Cognate Nonlytic Interactions Between CD8 T Cells and Breast

Published OnlineFirst January 28, 2019; DOI: 10.1158/0008-5472.CAN-18-0387

Cancer Tumor Biology and Immunology Research

Cognate Nonlytic Interactions between CD8þ T Cells and Breast Cancer Cells Induce Cancer Stem Cell–like Properties Roland G. Stein1,2,3, Stefan Ebert1,2, Laura Schlahsa1, Claus J. Scholz4,5, Matthias Braun3,6, Petra Hauck1,2, Evi Horn1, Camelia-Maria Monoranu7, Vincent J. Thiemann1, Michael P.Wustrow1, Sebastian F. Hausler€ 1, Itsaso Montalban del Barrio1, Tanja N. Stuber€ 1,3, Matthias Wol€ ﬂ4, Johannes Dietl1, Andreas Rosenwald7,8, Joachim E. Diessner1, Achim Wockel€ 1, and Jorg€ Wischhusen1,2,3

Abstract

Targeting of tumor immune escape mechanisms holds tumor-forming capacity in immunodeficient mice. Resulting enormous therapeutic potential. Still, most patients progress tumors were characterized by enhanced cell density, higher under immune checkpoint blockade and some even become proliferation rates, and increased propensity for lymphoid hyperprogressors. To investigate how cancer cells respond to metastasis. These findings describe a widely underappreciated activated but ineffective T cells, we challenged peptide-loaded pathway for immune escape, namely immune-mediated þ MCF-7 breast cancer cells with antigen-specific CD8 T cells in dedifferentiation of breast cancer cells, which is associated which lytic granules had been destroyed by pretreatment with with profound changes in gene expression and cellular behav- Concanamycin A. Gene expression analysis after coculture ior. As the enhanced malignant potential of cancer cells after þ revealed simultaneous induction of PD-L1, IDO1, CEACAM1, nonlytic cognate interactions with CD8 T cells enables and further immunoregulatory checkpoints in breast cancer increased tumor growth and metastasis in BALB/cnu/nu mice, cells. Strikingly, we further observed gene signatures charac- the described mechanism may provide a possible explanation teristic for dedifferentiation and acquisition of pluripotency for the clinical phenomenon of hyperprogression in response markers including Yamanaka factors. Cognate interaction with to unsuccessful immunotherapy. þ nonlytic CD8 T cells also increased the proportion of stem cell–like cancer cells in a cell-to-cell contact- or (at least) Significance: This study shows that ineffective immune proximity-dependent manner in various cell lines and in responses not only fail to clear a malignancy, but can also primary breast cancer cell cultures; this induction of activate pathways in cancer cells that promote stemness and stem cell–like properties was confirmed by enhanced tumor-seeding capacity.

þ Introduction confined to anti-HER2/neu antibodies for HER2/neu tumors. However, recent clinical trials indicate that effective tumor Treatments for breast cancer typically include surgery, irra- immunosurveillance may also be achieved in patients with diation, chemotherapy, and endocrine therapy, depending on breast cancer (2), provided that low antigenicity (3) and the the respective subtype (1). Immunotherapy had long been strongly immunosuppressive microenvironment can be over- come. High PD-1 (4) and low granzyme B/GZMB expression (5) fi 1Department of Obstetrics and Gynecology, Wurzburg€ University Hospital, indicate that in ltrating T cells in breast cancer have suffered a University of Wurzburg,€ Wurzburg,€ Germany. 2Interdisciplinary Center for sustained loss of cytotoxic activity. Still, T-cell–enriched tumor Clinical Research (IZKF), Wurzburg€ University Hospital, University of Wurzburg,€ areas and the invasive margin may display elevated CD274/PD- 3 Wurzburg,€ Germany. Else-Kroner-Fresenius-Forschungskolleg€ Wurzburg€ L1 and other traits of IFNg-inducible adaptive immunoresis- € € € (EKFK), Wurzburg University Hospital, University of Wurzburg, Wurzburg, tance (6–8). Early interactions with the immune system may Germany. 4Core Unit Systems Medicine, Interdisciplinary Center for Clinical Research, Wurzburg€ University Hospital, University of Wurzburg,€ Wurzburg,€ thus have shaped these tumors (9). Germany. 5LIMES Institute, Life & Medical Science Institute, University of Bonn, In rats (10) and in mice (11), immune responses against Bonn, Germany. 6Department of Pediatrics, Wurzburg€ University Hospital, epithelial breast cancer can result in T-cell–dependent outgrowth University of Wurzburg,€ Wurzburg,€ Germany. 7Institute of Pathology, Wurzburg€ of immunoedited tumors with altered properties including 8 University Hospital, University of Wurzburg,€ Wurzburg,€ Germany. Comprehen- HER2/neu (ERBB2) antigen loss (10, 11). In breast cancer (9) € sive Cancer Center Mainfranken Wurzburg University Hospital, University of and other malignancies, immunoediting further induces epithe- Wurzburg,€ Wurzburg,€ Germany. lial-to-mesenchymal transition (EMT). This program, which is Corresponding Author: Jorg€ Wischhusen, University of Wurzburg,€ Josef- orchestrated by inflammatory stimuli (9, 11, 12), converts epi- € Schneider-Str. 4, Wurzburg 97080, Germany. Phone: 49-931-201-25291; Fax: thelial-like MCF-7 breast cancer cells into highly immunosup- 49-931-201-25406; E-mail: [email protected] pressive mesenchymal stromal-like cells (13). In melanoma, doi: 10.1158/0008-5472.CAN-18-0387 dedifferentiation upon T-cell–induced EMT became apparent by 2019 American Association for Cancer Research. loss of pigmentation (12, 14).

