CARTh17/Tc17 cells with a RORg agonist augments their capacity tosecrete IL17 and conditions the cells for better persistencein vivo, which is associated with long-term regression ofmesothelin-positive tumors.

Th17 cells co-infused with Tc17 cells mediate potent anti-melanoma activity in vivo when primed in vitro with a RORgagonist

To assess if RORg agonist–treated Type 17 (Th17 and Tc17) Tcellsmediate durable antitumor responses inmice and the impact

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Priming human Type 17 T cells with a RORg agonist augments their function in vitro. A, Scheme of in vitro expansion of CAR T cells. Human T cells (both CD4and CD8 T cells) were enriched from the peripheral blood of healthy individuals and programmed to a Type 17 (Th17 and Tc17) phenotype. One dayafter activation, programmed cellswere genetically redirected to express a CAR that recognizesmesothelin. Redirected cellswere expanded for an additional 9 days.B–D, Ten-day expanded cells that were primed in the presence of RORg agonist (Type 17 þ LYC-54143) or vehicle (Type 17) were then tested for theirexpression of transcription factor RORg (B, top, Type 17 cells; bottom, Type 17 cells treated with LYC-54143), IFNg and IL17A (C, top, Type 17 cells; bottom,Type 17 cells treatedwith LYC-54143) by flow cytometry.D, LYC-54143–treated cells secretemore IL17A. IL17A levels secreted by CAR-directed Type 17 T cells duringexpansion were assayed by ELISA (P < 0.05). Data represent mean þ SD from five experiments. E, CAR-directed Type 17 cells primed with an agonist(gray bars) secrete more IL17 upon recognition of various mesothelin-expressing tumors (K652.Meso, M108, Ov79, and Panc1). P < 0.05. As controls, K562and K562 CD19 cells, which lack mesothelin, did not induce IL17A production. Data are representative of four experiments.






of a RORg agonist on the antitumor activity of IL2 expanded Tcells, traditionally used in adoptive cell therapy (ACT) clinicaltrials (22–24), we employed the MHCI-restricted transgenic TCRpmel-1 CD8þ T cell and MHCII-restricted TRP-1 CD4þ T celltumor ACT mouse models that recognize either gp100 or TRP-1antigens, respectively, on B16F10 melanoma. First, we deter-

mined the impact of a RORg agonist on IL2-expanded T cells(Th0) using the TRP-1 CD4 T cell tumor model. In these experi-ments, TRP-1 Th0 cells were generated in vitro from the MHCII-restricted transgenic TCR mice using antigen, irradiated antigenpresenting cells and IL2 without polarizing cytokines in thepresence or absence of RORg agonist LYC-54143. RORg agonisttreatment enhanced IL17A production in Th0 cells in a dose-dependent manner; however, the absolute titer was considerablylower than that observed from cytokine-polarized Th17 cells(Supplementary Fig. S2). The production of IFNg by IL2-expand-ed Th0 cells was not affected by RORg agonist treatment (Fig. 3A).Interestingly,when the in vitro–treated cellswere infused intomicewith established B16F10 tumors, LYC-54143 treatment improvedthe antitumor activity of Th0 cells (Fig. 3B; Supplementary Fig.S3A). Overall, more than half of the mice infused with agonist-treated Th0 cells had an extension of life for approximately 10more days. However, this therapy did not promote long-termcures and most animals relapsed and did not survive for morethan 1 month after ACT.

