Cell Responses to Self and Tumor Tissue the Inducible Costimulator

The Inducible Costimulator Augments Tc17 Cell Responses to Self and Tumor Tissue Michelle H. Nelson, Sreenath Kundimi, Jacob S. Bowers, Carolyn E. Rogers, Logan W. Huff, Kristina M. Schwartz, This information is current as Krishnamurthy Thyagarajan, Elizabeth C. Little, Shikhar of September 27, 2021. Mehrotra, David J. Cole, Mark P. Rubinstein and Chrystal M. Paulos J Immunol published online 9 January 2015

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 9, 2015, doi:10.4049/jimmunol.1401082 The Journal of Immunology

The Inducible Costimulator Augments Tc17 Cell Responses to Self and Tumor Tissue

Michelle H. Nelson,* Sreenath Kundimi,* Jacob S. Bowers,* Carolyn E. Rogers,* Logan W. Huff,* Kristina M. Schwartz,* Krishnamurthy Thyagarajan,† Elizabeth C. Little,† Shikhar Mehrotra,† David J. Cole,† Mark P. Rubinstein,† and Chrystal M. Paulos*,†

The inducible costimulator (ICOS) plays a key role in the development of Th17 cells, but its role in the development and antitumor activity of IL-17–producing CD8+ T cells (Tc17) remains unknown. We found that ICOS costimulation was important for the functional maintenance, but not differentiation, of Tc17 cells in vitro. Blocking the ICOS pathway using an antagonist mAb or by using recipient mice genetically deﬁcient in the ICOS ligand reduced the antitumor activity of adoptively transferred Tc17 cells. Conversely, activating Tc17 cells with an ICOS agonist in vitro enhanced their capacity to eradicate melanoma and induce autoimmune vitiligo when infused into mice. However, ICOS stimulation did not augment the antitumor activity of IL-2 expanded Downloaded from T cells. Additional investigation revealed that ICOS stimulation not only increased IL-2Ra, CXCR3, and IL-23R expression on Tc17 cells, but also dampened their expression of suppressive molecule CD39. Although Tc17 cells activated with an ICOS agonist cosecreted heightened IL-17A, IL-9, and IFN-g, their therapeutic effectiveness was critically dependent on IFN-g production. Depletion of IL-17A and IL-9 had little impact on antitumor Tc17 cells activated with an ICOS agonist. Collectively, our work reveals that the ICOS pathway potentiates the antitumor activity of adoptively transferred Tc17 cells. This work has major implications for the design of vaccine, Ab and cell-based therapies for autoimmunity, infectious disease, and cancer. The Journal http://www.jimmunol.org/ of Immunology, 2015, 194: 000–000.

nterleukin-17–producing CD8+ T cells (Tc17) have been by CD8+ T cells has recently been reported to support Th17 cell– identiﬁed in both mice and humans (1–3). Compared with mediated autoimmune encephalomyelitis (12), suggesting that I classical CTLs, Tc17 cells mediate a less cytotoxic effector Th17 and Tc17 cells cooperate to amplify immune responses to function toward antigenic targets, because of their diminished self tissue. Although Tc17 cells regulate immune responses to self, capacity to secrete IFN-g and granzyme B in vitro (4). Yet in vivo, foreign, and tumor tissue, the cues that control their noncytotoxic

Tc17 cells play a role in exacerbating autoimmune diseases, such and pathogenic states remain unclear. by guest on September 27, 2021 as multiple sclerosis, diabetes, colitis, and psoriasis (5–10). Tc17 Unlike Th17 cells, less is known about the role of Tc17 cells in cells also protect the host against lethal influenza challenge (2) disease pathogenesis. However, the cytokines that control IL-17– and mediate tumor regression (11). Interestingly, IL-17A secretion producing CD4+ and CD8+ T cell differentiation and maintenance appear to be similar. TGF-b1 and IL-6 differentiate naive CD8+ T cells into Tc17 cells, as evidenced by their induction of ROR-gt *Department of Microbiology and Immunology, Hollings Cancer Center, Medical and IL-17A, whereas IL-23 maintains their function and pheno- University of South Carolina, Charleston, SC 29425; and †Department of Surgery, Hollings Cancer Center, Medical University of South Carolina, Charleston, SC 29425 type in long-term cultures (13–15). Although the cytokines that Received for publication April 28, 2014. Accepted for publication December 3, 2014. regulate Tc17 cell development are known, the specific costimu- This work was supported by start-up funds at Medical University of South Carolina latory pathways that impact their differentiation, expansion, and and National Cancer Institute Grant R01 CA175061 (to C.M.P.), American Cancer maintenance remain to be fully elucidated. Society Institutional Research Grants 016623-004 and P01 CA15477804 (to C.M.P.), Ag-specific and costimulatory signals from APCs are needed South Carolina Translational Research KL2 Scholar Program (Grant UL1 TR000062 to C.M.P.), the Flow Cytometry and Cell Sorting Shared Resource for the Hollings for the activation and function of T cells (16). The costimula- Cancer Center (Grant P30 CA138313), a Jeane B. Kempner Postdoctoral Fellowship tory molecule CD28 is constitutively expressed on lymphocytes, (to M.H.N.), an American Cancer Society Postdoctoral Fellowship (Grant 122704- PF-13-084-01-LIB to M.H.N.), and the Melanoma Research Alliance (to M.P.R.). whereas the costimulatory protein ICOS (also known as CD278) is inducibly expressed on activated T cells and on a small cohort of M.H.N. designed and performed the experiments, analyzed the data, created the figures, and wrote the manuscript; S.K., J.S.B., and C.E.R. designed and performed resting regulatory T cells (Tregs) and Th17 cells (17–19). In ad- the experiments; L.W.H., K.M.S., K.T., and E.C.L. performed the experiments; S.M., dition to CD28, signaling via ICOS is required for optimal cyto- M.P.R., and D.J.C. designed experiments and edited the manuscript; and C.M.P. directed the project, designed experiments, and wrote the manuscript. kine secretion, because both costimulatory molecules are essential Address correspondence and reprint requests to Dr. Michelle H. Nelson and Chrystal for optimal IL-17A secretion by murine Th17 cells (20). In con- M. Paulos, Hollings Cancer Center, MSC 509, Medical University of South Carolina, trast with murine Th17 cells, however, ICOS augments the ex- 86 Jonathan Lucas Street, Charleston, SC 29425. E-mail addresses: nelsonmh@musc. pansion and function of human Th17 cells, whereas CD28 edu (M.H.N.) and [email protected] (C.M.P.) abrogates their function (18, 21). In a murine model, it has been The online version of this article contains supplemental material. reported that ICOS costimulation is not required for the differ- 2/2 Abbreviations used in this article: B6, C57BL/6; dLN, draining lymph node; ICOS , + ICOS-deficient; ICOSL, ICOS ligand; ICOSL2/2, ICOSL-deficient; IL-2-P, IL-2– entiation of naive CD4 T cells toward a Th17 phenotype. Rather, polarized CD8+ T cell; MUSC, Medical University of South Carolina; rh, recombi- ICOS costimulation is required for IL-23–driven expansion of + nant human; TBI, total body irradiation; Tc17, IL-17–producing CD8 T cell; Treg, already differentiated Th17 cells (19). Specifically, ICOS ampli- regulatory T cell; WT, wild-type. fies Th17 responses by inducing transcription factor c-MAF, Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 transactivating IL-21 production, and upregulating IL-23R

