Type I IFN Receptor Signaling Controls IL7-Dependent Accumulation and Activity of Protumoral IL17A-Producing Gdt Cells in Breast Cancer Emmanuel C

Published OnlineFirst October 25, 2017; DOI: 10.1158/0008-5472.CAN-17-1416

Cancer Tumor Biology and Immunology Research

Type I IFN Receptor Signaling Controls IL7-Dependent Accumulation and Activity of Protumoral IL17A-Producing gdT Cells in Breast Cancer Emmanuel C. Patin1, Daphnee Soulard1,Sebastien Fleury2,3, Maya Hassane1,4, David Dombrowicz2,3, Christelle Faveeuw1,Francois¸ Trottein1, and Christophe Paget1,5

Abstract

The protumoral activity of gdT17 cells has recently emerged tumorbedofapeculiarsubsetofgdT17 cells displaying a in a wide variety of solid malignancies, including breast cancer. CD27 CD3bright phenotype (previously associated with the þ These cells exert their detrimental functions by promoting invariant Vg6Vd1 TCR). Interestingly, this effect was indirect tumor growth, angiogenesis, and subsequent metastasis devel- and partially relied on the IFNAR1-dependent control of IL7 opment. However, the intratumoral factors that regulate the secretion, a factor that triggers proliferation and activating biology of gdT17cells within the tumor microenvironment are functions of deleterious gdT17 cells. Our work therefore iden- less well understood. Here, using two experimental models of tifies a key role of the type I IFN/IL7 axis in the regulation of breast cancer, we reinforced the concept that tumor-infiltrating intratumoral gdT17-cell functions and in the development of gdT17 cells are endowed with protumoral functions, which primary breast tumor growth and metastasis. promote tumor progression and metastasis development. More Significance: Tumor-derived IL7 can represent a therapeutic importantly, we demonstrated a critical role for type I IFN target to prevent accumulation of immune cells endowed with signaling in controlling the preferential accumulation in the potent protumoral activities. Cancer Res; 78(1); 195–204. 2017 AACR.

Introduction Among these cell subsets, tumor-infiltrating IL17A-producing gdT(gdT17) cells represent interesting candidates (3). Mouse Despite significant advances in treatment and earlier detection, gdT17 cells are characterized by the constitutive expression of cancer remains one of the most devastating diseases in industri- the orphan nuclear receptor RORgt (4) and the absence of the alized countries. Over the last decades, immunology has gained CD27 marker, a thymic regulator that conditions the cytokine bias considerable attention in the complex discipline of oncology. (IFNg vs. IL17A production) of gdT cells in the periphery (5). Indeed, it is now well established that leukocyte composition Moreover, many reports indicate that TCR chain usage is linked within tumors is considered significant for patient prognosis in a þ þ with specialized functions (6). In this context, Vg4 and Vg6 T wide variety of cancers, and therefore some immune populations cells have been described to preferentially produce IL17 (7); a are highly regarded by clinicians as new biomarkers for prognosis phenomenon that primarily relies on the acquisition of a specific and therapeutic success (1, 2). functional programming during their development in fetal thy- mus (8). Immune system evolves to avoid functional redundan- cies, and therefore it is likely that gdT17 cells bearing either Vg4or Vg6 TCR have unique properties. We recently reported that bright 1Universite de Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR levels of CD3 signals on gdT cells can be used as a surrogate marker 8204-CIIL-Center for Infection and Immunity of Lille, France. 2Universite de Lille, to specifically analyze the prototypic IL17 producer subset þ INSERM, Institut Pasteur de Lille, CHU Lille, U1011, EGID, Lille, France. 3European Vg6Vd1 T cells (9, 10). Genomic Institute of Diabetes, Lille, France. 4Laboratoire Microbiologie Santeet Over the past decade, numerous reports have consensually Environnement, Ecole doctorale en Sciences et Technologies/Faculte de Sante attributed protumoral functions to gdT17 cells in various exper- 5 Publique, Universite Libanaise, Tripoli, Liban. Universite de Tours, INSERM, imental transplantable or inducible models of cancer including Centre d'Etude des Pathologies Respiratoires (CEPR), UMR 1100, Tours, France. liver and lung carcinomas (11, 12), melanoma (13), breast cancer Note: Supplementary data for this article are available at Cancer Research (14), fibrosarcoma (15), and peritoneal/ovarian cancer (16). Online (http://cancerres.aacrjournals.org/). Similar observations have also been reported in tumor-bearing Current address for E.C. Patin: Retroviral Immunology, The Francis Crick patients such as colorectal cancer (17). Moreover, the frequency of Institute, London, United Kingdom. infiltrating gdT cells in breast cancer patients is a relevant predic- Corresponding Author: Christophe Paget, Institut Pasteur de Lille, 1 rue du Pr tive marker of clinical outcome; associated with poor prognosis Calmette, Lille 59000, France. Phone: 333-2087-7339; E-mail: and relapses (18). gdT17 cells exert their detrimental functions [email protected] through interactions with neighboring cells from the tumor doi: 10.1158/0008-5472.CAN-17-1416 environment, including suppressive immune cells (e.g., mye- 2017 American Association for Cancer Research. loid-derived suppressor cells, regulatory T cells, M2 macrophages)