We next compared the antitumor activity of Th17 with Th0cells using the TRP-1 system. Consistent with a previous report(4), we also found that Th17 cells provided better antitumoractivity over Th0 cells (Fig. 3C vs. Fig. 3B). However, superiorantitumor activity was mediated by Th17 cells treated in vitrowith LYC-54143 with long-term regressions observed (Fig. 3C;Supplementary Fig. S3B) and the majority of animals surviving2 months after ACT (Supplementary Fig. S3C). Interestingly, atthe time of infusion, agonist-treated Th17 cells produced onlyslightly more IL17A and IL22 than untreated Th17 cells likelydue to the robust cytokine cocktail used to polarize the cells invitro (Fig. 3D). Despite the modest effect on cytokine produc-tion in vitro, the addition of the RORg agonist to the Th17cultures imparted an improvement in antitumor activity in vivohighlighting the role of additional, cytokine-independentpathways induced by RORg activation. Similar to TRP-1 Th0and Th17 CD4 T cells, pmel-1 CD8 Tc17 cells also possessedenhanced antitumor properties following in vitro expansionwith RORg agonist LYC-54143 (Fig. 3E; SupplementaryFig. S3D).

The human CAR T experiment utilized pan T cells, to assessthe contributions of both Th17 and Tc17 subsets to mediatedurable antitumor responses we co-infused equal numbers ofTRP-1 Th17 cells and pmel-1 Tc17 cells into mice bearingestablished B16F10 melanoma. The coinfusion of both Th17and Tc17 cells resulted in an effective treatment (Fig. 3F;Supplementary Fig. S3E). These data are consistent with pre-vious publications showing that Th17 cells can augment theactivation of CD8þ T cells and adoptive transfer of CD4þ T cellshelps maintain the function of transferred CD8þ T cells (14,25). Even in this setting, expansion in the presence of RORgagonist LYC-54143 further augmented the antitumor effectwith all animals achieving complete or partial regressions.These data might imply that the most effective antitumorresponses are mediated by combination of Th17 and Tc17 cellsand that the addition of RORg agonists to even highly polarizedeffectors cells further augments their antitumor activity.

IL17 and IFNg, not IL22, production by agonist-treated T cellsmediates antitumor immunity

The plasticity of Th17 cell cytokine production and their abilityto produce IFNg have been reported to be important to their

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Human CAR Th17 and Tc17 cells regress tumors more effectively and persistlonger when primedwith a RORg agonist.A,RORg agonist–treated Type 17 CART cells mediated better tumor control when transferred into tumor-bearingmice. CAR Th17 and Tc17 cells primed or not with RORg agonist LYC-54143were infused into NGS mice bearing 30-day established mesothelioma.N ¼ 10 mice/group. P < 0.05 between CAR-Type 17 and CAR-Type 17 þ LYC-54143. B, More RORg agonist–treated CAR T cells were present in tumors.Frequency of donor cells (human CD45þ) in the tumor 30 days aftertransfer in satellite groups of mice. Black bar, mean value in each group.N ¼ 6/group. NT, no treatment; UT, untransduced T cells; T17,CAR-Type 17 T cells. P < 0.05 between T17 and T17 þ LYC-54143.

Hu et al.





antitumor activity (5). Thus, we next set out to determine theimportance of IL17, IL22, and IFNg to the antitumor efficacyobserved following coinfusion of agonist-primed Th17 and Tc17cells. To address this question, we programmed pmel-1 Tc17or TRP-1 Th17 cells in the presence or absence of the RORgagonist and infused them into lympho-depletedmelanoma-bear-ing mice. As expected, neutralizing IL17A or IFNg in mice infusedwithuntreated cells (Fig. 4AandB)or agonist-treated cells (Fig. 4Cand D) impaired their antitumor activity. The most impairedtreatment outcome in mice cytokine ablated of IL17A or IFNgoccurred in mice treated with the most effective therapy (i.e.,agonist-treated cells; Fig. 4CandD).Conversely, neutralizing IL22did not affect treatment outcome in mice, suggesting that thiscytokine does not alter tumor immunity, at least in the contextof adoptive T-cell transfer therapy. Collectively, our data revealthat IL17 and IFNg but not IL22 production by agonist-treatedco-infused Th17 and Tc17 cells are important for treatmentoutcome.