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1401082 2 ICOS AUGMENTS Tc17 CELL RESPONSES TO SELF AND TUMOR TISSUE expression (19). Although ICOS is important for regulating Th17 nm using a Multiskan FC plate reader (ThermoScientific) and the tested generation, the role of ICOS costimulation in controlling Tc17 dif- cytokines were quantified. ferentiation, expansion, and function remains to be identified. Flow cytometry. Data were acquired on a BD FACSCalibur or FACSVerse We report that ICOS is important for supporting the mainte- (BD Biosciences) and analyzed using FlowJo software (Tree Star, Ashland, OR). Abs specific for ICOS (CD278), IL-2Ra (CD25), IL-23R, CD44, nance, but not the differentiation, of memory Tc17 cells. In vivo, CD62L, ROR-gt, T-bet, Bcl-1, CD28, PD-1, CTLA-4, CXCR3, and CCR6 blockade of the ICOS–ICOS ligand (ICOSL) pathway impaired were purchased from Biolegend, and Abs specific for IFN-g, IL-17A, IL- their capacity to eradicate melanoma and induce autoimmune 10, CD26, CD39, CD73, Vb13, and IL-7Ra (CD127) were purchased vitiligo in mice. Conversely, activating Tc17 cells in vitro with an from BD Pharmingen. ICOS agonist augmented their capacity to mount immunity to self/ In vivo CTL assay tumor tissue in vivo in an IFN-g–dependent manner. ICOS stim- Tc17 cells from pmel-1 mice were sorted based on ICOS expression using ulation not only increased IL-2Ra, IL-7Ra, and IL-23R expres- a Dako MoFlo cell sorter (Beckman Coulter). A total of 1 3 106 in vitro– sion on Tc17 cell, but also heightened their in vivo cytotoxicity vaccinated cells (22) were transferred into irradiated (5 Gy; 4 h before cell and dampened their expression of suppressive/coinhibitory mol- transfer) B6 mice that were given 3 3 105 B16F10 tumor s.c. 5 d before ecule CD39. Collectively, these data reveal that ICOS augments transfer. All animals were given bolus IL-2 (i.p. daily at 3.6 g/dose, every 3 7 Tc17 responses to self and tumor tissue. 24 h, for a total of 4 doses). Ten d after adoptive T cell transfer, 2 10 target cells were i.v. transferred to assess cytolytic function. To generate the target cells, we split B6 splenocytes into two fractions; one fraction Materials and Methods was pulsed with 1 mM OVA peptide (SIINFEKL) for 45 min at 37˚C and then labeled with a low amount (0.25 mM) of CFSE (Molecular Probes), Mice and tumor lines

for use as an internal control, and the second fraction was incubated with Downloaded from To study the role of ICOS in tumor therapy with Tc17 cells, we used the 1 mM hgp10025–33 (KVPRNQDWL) and then labeled with a high level pmel-1 model of adoptive immunotherapy against the poorly immunogenic (2.5 mM) of CFSE. An equal number of cells from both fractions were B16F10 melanoma. pmel-1, C57BL/6 (B6), ICOS-deficient (ICOS2/2), and mixed and then delivered simultaneously. Mice were euthanized after 12 h ICOSL-deficient (ICOSL2/2) mice (Jackson Laboratory) were housed and of incubation, and the spleens were harvested and single-cell suspensions bred in the Medical University of South Carolina (MUSC) vivarium. In- made. Lymphocytes were isolated using Lymphocyte Separation Media stitutional Animal Care and Use Committee at the MUSC approved the (Mediatech), fixed, and analyzed by flow cytometric analysis. The per- 2 animal work. B16F10 tumors were obtained from the laboratory of centage of specific lysis was calculated as (1 ratio of untreated recipients / + 3 http://www.jimmunol.org/ Dr. Nicholas Restifo. ratio of activated CD8 T cell recipient) 100, where the ratio is % CFSElow/%CFSEhigh. T cell generation Statistical analysis methods Transgenic pmel-1 TCR or B6 or ICOS2/2 CD8+ splenocytes were cultured in IL-2–expanding conditions or in IL-17–polarizing conditions, as de- Comparison of average levels across groups or conditions was done using either a two-sample t test or an ANOVA model. Several of the conditions in scribed elsewhere (11), using 1 mM hgp10025–33 (KVPRNQDWL). In brief, pmel-1 cells were expanded with recombinant human (rh)IL-2 the tumor growth experiments resulted in prolonged periods of no tumor (100 IU/ml; National Institutes of Health). Tc17 cells were polarized us- growth or reduced tumor area from day zero, and therefore were analyzed ing rhIL-6 (5 ng/ml; National Cancer Institute preclinical repository), in two parts. First, the minimum tumor size over the course of the ex- rhTGF-b1 (10 ng/ml; BD Pharmingen) plus anti-mouse IFN-g and anti- periment for each mouse was noted, and the day of the first increase in mouse IL-4 (10 mg/ml; BD Pharmingen). rhIL-2 (50 IU/ml; National tumor size, post minimum, was determined for each mouse. Kaplan–Meier by guest on September 27, 2021 Institutes of Health) was added on the second day of culture. Cells were methods were then used to analyze the results of time to first tumor growth cultured for 6 d unless otherwise indicated. For secondary stimulation, the post minimum, and log rank tests were used to compare results across cells were restimulated with irradiated splenocytes coated with CD3 ag- groups. The second part of the analysis quantified the tumor growth rates onist and IL-23 (20 ng/ml; R&D Systems) for an additional 5 d. B6 or once tumor growth has started (postminimum area) and analyzed on the ICOS2/2 CD8+ T cells were cocultured with irradiated splenocytes and log scale in a mixed-effects linear regression model. Comparing their re- anti-CD3 (1 mg/ml; clone 145-2C11; Biolegend), with or without Th17 spective slopes, which were generated by various linear combinations of polarization. In some experiments, cells were treated with a soluble ICOS the model coefficients, we compared tumor growth rates across the groups. , agonist Ab (20 mg/ml; clone C398.4A; Biolegend), ICOSL blocker (20 mg/ml; For all the hypothesis tests, p 0.05 is considered statistically significant. clone HK5.3; Biolegend), or a control Ab on days 2, 4, and 6 of culture. Adoptive cell transfer and vitiligo score Results ICOS supports the maintenance, but not differentiation, of Adoptive transfer experiments have been described previously (22). In brief, recipient B6 mice were given 3 3 105 B16F10 melanoma tumor cells Tc17 cells s.c. on day 0. The mice were then irradiated with 5 or 6 Gy total body ICOS is critical for the generation of follicular helper, Th17, and irradiation (TBI), as indicated in the figure legends, 6 h before CD8+ T cell 3 6–7 + Tregs (17–19). Moreover, there is a reduced population of effector transfer. Mice received i.v. 1 10 pmel-1 CD8 T cells that were + + 2/2 in vitro vaccinated, in conjunction with bolus rhIL-2 (was administered to memory CD4 and CD8 T cells in ICOS mice and humans, mice i.p. once or twice daily at 3.6 mg/dose for a total of 4–6 doses). because ICOS signaling is important for sustaining their survival Vaccination involved coculturing the CD8+ T cells with irradiated B6 (23, 24). However, it remains unknown how ICOS regulates the splenocytes and 1 mM hgp10025–33 peptide for 6 h as in Ref. (22). In some differentiation of naive CD8+ T cells into IL-17–producing Tc17 experiments, mice were given 0.1 mg neutralizing Ab i.p. every other day for a total of five treatments. Anti–IL-9 (9C1), anti–IL-17 (17F3), and anti– cells and whether ICOS regulates their long-term function and IFN-g (XMG1.2) were purchased from BioXCell. Experiments were per- memory phenotype. We hypothesized that ICOS was important for formed in a blinded, randomized fashion, and tumor measurements were the differentiation of naive CD8+ T cells toward a Tc17 phenotype taken over time. Vitiligo on treated mice was scored on a scale of 0 to 5: 0, and subsequently for sustaining their function and effector mem- no vitiligo (wild-type [WT]); 1, depigmentation detected; 2, .10% de- . . ory profile after secondary expansion with IL-23. First, to deter- pigmentation; 3, 25% depigmentation; 4, 50% depigmentation; 5, + .75% depigmentation. Two different investigators who were unaware of mine the role of ICOS in these processes, we sorted naive CD8 hi lo 2/2 the treatment groups 5 wk after adoptive cell transfer scored mice. In one CD62L CD44 T cells from WT and ICOS C57BL6 mice set of experiments, ciprofloxacin (50 mg/ml for 1–2 wk; Bayer) was added with a flow sorter, programmed them to a Tc17 phenotype (with to the drinking water 2 d before irradiation. TGF-b, IL-6, anti–IFN-g, and anti–IL-4), and then activated them Secreted cytokine quantification with irradiated APCs cultured with a CD3 agonist (scheme; Fig. 1). In contrast with our hypothesis, after 5 d of in vitro ex- ELISA. ELISAs were performed according to manufacturer’s protocol 2/2 (DuoSet ELISA; R&D Systems or Biolegend) on supernatants from day 4 pansion, we found that WT and ICOS Tc17 cells expressed cultures. The absorbance values of the supernatants were obtained at 450 equally high levels of ROR-gt (the master transcription factor for The Journal of Immunology 3 Downloaded from