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Reversal of adaptive immune resistance by blocking PD-1/PD- negative bead-based magnetic selection (Miltenyi Biotec). Poly- L1 interaction (15) has major clinical impact in malignancies like clonally activated T-cell cultures were obtained from T cells or melanoma, non–small cell lung cancer, urothelial and renal cell PBMCs using the T Cell Activation and Expansion Kit (Miltenyi carcinoma, microsatellite-instable cancers, and Hodgkin lympho- Biotec). Cells were cultured in complete RPMI media supplemen- ma (16). However, even in highly mutated solid cancers, response ted with 20 U/ml rhIL2 (PeproTech) on days 3, 7, 11, and 14. rates to anti PD-(L)1 treatments remain below 40%, calling for T-cell purity (>70%) was controlled by FACS analysis. Cells were synergistic combination therapies. Moreover, in about 9% of used for coculture experiments between days 7 and 14. Natural patients (including patients with breast cancer), a paradoxical killer (NK) cells were grown on RPMI8866 feeder cells (19, 25). To acceleration of tumor growth in response to immunotherapy is obtain dendritic cells, PBMCs were allowed to adhere on plastic observed (17, 18). Understanding how surviving tumor cells for 1 hour before floating cells were removed by washing. Cells escape from and respond to activated T cells is thus critical for were then stimulated with IL4 (1,000 U/mL on days 0, 2, and 6) next-generation immunotherapies. and GM-CSF (800 U/mL on day 2, 1,600 U/mL on days 2 and 6, We previously showed that MCF-7 cells challenged with NK both from Immunotools). For maturation, 10 ng/mL LPS (Pepro- cells display enhanced tumor-forming capacity in vivo (19). This Tech) was added for 24 hours before the cells were loaded with 2.5 relates to a small subset of cells called tumor-initiating, metasta- mg/mL PRAME435-443 (NLTHVLYPV) or STEAP1292–300(293L) sis-initiating, or cancer stem cells (CSC). Their high malignant (MLAVFLPIV) peptide and used. potential is linked to stem cell–like features like unlimited self- renewal, resistance to apoptosis, and the ability to rebuild a Preparation of antigen-specific T cells against model antigens heterogeneous (tumor) tissue. As CSCs may survive chemo- and The detailed protocol has been published previously (26, 27). radiotherapy in a state of dormancy, they are held responsible for Dendritic cells (DC) from adherent monocytes were cultured in cancer relapse. Using dissociated tumor tissue from patients GM-CSF/IL4-containing medium (Cellgenix) for 72 hours. After with breast cancer, Al Hajj and colleagues found such CSCs to maturation with 100 ng/mL IL4, 800 IU/mL GM-CSF, 10 ng/mL high low reside within a CD44 CD24 cell population (20). In LPS, and 100 U/mL IFNg plus MELAN-A26–35(27L) (ELAGIGILTV) transformed breast epithelial cells, ganglioside GD2 expression peptide in a concentration of 2.5 mg/mL for 16 hours, DCs were further defines the CSC population (21). Strategies for targeting irradiated (30 Gy) and cocultured with CD45RO CD57 na€ve þ CSCs are being developed (22, 23) to improve the clinical CD8 T cells at a 1:4 ratio in medium containing 5% AB serum management of breast cancer. Little, however, is known about and 10 ng/mL IL21. On days 3, 5, and 7, fresh medium, IL7, and the effects of cancer immunotherapy on CSCs (24). While IL15 were added. All peptides were obtained from JPT. successful targeting of existing CSCs is desired, immune-medi- T-cell clones against the model antigens PRAME435-443 or ated dedifferentiation of previously differentiated cancer cells STEAP1292–300(293L) were generated from healthy donors. HLA- þ could lead to hyperprogression. A02:01 T cells (including na€ve cells).were primed and expanded To characterize changes in breast cancer cells induced by T cells, using mature DCs pulsed with 2.5 mg/mL PRAME435-443 we analyzed gene expression changes after challenge with cognate or STEAP1292–300(293L) peptide. After purification with APC- þ nonlytic antigen-specific CD8 T cells. Induction of key factors for conjugated dextramers (Immudex), T-cell clones were selected adaptive immune resistance like IDO1 and PD-L1 confirmed the by limiting dilution. Cells were seeded in 96-well plates and suitability of our experimental approach. Remarkably, stem cell– cultured with 2 105 feeder cells/well (PBMCs from different, associated gene expression patterns were also induced. Implan- HLA-mismatched donors at a 1:1:1 ratio) and OKT3 (30 ng/mL, tation of immunologically challenged MCF-7 breast cancer cells OrthocloneTM, Janssen-Cilag). Cells were cultured in CellGro DC in nude mice thus resulted in earlier tumor development and medium (CellGenix) with 5% human serum (Biochrom), 30 enhanced lymphogenic metastasis. IU/ml IL2, and 5 ng/mL IL15 (PeproTech). After 14 days, cells were restimulated with OKT3 and expanded for at least 10 days with irradiated feeder cells. MHC multimer staining (Immudex) Materials and Methods was used to check for antigen-specific (MELAN-A26–35(27L), Cell culture STEAP1292–300(293L), PRAME435-443) T cells. For further purifica- Cell lines were obtained from the Deutsche Sammlung fur€ tion, if required, Dextramer–APC conjugates (Immudex) and Mikroorganismen und Zellkulturen and cultured as recom- magnetic anti-APC Beads (Miltenyi Biotec) were used. mended. PlasmoTest (Invivogen) was used tri-monthly to exclude contamination with Mycoplasma. Identity of MCF-7, SK-Br-3, and Coculture and reporter gene experiments with tumor and MDA-MB-231 cells was confirmed by DNA fingerprinting. Pri- immune cells mary tumor cells were isolated from the ascites of patients with Tumor cells were detached with Accutase (Sigma Aldrich) when breast cancer as approved by the local ethics committee. Patients approximately 70% confluent. A total of 2 105 MCF-7 cells/well gave written informed consent. After centrifugation, cells were were cultured in a 6-well plate. For better discrimination, MCF-7 washed with PBS and cultured in complete RPMI with 10% FCS, cells destined for FACS analysis were stained with cell prolifera- penicillin (100 IU/mL), streptomycin (100 IU/mL), and 0.02% tion dye eFluor670 (eBioscience) before T cells were added at a sodium pyruvate solution (Gibco). T-cell to cancer cell ratio of 1:1 or 2:1, respectively. Technical replicates were always included. To inhibit their lytic capacity, T Preparation of polyclonal T cells, natural killer cells, and cells (or NK cells) were pretreated for 2 hours with 2 mmol/L dendritic cells Concanamycin A (CMA). Prior to coculture, free CMA was Whole blood was obtained from healthy volunteers. Peripheral removed by washing with medium. MCF-7 cells were treated blood mononuclear cells (PBMC) were isolated by density gra- with IFNg (1,000 IU/mL, PeproTech) for 24 hours to induce þ dient centrifugation (Biochrom). CD8 T cells were isolated via MHC I (HLA-A2) upregulation. Next, they were loaded with