Agonist-primed Th17 and Tc17 cells persist, co-secrete elevatedIL17 and IFNg, and possess a distinct memory profile

We have previously shown that higher numbers of RORgagonist–treated OT-1 Tc17 T cells can be found in the spleen andtumor of mice compared with untreated Tc17 cells (11). Asagonist-treated Th17 plus Tc17 cells control tumor growth inmice to a greater extent than untreated cells, we sought todetermine if these cells persisted in mice to a greater extent thanuntreated cells. To address this question, we analyzed the fre-quency, function, and memory profile of donor cells in mice thatexperienced long-term antitumor activity in vivo (>71 days afterACT). Aswe found that infusing 5�105untreateddonor cells intomice bearing 10-day established tumors was ineffective and didnot protect mice long term (Fig. 3F). Therefore, for these studies,we infused more donor cells (2 � 106) into mice with smallertumors so that both groups of mice receiving agonist-treated cellsand untreated cells would survive long term for us to test thisquestion in both treatment groups. As shown in Fig. 5A, 71 days

Figure 3.

Tumor-specific Th0 or Type 17 T cellsprimed with RORg agonist mediatepotent and durable antitumoractivity against established melanoma.A andB,Comparedwith vehicle-treatedcells, LYC-54143–primed,IL2-expanded TRP-1 Th0 cells do notproduce more IL17A or IL22 (A) butmore effectively control tumor growthwhen infused into mice with melanoma(B). C and D, TRP-1 Th17 cells primedwith the RORg agonist effectivelyregress tumors compared withuntreated Th17 cells and produce moreIL17 and IL22 (C) but similarIFNg in vitro (D). E, Pmel-1 Tc17 cellsregress melanoma to a greater extentwhen primed with a RORg agonistcompared with untreated cohorts.F, Co-infused agonist-primedTh17 and Tc17 cells mediate robust andlong-lived antitumor activity in micewith melanoma. In B, C, E, andF, mice-bearing 10-day establishedB16F10 melanoma were preconditionedwith a 5 Gy total body irradiation andthen infused with 0.25 � 106 cells. n ¼9–11/group. Tumor diameters weremeasured with a caliper and expressedin area. Data are representative of twoindependent experiments.






after infusion, TRP-1 Th17 cells primed with a RORg agonistpersisted in the tumor at 2-fold higher levels than untreated Th17cells. Likewise, pmel-1 Tc17 cells primedwith RORg were detectedin the tumors at approximately 4-fold greater levels than untreat-ed cohorts (Fig. 5A). On the day of transfer approximately 10%of cells were producing IFNg regardless of LYC-54143 treatmentduring in vitro priming (Fig. 3D); however 71 days after transfer,the percentage of IFNgþ cells is increased (Fig. 5B). Interestingly,in vitro treatment with RORg agonist 71 days prior resultedin maintenance of more IL17þ cells (2.8% vs. 8.3%) and thedevelopment of more IL17þIFNgþ double positive population(Fig. 5B).

Moreover, both Th17 and Tc17 cells, when in vitro primedwith an agonist 71 days prior, secreted more IL17A when reacti-vatedwith their cognate antigens (Fig. 5C). Interestingly,when thememory phenotype of the transferred cells in the tumor wasexamined by flow cytometry, we found that the agonist-primedTh17 and Tc17 cells possessed a wider repertoire of centralmemory (CD44þCD62Lþ) and stem-like memory (CD44-CD62Lþ) 71 days after infusion into tumor-bearing mice(Fig. 6A and B). Conversely, untreated cells mainly consisted ofcentral and effector memory cells (CD44þ CD62L�; Fig. 6A andB). Of note, this finding was more striking with Th17 cells thanTc17 cells (Supplementary Fig. S4). Additional investigationrevealed that agonist-primed Th17 cells in the tumor expressedmore Tcf7 than untreated donor cells (Fig. 6C), suggesting thatagonist therapy supports the generation of T cells with durablestem memory. The memory phenotype of transferred cells is insharp contrast to in vitro–activated cells, which predominantly areCD44þCD62L� effector cells. Collectively, our data reveal that ashort in vitro exposure to RORg agonist bolsters Th17 and Tc17cells, which co-secrete more cytokines, have superior in vivopersistence, and develop a distinct memory phenotype duringresponses to tumors in vivo.