FIGURE 1. ICOS does not regulate Tc17 differentiation. As shown in the schematic, naive CD8+CD62L+CD44lo T cells were sorted with the MoFlo instrument from either WT or ICOS2/2 mice. These cells were then activated with irradiated C57BL6 splenocytes coated with a CD3 agonist (1 mg/ml, clone 145-2C11 mAb; Biolegend) and programmed toward a Tc17 phenotype with IL-6, TGF-b, anti–IL-4, and anti–IFN-g as described in Materials and http://www.jimmunol.org/ Methods. IL-17A and IFN-g production by these cells were assessed 5 d after primary expansion. Representative ﬂow plots (A) and mean (B) are shown (mean 6 SEM, n = 3). (C and D) The central and effector memory phenotype of Tc17 cells from WT and ICOS2/2 mice were determined 5 d after their expansion via their CD44 and CD62L expression by ﬂow cytometry. Tc17 cells from ICOS2/2 mice contain a higher frequency of lymphocytes with an effector memory-like phenotype (mean 6 SEM, n = 3). (E) After 5 d of expansion, the expression of IL-23R expression was assayed on Tc17 cells from WT and ICOS2/2 mice (n = 4). Student t test was performed on the combination of three to four experiments. *p , 0.05.

Th17 and Tc17 cells [25, 26]; data not shown) and secreted simi- cells. Similar to ICOS2/2 Th17 cells (Supplemental Fig. 1C), we larly high amounts of IL-17A, but very little IFN-g (Fig. 1A, 1B). consistently found that ICOS2/2 Tc17 cells expressed less IL-23R Our findings with Tc17 cells are in alignment with work by Bauquet on their cell surface than WT counterparts (mean fluorescent in- by guest on September 27, 2021 and coinvestigators (19) with Th17 cells, who found that naive tensity of IL-23R+ cells that are either WT (2747 6 143) or CD4+ T cells from ICOS2/2 mice expressed comparable ROR-gt ICOS2/2 Tc17 cells (820 6 84; p , 0.05; Fig. 1E). Collectively, (data not shown) and IL-17A (Supplemental Fig. 1A) as WT CD4+ these data reveal that ICOS signaling is important for inducing T cells when initially differentiated to a Th17 phenotype. IL-23R on Tc17 cells. Next, we assessed the role of ICOS in regulating the memory- We therefore posited that after IL-23 restimulation, ICOS2/2 like profile of Tc17 cells in vitro, which may affect the cells’ in vivo cells would display a weakened capacity to secrete IL-17A be- persistence and effector function. The central memory phenotype cause of a reduced ability for IL-23 to bind and signal through the of Tc17 cells was identified as CD44hiCD62Lhi T cells, whereas IL-23R. To address this idea, we reactivated 5-d ICOS2/2 and WT their effector memory phenotype was discerned by CD44hi differentiated Tc17 cells with IL-23 and anti-CD3–coated APCs, CD62Llo expression. We detected a significantly lower frequency and analyzed their capacity to secrete IL-17A and/or IFN-g on day of effector memory cells in ICOS2/2 Tc17 (45%) compared with 10 (scheme; 2-degree expansion; Fig. 2). As a control, the role of WT Tc17 (63%) cells (Fig. 1C, 1D). There was also a slightly (but ICOS on Th17 function was also tested. By flow cytometry, we not significantly) higher frequency of naive (23 versus 14%) and found that ICOS2/2 Tc17 cells cosecreted less IL-17A and IFN-g central memory cells (28 vs 20%) from ICOS2/2 versus WT Tc17 than their WT counterparts (Fig. 2A, 2B). Moreover, compared cells. These phenotypic data are displayed representatively in dot with type 17 CD8+ T cells, ICOS2/2 CD4+ Th17 cells secreted plots (Fig. 1C) and as an average of three separate experiments in even less IL-17A than WT cells (Supplemental Fig. 1D, 1E), as Fig. 1D. Similar to Tc17 cells, a reduced pool of effector memory reported by Bauquet and coworkers (19). We next quantitated the cells was detected from Th17 cells deficient in the ICOS receptor amount of IL-17A and IFN-g produced by WT versus ICOS2/2 compared with WT counterparts (Supplemental Fig. 1B). These Tc17 cells via ELISA analysis. Although WT and ICOS2/2 Tc17 in vitro data suggest that ICOS costimulation is important for cells secreted comparable amounts of IL-17 and IFN-g during supporting the generation of effector memory Tc17 cells. their primary expansion (Fig. 2C), ICOS2/2 Tc17 cells secreted

+ + less IL-17A and IFN-g than WT Tc17 cells when reactivated with ICOS augments the generation of IL-17 IFN-g Tc17 cells IL-23 during their secondary expansion (Fig. 2D). ICOS2/2 Tc17 upon IL-23 restimulation cells also expressed slightly reduced (not signiﬁcant) levels of the ICOS induces IL-23R expression on differentiated Th17 cells (18, ROR-gt, CD107A, granzyme B, and antiapoptotic Bcl-2 molecule 19) and is important for supporting their long-term function (19). compared with WT Tc17 cells (Fig. 2E). Further, ICOS2/2 Tc17 Therefore, we hypothesized that ICOS also induces IL-23R on cells expressed slightly higher T-bet levels (a transcription factor Tc17 cells and that the IL-23/IL-23R pathway is important in for Tc1 cells) (27) and secreted signiﬁcantly less IL-10 than WT fostering Tc17 function. To test this idea, we assessed IL-23R Tc17 cells, which we expected, because ICOS promotes IL-10 expression on differentiated WT and ICOS2/2 Tc17 or Th17 secretion by T cells (28). Collectively, our data show that ICOS, 4 ICOS AUGMENTS Tc17 CELL RESPONSES TO SELF AND TUMOR TISSUE

