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Differential Suppression of Tumor-Specific CD8 + T Cells by Regulatory T Cells Edward James, Alex Yeh, Cathy King, Firouzeh Korangy, Ian Bailey, Denise S. Boulanger, Benoît J. Van den Eynde, This information is current as Nicholas Murray and Tim J. Elliott of October 2, 2021. J Immunol 2010; 185:5048-5055; Prepublished online 4 October 2010; doi: 10.4049/jimmunol.1000134 http://www.jimmunol.org/content/185/9/5048 Downloaded from

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References This article cites 42 articles, 19 of which you can access for free at: http://www.jimmunol.org/ http://www.jimmunol.org/content/185/9/5048.full#ref-list-1

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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 © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Differential Suppression of Tumor-Specific CD8+ T Cells by Regulatory T Cells

Edward James,* Alex Yeh,*,1 Cathy King,* Firouzeh Korangy,† Ian Bailey,* Denise S. Boulanger,* Benoıˆt J. Van den Eynde,‡ Nicholas Murray,* and Tim J. Elliott*

In the CT26 BALB/c murine model of colorectal carcinoma, depletion of regulatory T cells (Tregs) prior to tumor inoculation results in protective to both CT26 and other BALB/c-derived tumors of diverse histological origin. In this paper, we show that cross-protection can be conferred by adoptively transferred CD8+ CTLs. Other schedules for inducing immunity to CT26 have been described, but they do not lead to cross-protection. We show that Treg ablation facilitates the development of new CTL specificities that are normally cryptic, and have mapped the root of one of these responses. This work has allowed us to demonstrate how the specificity of CTL responses to tumor Ags can be controlled via differential suppression of CTL specificities by Tregs, and how this can result in very different physiological outcomes. The Journal of Immunology, 2010, 185: Downloaded from 5048–5055.