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peptide (5 mg/mL STEAP1292–300(293L),PRAME435-443,or Quantitative real-time PCR analysis MELAN-A26–35(27L)) for 24 hours. Cancer cells were then cocul- Total RNA was prepared using TRI reagent (Sigma). cDNA was tured with T cells overnight in complete RPMI1640 medium transcribed with iScript cDNA Synthesis Kit (Bio-Rad). For real- (16–24 hours). Prior to further functional assays, the superna- time PCR analysis, SYBR Select Master Mix (Thermo Fisher) and tant was discarded, cells were washed twice with PBS, and the following primers were used: Slug_frw: 50-GGGGAGAAGC- adherent cells were detached with Accutase. For FACS analysis, CTTTTTCTTG-30, Slug_rev: 50-TCCTCATGTTTGTGCAGGAG-30; all cells were used and stained as indicated. For reporter gene Snail_frw: 50-CCCCAATCGGAAGCCTAACT-30, Snail_rev: 50-GC- assays, MCF-7 cells were treated with IFNg and loaded with TGGAAGGTAAACTCTGGATTAG-30; Twist_frw:50-GGAGTCCG- peptide before being batch-transfected with reporter plasmid CAGTCTTACGAG-30, Twist_rev: 50-TCTGGAGGACCTGGTAGA- (pGL-Oct4-Luc, pGL4.14-Nanog-Luc) using FuGene Transfec- GG-30; vimentin_frw: 50-GAGAATTTGCCGTTGAAGC-30, vimen- tion reagent (Promega). On the next day, cells were distributed tin_rev: 50GCTTCCTGTAGGTGGCAATC-30; E-cadherin_frw: 50- in Opaque 96-well plates (Greiner BioOne). Twenty-four hours GTGTATGTGGCAATGCGTTC-30, E-cadherin_rev: 50TGCCCAG- 0 after transfection, CMA-treated STEAP1292–300(293L)-specificT AAAATGAAAAACG-3 . cells were added for 1–24 hours. Culture conditions were set up Data were collected on a StepOne Plus Sequence Detection in triplicates. System (Thermo Fisher Scientific) applying a two-step PCR protocol (95C for 15 seconds, 60C for 1 minute). All conditions cDNA microarray analysis of gene expression were set up in duplicate and controlled by melting curve analysis. RNA integrity of microarray samples was determined with a Relative quantification was performed by the DDCt method using Bioanalyzer 2100 (Agilent) revealing RNA integrity numbers 18S rRNA (frw:50-CGGCTACCACATCCAAGGAA-30, rev:50- GCT- between 8.8 and 9.7 (with 10 being the best possible value). For GGAATTACCGCGGCT-30) as reference. RNA amplification, cDNA generation, fragmentation, and label- ing, the GeneChip WT PLUS kit (Affymetrix) was used. Sample Clonogenicity assay hybridization to Human Transcriptome Arrays 2.0 (Affymetrix) Following coculture or cell sorting on a FACSAria III (BD was performed using a Fluidics Station 450 (Affymetrix); micro- Biosciences), 100 viable CD3 cells per well, as measured by array signals were detected with a GeneChip Scanner 3700 G FACS analysis, were seeded in 6-well plates (Corning). After about (Affymetrix). Microarray read-out was processed in R version 10 days, the spheroids had reached a size allowing visual inspec- 3.3.2; briefly, raw data stored in CEL files were read, background tion and could thus be counted manually. As MCF-7 spheroids corrected, normalized, and subjected to gene-level expression gradually become more adherent, we selectively focused on values using the Bioconductor package "oligo" version 1.38.0. nonadherent spheroidal colonies. Expression changes between analysis groups were quantified as log2 fold changes (logFC) with absolute logFCs of at least 1 being In vivo tumor formation assay classified as strong change. Functional categorization and enrich- Peptide-loaded MCF-7 cancer cells were cocultured overnight þ ment analysis of strongly changed genes was performed with the with antigen-specific CD8 T cells as described above. T cells were package "clusterProfiler" version 3.2.11; protein–protein interac- removed by gentle washing with PBS. Adherent cells were tion network analysis and definition of highly connected sub- detached with Accutase. A total of 103 or 104 cells were resus- clusters was done with package "STRINGdb" version 1.18.0. pended in 25 mL RPMI1640, mixed 1:1 with Matrigel (Corning) Microarray data were deposited at Gene Expression Omnibus in and inoculated into the mammary fat pad of CAnN.Cg-Foxn1nu entry GSE108236. mice. Mice inoculated with untreated MCF-7 cells served as controls. Tumor growth at the injection site as well as the size Flow cytometric analysis and cell sorting of regional subcutaneous lymph nodes in mm was measured Cells were detached with Accutase and blocked with 2% FCS twice per week. In accordance with local guidelines, animals in PBS. The following antibodies were used for flow cytometry: suffering from bite injuries were removed from the experiment. anti-CD44-PE (clone 2BJ18, BioLegend), anti-CD24-FITC All other mice were sacrificed after 48 days in the pilot study (clone SWA-11), anti-CD3-PE, anti-CD4-FITC, anti-CD8-FITC (3 mice/group) or in the more comprehensive experiment (n ¼ 7 (clones UCHT-1, EDU-2, HIT8a, Immunotools). Dead cells mice/group) after 11 weeks. No signs of impairment by the were excluded by costaining with 7-aminoactinomycin D (7- growing tumors were apparent. Tumors, regional subcutaneous AAD, Sigma). Cells were analyzed on either a FACSCalibur flow lymph nodes, and selected organs (brain, liver, lung, heart) cytometer (BD Biosciences) or an Attune Acoustic Focusing were explanted. Tumor size and weight were measured fol- Cytometer (Life Technologies). Where indicated, MHC Class I lowed by formalin fixation and embedding in paraffin. Samples and II molecules were blocked using 10 mg/mL of either W6/32 were analyzed as 4-mm slices stained with hematoxylin/eosin (class I) or Tu39€ (class II) antibodies. For cytotoxicity assays, (Carl Roth). Animal experiments had been approved by the control or peptide-loaded MCF-7 cells were cocultured with Institutional Animal Care and Use Committee (#AZ 55.2- effector cells at a 1:2 ratio for 18 hours prior to FACS-based 2531.01-123/13). identification of 7-AADpositive cells. For T-cell activation assays, T cells were likewise cocultured with peptide-loaded MCF-7 IHC analysis cells for 18 hours. Then, they were stained with anti-CD8a-PE- Four-micron–thick tissue sections were cut, deparaffinized, and Cy7 and anti-CD137-Super Bright600 (clones SK1 and 4B4-1, stained with hematoxylin & eosin according to the standard eBioscience) or treated with brefeldin A (eBioscience), followed protocols. Following deparaffinization and retrieval in citric acid, by surface staining with anti-CD8a-PE-Cy7 and intracellular tissue sections were treated with liquid coverslip (LCS, Ventana staining with anti-IFNg-FITC (Clone 4S.B3, BioLegend) after Medical Systems, Roche) and stained for estrogen receptor (Clone 6hours. SP1, Ventana), progesterone receptor (clone 1E2, Ventana), Ki67