FIGURE 2. ICOS regulates the effector function of Tc17 cells. WT or ICOS2/2 Tc17 were restimulated with irradiated splenocytes coated with CD3 agonist and IL-23 for an additional 5 d. The ability of already differentiated Tc17 cells to produce IL-17A and IFN-g after restimulation with IL-23 was detected by (A) ﬂow cytometry, and (B) the percentage of cells producing IL-17A and/or IFN-g was graphi- cally represented (mean 6 SEM, n = 3). The cells ability to secrete IL-17A and IFN-g after (C) primary and (D) secondary stimulation was also analyzed by ELISA (mean 6 SEM, n = 6). (E) ROR-gt, T-bet, IL-10, granzyme B, CD107A, and Bcl-2 expression were analyzed on restimulated Tc17 cells from WT and Downloaded from ICOS2/2 mice via ﬂow cytometry (mean 6 SEM, n = 6). Student t test was performed on the combination of three to six repeat experiments. *p , 0.05. http://www.jimmunol.org/

although not essential for Tc17 differentiation, is important for CD39 is elevated, whereas CXCR3 is reduced, on ICOS2/2 maintaining their polyfunctionality, because ICOS2/2 Tc17 cells Tc17 cells had a defect in their ability to cosecrete IL-17A and IFN-g after Next, we sought to determine whether there existed a difference in restimulation with IL-23. 2/2 the expression of costimulatory and coinhibitory molecules on We found that ICOS Tc17 cells not only express lower ICOS2/2 Tc17 cells, because these molecules are critical for by guest on September 27, 2021 IL-23R, but fewer receptors for homeostatic cytokines IL-2 regulating T cell activation and expansion (30–32). We found that (CD25) and IL-7 (CD127) than WT Tc17 cells (Supplemental 2/2 2/2 ICOS Tc17 cells expressed comparable amounts of CD28, Fig. 2A). Thus, we judged that ICOS Tc17 cells would have CD27, CD26, PD-1, and CTLA-4 on their cell surface as WT cells a reduced responsiveness to IL-2 and IL-7, and in turn a re- (Fig. 3B). These data may explain, at least in part, how Tc17 cells duced capacity to secrete IL-17A and/or IFN-g.Totestthis 2/2 that lack ICOS are still able to expand as effectively as their WT idea, we restimulated 5-d ICOS or WT differentiated Tc17 counterparts (Fig. 3A). cells with either IL-2 or IL-7 and measured their cytokine We then sought to investigate whether ICOS regulates the production compared with those stimulated without cytokines expression of chemokine receptors on CCR6 and CXCR3, as well or with IL-23 on day 10 (scheme in Supplemental Fig. 2B). as immunosuppressive ectoenzymes CD39 and CD73 on Tc17 Similar to our findings with IL-23, Tc17 cells devoid of ICOS cells. Although ICOS signaling did not alter CCR6 on Tc17 cells, had a slightly reduced capacity to cosecrete IL-17A and IFN-g we observed an ∼20% reduction of CXCR3 on ICOS2/2 Tc17 when reactivated with either IL-2 or IL-7 (Supplemental Fig. 2/2 cells compared with WT cells (Fig. 3C, upper panel). Moreover, 2B). In addition, ICOS Tc17 cells stimulated in the absence CD39, but not CD73, was expressed at a higher level on ICOS2/2 of cytokine cosecreted comparable yet nominal IL-17A and Tc17 cells (Fig. 3C, lower panel). Our data reveal that ICOS IFN-g as WT cells. These data suggest that ICOS augments the + + + signaling partially regulates chemokine receptors and ectoenzyme generation of IL-17A IFN-g CD8 T cells, perhaps by induc- molecules on Tc17 cells, which may impact their migration to ing the expression of cytokine receptors. inflamed tissue and immune tolerance mediated by CD39/CD73- ICOS differentially regulates the expansion capacity of Th17 induced adenosine (33, 34). But it remains unclear whether ICOS versus Tc17 cells plays a role in Tc17 cell–mediated immunity to self and tumor tissue. How ICOS regulates Th17 versus Tc17 cell expansion is unknown. Given that unprogrammed CD8+ T cells deficient in Ab blockade of ICOS impairs the antitumor activity of infused ICOS expand to a greater extent than WT counterparts in a Tc17 cells GVHD model (29), we hypothesized that Tc17 (but not Th17) Given that Tc17 cells mediate robust regression of melanoma in cells deficient in ICOS would expand to a greater extent than WT mice (11, 35), we wanted to determine the role of the ICOS/ Tc17 cells. Indeed, ICOS2/2 Tc17 cells expanded to a slightly ICOSL pathway on Tc17-mediated tumor immunity. We hypoth- greater extent than WT Tc17 cells (Fig. 3A). Conversely, esized that blockade of the pathway would impair the antitumor ICOS2/2 Th17 cells were compromised in their capacity to activity of Tc17 cells in vivo. To test this idea, we programmed expand (Supplemental Fig. 2C). Our data suggest that Th17 and melanoma-specific pmel-1 transgenic CD8+ T cells toward a Tc17 Tc17 cells respond differently in their capacity to proliferate phenotype, expanded them with a hgp10025–33 peptide, and then when engaged by the ICOS signal. cultured them in the presence of an ICOSL blocking mAb (clone The Journal of Immunology 5

from the radiation-injured gut (37), decreased the absolute number of CD11chi cells expressing ICOSL and the number of infused T cells in the animal (Supplemental Fig. 2E). These data imply that, at least in part, microbes liberated from lymphodepletion induce ICOSL on APCs. Given these ﬁndings and that lymphodepletion augments the function and antitumor activity of transferred T cells in patients (37), we surmised that the antitumor/self-activity of Tc17 cells would be compromised in ICOSL2/2 mice lymphodepleted with 5 Gy TBI. To test this concept, we infused pmel-1 Tc17 cells into 5 Gy irradiated WT or ICOSL2/2 C57BL6 mice bearing established B16F10 melanoma. Indeed, Tc17 cells were less effective in mediating tumor immunity (p , 0.001; Fig. 4A) and autoimmune vitiligo (p , 0.05; Fig. 4B) when infused into ICOSL2/2 mice compared with WT hosts. Collectively, these data support the notion that engaging ICOS via its ligand (upregulated by lymphodepletion) is important for potentiating the Tc17 cell–mediated immunity to self and tumor tissue. Downloaded from Tc17 cells express ICOS and regress melanoma to a greater extent than classic CTLs We and other investigators reported that Tc17 cells (which secrete IL-17A and nominal levels of IFN-g; Fig. 5A) mediate superior melanoma regression than IL-2–programmed tumor-speciﬁc + 2 2