tudies in mouse models of have shown that the ac- and other factors known to affect survival (5). Patients with high quired can prevent both onset and pro- numbers of Tregs in ovarian tumor tissue were observed to have S gression of tumors and that Ag-specific T cells may confer a mortality of 100% at 5 y, compared with 50% in the low-Treg lifelong protection against malignancy (1). Descriptive studies in group. In addition, Tregs capable of inhibiting tumor-associated http://www.jimmunol.org/ human , particularly melanoma, ovarian cancer, and co- Ag-specific immune responses were increased in patients with lonic cancer, have strongly suggested that the presence of tumor- colorectal cancer (10). These data indicate an important role of infiltrating is associated with better survival outcomes Tregs in modulating the antitumor , and the in- (2–4). Tumors, however, possess many strategies for evading the duction of Tregs may present a significant barrier to the successful immune system. One critical pathway is the induction of tumor application of anticancer . Ag-specific mediated through the generation of In mouse models, depletion of Tregs generally results in better T regulatory cells (Treg), and several lines of evidence support the immune protection against tumors (11–14), and we have shown immunosuppressive role of Tregs in influencing cancer prognosis that mice depleted of Tregs can reject the colorectal tumor line + + (5, 6). There is an increase in the number of CD4 CD25 Tregs in CT26, which is not normally immunogenic. The rejection of the by guest on October 2, 2021 the peripheral of cancer patients in parallel with advancing primary tumor was accompanied by development of protective stage of disease, and this plays a major part in the failure of many immunity, because mice vaccinated with CT26 were able to reject against tumors. Patients with early non-small cell the same tumor when rechallenged 50–74 d later. Both CD4+ and lung and breast cancer with higher proportions of tumor Tregs CD8+ T cells could mediate immunity to the second CT26 chal- relative to tumor-infiltrating lymphocytes had a significantly higher lenge. In addition, mice rendered CT26 immune following anti- risk of recurrence, and in melanoma this predicted reduced patient CD25 mAb treatment were able to reject other tumor lines of di- survival (7–9). In ovarian cancer, the absolute numbers of Tregs verse histological origin (12). in tumor tissue was shown to be a significant, independent predictor Our results indicate, therefore, that depletion of Tregs uncovers of mortality even after controlling for stage, surgical debulking, cryptic responses to Ags that are shared among different tumor cell lines. CT26-specific responses can be elicited by different forms of vaccination in the presence of regulatory cells, but in these *Cancer Sciences Division, School of Medicine, University of Southampton, South- ampton, United Kingdom; †Department of Gastroenterology, Hepatology, and Endo- cases T cell responses are highly focused on a single tumor-specific crinology, Hannover Medical School, Hannover, Germany; and ‡Brussels Branch and epitope, AH1 (corresponding to amino acids 423–431 of the en- Cellular Genetics Unit, Ludwig Institute for Cancer Research, Catholic University of dogenous murine leukemia [MuLV], gp90 gene), presented Louvain, Brussels, Belgium by MHC class I H-2Ld (15). 1Current address: Department of Surgery, Kaohsiung Veterans General Hospital, Kaohsiung, Taiwan, Republic of China. Taken together, these data suggest that immune responses to Received for publication January 21, 2010. Accepted for publication August 30, some Ags are more tightly regulated than others. This may be a 2010. consequence of the types of Ags that have the ability to activate This work was supported by grants from Cancer Research UK (to T.E. and E.J.), the Tregs. We therefore undertook to identify the Ag or Ags recognized Medical Research Council (T.E.), and the Wellcome Trust (E.J.). by cross-protective CD8+ CTLs and to determine the immuno- Address correspondence and reprint requests to Dr. Edd James, University of South- logical correlates of cross-protection. ampton, Somers Cancer Research Building, MP824 Tremona Road, Southampton, Hampshire SO166YD, United Kingdom. E-mail address: [email protected] Materials and Methods The online version of this article contains supplemental material. Mice, Abs, and in vivo depletion Abbreviations used in this paper: CCD2, CT26-specific Balb/c H2-d restricted T cell clone 2; HERV, human endogenous ; lacZ, b-galactosidase; MuLV, murine BALB/c mice were bred locally under specific -free conditions in leukemia virus; Treg, . Southampton, U.K. Female or male mice (6–8 wk old) were used in all experiments. During experimental procedures, mice were housed in con- Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 ventional facilities. Hybridomas secreting CD25 (PC61, rat IgG1)-specific www.jimmunol.org/cgi/doi/10.4049/jimmunol.1000134 The Journal of Immunology 5049 mAb have been described previously (16, 17) and were grown in culture specific forward primer and a P20E8A1-specific reverse primer C2 (59- and mAb purified by precipitation in saturated ammonium sulfate. For GACTCTAGATTACTGTGATGTAGTCCCAG-39); C3 (59-GACTCTAGA- depletion, mice received 1 mg mAb PC61 injected i.p. in volumes of 100– AGTTTTTGGTCTGGTGCCGG-39), C7 (59-GACTCTAGAGAAGGATT- 200 ml, twice with a 1-d interval. The Ab was administered 3 and 1 d prior CTGGACCACCAC-39), and C8 (59-GACTCTAGAGTGGTTTCGCAGC- to the injection of tumor cells. CCCAAGA-39) with KOD polymerase (Merck, Darmstadt, Germany). PCR fragments were digested with EcoRI and XbaI and cloned into the corre- Tumor cells sponding sites of pcDNA3. The synthetic peptides GSA9 (GGPESFYCA), Cultures of the cell lines, CT26, CT26/GM-CSF, P815, MCFL, Renca, and GSS10 (GGPESFYCAS), GSW11 (GGPESFYCASW), GSG12 (GGPES- A20 (American Type Culture Collection, Manassas, VA), were maintained FYCASWG), GFC13 (GGPESFYCASWGC), GSI11 (GGPESFYCASI), GSF11 (GGPESFYCASF), AH1 (SPSYVYHQF) (15), QL9 (QLSPFPFDL) in RPMI 1640 (Sigma-Aldrich, St. Louis, MO) supplemented with 10% (25), and YF9 (YGPSLYRRF) (26) were purified by HPLC and confirmed by FCS (Globepharm, Surrey, U.K.), 2 mM L-glutamine, penicillin/streptomycin (Sigma-Aldrich), 50 mM 2-ME, 1 mM sodium pyruvate (Life Technologies- mass spectrometry (GL Biochem, Shanghai, China). BRL, Rockville, MD), and 1mM HEPES (PAA Laboratories, Dartmouth, Extraction and HPLC analysis of endogenously processed MA). CT26/GM-CSF was derived as described (18). BCL1 was passaged peptides in vivo only (19). MCFL was a generous gift from Dr. Mario Colombo (Instituto Nazionale Tumori, Milan, Italy). In all experiments, mice were Total acid-soluble peptide pools from tumor cells CT26, CT26/GM-CSF, A20, 5 injected s.c. with 10 tumor cells in PBS. All these cell lines were tested and and MCFL were extracted as described (27). Briefly, 108 CT26 and CT26/ were found to be mycoplasma free. GM-CSF, 4 3 108 MCFL, 109 A20, 109 Renca, and 109 P815 cells were washed with PBS and extracted with 800 mL 10% boiling formic acid in the Isolation and adoptive transfer of subsets presence of a 10-mM “martyr” peptide ASNENMETM for 10 min. Cellular Single-cell suspensions were prepared from the spleens of donor mice. debris was removed by centrifugation and fractionated by HPLC after fil- The cells were labeled with MACS Abs for CD4+ and CD8+ T cells and tration through a 10-KDa filter (Millipore, Billerica, MA). Reverse-phase C18