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(clone MIB-1 Dako), or Cytokeratin 7 (CK7, bs-1610R-Biotin, were specifically or primarily deregulated in noncognate cocul- Bioss) using a BenchMark ULTRA IHC/ISH Staining module. For tures. Cognate interactions, in contrast, induced stronger detection, Ultra View Universal DAB Detection Kit was used for changes in TNFa/NF-kB signaling, IFNg response, IL2/STAT5 ER and PR staining (Roche) while Liquid DAB þ Substrate signaling, and inflammatory response genes. Hallmark signa- Chromogen System with ADVANCE HRP was used for Ki67 (both tures for allograft rejection, the complement cascade, late and from Dako). Slides were counterstained with hematoxylin. early estrogen response, apoptosis, G2–M cell-cycle checkpoint, IL6/JAK/STAT3 signaling, the p53 pathway, E2F targets, type I Statistical analysis IFN response, the mitotic spindle, and epithelial-to-mesenchy- Microarrays were meant to be hypothesis-generating and thus mal transition (EMT) were only found upon cognate interac- only controlled internally and by testing related, though not tion (Fig. 2A; Table 1). Immunoregulatory genes including identical conditions. All other in vitro experiments were performed indoleamine-2,3-dioxygenase IDO1, the granzyme B inhibitor at least three times with similar results. Summit software (Beck- SerpinB9/PI9, the TIM-3–interacting protein CEACAM1/ man Coulter) and Attune cytometric software (Applied Biosys- CD66A, TNFSF14/LIGHT, the mucin MUC4, and CD274/ tems) were used to analyze FACS data. ImageJ (Wayne Rasband, B7H1/PD-L1 (37–42) were primarily induced in response to NIH, Bethesda, MD) was used to analyze staining intensity in cognate interaction with T cells (Fig. 2B). Thus, known med- immunofluorescence and to count colonies. Unless indicated iators of adaptive immune resistance could be identified by otherwise, significance was assessed by two-sided unpaired Stu- analyzing nonlytic antigen-specific interactions. Strikingly, dent t test. P values <0.05 are indicated as P < 0.01 ( and ) and CHEA and ENCODE analyses, which connect gene expression P <0.001 (). changes to associated transcription factors revealed that pluripotency-related factors were rather selectively activated by cognate interaction. In addition to the Yamanaka factors Sox2, Results KLF4, and OCT4/POU5F1 (43), NANOG, STAT3, TP63, cDNA Microarray analysis reveals marked changes in cancer cell GATA2, RELA, and RUNX (Fig. 2C and D) were also identified gene expression upon cognate interaction with nonlytic, via increased expression of target genes. Notably, all these þ antigen-specific CD8 T cells transcription factors are associated with cellular differentiation Any experimental setting to analyze adaptive immune resis- or dedifferentiation (44). Likewise, changes in E2F targets, JAK/ tance in vitro must meet two requirements: Target recognition is STAT3 signaling, glycolysis, and EMT found by hallmark needed to boost IFNg production in cytotoxic T cells and to induce analysis (Fig. 2A) point toward significant changes in stem the full range of defense mechanisms in cancer cells. Target cell cell–related properties. Because of the use of largely killing- killing must, however, be limited to enable further analysis of deficient T cells, these changes in gene expression cannot be target cells. Uncoupling of tumor cell recognition and killing may ascribed to selective survival of more immunoresistant subsets also occur in the tumor microenvironment where cytotoxic effec- of cancer cells. Instead, induction of pluripotency-related tran- tor mechanisms are often blunted (28–31). We simulated this scripts by coculture with cognate T cells appeared likely. þ behavior by using antigen-specific CD8 T-cell clones that had þ been pretreated with the vacuolar type H -ATPase inhibitor Nonlytic immune cell interactions increase pluripotency gene Concanamycin A. As MCF-7 cells are quite resistant toward expression in cancer cells FasL (32), destruction of cytotoxic granula by pretreatment with MCF-7 cells are a well-established model for research on Concanamycin A (33, 34) resulted in greatly reduced lysis of MCF- breast cancer stem cells. Having found that cognate interactions þ 7 targets by cognate T cells and by NK cells (Fig. 1A-C). Induction with killing-deficient CD8 T cells induce gene signatures of CD137/4-1BB surface expression (Fig. 1D) and intracellular associated with pluripotency (Fig. 2C and D), we sought to IFNg production (Fig. 1E) upon T-cell activation were, in contrast, validate these microarray-based analyses. We thus performed unimpaired. Hence, this experimental set-up allows for analysis of luciferase-based reporter gene assays from cognate or noncog- viable tumor cells after cognate interactions with activated nate cocultures between peptide-loaded MCF-7 cells and immune effector cells. STEAP1292–300(293L)-specific T cells. While induction of OCT4- During an initial hypothesis-generating part of the project, and NANOG-responsive reporter genes was moderate and microarrays were performed from immunologically challenged transient, statistically significant changes at early time points breast cancer cells. Particularly interesting results were obtained confirmed the array-based findings(Fig.3A).Becauseacquisi- from MCF-7 cells that had been loaded with STEAP1292–300(293L) tion of stem cell–like features in breast cancer cells has previ- or PRAME435-443 peptide before being challenged overnight ously been linked EMT (11), we also validated the induction of with primed tumor-reactive (26) Concanamycin A–treated EMT-related genes using qRT-PCR (Fig. 3B). While Snail was þ STEAP1292–300(293L)-specificCD8 T cells (35). In this setting, consistentlyinducedbycognateandtosomeextentalsoby the same cells engage in cognate or noncognate interactions noncognate interactions, Slug and Twist mRNA levels were only (depending on the added peptide), which ensures maximum induced upon cognate interaction and not in all assays. This comparability. For gene expression analysis, challenged MCF-7 may indicate that upregulationonmRNAleveloccursvery cells were purified by magnetic bead-based removal of T cells transiently and is not the main regulatory mechanism for these from the cocultures. IFNg-treated, peptide-loaded MCF-7 cells transcription factors. The transcriptional downstream targets were included as controls. As expected, the number of signif- vimentin and E-cadherin were, however, strongly regulated across icantly deregulated genes was greatly increased upon cognate 5 assays. Hence, cognate interactions with killing-deficient T cells interaction (613 vs. 76). Hallmark enrichment analysis (36) may not only promote adaptive immune resistance, but also nevertheless showed that gene sets implicated in mTORC1 induce stem cell–like traits like the ones found in highly malig- signaling, cholesterol homeostasis, glycolysis, and hypoxia nant cancers (45).

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Figure 1. Treatment with Concanamycin A inhibits the lytic capacity of NK and cytotoxic T cells without preventing T-cell activation. A and B, MCF-7 breast cancer cells were stained with Cell Proliferation Dye eFluor 670 before being seeded into 6-well plates at a density of 2 105 cells/well. MCF-7 cells were treated with IFNg þ for 24 hours before being loaded with either PRAME435-443 (NLTHVLYPV) or STEAP1292–300(293L) (MLAVFLPIV) peptide. STEAP1292–300(293L)-specific CD8 T cells that had either been pretreated or not with CMA were then added for 16 hours. The proportion of 7-AAD-positive breast cancer cells was assessed by flow cytometry. Representative FACS plots are shown in A, while a statistical analysis across four independent experiments is displayed in B. C, MCF-7 breast cancer cells were stained with Cell Proliferation Dye eFluor 670 before being seeded into 12-well plates at a density of 1 105 cells/well. A total of 2 105 polyclonal NK cells that had either been pretreated or not with CMA were added to each well. After 16 hours, the proportion of dead tumor cells was measured by flow cytometric analysis using 7-AAD. MCF-7 cells that had not been challenged with NK cells were used as controls. P values were calculated by two-sided unpaired Student t test from four independent experiments. D, Using the same coculture settings as in A and B, induction of CD137/4-1BB on T cells was assessed by flow þ cytometry. The filled gray curves show the signal obtained from unstimulated STEAP1292–300(293L)-specific CD8 T cells that did not express CD137/4-1BB (as þ confirmed by isotype controls). The black curves in the overlay diagrams represent STEAP1292–300(293L)-specific CD8 T cells that had been pretreated or not with CMA prior to coculture with MCF-7 cells presenting STEAP1292–300(293L) or PRAME435-443 peptides (n ¼ 3). E, Using the same experimental set-up as in A, B,and þ D, STEAP1292–300(293L)-specific CD8 T cells were stained for intracellular IFNg expression (n ¼ 3).