/ CD8 T cells (IFN-g–producing cells; Fig. 5A, IL-2–polarized http://www.jimmunol.org/ FIGURE 3. ICOS Tc17 cells are compromised in their ability to + induce tumor in vivo. Naive CD4+ and CD8+ T cells (i.e., sorted by CD8 T cells [IL-2-P]), as shown in Fig. 5B and published else- CD44loCD62L+) were isolated from WT or ICOS2/2 mice, programmed where (11). To explain why Tc17 cells and IL-2–programmed + toward a type 1 or type 17 phenotype, and activated with irradiated CD8 T cells mediate differential antitumor responses, we as- splenocytes that were coated with a CD3 agonist. (A) The expansion of sessed their ICOS expression level, because it has been reported Tc17 cells was monitored, and their relative yield on day 10 is shown. (B as a biomarker for enhanced survival in melanoma patients (38). and C) ICOS, CD28, CD27, CD26, PD-1, CTLA4, CCR6, CXCR3, Indeed, we found higher ICOS expression on Tc17 cells versus IL- 2/2 CD39, and CD73 expression on WT and ICOS Tc17cellsasassayed 2–programmed cells 5 d after their infusion (Fig. 5C, 5D). No by ﬂow cytometry on day 10 postexpansion. Student t test was performed difference in ICOS was detected on host CD8+ T cells from either on the combination of two experiments. ***p , 0.001. (D)Pmel-1CD8+ treatment group (Fig. 5D). These data may explain why there is by guest on September 27, 2021 Tc17 cells were expanded in the presence, or not, of an ICOS antagonist for 8 d; then 1 3 106 cells were infused into WT mice bearing 10-d a higher frequency of Tc17 cells in the blood, tumor, draining established B16F10 melanomas. Recipient mice were pretreated with lymph nodes (dLNs), and mesenteric lymph nodes compared to + 5 Gy TBI and then received in vitro–vaccinated Tc17 cells cultured with mice receiving IL-2–expanded CD8 T cells (Fig. 5E). These data an ICOS antagonist or IgG control in conjunction with bolus IL-2. Tu- reveal that ICOS is preferentially expressed on transferred Tc17 mor areas were calculated over time. Error bars indicate the SEM (n =7 cells in vivo, but whether ICOS expression augments their in vivo mice/group). Tc17 (IgG) compared with Tc17 (anti-ICOS) time to cytotoxicity is unknown. growth and slope of growth, p , 0.001, log rank test. All experiments were repeated twice.

HK5.3), compared with a negative control IgG, for 8 d in vitro. These cells were then infused into mice bearing an established B16F10 melanoma that were lymphodepleted with 5 Gy TBI. In vitro, we found that ICOSL blockade impaired the capacity of Tc17 cells to secrete IL-17A and IFN-g (data not shown), similar to our findings with ICOS2/2 Tc17 cells (Fig. 2D). Moreover, in vitro blockade of ICOSL reduced the antitumor activity of Tc17 cells in vivo (p , 0.001; Fig. 3D). Although these data show that transient blockade of ICOS signaling impairs the antitumor activity of Tc17 cells, it remains unclear whether permanent defi- FIGURE 4. Tc17 cell–mediated tumor regression and autoimmune ciency in components of the ICOS/ICOSL pathway impacts Tc17 vitiligo is reduced in mice genetically deficient in the ICOSL. Ten + 2/2 responses to self and tumor tissue. million pmel-1 CD8 Tc17 cells were infused into WT or ICOSL mice bearing 10-d established B16F10 melanomas. Recipient mice were Tc17-mediated tumor regression is impaired when infused into pretreated with 5 Gy TBI and then received in vitro–vaccinated Tc17 2 2 ICOSL / mice cells in conjunction with bolus IL-2. (A) Tumor areas were calculated over time. Error bars indicate the SEM (n = 7 mice/group). Tc17 com- The ICOS signal is induced by interactions with its partner, the pared with Tc17 + ICOSL time to growth and slope of growth. p , ICOSL (CD275), a molecule expressed on APCs, such as B cells B hi 0.001, log rank test. ( ) Vitiligo scores 1 mo after cell transfer from two and dendritic cells (16, 36). We found that Ag-presenting CD11c independent experiments in mice receiving pmel-1 Tc17 cells. Circles cells from the spleens of irradiated mice expressed higher levels of indicate experiment 1 and triangles experiment 2. All experiments were ICOSL on their cell surface (Supplemental Fig. 2D). Irradiated repeated twice. Student t test was performed on the combination of two mice treated with ciprofloxacin, which ablate microbes liberated experiments. *p , 0.05. 6 ICOS AUGMENTS Tc17 CELL RESPONSES TO SELF AND TUMOR TISSUE

than bulk or ICOSneg Tc17 cells. To explore this question, as detailed in Fig. 6A, we sorted pmel-1 Tc17 cells by flow cytometry via relative ICOS expression and examined their ability to lyse tumor targets 12 h after being infused into mice using an in vivo CTL assay. Sorted Tc17 cells with high expression of high ICOS (ICOS ) lysed target cells presenting hgp10025–33 peptide to a significantly greater extent than ICOS-negative (ICOSneg)or bulk Tc17 cells (ICOShigh to Bulk, p = 0.002; ICOShigh to ICOSneg, p , 0.001; Fig. 6B, 6C). However, there was no significant difference between bulk and ICOSneg Tc17 cells in their ability to lyse target and, as expected, mice not infused with donor T cells (i.e., no treatment) were unable to lyse labeled targets. Collectively, our data show that ICOShigh Tc17 cells possess a greater cytotoxic potential than ICOSneg or bulk Tc17 cells. ICOS agonists augment the multifunctionality and antitumor activity of Tc17 cells Because ICOS is expressed on Tc17 cells to a greater extent than

IL-2–expanded cells and because ICOShigh Tc17 cells lyse targets Downloaded from superior to other cells, we surmised that activating Tc17 cells with an ICOS agonist in vitro would augment their capacity to secrete cytokines and their therapeutic effectiveness in vivo. To test this question, melanoma-speciﬁc pmel-1 CD8+ T cells were activated with hpg10025–33 peptide with or without ICOS agonist Ab and

then polarized toward a Tc17 subset. On day 3 after reactivation, http://www.jimmunol.org/ we collected supernatant and assessed for the ability of the cells to secrete various cytokines. We found that the addition of ICOS agonists to the culture bolstered Tc17 function, as indicated by their increased ability to secrete IL-17A, IL-17A/F, IL-9, CCL20, IL-22, IFN-g, TNF-a, and IL-10 (Fig. 7A). IL-2–expanded cells secreted more IFN-g when activated with an ICOS agonist, but secreted nominal IL-17A, IL-17A/F, CCL20, and IL-22. Further, we investigated the transcription factor proﬁle of these cells on day 7 after primary stimulation. As expected, IL-2–expanded cells by guest on September 27, 2021 (with or without the addition of the ICOS agonist) expressed signiﬁcantly greater levels of T-bet, but nominal expression of FIGURE 5. Tc17 cells express ICOS, engraft in multiple organs, and ROR-gt by Western blot analysis, compared with Tc17 cells (data mediate tumor immunity to a greater extent than IL-2–polarized cells. not shown). Conversely, Tc17 cells expressed high amounts of Pmel-1 CD8+ T cells were primed in IL-17–polarizing conditions (Tc17) ROR-gt, but very little T-bet. ICOS agonist did enhance the ROR- as detailed elsewhere (39) or in nonpolarizing conditions (IL-2-P) and then gt expression on Tc17 cells. Additional investigation revealed that expanded with IL-2. (A) Representative intracellular cytokine expression ICOS stimulation increased the coexpression of IL-7Ra and IL- + of IL-17A and IFN-g in Vb13 IL-2–polarized and Tc17-polarized cells 2Ra on Tc17 cells, but not on IL-2-P (Fig. 7B), suggesting that B before adoptive transfer. ( ) Recipient mice, bearing B16F10 melanoma, in vitro ICOS-stimulated cells may have greater persistence. were pretreated with 5 Gy TBI. They then received 1 3 107 pmel-1 CD8+ To determine whether ICOS agonist treatment enhanced persis- in vitro–vaccinated IL-2–polarized or Tc17 cells in conjunction with bolus i.v. IL-2, as described elsewhere (22). The slope of tumor regression (mm2 tence in vivo of Tc17 cells compared with Tc0 cells, we transferred /day) for individually treated mice is shown. Error bars indicate the SEM in vitro ICOS agonist-stimulated pmel-1 Tc17 or IL-2–expanded + (n = 7 mice/group). The slope of IL-2-P compared with Tc17, p , 0.001. CD8 T cells into a B16F10-bearing host. Consistently, we found (C) Flow cytometric and (D) graphical representation of ICOS expression that ICOS agonist treatment enhanced the persistence of Tc17 cells on donor Vb13+ IL-2–polarized or Tc17 cells after 18 d in vivo (n =5 over IL-2–expanded cells (Fig. 7C, 7D). This was also demon- mice/group). Tc17 donor versus host, **p , 0.001. Tc17 versus IL-2-P strated in the frequency of cells on day 21 after T cell infusion in within host, p = 0.002. Tc17 versus IL-2-P of donor cells, **p , 0.001, both the spleen and the inguinal lymph node (Fig. 7E). Yet, similar E ANOVA. ( ) Recipient mice were pretreated with 5 Gy TBI, bearing frequencies of Tc17 and IL-2–primed cells stimulated with ICOS established B16F10 melanomas, and received in vitro–vaccinated non- were detected in the tumor (data not shown). polarized or Tc17 pmel-1 CD8+ T cells (1 3 107) in conjunction with Further, to examine whether the addition of ICOS agonist bolus IL-2. Engraftment of adoptively transferred Vb13+ nonpolarized or , , therapy would augment T cell–mediated tumor immunity, we Tc17 cells in the spleen (**p 0.001), tumor (**p 0.001), dLN + (*p = 0.003), and mesenteric lymph node (**p , 0.001) were assayed on treated pmel-1 Tc17 or IL-2–expanded CD8 T cells with an day 21 (n = 3 mice/group), t test. All experiments were repeated twice. ICOS agonist in vitro and subsequently transferred them into melanoma-bearing hosts. We found that Tc17 cells activated with