3 ˚ Downloaded from purified using MACS VS+ separation columns according to the manufac- column (Grace-Vydac, Hesperia, CA, 2.1 250 mm, 5 mM, 300 A)wasrun turer’s instructions (Miltenyi Biotec, Bisley, U.K.). Following elution of the in 0.1% trifluoroacetic acid in water (solvent A) and 0.1% trifluoroacetic acid purified cells off the columns, the purity of the cells (.90%) was confir- in acetonitrile (solvent B). The gradient was from 17 to 45% (solvent B) at med by FACS analysis. Cells were resuspended in PBS at a concentration of a rate of 1%/min. Flow rate was maintained at 0.25 ml/min, and 150-ml 5 3 107/ml. Mice were injected i.v. with 200 ml cell suspension. fractions were collected and dried in a vacuum centrifuge. Mock injections with sample buffer alone were performed prior to each extracted sample, Cell lines and hybridomas using the same column and identical run conditions. The collected fractions were assayed in the same experiment, using the same APCs and T cells, in Generation of CTL lines, clones, b-galactosidase (lacZ)-inducible T cell parallel with fractions from the cell extracts and synthetic peptide standards. http://www.jimmunol.org/ hybrids has been described (20, 21). Briefly, spleen cells from CT26- immune Treg-depleted BALB/c mice were removed of CD4+ Tcells,as RMA-S stabilization described above, and stimulated in vitro every 12–14 d with irradiated d d CT26 cells and 20 IU/ml rIL-2. CTL clones were generated using limiting RMA-S–D ,–L cells were cultured overnight at 26˚C and then for 60 min dilution of the parental CTL lines. The lacZ-inducible T cell hybridoma in the presence of a saturating concentration of peptide (100 mM). The- CCD2Z was generated by fusing the CTL clone CT26-specific Balb/c H2-d cells were washed twice with PBS, then cultured for the indicated times at 37˚C. Cells were taken at the indicated times and stained with either anti- restricted T cell clone 2 (CCD2) with the BWZ.36/CD8a fusion partner, as d d described (21). D Ab (34-5-8S) or anti-L Ab (30-5-7S) mAbs conjugated with PE (BD Biosciences, San Jose, CA). The cells were washed twice and analyzed by T cell activation assays FACSCanto (BD Biosciences). Flow cytometry analysis was performed using FlowJo software (Tree Star, Ashland, OR). by guest on October 2, 2021 LacZ-inducible T cell hybrids were cocultured with APCs that either were transfected with Ag cDNAs or were pulsed with exogenous peptides. The CD8 T cell responses lacZ activity was measured as described previously (22). CTL lysis assays Spleen cells from CT26-challenged mice (Treg depleted or replete) were were performed with 51Cr-labeled tumor cells. WEHI killing was carried out harvested between days 12 and 14. The CD8+ T cell responses to the CT26 using supernatant from CTL lines/clones cultured in the presence of tu- Ags GSW11 and AH1 were measured by assay of inducible IFN-g by mor cells overnight. WEHI cells (TNF sensitive) were exposed to the su- those cells in response to incubation with peptide or CT26. CD8+ T cells, pernatant and the level of killing measured. APCs, and peptides/tumors were cultured together for 4 h in the presence of Ab blocking brefeldin A before being stained for surface CD8 and intracellular IFN-g (BD Biosciences). Cells were then analyzed on a FACSCanto (BD Bio- For inhibition of presentation by MHC class I molecules, tumor cells were sciences) and data analyzed with FlowJo Software (Tree Star). Numbers are incubated for 1 h at 4˚C with anti–H-2Dd (34-5-8S), -Ld (B22.249) or Kd reported as above the background response of T cells alone. For in vitro (SF1.1) before being cultured together with CCD2Z hybridoma cells, as restimulation, 5 3 106 spleen cells were cultured with 20 IU/ml rIL-2 described above. (PeproTech, Rocky Hill, NJ) and 1 mM peptide (AH1 or GSW11). After 7 d, cells were harvested and assessed for AH1- and GSW11-specific CD8 cDNA library and expression screens T cell responses, as described above. For the second round of restimulation, A cDNA library using mRNA from CT26 cells was cloned into the EcoRI cultures were harvested and live cells purified using Ficoll-Paque (GE 3 6 site of the mammalian expression vector pcDNA3 (Invitrogen, Carlsbad, Healthcare, Chalfont, U.K.). A total of 1 10 purified cells were in- 3 6 CA). The cDNA library was screened in pools of 50–100 CFUs by trans- cubated with 5 10 irradiated (10 Gy) BALB/c spleen cells, 20 IU/ml rIL- forming with recombinant plasmids, as described previously (23, 24). 2, and 1 mM AH1 or GSW11 peptide. As for the first round of stimula- Briefly, cDNA pools were cultured, isolated, and screened in 96-well plates by tion, cells were harvested at day 7 and analyzed for AH1- and GSW11- transient transfection into recipient COS cells cotransfected with the relevant specific CD8 T cells. 5 MHC class I cDNA. At 2 d later, 10 CTL clones were added to the wells lysis and cocultured overnight. Supernatant from the overnight cultures was incu- bated with the TNF-b–sensitive cell line, WEHI, and the level of cell death CT26 cells were stained with PKH26 (Sigma-Aldrich) according to the measured by the MTT colorimetric assay (Sigma-Aldrich). The level of manufacturer’s instructions and incubated with AH1-specific CTLs at WEHI cell death is directly related to the amount of TNF-b present in the 37˚C for 2 h; then they were stained for 30 min on ice with 1 mM YoPro-1 supernatant and thus indicates the level of stimulation of the CTL clones. (Molecular Probes, Eugene, OR) and analyzed by flow cytometry. Specif- The positive pool was identified by repeating the screening with individual ic lysis of the PKH+ target cells was calculated as 100p[%YoPro1+ 2 cDNA clones from the positive cDNA pool. The P20E8A1 cDNA sequence %YoPro1+E:T = 0]/[100 2 %YoPro1+E:T = 0]. As negative controls, we is available from the NCBI Genbank (http://www.ncbi.nlm.nih.gov/genbank/) included AH1-specific CTLs with BALB/c splenocytes. with the accession number GU441834. Statistical analysis Expression constructs and peptides Analyses were performed using the Prism software (GraphPad, San Diego, All plasmid constructs were in the expression vector pcDNA3. Nested CA). The p values were calculated using either the two-tailed unpaired t deletions were generated by PCR of the P20E8A1 cDNA with a vector- test or the paired t test. 5050 SUPPRESSION OF TUMOR-SPECIFIC CD8+ T CELLS