RORg agonist treatment in vitro induces long-term T-cellmemory and drives durable protection after ACT

As agonist-primed cells possessed a stem-like memory phe-notype, we hypothesized that mice receiving this therapy wouldbe protected from tumor rechallenge. To address this question,we co-infused TRP-1 Th17 and pmel-1 Tc17 cells generated invitro in the presence or absence of the RORg agonist LYC-54143to mice bearing B16F10 melanoma tumors as above. Sufficientnumbers of cells were infused to mediate full tumor regressionin both groups (Supplementary Fig. S5A). We then rechal-lenged these mice that had survived long term from cellulartherapy with a second subcutaneous injection of B16F10 mel-anoma 45 days after adoptive transfer. As a control, we gavemelanoma to previously untreated (na€�ve) mice. As shownin Fig. 7A, we found that tumors grew rapidly in na€�ve mice.In contrast, mice previously infused with anti-melanoma Th17and Tc17 cells without agonist treatment were initially pro-tected from tumor rechallenge. However, 2 weeks after rechal-lenge, tumors began to grow in these animals. In contrast, micewere protected for more than 1 month after tumor rechallengeif they had originally received agonist-primed Th17 and Tc17cells (Fig. 7A). Also, 75 days after adoptive transfer of T cells,when we rechallenged these mice a third time with B16F10tumor cells, they remained protected from melanoma forapproximately 20 days with 2 of 4 mice remaining tumorfree. Conversely, if these mice were rechallenged with EL4tumors, the malignancy grew (Fig. 7B), showing that thememory response was antigen specific. Consistent with theantitumor effect in each animal, blood levels of donor cellsinversely correlated with tumor growth (SupplementaryFig. S5B). Thus, collectively RORg agonists can dramaticallypotentiate the stem-like memory phenotype of Type 17 T cellsand provide superior, long-term protection against tumorchallenges in vivo.

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IL17 and IFNg but not IL22 are important inmediating durable tumor immunity in vivofollowing transfer of Th17 and Tc17 cells.Tumor growth inhibition (A and C) andsurvival (B and D) benefits of celltherapy were compromised when IL17A- orIFNg-neutralized mice bearing B16F10tumors were preconditioned with 5 GyTBI and then infused with untreated TRP-1Th17 plus pmel-1 Tc17 cells (A and B) oragonist-treated cells (C and D). In bothgroups, IL17A, IL22, or IFNg wereneutralized with an antibody 5 times everyother day, starting from the day of infusion.P < 0.05 for anti-IL17A and anti-IFNgversus IgG.

Hu et al.





DiscussionHerein, we report that a novel RORg agonist can potentiate the

function, persistence, and antitumor activity of both human CART cells and murine Th0, Th17, and Tc17 cells in two distinct andclinically relevant mouse tumor models.

We found that the antitumor activity was improved in miceco-infused with agonist-treated T cells. Interestingly, RORgagonist treatment not only supported survival of T cells butalso helped them co-secrete IL17A, IL22, and IFNg . It is impor-tant to appreciate that while Type 17 cells mediated the mostpotent antitumor properties in vivo, the IL2-expanded, unpro-grammed Type 0 cells (normally used in the clinic) were alsoaugmented by RORg agonist treatment. These data suggest thatthe addition of a small-molecule RORg agonist can be rapidlytranslated into ACT expansion protocols currently used in theclinic.

In mice surviving from agonist-Type 17 therapy for morethan 2 months, we found that donor cells were composed ofdiverse populations ofmemory cells (including stem, central, andeffector). While mice infused with untreated cells were mainlyeffectors with some central memory lymphocytes. Remarkably,agonist-primed cells within tumors expressed heightened Tcf7, atranscription factor downstream of the canonical Wnt pathwayessential for stemness (20) and directly associated with lympho-cytes with durable immunity to cancer (19). Importantly, thisaltered memory phenotype of cells treated in vitro with a RORgagonist several months prior were functionally superior tountreated cells and provided protection from repeated tumorchallenges. Collectively, our work reveals that antitumor Type17 cells provide durable memory responses in vivo when treated

in vitro with a RORg agonist that bolsters, at least in part, theWnt/TCF7 pathway.