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Figure 2. Cognate interactions with nonlytic T cells induce signatures characteristic for adaptive immune resistance and for tissue regeneration in MCF-7 breast cancer cells. Dye-labeled MCF-7 cells presenting either mismatched

PRAME435-443 or matched STEAP1292–300(293L) peptide were cocultured with CMA–treated þ STEAP1292–300(293L)-speciﬁc CD8 T cells. After 16 hours of coculture, tumor cells were puriﬁed, RNA was extracted, reverse-transcribed, and microarray analysis was performed using Affymetrix Human Transcriptome Arrays 2.0. MCF-7 cells that had also been loaded with peptide (but not cocultured) served as controls. For each condition, a single array with approximately ten probes per exon was performed. A, Functional pathway analyses using Hallmark gene categories (36) are presented in Table 1. False

discovery rates were -log10(P) transformed and plotted. An amplitude of 5 thus corresponds to a false discovery rate of 105. Enrichment statistics were connected by lines, forming proﬁles for cognate (light gray area) and noncognate (dark gray area) interactions. B, A subset of immunomodulatory genes was selected and their respective expression level after coculture is indicated for conditions with (STEAP) or without (PRAME) antigen recognition. C, Transcription factor activity under coculture conditions was inferred from the number of signiﬁcantly deregulated transcription factor targets. Shown are the respective in silico analyses based on ChIP Enrichment Analysis (ChEA; ref. 49) and on the algorithms provided by the Encyclopedia of DNA Elements (ENCODE; ref. 50; D). The total number of response genes for each transcription factor is shown in brackets. Data were obtained from a single high-density cDNA array.

Coculture with activated nonlytic T cells increases the stem cell– T cells had, in contrast, little effect. This enrichment for CSCs like population in MCF-7 breast cancer cells was not only observed both with a STEAP1292–300(293L)-specific We had previously observed that MCF-7 breast cancer cells T-cell clone (Fig. 3C and D), but also with a PRAME435-443- high low with a CD44 CD24 CSC phenotype preferentially survive specific T-cell clone and with polyclonal MELAN-A26–35(27L)– coculture with NK cells and HER2-specific antibody (19). How- specific T cells, while STEAP1292–300(293L)-specificTcellshada ever, to obtain an enrichment of CSCs in the absence of target negligible effect on MCF-7 cells loaded with mismatched anti- cell lysis, an increase in the absolute number of CD44high gen (Fig. 3E). Hence, it does not seem to be a unique property CD24low CSC is required. Flow cytometry indeed revealed an of a specific T-cell clone. The more stem cell–like behavior of induction of CD44highCD24low CSC upon coculture with Con- MCF-7 cells with a CD44highCD24low phenotype was further canamycin A–pretreated antigen-specific T cells (Fig. 3C and confirmed by clonogenicity assays performed from FACS-sorted D). Noncognate interactions with mismatched antigen-specific cells (Fig. 3F).

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Table 1. Functional characterization of genes regulated during cognate and noncognate interaction Hallmarks regulated by cognate (STEAP/STEAP) and noncognate (STEAP/PRAME) interaction False discovery rates Hallmark Cognate interaction Noncognate interaction TNFa Signaling via NF-kB 6.04 10 28 0.002 Hypoxia 1.89 1012 9.24 1023 Glycolysis 0.029 1.28 109 IFNg Response 4.75 106 0.009 IL2 STAT5 Signaling 1.22 105 0.031 Inﬂammatory response 7.87 105 0.031

Hallmarks regulated by cognate (STEAP/STEAP) interaction only False discovery rates Hallmark Cognate interaction Noncognate interaction Allograft rejection 1.50 107 0.26 Complement 7.87 105 0.33 Estrogen response (late) 0.0002 0.63 Estrogen response (early) 0.0005 0.63 Apoptosis 0.001 0.45

G2–M Checkpoint 0.001 0.63 IL6 JAK STAT3 Signaling 0.001 0.12 P53 pathway 0.001 0.33 E2F targets 0.009 0.63 IFNg Response 0.016 0.33 Mitotic spindle 0.017 0.63 Epithelial-to-mesenchymal transition 0.029 0.33

Hallmarks regulated by noncognate (STEAP/PRAME) interaction only False discovery rates Hallmark Cognate interaction Noncognate interaction mTORc1 Signaling 0.05 1.20 107 Cholesterol homeostasis 0.30 2.62 105 NOTE: Differentially expressed genes were tested for overrepresentation in Hallmark gene categories (36) using hypergeometric tests. False discovery rates were determined from nominal P values across all tested categories.

CSC induction by cognate interaction with antigen-specificT the coculture experiments described in Fig. 3. As allogeneic cells is cell-to-cell contact- or proximity-dependent tumor cell recognition leads to much less killing than cognate þ In melanoma cells, a cytokine-driven dedifferentiation has recognition by primed tumor-reactive CD8 T-cell clones, we been reported (12). The requirement for antigen recognition tested whether allogeneic coculture experiments could lead to shown in Figs. 3C–E could thus simply be due to the triggering similar effects in the absence or presence of Concanamycin A. of cytokine production upon T-cell receptor engagement. To test Infact,anincreaseinCD44highCD24low CSC was also observed þ the requirement for cell-to-cell contact, further assays were per- after allogeneic coculture with polyclonal CD8 T cells þ formed in transwell chambers where STEAP1292–300(293L)- (Fig.4A).PretreatmentofpolyclonalCD8 TcellswithCMA or PRAME435-443-peptide loaded MCF-7 cells were seeded improved the robustness of the experimental system without into both the top and the bottom well while Concanamycin being always required (Fig. 4B). Induction of CD44highCD24low þ A–treated matched or mismatched STEAP1292–300(293L)-or, cells by CMA-treated or untreated polyclonal CD8 T cells in respectively, PRAME435-443-specific T cells were added to bothcasestranslatedintoahighlysignificant increase in colony one compartment only. This crossover transwell coculture setting formation (Fig. 4C). In line with the need for cognate interac- þ suggested that induction of CSCs requires cognate recognition and tion with CD8 T cells, induction of CD44highCD24low MCF-7 þ þ works best with direct cell-to-cell contact (Fig. 3G and H). However, breast cancer cells by polyclonal CD4 and CD8 T cells could when STEAP1292–300(293L)-presenting dendritic cells were added, be abrogated by the blocking anti-MHC class I antibody W6/32. high low € induction of CD44 CD24 CSC by STEAP1292–300(293L)- Antibody-mediated blockade of MHC class II with Tu39 had, in specific T cells was also observed when MCF-7 cells presented the contrast, no effect (Fig. 4D). Having explored CSC induction in mismatched antigen (Fig. 3I). Short-lived scanning interac- the luminal A-type–like MCF-7 cell line, we wondered whether tions (46), recognition of very low levels of endogenously expressed the concept of immune-mediated reprogramming might also (or transferred) antigen, or close proximity between tumor cells and apply to other subtypes of the disease. We hence performed optimally activated T cells sensing their antigen on dendritic cells similar coculture assays with the HER2/neu-amplified cell line may thus bypass the need for interaction with antigen-loaded SK-BR-3, with triple-negative MDA-MB-231 breast cancer cells tumor cells. and with primary ascites-derived breast cancer cell cultures. These assays revealed that induction of CD44highCD24low cells Immune-induced CSC induction can also be observed with by polyclonally activated T cells is not restricted to the MCF-7 polyclonal allogeneic T cells and in primary breast cell line. Effects were again more pronounced when T cells had cancer cells been pretreated with CMA (Fig. 4E). Consistently significant Given the remarkable killing capacity of primed tumor- differences, however, were only observed with primary ascites- þ reactive CD8 T-cell clones, Concanamycin A was required for derived breast cancer cells.