Results Derivation of cross-reactive CD8+ T cells in mice primed to CT26 in the absence of Tregs We have previously shown that both CD4+ and CD8+ effector T cells generated to the transplantable colon carcinoma CT26 in the absence of Tregs can mediate cross-protection and possibly act synergistically (12). Because, in the presence of Tregs, other forms of vaccination to CT26 using whole cells (either irradiated or transfected with GM-CSF) result in a protective CTL response that is highly focused on a single epitope (Ld-restricted AH1) and are not cross protective, our results suggested that depletion of Tregs uncovers responses to cryptic Ags that are shared among different tumor cell lines. We therefore sought to identify the source Ag(s) recognized by cross-protective CD8+ T cells, to improve our un- derstanding of their apparent suppression by Tregs. To this end, we set out to establish CD8+ T cell lines that were representative of the cross-protective response in terms of their pattern of tumor recognition. T cell lines were generated from pooled purified CD8+ splenocytes from three mice immunized with live CT26 after Treg Downloaded from ablation. The splenocytes were shown to confer cross-protection to tumors of diverse origin when transplanted into SCID mice with a high potency (Fig. 1A). Several lines were generated in this way and found to contain predominantly H-2Dd- and H-2Kd-restricted reactivities (data not shown). H2-Ld-restricted responses, which

would include responses to the previously described immunodo- http://www.jimmunol.org/ minant epitope from CT26, AH1 (SPSYVYHQF) derived from an endogenous ecotropic leukemia virus, were rarely seen, although in one case, the H-2Ld-restricted AH1 reactivity, which we detected with fluorescent tetramers, appeared after months in culture and came to dominate after a period of 6 mo in continuous culture (data not shown). The anti-AH1 response was, however, the dominant reactivity when splenocytes from CT26 tumor-bearing, Treg- replete mice were cultured in the same way (data not shown). + Thus, the CD8 responses elicited to CT26 in the absence of Tregs by guest on October 2, 2021 appeared qualitatively different from those previously described, FIGURE 1. CD8 T cell adoptive transfer confers cross-protection in vivo. A, CD8 T cells isolated from CT26-immune Treg-depleted BALB/c even at this low level of resolution. One T cell line, CCD, which 6 d mice and 10 cells injected into SCID recipients. Following transfer, mice contained reactivities that were inhibited by anti–H2-K and anti– 5 d d were challenged with 10 CT26 (n = 5), BCL-1 (n = 5), or A20 (n =5) H2-D but not anti–H2-L Abs, was selected for its ability to tumor cells. B, CTL lines were generated from CT26-immune Treg-de- recognize CT26, A20, and Renca, but not the mastocytoma cell pleted BALB/c mice by in vitro restimulation with 20 IU/ml rIL-2 and 104 line P815, in both IFN-g secretion and killing assays (Fig. 1B), irradiated CT26. CTL lines were assessed for stimulation by CT26, A20, therefore displaying a pattern of reactivity that closely matched Renca, and P815 by assay (top panel) and IFN-g production the bulk culture of CT26-stimulated splenocytes for Treg-depleted (bottom panel). C, CTL clones were generated from CCD CTL lines by mice. We generated CTL clones from this line, of which one, limiting dilution of the CTL lines. One clone, CCD2, was assayed for designated CCD2, was prosecuted in detail. Clonality was con- stimulation by CT26, A20, BCL-1, and Renca, using the WEHI killing 6 d firmed with a panel of anti-TcRVa and Vb Abs (Va8.3b8.3 and assay. Results are the mean SEM. D, MHC blocking Abs against L (B22) and Dd (34-5-8S) were used to assess the MHC restriction of the continuously monitored by nested-set PCR). CCD2 reacted CCD2 response to CT26 in a cytotoxicity assay. Data are representative of strongly against CT26, A20, and BCL-1; however, the response to two or three independent experiments. Renca was significantly lower (Fig. 1C; p = 0.001). Further characterization showed the response to CT26 was inhibited by anti-Dd and -Dd/Kd but not anti-Ld or control Abs (Fig. one of twenty 96-well plates show two cDNA pools with above 1D). Thus the CCD2 CTL displayed an H-2Dd–restricted pattern background killing of WEHI cells (Fig. 2A). Indeed, several stim- of cross-reactivity that was representative of the cross-protective ulatory plasmid clones were found after fractionating the mixture CD8+ T cell response in Treg-depleted, CT26-immunized mice. into individual colonies (Fig. 2B). One of these plasmids, P20E8A1, was further tested for its ability to stimulate CCD2 in an Ag-specific Identification of a cross-reactive Ag by expression cloning and MHC-restricted manner. COS-7 cells transfected with P20E8A1 To identify the Ag recognized by the cross-reactive CTL CCD2, we generated the T cell epitope in a dose-dependent manner—only employed an expression cloning technique (20). A cDNA library when P20E8A1 cDNA was cotransfected with cDNA encoding the was prepared from CT26, composed of inserts with a size range of MHC molecule Dd, but not Kd (Fig. 2C,2D). In addition, the re- 0.4–5 kb cloned nondirectionally into pcDNA3 and organized into sponse to P20E8A1 was inhibited by anti-Dd Abs, as seen previously pools of 100 clones. The CCD2 T cell clone was used as a probe with the CCD2 anti-CT26 response. To facilitate the subsequent for expression of the cognate in Dd-expressing COS-7 cells identification of the immunostimulatory epitope, we generated that were transiently transfected with pools of a plasmid cDNA a lacZ-inducible hybridoma, CCD2Z, from fusion of the CCD2 expression library prepared from CT26. Representative data from T cell and the BWZ.36/CD8+ fusion partner (21), which displayed The Journal of Immunology 5051 Downloaded from http://www.jimmunol.org/