Type 17 T cells mostly provide antitumor immunity whentransferred into mice with established tumors as shown in thiswork and many other reports (4, 10, 14, 21); however, both pro-and antitumor activities of IL17A have been reported, with mostprotumor activities observed in inflammation-induced tumori-genesis in the gut (7, 26). In our ACT model, blocking IL17A atleast partially reduced the antitumor activity of transferred Type17 T cells, suggesting an antitumor role of IL17A (Fig. 4). Inter-estingly, a recent report indicates that treatment of anti-PD1–induced autoimmune toxicity by anti-IL17A secukinumabresulted in loss of antitumor activity in a patient with metastaticcolon cancer (8). Similarly, in patients with melanoma receivingPD-1 therapy, the frequency of IL17-producing T cells wasincreased in responders versus nonresponders (27). Togetherthese results suggest that RORg and Type 17 cells play importantroles in cancer immunotherapy and may mediate the antitumoractivity of checkpoint inhibitors, such as anti-PD1.

There is a significant need to generate durablememory in T cellsin the field of cancer immunotherapy. Persistence of transferredlymphocytes correlates with cancer regression in patients receiv-ing adoptive cell therapy (28, 29). Many investigators are enrich-ing CD62Lþ T cells from bulk TILs or CAR T-cell cultures, as thesecentral memory T cells are more efficacious than the effectormemory CD62L� T cells normally expanded with high dose IL2under rapid expansion protocols. Other researchers are addingdistinct cytokines (such as IL15; ref. 30) or pharmacologicreagents (such as Akt inhibitors; ref 31) to cultures to generatecentral or stem memory T cells.

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Agonist-treated cells persist long-termand are multifunctional in vivo.A,More agonist-treated donor Type 17 Tcells were recovered 71 daysafter transfer. The frequency of TRP-1Th17, and pmel-1 Tc17 donor cells intumors was assayed (n¼ 4; P < 0.05). B,These cells produced more IL17 andcoproduced more IL17A and IFNg whenreactivated ex vivo with PMA andionomycin. C, When reactivated withTRP-1 peptide (left; P < 0.05) or hgp100peptide (right; P <0.01), donor cells fromspleen also produced more IL17A. Dataare representative of two independentexperiments.






To our knowledge, our work is the first demonstration thatsmall molecules stimulating the activity of one transcriptionfactor—RORgt—can profoundly affect the persistence and func-tion of T cells. We also have shown that LYC-54143, as a repre-sentative RORg agonist, affects multiple pathways through ele-vating cytokines, enhancing survival of T cells and expression ofcostimulatory receptors, and blocking Treg generation anddecreasing multiple coinhibitory receptors (11). All of theseactivities could also contribute to the improved antitumorimmune responses observed here. Indeed, when TRP-1 andpmel-1 Type 17 T cells in the tumor were analyzed 71 days aftertransfer, reduced percentage of Treg cells and decreased PD-1expression were found in agonist-primed cells (Supplementary

Fig. S6). It is remarkable that a short in vitro exposure of tumor-specific T cells to a small-molecule RORg agonist not onlyenhances the in vivo survival of cells, but also results in persistenteffects on cytokine production and the generation of long-livedmemory cells following adoptive transfer. These results suggestthat RORg agonist treatment likely induces long-lasting epigeneticchanges in T cells. Future studies will assess possible epigeneticeffects of RORg agonists.

Our findings have immediate translational implications asadditional investigation revealed that small-molecule RORg

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RORg agonist induces stemmemory Tcf7þType 17 cells in vivo. Donor TRP-1 Th17and pmel-1 Tc17 cells primed with a RORg agonist possess stem, central,and effector memory cells in the tumor, while those treated with vehicle aremainly effector memory cells, as represented by flow cytometry plot (A) orby a pie chart (n ¼ 3; B). Seventy-one days after transfer, cells were analyzedafter antigen rechallenge with corresponding peptides. Donor cells in thetumor of mice that received LYC-54143–treated T cells expressed significantlymore Tcf7 than those that received untreated cells (C; P < 0.05).