high an ICOS agonist in vitro regressed tumors (p = 0.003; Fig. 8A) ICOS Tc17 cells efﬁciently lyse melanoma peptide-loaded and drove autoimmune vitiligo in lymphodepleted mice to a targets in vivo greater extent than Tc17 cells treated with an IgG control Although our data reveal that ICOS is expressed on Tc17 cells to (Fig. 8B). Conversely, ICOS agonist treatment did not augment a greater extent than IL-2–programmed cells, it remained unknown the antitumor activity of transferred IL-2–programmed pmel-1 whether ICOShigh Tc17 cells lyse target cells to a greater extent CD8+ T cells (Fig. 8A). Additional investigation revealed that The Journal of Immunology 7

FIGURE 6. Tc17 cells expressing high con- centrations of ICOS mediate cytotoxicity to tumor Ag-expressing targets in vivo. Pmel-1 Tc17 cells were sorted via MoFlo cytometry based on their expression level of ICOS. This strategy yields ICOShigh Tc17 or ICOSneg Tc17 cells. As a control, Tc17 cells were run through the MoFlo instrument, but not sorted on ICOS expression, called bulk Tc17 cells. (A) One million cells from each group were then transferred to irradiated B6 mice with palpable B16F10 melanoma tumor followed by bolus IL-2 therapy. Ten days after transfer, mice received target cells (splenocytes pulsed with either tumor-specific hgp10025–33 peptide or a control peptide [OVA]) and were analyzed, 12 h later, for specific lysis as represented by Downloaded from flow cytometry (B) and shown by bar graph (C) with five mice per group. Generation of target cells and percent specific lysis was calculated as described in Materials and Methods.Com- paring ICOShigh with bulk: *p = 0.002, ICOShigh with ICOSneg:*p , 0.001, t test. Representative http://www.jimmunol.org/ of three experiments.

Tc17 cells engraft in melanoma-bearing mice to a greater extent neutralizing IFN-g impaired Th17-mediated tumor regression, we when in vitro primed with an ICOS agonist (Fig. 8C). Yet, ICOS surmised that IFN-g was responsible for tumor regression by agonists did not augment the engraftment of IL-2–primed CD8+ ICOS-stimulated Tc17 cells. Indeed, we found that blocking IFN-g by guest on September 27, 2021 T cells in mice (data not shown). We also found that Tc17 cells dramatically retarded their ability to eradicate melanoma (Fig. 9 stimulated with an ICOS agonist expressed less KLRG-1 (an ex- and Supplemental Fig. 3; p = 0.04). Collectively, our data suggest haustion marker) than Tc17 cells stimulated with an IgG control a temporal influence of IL-17A in the induction of ICOS+ CTL, (data not shown), suggesting that ICOS preserve the antitumor and that the ICOS–ICOSL pathway is important for enhancing activity of Tc17 cells. These data reveal that ICOS agonists aug- the cytotoxicity of Tc17 cells in an IFN-g–dependent manner. ment the engraftment and antitumor activity of Tc17 cells, but Moreover, ICOS signaling increases the expression of CD25, how the cytokines secreted by ICOS-stimulated Tc17 cells regress CD127, and IL-23R, which may help the infused Tc17 cells melanoma remains incompletely elucidated. consume more cytokines, thereby bolstering their persistence. Because ICOS ligation increases IL-17A, IL-9, and IFN-g secretion by Tc17 cells, we sought to determine which cytokine(s) Discussion were responsible for mediating the enhanced antitumor immunity Cytokines are not the only cues important in supporting the gen- by this treatment. Thus, we blocked these cytokines in vivo using eration and function of Tc17 cells. We found that ICOS stimulation neutralizing Abs. Given the pronounced secretion of IL-17A by augments Tc17 cell responses to tumor/self tissue in a clinically Tc17 cells stimulated with ICOS, we rationalized that IL-17A was relevant adoptive cell transfer therapy model of murine melanoma responsible for enhancing the antitumor activity of ICOS-stimulated and autoimmune vitiligo. We identified that ICOS signaling is not Tc17 cells. This hypothesis was based on previous reports showing needed for the in vitro differentiation of naive CD8+ T cells to that IL-17A2/2CD4+ Th17 cells were unable to eradicate large a Tc17 subset. Rather, ICOS signaling is important for main- tumors, as well as their WT counterparts (39). In contrast with our taining the multifunctionality of Tc17 cells in vitro. We also ob- hypothesis, we found that blocking IL-17A in vivo did not impair served a reduced pool of effector memory-like CD44hiCD62Llo the antitumor response of ICOS-engaged Tc17 cells (Fig. 9 and Tc17 cells from ICOS2/2 mice. Of clinical significance, we found Supplemental Fig. 3; p = 0.75). These data suggest that IL-17A that ICOS signaling was important for enhancing Tc17 cell–me- plays a role as priming factor needed in the survival of ICOS- diated tumor regression and autoimmunity in vivo, as Ab or ge- activated Tc17 cells, but is not necessary for effector function and netic blockade of the ICOS pathway impaired their capacity to antitumor activity. We also found that IL-9 did not alter the anti- eradicate melanoma and induce autoimmune vitiligo in mice. tumor response of ICOS-stimulated Tc17 cells, because its blockade Conversely, activating Tc17 cells in vitro with a soluble ICOS did not impact treatment outcome (Fig. 9 and Supplemental Fig. 3; agonist further augmented their capacity to secrete multiple p = 0.96). This finding was unexpected because IL-9 was reported proinflammatory cytokines, eradicate tumors, and induce auto- to augment Tc9 cell–mediated antitumor immunity (40, 41). immune vitiligo in vivo, which was found to be IFN-g dependent. Because Th17 cells convert to IFN-g producers when infused Our finding that activating Tc17 cells with an ICOS agonist into an irradiated host and Muranski et al. (42) found that in vitro augments their in vivo effectiveness has clinical 8 ICOS AUGMENTS Tc17 CELL RESPONSES TO SELF AND TUMOR TISSUE