FIGURE 2. Expression cloning and mapping of a cross-protective tumor Ag. A, Primary screen of a CT26 cDNA plasmid library in which the cDNA pools were transiently transfected into recipient COS cells along with Dd MHC class I. The WEHI killing assay was used to assess CCD2 stimulation. Dashed line represents background WEHI killing. B, A sec- ondary screen of individual colonies from the cDNA pool P20E8 trans- FIGURE 3. Genetic mapping of a cross-protective Ag and its charac- terization. A, Schematic of the alignment of a P20E8A1 cDNA insert with fected into recipient cells was performed as above. The full-length clone, by guest on October 2, 2021 P20E8A1, was selected for further study. Dashed line represents back- the endogenous ecotropic MuLV.Nested deletions C2, C3, C7, and C8 of the ground WEHI killing. C, Recipient COS cells were transfected with 1 mg P20E8A1 cDNA were generated with P20E8A1-specific primers C2, C3, of the P20E8A1 plasmid DNA or vector alone and cDNA encoding either C7, and C8, respectively. Response of CCD2Z to truncated P20E8A1 the Dd or the Kd MHC I molecule. In addition, incubation with an a-Dd constructs is indicated. B, The minimal region encoding the cross-protective blocking Ab was also used. Stimulation of CCD2 was assessed by WEHI Ag between the C7- and C8-truncated P20E8A1. The peptide epitope is killing. D, COS cells were transfected as above and assayed for stimulation highlighted in the gray box. C, CCD2Z responses to COS cells transfected of CCD2Z measured by the LacZ response. Data are representative of two with C2, C3, C7, and C8 reveal the minimal region encoding the cross- d or three independent experiments. protective Ag. D, CCD2Z responses to candidate D restricted synthetic peptides titrated on P815 cells. The peptide identities are shown below the graph. E,Dd and Ld MHC binding by GSW11 (Dd) or AH1 (SPSYVYHQF; d d d exactly the same recognition properties (Supplemental Fig. 1A). L ) was measured by RMA-S stabilization assay with D or L mAb. YF9 d d In addition, CCD2Z was stimulated by most tumor cell lines, (YGPSLYRRF) and QL9 (QLSPFPFDL) served as D and L binding apart from P815, which also did not stimulate CCD2 (Supplemental controls, respectively. Data are representative of three independent experi- ments. Fig. 1B).

The cross-reactive epitope is encoded by the endogenous coded for the optimal H-2Dd binding motif of XGPXXXXX[I,L,F], ecotropic murine leukemia virus although one sequence contained the N-terminal XGP motif Sequencing of the P20E8A1 cDNA insert revealed 99% sequence without a typical hydrophobic C terminus. We therefore synthe- identity with the final 600 bp of pol and entire env (gp90) genes of an sized the 13-mer GGPESFYCASWGC (GFC13), as well as se- endogenous ecotropic MuLV, emv-1 (Fig. 3A). The minimal se- quential C-terminal truncations, down to the 9-mer GGPESFYCA quence required to confer antigenic activity within P20E8A1 was (GSA9), and measured their ability to stimulate CCD2Z when determined by testing the functional activity of 39 deletion con- added to P815. All peptides assayed showed a degree of stimula- structs. As determined by the stimulation of CCD2Z, the antigenic tion, but one peptide, GGPESFYCASW (GSW11), stimulated peptide was encoded within the gp90 , because the C3 CCD2Z at 5- to 10-fold lower concentrations (∼10 nM) than the truncation encoding the 39 600 bp of pol failed to stimulate (Fig. 3A, other peptides assayed. Thus, GSW11 is likely to represent the 3C). Interestingly, the Ld-restricted AH1 epitope is encoded within naturally processed cross-protective tumor Ag. the same MuLV gene, gp90 (800 bp downstream of GSW11) in It is of particular note that GSW11 does not conform to the ca- reading frame +1. Further truncations indicated that the epitope was nonical Dd binding peptide, having tryptophan as the C-terminal encoded within or overlapped the 51 nucleotides represented by the anchor residue rather than the more typical isoleucine, leucine, or C7 and C8 constructs (Fig. 3B,3C). There was no contiguous se- phenylalanine. To measure binding of GSW11 to MHC class I quence in any of the three reading frames within this region that molecules, Dd-expressing TAP2/2 RMA-S cells were incubated 5052 SUPPRESSION OF TUMOR-SPECIFIC CD8+ T CELLS with peptide at saturating concentrations overnight at 26˚C. Cells were washed of unbound peptide and incubated at 37˚C. Peptide binding was measured as the rate at which MHC expression was lost at the cell surface: a commonly used surrogate measurement of peptide off-rate. As a further comparison, binding of the previously characterized AH1 Ag was assessed using Ld-expressing RMA- S cells. GSW11 was found to have a very rapid half-life for stabi- lization of Dd (20 min) and was faster than a second known Dd epitope, YGPSLYRRF (YF9) (26), which had a half-life of 50 min (Fig. 3E). AH1 binding to Ld had a slower half-life still of 60 min, yet this was significantly faster than that of the control high-affinity peptide QLSPFPFDL (QL9), derived from P1 mastocytoma cells (25), which had a half-life of .120 min. Surprisingly, when we made C-terminal variants of GSW11 to include either the “classical” anchor variants I or F, neither of these, GSI11 or GSF11, showed improved affinity for Dd (half-life = 20 min; Supplemental Fig. 2). This finding indicated that the unstable of the interaction with Dd was not due to the C-terminal tryptophan residue.