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In vivo, immunologically challenged MCF-7 breast cancer cells cocultured overnight with CMA-treated STEAP1292–300(293L)- show enhanced capacity for formation of primary tumors and speciﬁc T cells. T cells were removed from the cocultures and lymphoid metastases 103 or, respectively, 104 tumor cells were injected in CAnN.Cg- CSC-like properties are best tested in vivo. Thus, IFNg-stimulated Foxn1nu mice. In a pilot experiment (n ¼ 3 mice per group), tumor and STEAP1292–300(293L) or PRAME435-443-loaded MCF-7 cells were cells were injected contralaterally in both sides of the mammary fat

Figure 3. Cognate interaction with CD8þ T cells induces stem cell–like properties in MCF-7 breast cancer cells. A, pRL-CMV and luciferase-based reporter plasmids for Oct-

4 or NANOG activity were cotransfected into STEAP1292–300(293L) or PRAME435-443 peptide-loaded MCF-7 cells, which were then cocultured with CMA-treated STEAP1292–300(293L)-speciﬁc T cells. Oct-4 reporter gene activity was assessed after 1 hour of coculture, NANOG after 2 hours. Normalized values for luciferase activity in control or immune-challenged tumor cells were pooled from three independent experiments. B, STEAP1292–300(293L) or PRAME435-443 peptide-loaded MCF-7 cells were cocultured or not with CMA-treated STEAP1292–300(293L)-speciﬁc T cells. After 16 hours, T cells were removed before RNA from tumor cells was extracted and reverse-transcribed. Expression of EMT markers Snail, Twist, Slug, E-cadherin, and vimentin was assessed by SYBR Green-based qRT-PCRand

quantiﬁed according to the DDCt method using MCF7 control cells as reference. Statistical signiﬁcance is indicated for the comparison between cognate and noncognate interactions. Compared with the untreated control, all EMT markers were highly induced by cognate interaction while noncognate interactions only

upregulated Snail and vimentin. A representative experiment is shown (n ¼ 3). C, MCF-7 cells presenting either mismatched PRAME435-443 or matched þ high low STEAP1292–300(293L) peptide were cocultured with CMA–treated STEAP1292–300(293L)-specific CD8 T cells. The proportion of CD44 CD24 breast cancer cells was assessed by flow cytometry after staining with fluorescence-labeled antibodies. A representative experiment is shown. D, Results of 9 independent FACS experiments corresponding to C are shown. E, MCF-7 breast cancer cells were treated with IFNg (to upregulate MHC I) and subsequently loaded with either þ PRAME435-443 or MELAN-A26–35(27L) peptide. PRAME435-443, MELAN-A26–35(27L) and STEAP1292–300(293L)-specific CD8 T cells were treated with CMA (to inhibit target cell killing) before being added to the peptide-loaded cancer cells. After overnight coculture, CD44 and CD24 expression was analyzed by FACS and the respective percentages of CD44highCD24low MCF-7 CSCs were determined for the various cocultures. F, CD44highCD24low and CD44highCD24normal/high MCF-7 cells were sorted on a FACS Aria III and seeded at 100 cells/well in low-adherence cell culture plates. On day 18, cells were stained with crystal violet and the number of colonies was counted (6 wells per group). A representative experiment is shown. G, MCF-7 cells were seeded into both chambers of a transwell slide þ where they were treated with IFNg and loaded either with PRAME435-443 or STEAP1292–300(293L) peptide. PRAME435-443- or STEAP1292–300(293L)-specific CD8 T cells were added to the top and bottom compartment of the transwell slide to have cocultures in matched (bottom chamber) and mismatched configuration in the same transwell slide. After removal of T cells, the percentage of CD44highCD24low CSC was determined by flow cytometry as in C (n ¼ 3). An overview across all transwell experiments (minimum n ¼ 3, maximum n ¼ 6 per condition) is provided in H. I, A transwell experiment was set up as in G. However, while only 4 MCF-7 cells (2.5 10 cells/well) were seeded in the bottom chamber, the top chamber contained equal numbers of peptide-loaded MCF-7, STEAP1292–300(293L)- loaded dendritic, and STEAP1292–300(293L)-specific T cells. After 16 hours, MCF-7 cells from both compartments were analyzed as in C. Results were pooled from three independent experiments.

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pads. When the animals inoculated with 104 tumor cells were sacrificed on day 48, tumors were significantly larger on the side where tumor cells from cognate cocultures had been injected (Fig. 5A, P ¼ 0.03). In the subsequent experiment, mice (n ¼ 7 animals per group) were again injected with 103 or, respectively, 104 MCF-7 cells that had been cocultured or not with cognate or noncognate T cells. On day 28, tumors were clearly palpable in all except one mouse inoculated with MCF-7 cells from matched cocultures. In the other groups, tumor development was delayed (Fig. 5B, compare also Fig. 6C). This again supports the presence of more tumor-initiating cells after antigen-specific þ cognate interaction with functionally impaired CD8 Tcells. On day 42, when most of the mice injected with 104 cells had developed tumors (and just before the firstanimalshadtobe sacrificed for nontumor-related reasons), tumors formed from MCF-7 cells after cognate interaction still showed a nonsignificant trend toward a larger size (Fig. 5C, P ¼ 0.12). Kaplan– Meier analysis (Fig. 5D) confirms that inoculation with a limiting number of tumor cells results in earlier tumor formation when MCF-7 cells have undergone cognate interactions with nonlytic T cells (P ¼ 0.022 for 103, P ¼ 0.35 for 104 injected cells, analyzed by Mantel–Cox test). When the mice injected with 104 cells were sacrificed after 11 weeks, tumors had formed in the vast majority of animals. While "matched" tumors still þ showed a slight tendency toward more Ki67 cells (P ¼ 0.13), tumors were phenotypically similar. Also estrogen and progesterone receptor expression were unaltered (Fig. 5E). Represen- tative pictures from tumors formed by MCF-7 cells after cognate or noncognate interaction are displayed in Fig. 5F.