FIGURE 7. Tc17 cells stimulated with an ICOS agonist in vitro secrete heightened levels of inflammatory cytokines, coexpress high receptor levels of IL-2 (CD25) and IL-7 (CD127), and persist better than Tc0 cells. Pmel-1 CD8+ T cells were primed in IL-17–polarizing conditions (Tc17) or not (IL-2-P), expanded with IL-2, and treated in vitro with an ICOS agonist or Ab control (20 mg/ml on days 2, 4, and 6 of culture). (A) Cytokine and chemokine secretion by reactivated IL-2–polarized or Tc17 cells was determined by ELISA (day 4). Student t test was performed, *p , 0.05, **p , 0.01. (B) In vitro expression of IL-7Ra and IL-2Ra on Vb13+ cells by flow cytometry (day 7). Recip- ient mice, bearing established B16F10 mela- Downloaded from noma, were pretreated with 5 Gy TBI and then received 3 3 106 pmel-1 in vitro–vaccinated nonpolarized IL-2-P + ICOS agonist or Tc17 + ICOS agonist cells in conjunction with bolus IL-2. (C and D) Blood was taken to assess persistence. (C) Representative plots from day 7 are shown, and (D) averages are graphed on http://www.jimmunol.org/ the days indicated. Engraftment of adoptively transferred CD8+ Vb13+ nonpolarized or Tc17 cells + ICOS agonist in the blood on days 3 (**p = 0.0016), 7 (*p = 0.016), 14 (p = 0.0508), and 21 (NS) were examined (n = 3 mice/group), t test. (E) Surviving mice were sacrificed on day 21, and the inguinal lymph node, spleen, and tumors were harvested. Engraftment of Vb13+ +

CD8 T cells in the spleen (**p = 0.0049), tu- by guest on September 27, 2021 mor (NS), and dLN (**p , 0.001) were assayed (n = 3 mice/group), t test. Data are the combination of two separate experiments.

implications. Along with bolstering their cytotoxicity, the addition antitumor activity ICOS-activated Tc17 cells. Given that we also of an ICOS agonist to the Tc17 culture augmented their multi- found that ICOS engagement increased IL-17A/F and IL-17F by functionality, as indicated by their increased ability to secrete IL-9, Tc17 cells, it is also possible that these cytokines additionally IL-17A, IL-17A/F, CCL20, IL-22, and IFN-g. Despite the fact that contribute to their effectiveness. Experiments in our laboratory are these cells secreted numerous cytokines before transfer, it has ongoing to understand how these multiple cytokines contribute to been shown that Tc17 cells are plastic, because they are able to the effectiveness of ICOS-activated Tc17 cells. Regardless, it is convert from a cell that mainly secretes IL-17A to one that mainly clear that Tc17 cells convert in vivo into IFN-g–producing cells produces IFN-g (43–46). Previous studies demonstrate that IL- (11, 18, 47, 48) and that ICOS increases IFN-g secretion by Tc17 17A and IFN-g are both important for driving antitumor re- cells, a type 1 cytokine with cytotoxic properties that impacted sponse of Th17 cells (42). In our work, we found that Tc17 cells treatment outcome by ICOS-stimulated Tc17 cells. stimulated with an ICOS agonist mediate tumor regression via We found that ICOS agonist greatly increased coexpression of their secretion of IFN-g, despite the fact that very little of the IL-7Ra and IL-2Ra on Tc17 cells, but less so on IL-2–expanded master transcription factor T-bet is expressed and that only a CD8+ T cells. Given that IL-7 and IL-15 are elevated after host nominal amount of IFN-g is secreted by these cells on the day of lymphodepletion (49), we posit that infusion of Tc17 cells into transfer. Conversely, we found that blocking IL-17A did not im- irradiated mice would promote their engraftment. Indeed, ICOS+ pair the antitumor activity of ICOS-stimulated Tc17, even though Tc17 cells engrafted in the spleen, blood, and lymph nodes of ICOS dramatically increased IL-17A by these cells. However, it is mice better than IL-2–expanded CD8+ T cells. Given that the important to appreciate that it is possible that neutralizing IL-17A persistence of tumor-speciﬁc T cells is critical for durable anti- in vivo did not sufﬁciently remove all of the IL-17A secreted by tumor responses, further studies in our laboratory are under way to ICOS-stimulated Tc17 cells. Follow-up studies with IL-17A2/2 investigate the effects of ICOS stimulation on the persistence of Tc17 cells versus WT Tc17 cells will provide deeper insight into memory phenotype of Tc17 cells and the importance of the IL- whether IL-17A contributes to augmenting the persistence and 2Ra and IL-7Ra in shaping Tc17 immunity in tumor-bearing The Journal of Immunology 9

ICOSL expression on APCs is beyond the scope of this study, we are excited about the data because it leads us down a new and important line of study. It has been reported that ICOSL expression on melanoma cells increases the Treg population in mice (52), but based on our ﬁndings, the expression of ICOSL on the tumor may also have a positive effect on Tc17 cells in vivo. Our studies did not investigate the expression of Foxp3, but we did see an increase of CD25 expression in Tc17 cells given the ICOS agonist. CD25 expression did not correlate with a regulatory phenotype, as these cells secreted more effector molecules and had increased antitumor potential in vivo. It is interesting to consider the impact of coinhibitory blockade therapies for cancer. Many investigators use these therapies to induce robust lymphocyte activation and cytotoxicity to tumor Ags (53). Yet, melanoma patients treated with CTLA-4 blockade therapy (ipilimumab) do not merely have an increased frequency of functional tumor-speciﬁc T cells, but it has been reported that