GSW11 is generated in multiple tumor cell lines Downloaded from We next wanted to confirm that GSW11 was endogenously gen- erated in tumors recognized by the cross-protective CTL response. To achieve this, we generated peptide extracts from live tumor cells, fractionated them by reverse-phase HPLC, and assayed for the presence of GSW11 in the fractionated mixture by its ability to http://www.jimmunol.org/ stimulate CCD2Z. Calibration with synthetic peptides indicated FIGURE 4. Endogenous processing of the cross-protective Ag. A, GSA9 that differential identification of GSA9 and GSW11 peptides was and GSW11 peptides were fractionated by HPLC and fractions assessed possible (Fig. 4A). The profile obtained from CT26 peptide extracts for the presence of CCD2Z-stimulating activity, using P815 as APCs. The revealed one significant peak that coeluted with synthetic GSW11 arrows highlight the retention times of GSA9 and GSW11 peptides. (Fig. 4B). A minor peak eluting ahead of GSW11 was shown to “Mock” indicates fractions collected in an identical run with sample buffer be an oxidized adduct (or dimer) that probably formed during the alone prior to the peptides. B–E, The naturally processed cross-protective extraction process, because treatment of peptide extracts before Ag coelutes with GSW11. CT26 (B), CT26/GM-CSF (C), A20 (D), and and following fractionation with DTT, or treatment of cells with MCFL (E) peptide extracts were fractionated by HPLC and fractions assayed for CCD2Z activity, as above. Data are representative of three methyl methanethiosulfonate (which binds to free sulfhydryl independent experiments. by guest on October 2, 2021 groups on cysteine side chains and prevents interaction with other sulfhydryl groups) prior to extraction, resulted in loss of this peak and recovery of a single peak corresponding to the synthetic tracellular staining after 12 d. This schedule allowed us to GSW11 (Supplemental Fig. 3). Extraction of peptides using a measure T cell responses in Treg-replete, tumor-bearing mice— freeze/thaw method also led to the disappearance of the additional even though the majority of these would eventually succumb to minor peak. We were able to determine the relative quantity of tumor growth. In the presence of Tregs, we were able to detect GSW11 generated by different tumor cell lines by comparing the GSW11-specific T cells in only 4 of 11 mice investigated, with only activity of the recovered natural peptide with synthetic GSW11. ∼0.04% of CD8 T cells being GSW11 specific in the four mice Thus, we estimated ∼500 pM GSW11 was generated per 106 that responded. These were not considered genuine anti-GSW11 CT26 cells; 750 pM/106 cells from CT26/GM-CSF (Fig. 4C); and responses because they failed to expand when stimulated in vitro only 10 pM/106 cells from A20 (Fig. 4D). MCFL cells were ob- (Supplemental Fig. 5). By contrast, all mice mounted a strong re- served to generate GSW11 at ∼5-fold lower quantity than were sponse to AH1, with 0.2–1% of CD8+ T cells being AH1 specific CT26 (∼100 pM/106 cells; Fig. 4E). However, we were unable to (Fig. 5A). This AH1-specific response is nevertheless insufficient to detect GSW11 peptide in extracts from Renca and P815 tu- reject the tumor. A similar response is seen in mice vaccinated with mor cell lines (Supplemental Fig. 4). These results revealed that irradiated CT26/GM-CSF. However, a different response profile GSW11 could be detected only in tumor cell lines that strongly was observed in Treg-depleted mice. In this case, all mice con- stimulate either CCD2 or CCD2Z T cells. It is important to note tained GSW11-specific CD8+ T cells, with an average of ∼1.5%, that the amount of GSW11 generated in the different tumor cell representing an ∼300-fold increase in numbers compared with lines did not correlate to the level Dd cell surface expression (data Treg-replete mice. We also observed a 2- to 3-fold elevation of not shown). the anti-AH1 response in Treg-depleted mice (Fig. 5A). Overall,

+ GSW11-specific T cells were seen to be codominant with AH1- Anti-GSW11 and anti-AH1 CD8 T cells are differentially specific T cells (∼1.5% versus ∼1.3%). A similar response pattern suppressed by Tregs was seen in CT26/GM-CSF primed in Treg-depleted mice. Codo- Having identified GSW11 as the source epitope of a cryptic re- minance between AH1- and GSW11-specific T cells was also seen sponse to CT26, we wanted to determine whether GSW11-specific at an earlier time point, day 8; however, the responses were much CD8+ T cells are important in the cross-protective responses ob- lower (data not shown). Because Treg-depleted mice reject tumor, served in the absence of Tregs; to achieve this, we investigated the correlation of protection could be either the appearance of new CD8+ responses in BALB/c mice inoculated with CT26 in the specificities (exemplified by GSW11), broadening of the response presence and absence of Tregs. Spleens and draining lymph nodes to allow interclonal synergy, or enhancement of the immunodo- were assayed for GSW11- and AH1-specific CD8+ T cells, using in- minant AH1 response. The last option would seem unlikely, given The Journal of Immunology 5053 Downloaded from FIGURE 6. AH1-specific CD8 T cells are functionally identical when primed in the presence or absence of Tregs. A, At 12 d after CT26 chal- lenge, AH1-specific CD8 T cell responses from Treg-replete (left)or -depleted (right) BALB/c mice were measured with intracellular IFN-g staining. The mean fluorescence intensity of IFN-g–producing AH1-spe- cific CD8 T cells is indicated on the dot plots. B, The comparative mean

fluorescence intensity of AH1-specific CD8 T cells from CT26-challenged http://www.jimmunol.org/ Treg-depleted or -replete BALB/c mice was assessed. Results are the mean 6 SEM of six mice from two separate experiments. NS, p = 0.22. C, Following in vitro stimulation with AH1 pulsed irradiated BALB/c splenocytes, specific lysis of CT26 cells by AH1-specific CD8 T cells was measured. Data are representative of two independent experiments.