þ Nonlytic cognate interaction with CD8 T cells enhances formation of lymph node metastases In the pilot study, animals were contralaterally inoculated with 104 tumor cells from "matched" or "mismatched" cocultures. When these mice were sacrificed on day 48, enlarged lymph nodes were only observed on the side where tumor cells from cognate cocultures had been injected (Fig. 6A). Mice from the subsequent study were only injected with a single tumor. When these animals were sacrificed after 11 weeks, ipsilateral and contralateral inguinal lymph nodes were collected and analyzed for the presence of CK7-positive breast cancer cells. Representative stainings are shown in Fig. 6B. Strikingly, 4 of 5 evaluable mice injected with 103 MCF-7 cells after cognate coculture displayed a highly þ CK7 contralateral lymph node while this was not found in any of the other groups. Likewise, ipsilateral lymph nodes were Figure 4. more strongly CK7-positive when MCF-7 cells had interacted high low Polyclonal allogeneic T cells can also induce clonogenic CD44 CD24 in with cognate T cells prior to inoculation. Control tumors that MCF-7 and other cancer cell lines. A, MCF-7 cells were cocultured with bead- purified activated allogeneic T cells that had been pretreated or not with had not been cocultured showed, in contrast, poorer tumor take CMA. The proportion of CD44highCD24low breast cancer cells was assessed by (P ¼ 0.022, compare Fig. 5D) and lesser lymph node involvement. flow cytometry after staining with fluorescence-labeled antibodies. A representative experiment is shown in A, an analysis across three W6/32 or the MHC Class II blocking antibody Tu39€ was added to the independent experiments is provided in B. To adjust for the different baseline coculture impairing the T-cell–cancer cell interaction. FACS analysis for levels displayed by the respective controls statistics were calculated by two- CD44highCD24low CSC was performed as described previously. The sided paired Student t test. Statistical analysis was done by two-sided paired respective percentages of CSCs are presented compared with control Student t test to adjust for the different baseline percentage of coculture conditions (n ¼ 3). Significance was calculated versus control CD44highCD24low cells displayed by the respective controls on different days. conditions without antibody. E, The Her2-expressing SK-BR-3, the basal-like C, MCF-7 cells were cocultured with bead-purified activated allogeneic CD8þ MDA-MB-231, and primary cells from malignant ascites from a patient with T cells that had been pretreated or not with CMA. After removal of T cells breast cancer were challenged with labeled and (where indicated) CMA- by washing, 100 MCF-7 cells per well were seeded in 6-well plates. The treated T cells. After removal of T cells, tumor cells were stained for CD44 number of spheroids was counted after 10 days and the number of colonies and CD24 expression and the percentage of CD44highCD24low CSCs was formed from 100 cells is indicated (n ¼ 3). D, MCF-7 cells were challenged determined. Dead cells were excluded by 7-AAD. P values were calculated with polyclonal T cells as described above. The MHC Class I blocking antibody versus controls without T cells (n ¼ 3).

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Figure 5. þ Nonlytic cognate interaction with CD8 T cells enhances the tumor-forming potential of MCF-7 cells. A, MCF-7 cells presenting either STEAP1292–300(293L) or þ 4 PRAME435-443 peptide were challenged with STEAP1292–300(293L)-specific CD8 T cells. Contralateral injections (10 tumor cells each) were administered into the mammary fat pad of BALB/cnu/nu mice (n ¼ 3). The animals were thus simultaneously implanted with both groups of differently pretreated MCF-7 cells at opposite sides. Animals were sacrificed on day 48, tumor volumes were determined by caliper measurement, and compared by two-sided unpaired Student t þ test. B–F, MCF-7 cells presenting either STEAP1292–300(293L) or PRAME435-443 peptide were challenged or not with STEAP1292–300(293L)-specific CD8 T cells. T cells were removed by washing. The 103 or 104 tumor cells were then injected into the mammary fat pad of BALB/cnu/nu mice (n ¼ 7 mice per group, one tumor per mouse). B, The number of mice showing palpable tumors is indicated for the different groups on day 28. C, Tumor volumes are displayed as measured by caliper at day 42, that is, just before the first animals had to be taken out of the experiment for nontumor-related reasons. D, The number of tumor-free mice per group is shown as a Kaplan–Meier plot (P ¼ 0.022 when mice were injected with 103 cells, P ¼ 0.35 for inoculation with 104 cells, as analyzed by Mantel–Cox test). When mice were finally sacrificed after 11 weeks, tumors were resected and stained (E and F) for estrogen receptor, progesterone receptor, and Ki-67 expression. Stainings were quantified on the basis of the percentage of positive cells. False discovery rates as calculated by multiple t tests are indicated (E). Representative images from tumors formed after noncognate or cognate interaction are shown in F. H&E, hematoxylin and eosin.

Differences in CK7-positive cells in lymph nodes were statistically tion may, however, also induce cytokine-driven adaptive significant between controls and tumors after cognate interaction, immune resistance (6). Such resistance and immune-inhib- while tumors after noncognate interaction showed intermediate itory cues from the tumor microenvironment may result in þ CK7 lymph node colonization with nonsignificant differences nonlytic interactions between immune cells and target cells. þ toward either the control or the cognate interaction group In vitro, however, activated antigen-specificCD8 T cells (Fig. 6C). No distant metastases were detected in lung, liver, induce death of target cells too efficiently to allow for further spleen, or myocardium. investigation. To analyze adaptation processes, we thus pretreated matched or mismatched antigen-specific T cells with Concanamycin A, which destroys their cytotoxic granules. Discussion T cells thus become transiently incapable of lysing their Cancer immunotherapy seeks to reinvigorate impaired target while other functions like IFNg production remain immune functions in patients with cancer. Immune activa- fully intact (Fig. 1).

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Figure 6. Nonlytic cognate interaction with CD8þ T cells enhances formation of lymph node metastases. Animals inoculated with tumor cells as described in Fig. 5A were further assessed for lymph node metastases. A, MCF-7 cells presenting either STEAP1292–300(293L) or PRAME435-443 peptide were challenged or not with þ þ STEAP1292–300(293L)-specific CD8 T cells. The tumor cells that had undergone a noncognate or cognate interaction with CD8 T cells as well as the control cells were then injected into the mammary fat pad of BALB/cnu/nu mice (n ¼ 3). Mice were sacrificed on day 48. The ipsilateral inguinal lymph nodes corresponding to the respective contralateral tumors were resected and lymph node volume was measured. B, Mice bearing a single tumor (n ¼ 7 mice per group) were sacrificed after 11 weeks (compare Fig. 5B–F). Ipsi- and contralateral inguinal lymph nodes were formalin-fixed, paraffin-embedded, sliced (4 mm), and stained for human CK7. The resulting cytoplasmic DAB staining is shown. Size bars, 50 mm. C, MCF-7 breast cancer cells were loaded with mismatched PRAME435-443 or matched þ STEAP1292–300(293L) peptide before being cocultured for 16 hours with CMA–treated STEAP1292–300(293L)-specific CD8 T cells. Afterwards, T cells were removed by washing. MCF-7 cells that had only been loaded with peptide were used as control. The 103 tumor cells were injected in Matrigel into the mammary fat pad of BALB/cnu/nu mice (7 mice per group, one tumor per mouse). After 11 weeks, mice were sacrificed and ipsilateral inguinal lymph nodes were resected and stained for cytokeratin 7 (CK7). Mice that could not be evaluated or that had to be removed from the experiment at an earlier time point (one mouse in the control group, two mice in the "matched" group) were censored. The following score was applied: the value 0 was assigned when no tumor had grown; 1 indicates primary tumor growth in the absence of lymph node metastases; 2 stands for single CK7þ cells in lymph nodes of tumor-bearing mice; 3 was given when the proportion of CK7þ cells was 1%–3%. Stronger CK7þ infiltration of lymph nodes was rated as 4. Using a nonparametric Kruskal–Wallis, paired comparisons were performed both for the ipsilateral and the contralateral lymph node. H&E, hematoxylin and eosin.