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Type 17 cells primed with a RORg agonist possess memory and protect micerechallenged with B16F10 melanoma tumor. A total of 2 � 106 TRP-1 Th17 andpmel-1 Tc17 cells (1:1) were transferred into B16F10-bearing mice to eradicateprimary tumors (Supplementary Fig. S5A). Forty-five days after T-cell infusion,mice were rechallenged with B16F10 tumor cells. Mice originally infused withLYC-54143–treated donor TRP-1 Th17 and pmel-1 Tc17 cells were protected fromsecond tumor challenge (A; n ¼ 6/group). Data are representative of twoexperiments. B, Thirty days after second challenge, third tumor challenge wasgiven andmice that originally received LYC-54143–treated cells were protectedfrom B16F10 (n ¼ 4) but not EL4 tumors (n ¼ 2). Numbers on the graph areanimal numbers and correspond to those in Supplementary Fig. S5B.

Hu et al.





agonist can also enhance the antitumor activity of IL2-expanded,nonpolarized T cells in vivo. This finding is particularly exciting, asthese cells are commonly used in clinical trials for adoptive T-celltransfer therapy. Nonpolarized T cells do express RORgt albeit atlower levels than T cells polarized under Type 17 conditions. Inhumans, RORgt is expressed in some circulating T cells and in TILs(13). Thus, it is conceivable that a RORg agonist could be easilyadded to nearly any tumor-specific T-cell culture such as TILexpansions or bulk lymphocytes redirectedwith antigen receptors(TCRs or CARs) to endow long-lived memory responses totumors. In addition to the potential utility of RORg agonists inin vitro priming of T-cell therapies, clinical studies of an oral RORgagonist LYC-55716 in patients with cancer are currently ongoing(NCT02929862). Collectively, our findings have importantimplications in the field of cancer immunotherapy, includingadoptive T-cell transfer therapy as well as other T-cell–basedtherapies such as vaccines, cytokines, and immune checkpointinhibitors.

Disclosure of Potential Conflicts of InterestX. Hu has ownership interest (including patents) in Lycera Corp. Y. Liu has

ownership interest (including patents) in and is a consultant/advisory boardmember for Lycera Corp. L.L. Carter has ownership interest (including patents)in Lycera Corp. C.M. Paulos reports receiving commercial research grant fromLyceea. No potential conflicts of interest were disclosed by the other authors.

Authors' ContributionsConception and design: X. Hu, X. Liu, C. Spooner, J. Moisan, W. Zou,L.L. Carter, C.M. PaulosDevelopment of methodology: K. Majchrzak, L.L. Carter, C.M. PaulosAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): K. Majchrzak, X. Liu, M.M. WyattAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): X. Hu, X. Liu, M.M. Wyatt, J. Moisan, L.L. Carter,C.M. PaulosWriting, review, and/or revision of the manuscript: X. Hu, X. Liu, C. Spooner,J. Moisan, L.L. Carter, C.M. PaulosAdministrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): X. Hu, C.M. PaulosStudy supervision: X. Hu, L.L. Carter, C.M. Paulos

AcknowledgmentsWe thank all chemists at Lycera Corp. for making RORg agonist compounds.

This work was supported by funding from Lycera Corp.

The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indicatethis fact.

ReceivedDecember 22, 2017; revisedMarch 16, 2018; acceptedMay10, 2018;published first May 16, 2018.

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Hu et al.




2018;78:3888-3898. Published OnlineFirst May 16, 2018.Cancer Res Xiao Hu, Kinga Majchrzak, Xikui Liu, et al.

In VivoAgonist Confers Durable Memory and Stemness γ Priming of Adoptively Transferred T Cells with a RORIn Vitro

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