FIGURE 5. GSW11-specific CD8 T cells are observed only in Treg- CT26-stimulated T cell cultures from Treg-depleted CT26- depleted mice. A, Treg-depleted or -replete BALB/c mice were challenged immunized mice. In this instance, the AH1 response was not with CT26. After 12 d, CD8 T cell responses to AH1 and GSW11 were d

easily detected, with GSW11 and other non–L -restricted responses by guest on October 2, 2021 measured with intracellular IFN-g staining following in vitro stimulation with either AH1 or GSW11 peptides. B, Anti-AH1 and -GSW11 CD8 predominating. A clue to why this might be the case came from T cell responses of individual Treg-depleted mice challenged with CT26 closer examination of each response in individual mice in which are indicated by joining lines. Each data point is from one mouse, with there was a suggestion that high responders for GSW11 tended to be results pooled from three separate experiments. lower responders for AH1 and vice versa (Fig. 5B). This finding suggests a dynamic relationship between competing reactivities as they are established within the responding individual and may the small increase in AH1-specific CD8 T cells observed in Treg- represent the process that is targeted by Tregs, resulting in differ- depleted mice, but it is still possible that in the absence of Tregs, ential suppression of the two responses. T cells (including anti-AH1) develop more potent effector function. To investigate this further, we analyzed AH1-specific CD8 T cells Discussion for their level of IFN-g production in response to peptide stimu- We have used an expression cloning technique to reveal the mo- lation and their ability to kill CT26 tumor cells from both Treg- lecular identity of what we believe is a novel murine cross- depleted and -replete CT26-challenged mice. The level of IFN-g protective tumor Ag. We have previously shown that removal of produced from individual CD8 T cells derived from Treg-depleted Tregs through Ab depletion induced a robust protective immunity or -replete splenocytes was found to be the same following stim- upon subsequent CT26 tumor challenge. More importantly, the ulation by AH1 peptide (Fig. 6A,6B). Owing to the lower quantity antitumor immunity induced was found to be cross protective to of AH1-specific CD8 T cells in Treg-replete mice, we were unable challenge with histologically distinct tumors (12). In this paper, to purify enough to ascertain their ex vivo CTL killing capacity. To we describe the characterization of one of the tumor Ags re- address this, we assessed the CTL cytotoxicity of splenocyte cul- sponsible for the cross-protective immunity, GGPESFYCASW tures from Treg-depleted and -replete CT26-challenged mice (GSW11), derived from the gp90 gene of an endogenous ecotropic stimulated in vitro for 7 d in the presence of AH1. These cultures MuLV, emv-1; this has provided valuable insights into the role of showed expansion of AH1-specific CD8 T cells (Supplemental Fig. Treg suppression in the generation of a protective antitumor re- 5) and did not contain any detectable GSW11-specific CD8 T cells sponse. (data not shown). The ability of these AH1 CTL lines to kill CT26 The binding of GSW11, which does not conform to the con- target cells was found to be the same (Fig. 6C). These results sensus Dd binding motif XGPXXXXX[I,L,F] (28), to Dd was support the interpretation that the protection observed in the ab- relatively weak compared with other Dd-restricted peptides. The sence of Tregs is due to a broadening of the immune response rather precise structural basis for this is not known because substitu- than the acquisition of a more potent effector function. Inter- tion of the C-terminal tryptophan to residues that conform to the estingly, the codominance of AH1 and GSW11 responses seen in canonical Dd binding motif (isoleucine or phenylalanine) did not these ex vivo experiments was rarely recapitulated in short-term enhance binding (Supplemental Fig. 2). The extended length of 5054 SUPPRESSION OF TUMOR-SPECIFIC CD8+ T CELLS