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Microarrays showed that cognate nonlytic interactions with have acquired ways to inhibit target cell lysis could become þ CD8 T cells not only induce mediators of adaptive immune hyperprogressors under immunotherapy (17, 18). Understanding resistance (Fig. 2B), but also pluripotency-associated genes in the conditions that may promote such an undesirable outcome MCF-7 breast cancer cells (Fig. 2C and D; Table 1). Reporter gene could thus help in selecting patients for specific immunothera- assays, qRT-PCR, and FACS-based analyses confirmed induction pies. Unraveling the signals involved in immune-mediated dedif- of EMT and stem cell–like traits (Figs. 3 and 4). ferentiation of cancer cells may also reveal new targets for improv- The identified hallmarks showed a very low likelihood for ing cancer immunotherapy. Further exploration of the described false discoveries (Table 1; ref. 36). Still, only some of the genes effects thus holds great promise. in the respective pathways were induced. Others may have increased their activity without being significantly induced on Disclosure of Potential Conflicts of Interest mRNA level. Nevertheless, the interplay of numerous partly R.G. Stein reports receiving other commercial research support from Else- activated pathways implies a well-orchestrated cellular pro- Kroener-Foundation and IZKF Wuerzburg University. T.N. Stueber reports receiving other commercial research support from Else-Kroner-Fresenius gram, which may constitute a pathway of its own. Mechanis- fi fi and has received speakers bureau honoraria from Roche Pharma and P zer. tically, antigen-speci c recognition of peptides presented on J. Wischhusen reports receiving a commercial research grant from Daiichi MHC class I is required. Still, a bystander effect, where imme- Sankyo (TaneDS grant). No potential conflicts of interest were disclosed by diately adjacent cells acquire some stem cell–like traits cannot the other authors. be ruled out. Inoculation of control and immunologically challenged MCF-7 Authors' Contributions in nude mice showed that interactions with Concanamycin A– Conception and design: R.G. Stein, J. Wischhusen þ treated antigen-specific CD8 T cells had not functionally inca- Development of methodology: R.G. Stein, S. Ebert, P. Hauck, E. Horn, M. Wol€ fl, pacitated MCF-7 cells. Instead, tumor cells had become more J.E. Diessner, J. Wischhusen aggressive after cognate interaction. Initially, this became appar- Acquisition of data (provided animals, acquired and managed patients, fi provided facilities, etc.): R.G. Stein, S. Ebert, L. Schlahsa, M. Braun, ent by signi cantly increased tumor take and earlier tumor onset P. Hauck, E. Horn, V.J. Thiemann, S.F. H€ausler, I.M. del Barrio, M. Wol€ fl, after inoculation of a limiting number of tumor cells (Fig. 5), J. Dietl, A. Rosenwald which reflects the enhanced frequency of CD44highCD24low Analysis and interpretation of data (e.g., statistical analysis, biostatistics, tumor-initiating cells. After prolonged outgrowth of nonlimiting computational analysis): R.G. Stein, S. Ebert, C.J. Scholz, C.-M. Monoranu, cell numbers tumors became, however, more similar, suggesting V.J. Thiemann, M.P. Wustrow, A. Rosenwald, J. Wischhusen that cognate interactions with T cell increased the number of Writing, review, and/or revision of the manuscript: R.G. Stein, S. Ebert, C.J. Scholz, M. Braun, S.F. H€ausler, T.N. Stueber, M. Wol€ fl, A. Rosenwald, tumor-initiating cells without altering their phenotype. Still, J. Wischhusen tumor cells that had been challenged with killing-deficient cog- Administrative, technical, or material support (i.e., reporting or organizing þ nate CD8 T cells showed a greater tendency to metastasize into data, constructing databases): R.G. Stein, S. Ebert, M. Braun, P. Hauck, E. Horn, lymph nodes (Fig. 6C). M.P. Wustrow, S.F. H€ausler, M. Wol€ fl, A. Rosenwald, A. Wockel,€ J. Wischhusen þ In vitro, this induction of CSCs was achieved by various CD8 T Study supervision: R.G. Stein, J. Wischhusen cells. Inhibitory signals that may mimic the effect of Concana- Other (examination and evaluation of immunohistological stainings): C.-M. Monoranu mycin A in vivo remain to be systematically investigated. Impor- high low tantly, induction of the clonogenic CD44 CD24 CSC-like Acknowledgments phenotype does not require Concanamycin A, but may also been R.G. Stein was supported by a clinician scientist fellowship from the Else seen when untreated T cells show poor killing capacity. Flow Kroner€ Research program for Interdisciplinary Translational Immunology cytometric analyses further suggest that primary breast cancer cells fundedbytheElseKroner-Fresenius€ Stiftung. R.G. Stein received further show a similar behavior. grant support from the Wurzburg€ Interdisciplinary Center for Clinical Taken together, these findings suggest that T cells capable of Research (IZKF). J. Wischhusen received a TaNeDS grant from Daiichi Sankyo. Technical assistance with regard to cell sorting was provided by cognate interaction with tumor cells, but incapable of their lysis, Christian Linden at the IZKF core facility for confocal microscopy and flow can reshape cancer cells' fate toward stemness by activating a cytometry–based cell sorting. Microarrays were run by Margarete Gobel€ at complex cellular dedifferentiation program. While this is most the IZKF core facility for systems medicine. We thank Prof. Stefan Stevanovic undesirable in cancer tissue, it could, however, be beneficial (Tubingen,€ Germany) for kindly providing W6/32 and Tu39€ antibodies. when nonmalignant tissues are damaged. Recruited immune cells SWA-11 antibody was a kind gift of Prof. Peter Altevogt (German Cancer recognizing somatic cells as "self" could then promote tissue Research Centre, Heidelberg, Germany). regeneration. The costs of publication of this article were defrayed in part by the payment of In solid cancers, however, where tumor-promoting inflamma- page charges. This article must therefore be hereby marked advertisement in tion was proposed as hallmark (47, 48), induction of stemness accordance with 18 U.S.C. Section 1734 solely to indicate this fact. may contribute to adaptive resistance, interfere with successful cancer immunotherapy, and convert immunotherapy into a dou- Received February 13, 2018; revised August 6, 2018; accepted January 23, ble-edged sword: Treatment-refractory or recurrent tumors that 2019; published first January 28, 2019.

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www.aacrjournals.org Cancer Res; 79(7) April 1, 2019 1519

Cognate Nonlytic Interactions between CD8+ T Cells and Breast Cancer Cells Induce Cancer Stem Cell−like Properties

Roland G. Stein, Stefan Ebert, Laura Schlahsa, et al.

Cancer Res 2019;79:1507-1519. Published OnlineFirst January 28, 2019.

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