the patients who experience the best treatment outcome also have Downloaded from a greater frequency of ICOS+ T cells (54). Likewise, preclinical B16F10 melanoma models reveal that ICOS expression on T cells is vital for the effectiveness of CLTA-4 blockade therapy (55). FIGURE 8. ICOS-stimulated Tc17 cells mediate immunity to self and Given that ICOS and ICOSL mediate autoimmunity via Tc17/ tumor tissue to a significantly greater extent than IL-2–expanded T cells Th17 cells and enhance tolerance by supporting Treg develop- A stimulated with ICOS. ( ) Recipient mice, bearing established B16F10 ment (56), our data may suggest that this pathway bolsters the http://www.jimmunol.org/ 7 melanoma, were pretreated with 5 Gy TBI and then received 1 3 10 antitumor activity of Tc17 or Th17 cells in patients treated with pmel-1 in vitro–vaccinated nonpolarized (IL-2-P), IL-2-P + ICOS agonist, ipilimumab. However, recent work by Fan et al. (57) revealed that Tc17, or Tc17 + ICOS agonist cells in conjunction with bolus IL-2. ICOS this might not be the case. They found that combining CTLA-4 agonist or Ab control (20 mg/ml) was given during in vitro expansion on blocking therapy with a vaccine incorporating ICOSL augmented days 2, 4, and 6 of culture. Tumor areas were calculated over time. Error + + bars indicate the SEM (n = 7 mice/group). Comparing time to tumor the antitumor activity of host type 1 CD8 and CD4 T cells. growth of Tc17 with Tc17 + ICOS: p = 0.003, Tc17 with IL-2-P: p = 0.01, Interestingly, this combination therapy did not appear to potentiate IL-2-P + ICOS with Tc17 + ICOS: p = 0.003, log rank test. (B) Vitiligo the function of immune cells classically known to express ICOS, scores from two independent experiments in mice receiving pmel-1 Tc17 that is, Tregs, follicular helper T, Th17, or Tc17 cells (17–19). It is cells with or without ICOS agonist treatment. Circles indicate experiment by guest on September 27, 2021 1 and triangles experiment 2. Student t test was performed on the combination of two experiments, p , 0.01. (C) The persistence of Tc17 cells (treated, or not, with ICOS agonist in vitro) in the spleen of tumor-bearing mice 7 d after T cell infusion. All experiments were repeated twice. Stu- dent t test was performed, *p = 0.0123. mice. Moreover, it has recently been reported that lymphodepletion induces APCs to secrete heightened IL-12 and IL-23 in patients with melanoma (50). Because ICOS induces IL-23R expression of Tc17 cells, it is possible that IL-23 (and perhaps IL- 12) also augments anti-self/tumor immunity by ICOS-activated Tc17 cells in vivo. It will be informative to evaluate how ICOS impacts the for- mation of long-lived memory Tc17 and Th17 cells in tumor immunity. Muranski, Gattinoni, Restifo, and coworkers (39, 51) found that Th17 cells have phenotypic and molecular signatures that are distinct from Th1 cells and that are reminiscent of CD8+ T cell with stem cell–like memory phenotype. Indeed, recent FIGURE 9. ICOS-stimulated Tc17 cells mediate immunity in an IFN-g findings suggest that T cells with this phenotypic signature elicit (not IL-17)–dependent manner. Recipient mice treated with 6 Gy TBI robust and potent tumor immunity in murine and humanized bearing established B16F10 melanoma received 5 3 106 in vitro–vacci- mouse models of melanoma, ovarian cancer, and mesothelioma nated Tc17 pmel-1 cells treated during in vitro expansion with 20 mg/ml (39, 45). ICOS agonist on days 2, 4, and 6. Mice were then given 0.1 mg neutral- Irradiated mice treated with a broad-spectrum antibiotic, cip- izing Ab (IL-17A, IFN-g, or IL-9) every other day for five treatments and rofloxacin, triggered and systemically liberated from the gut via IL-2 (50,000 IU) every day for the first 3 d after T cell transfer. Tumor measurements were recorded twice weekly. Average tumor curves are TBI (37), dramatically decreased the absolute number of CD11chi displayed. Error bars denote SEM (n = 10–13 mice/group). Comparing cells expressing ICOSL. We likewise found a decrease in the time to tumor growth of Tc17 + ICOS with no treatment: p = 0.01, Tc17 + number of donor pmel-1 T cells in the blood of these mice. ICOS with Tc17 + ICOS (IFN-g depletion): p = 0.04, Tc17 + ICOS with Collectively, these data suggest that microbes might, at least in Tc17 + ICOS (IL-17 depletion): p = 0.75, Tc17 + ICOS with Tc17 + ICOS hi part, be responsible for regulating ICOSL expression on CD11c (IL-9 depletion): p = 0.96, log-rank test. These experiments were repeated APCs. Although the exact microbes responsible for regulating three times. 10 ICOS AUGMENTS Tc17 CELL RESPONSES TO SELF AND TUMOR TISSUE possible that CTLA-4 blockade induction of ICOS does impact 13. Manel, N., D. Unutmaz, and D. R. Littman. 2008. The differentiation of human T(H)-17 cells requires transforming growth factor-beta and induction of the Treg and Th17/Tc17 subsets, because of downstream signals in- nuclear receptor RORgammat. Nat. Immunol. 9: 641–649. duced by this CTLA-4 blockade or the tumor microenvironment. 14. Yang, L., D. E. Anderson, C. Baecher-Allan, W. D. Hastings, E. Bettelli, Such signals may drive Th17/Tc17 or Tregs to convert to a Th1- M. Oukka, V. K. Kuchroo, and D. A. Hafler. 2008. IL-21 and TGF-beta are required for differentiation of human T(H)17 cells. Nature 454: 350–352. like cell. It is also possible that CTLA-4 blockade therapy induces 15. Volpe, E., N. Servant, R. Zollinger, S. I. Bogiatzi, P. Hupe´, E. Barillot, and signals that override ICOS-induced polarization of cells to a V. Soumelis. 2008. A critical function for transforming growth factor-beta, in- committed regulatory or type 17 phenotype. Regardless, the find- terleukin 23 and proinflammatory cytokines in driving and modulating human T(H)-17 responses. Nat. Immunol. 9: 650–657. ing that ICOSL vaccines augment CTLA-4 blockade therapy is 16. Greenwald, R. J., G. J. Freeman, and A. H. Sharpe. 2005. The B7 family promising and has impactions for cell-based therapies for cancer, revisited. Annu. Rev. Immunol. 23: 515–548. particularly Th17 or Tc17 therapies. 17. Ito, T., S. Hanabuchi, Y. H. Wang, W. R. Park, K. Arima, L. Bover, F. X. Qin, M. Gilliet, and Y. J. Liu. 2008. Two functional subsets of FOXP3+ regulatory In conclusion, our work reveals that ICOS augments the function T cells in human thymus and periphery. Immunity 28: 870–880. and anti-self/tumor activity of Tc17 cells. Our findings are important 18. Paulos, C. M., C. Carpenito, G. Plesa, M. M. Suhoski, A. Varela-Rohena, T. N. Golovina, R. G. Carroll, J. L. Riley, and C. H. June. 2010. The inducible for the design of next-generation cell therapies for cancer, such as costimulator (ICOS) is critical for the development of human T(H)17 cells. Sci. those using genetically engineered T cells that recognize tumor using Transl. Med. 2: 55ra78. TCRs or chimeric Ag receptors (58–62). This work also suggests 19. Bauquet, A. T., H. Jin, A. M. Paterson, M. Mitsdoerffer, I. C. Ho, A. H. Sharpe, and V. K. Kuchroo. 2009. The costimulatory molecule ICOS regulates the ex- that expanding redirected Tc17 with vaccines incorporating ICOSL pression of c-Maf and IL-21 in the development of follicular T helper cells and could augment treatment outcome in cancer patients. Collectively, TH-17 cells. Nat. Immunol. 10: 167–175. our work suggests that targeting this pathway may have therapeutic 20. Park, H., Z. Li, X. O. Yang, S. H. Chang, R. Nurieva, Y. H. Wang, Y. Wang, L. Hood, Z. Zhu, Q. Tian, and C. Dong. 2005. A distinct lineage of CD4 T cells regulates tissue merit for patients with advanced diseases. inflammation by producing interleukin 17. Nat. Immunol. 6: 1133–1141. Downloaded from 21. de Wit, J., Y. Souwer, A. J. van Beelen, R. de Groot, F. J. Muller, H. Klaasse Bos, T. Jorritsma, M. L. Kapsenberg, E. C. de Jong, and S. M. van Ham. 2011. CD5 Acknowledgments costimulation induces stable Th17 development by promoting IL-23R expression We thank Colleen Cloud, Caitlin Moore, and Tracy Vandenberg of the De- and sustained STAT3 activation. Blood 118: 6107–6114. partment of Surgery at MUSC and Marshall Diven and Danh Tran of the 22. Klebanoff, C. A., Z. Yu, L. N. Hwang, D. C. Palmer, L. Gattinoni, and Department of Microbiology & Immunology at MUSC for assistance. We N. P. Restifo. 2009. Programming tumor-reactive effector memory CD8+ T cells in vitro obviates the requirement for in vivo vaccination. Blood 114: 1776–1783. thank Adam Soloff at the MUSC Flow Cytometry and Cell Sorting Core 23. Takahashi, N., K. Matsumoto, H. Saito, T. Nanki, N. Miyasaka, T. Kobata, http://www.jimmunol.org/ for sorting cells. In addition, we thank Kent Armeson for support with M. Azuma, S. K. Lee, S. Mizutani, and T. Morio. 2009. 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