GSW11 may be a factor, although this is likely to be a minor Being encoded within gp90, which is ubiquitously expressed in consideration because there are multiple Dd-restricted peptides BALB/c tumors, means GSW11 is a shared tumor Ag (data not that are longer than the typical 9-mers suggested (29–31). A shown and Ref. 35) and is generated in all tumor cell lines assayed further anchor residue is suggested at position 5 (28); manipula- (Fig. 3). However, the generation of a cross-protective response is tion of this residue may enhance its binding to Dd. CD8+ T cell not reciprocal; challenge with A20 in the absence of Tregs does responses to GSW11 are absent in naive mice following CT26 not lead to cross-protection against CT26 (Supplemental Fig. 6). challenge; GSW11-specific responses are observed only when One explanation is the finding that the amount of GSW11 gen- Tregs are depleted prior to challenge. This finding is in contrast erated by tumor cell lines differs significantly. CT26 generates to AH1-specific T cell responses, which are present in naive and .50 times more GSW11 than A20, resulting in lower cell surface Treg-depleted mice. The appearance of GSW11-specific T cells expression of GSW11/Dd. This is also reflected in the ability of indicates a broadening of the antitumor response in the absence of the tumor cell lines to stimulate CCD2Z, which is independent Tregs. Indeed, in the absence of Tregs, the anti-CT26 response of costimulatory molecules and thus responds in a way directly consists of at least an additional two Ags, apparent from CTL linked to the number of GSW11/Dd complexes present (Supple- clones generated from spleens of Treg-depleted CT26-challenged mental Fig. 1B). The lower level of cell surface GSW11/Dd com- mice; in addition to anti-GSW11–specific T cells, further Dd- and plexes may prevent induction of GSW11-specific T cells and thus Kd-restricted CD8+ T cell responses are observed (data not failure to induce a cross-protective response. shown). These responses are likely to account for the inability of Despite its generation of GSW11, the ability to generate anti- CCD2 T cells to be stimulated by Renca (Fig. 1C). The anti- GSW11 CD8+ T cells following CT26 challenge is observed GSW11 response is, therefore, a surrogate marker for the in- only in Treg-depleted mice. AH1-specific T cells are observed in duction of a broader antitumor response in Treg-depleted mice, all mice; therefore, differential suppression of GSW11 and AH1 Downloaded from which is not solely restricted to anti-AH1 T cells and is ultimately T cell responses is apparent in Treg-replete mice. An explanation protective. This broadening of the T cell response has also been for this differential suppression of T cell responses may be linked confirmed from spectrotyping studies (32). Depletion of Tregs in to the low affinity GSW11 has for binding to Dd. GSW11/Dd mice has been shown to result in enhancement of T cell responses complexes are very short lived, as determined by MHC binding directed to many pathogen- and tumor-derived (11–13, and from the observation that we have been unable to generate 33). These studies showed stronger responses and, in most cases, GSW11/Dd tetramers; the interaction between GSW11 and Dd is http://www.jimmunol.org/ a better outcome against or tumors. However, none of not strong enough to promote their formation. As these complexes these investigations distinguished between a quantitative and a have a short half-life at the cell surface, GSW11-specific CD8+ qualitative mechanism for improved immunity. In the majority of T cells might be expected to require a high-affinity interaction these studies, peptide-specific T cell responses were not measured, between the TCR and GSW11/Dd to reach an activation threshold. and in those that did, no new T cell reactivities were observed. In Differences in T cell avidity have been shown to correlate with fact, examination of antiviral CD8+ T cell responses directed to differential ability to secrete , such as IL-2, and may influenza A-, vaccinia virus-, or SV40-transformed cells by Haer- correlate with their dependence on them (36–38). We have ob- yfar et al. (33) identified that the greatest Treg suppression was served this difference in GSW11- and AH1-specific T cell lines. by guest on October 2, 2021 observed to the most immunodominant CD8+ T cells, which in GSW11-specific CTLs are more dependent on IL-2 for their ac- some instances altered the immunodominance of the response. Our tivation and growth in culture than are AH1-specific lines, even study is, therefore, the first to our knowledge to isolate the source though they expand more rapidly (data not shown). Because epitope of a cryptic CTL response that is uncovered by Treg ab- GSW11-specific T cells are suppressed by Tregs, the IL-2 de- lation. pendence of these CTLs may provide a mechanism of differential AH1-specific CD8+ T cells are immunodominant in naive mice suppression. Tregs have been shown to facilitate suppression of challenged with CT26, with no other CD8+ T cell response de- CTL responsiveness through acting as an IL-2 “sink,” thus in- tectable (Fig. 4; Ref. 15). CT26-challenged Treg-depleted mice hibiting their proliferation (39–41). It is likely, therefore, that in show a codominant phenotype, with anti-GSW11 T cells being situ Tregs could lower local IL-2 concentrations below a critical equivalent to those directed against AH1. However, upon closer threshold required for the activation of some T cell clones, such as examination, individual mice have a tendency toward clonal im- GSW11-specific ones, but not others, thereby resulting in differ- munodomination of either GSW11 (4 of 10) or AH1 (3 of 10), ential suppression of T cell responses within “reach” of Tregs. It with 3 of 10 mice maintaining the codominant phenotype, sug- will be interesting to identify whether the Tregs are tumor specific. gesting that competition between GSW11- and AH1-specific The emv-1 is not active in the neonatal or juvenile CD8+ T cells exists in vivo. Nevertheless, all mice elicit a GSW11 but is reactivated in 80% of mice at 6 mo of age (42). response that is at least 50 times higher in the absence of Tregs Similarly, human endogenous retroviruses (HERV) are present than in their presence. We do not know the precise mechanism within the human genome and reactivate late in life and are often underlying this competition for dominance, but we note that in expressed ectopically in human tumors (43). It is likely that T cell medium-term cultures of splenocytes from Treg-depleted CT26- responses to HERV are controlled in the same way as we have immune mice, the GSW11 response dominates, even though long- described in this paper, perhaps with the development of suppres- term cultures permit outgrowth of the AH1 response (data not sive immunity accompanying endogenous expression. Therefore, shown). Interestingly, we have found that AH1-specific lines understanding how to break these immunosuppressive networks grown from these cultures following peptide stimulation expand may offer a therapeutic avenue for HERV-expressing tumors. more slowly than similarly derived GSW11-specific lines, which may account for the dominance observed in vitro. The presence of Acknowledgments codominance between AH1- and GSW11-specific responses is an We thank Nasia Kontouli for invaluable assistance and technical support. unexpected finding. In the vast majority of cases, Ags with low MHC binding induce only subdominant T cell responses and are outcompeted by those responses directed against Ags with high Disclosures MHC binding (34). The authors have no financial conflicts of interest. The Journal of Immunology 5055

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