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and endothelial cells. This ultimately leads (i) to the inhibition of conditions. Animal studies have been conducted in accordance þ the protective CD8 T-cell response and (ii) to the initiation with an Institutional Animal Care and Use Committee. All animal of angiogenesis, subsequent increase of tumor growth, and metas- work conformed to the French "Comite d'Ethique en Experimen- tasis development. tation Animale" (C.E.E.A. 75; #00357.01) and committee The factors that regulate gdT17 cell accumulation and activity guidelines. within the tumor have been mainly attributed to Th17-driving cytokines such as IL1b, IL6, IL23, and TGFb (11, 15). IL7 has been Reagents recently involved in selective expansion and functions of IL17A- Monoclonal antibodies against mouse CD45 (APCCy7- or producing innate-like T cells, including type I natural killer T AF700-conjugated), CD3 (Pacific Blue-conjugated), TCRd (NKT)17 and gdT17 cells (19, 20). Interestingly, the level of IL7 (PerCpCy5.5-conjugated), TCRb (FITC-conjugated), Vg1 (APC- increases with tumor progression in peritoneal exudates from conjugated), Vg4 (PECy7-conjugated), CD27 (PECy7-conjugat- ovarian cancer-bearing mice (16). Moreover, levels of IL7 have ed), CD127 (FITC- or PECy7-conjugated), IL17A (PE-conjugat- been associated with poor prognosis in patients with prostate ed), CD69 (AF700-conjugated), CD44 (PE-conjugated), and cancer (21, 22) and positively correlated with tumor aggres- appropriated isotype controls were purchased from BioLegend, siveness in patients with breast cancers (23). In view of this, it BD Pharmingen, and eBioscience. PBS-57 glycolipid-loaded and is tempting to speculate that the putative regulatory functions of -unloaded control CD1d tetramers (APC-conjugated) were from tumor-derived IL7 on gdT17 cell biology can somehow explain the National Institute of Allergy and Infectious Diseases Tetramer the phenotypes stated above. Facility (Emory University, Atlanta, GA). Propidium iodide was Type I IFNs are composed of several proteins (IFNa, IFNb, purchased from BD Pharmingen. Mouse monoclonal anti-IL17A and atypical IFNs) that exert major antimicrobial functions (24). (clone 17F3) and anti-IL7 (clone M25) antibodies and their There is also accumulating evidence, indicating that type I IFNs are respective isotype controls [MOPC-21 (mouse IgG1) for anti- pivotal cytokines in cancer immunosurveillance and response to IL17A and MCP-11 (mouse IgG2b) for anti-IL7] were purchased anticancer therapies (25). Type I IFNs have been shown to regulate from Bio X Cell. Recombinant mouse IFNb and IL7 were pur- gdT-cell response. Although type I IFNs increase activity of cyto- chased from R&D Systems. Recombinant mouse IL1b and IL23 toxic/IFNg–producing gdT cells (26, 27), they constrain IL17 were purchased from Peprotech. Phorbol 12-myristate 13-acetate production by gdT17 cell subsets in various models of infection (PMA) and ionomycin were from Sigma-Aldrich. (28–30). For instance, this effect is associated with the suppressive activity of type I IFN-induced IL27 (31–36). However, the regu- Mouse tumor models latory role of type I IFNs on gdT17-cell activity in cancer is The C57BL/6 mouse breast carcinoma cell line AT-3 was currently unknown. obtained from Trina Stewart (Griffith University, Brisbane, Aus- We have here analyzed the role and regulatory mechanisms of tralia; ref. 38), 4T1.2 tumor cells were kindly provided by Robin gdT17 cells in cancer immunosurveillance using two syngeneic Anderson (Peter MacCallum Cancer Centre, Melbourne, Austra- mouse models of breast cancer. Similar to the human disease, lia). Cells were cultured and maintained in DMEM supplemented both cell lines enable growth of highly metastatic primary tumors with 10% FCS, 2mmol/L L-glutamine, penicillin/streptomycin (37). We demonstrate that gdT cells are a prime source of IL17A and sodium pyruvate and 55nmol/L 2-mercaptoethanol. For within the tumor and that T-cell receptor (TCR) d-deficient mice as mouse cancer models, mice were inoculated subcutaneously with well as IL17A depletion (deficient mice or neutralizing mAb 5 105 AT-3 or 4T1.2 cells on the right flank. For tumor growth treatment) significantly impaired tumor growth. Analysis of studies, tumors were monitored every 2/3 days using a caliper. intratumoral gdT-cell subsets indicated that IL17 production was almost exclusively due to a subset harboring intense CD3 signals. Quantification of metastatic burden This subset has recently been associated in the lung tissue with the Metastasis of AT-3 cells were determined in sections of the lung þ expression of the clonal Vg6Vd1 TCR (9, 10). Accumulation of stained with hematoxylin and eosin. The number of metastases CD3bright gdT cells in the tumor bed was largely controlled by type per section was counted. I IFNs. Interestingly, blockade of type I IFN signaling in host led to an increase level of the IL17-promoting factor IL7 within the Cytokine detection by enzyme-linked immunosorbent assay tumor. Early neutralization of IL7 significantly delayed tumor At time-point as indicated, tumors were weighted and collected growth in both experimental syngeneic tumor models. Moreover, in cold PBS until processing. Tumors were then transferred to glass IL7 depletion in interferon a/b receptor 1 (IFNAR1)-deficient tubes and homogenized in 150 mL PBS. Tubes were centrifuged mice reduced IL17A production by gdT cells as well as their and supernatants were collected for ELISAs. Cytokine detections accumulation in the tumor bed. Our study identifies for the first were performed using IL17A, IL1b, IL23p19 (eBioscience), and time a cytokine network that strongly regulates the accumulation IL7 (R&D Systems) ELISA kits following the manufacturer's of protumoral gdT17 cells in breast cancer. instructions.

Flow cytometry Materials and Methods At time as indicated, tumors were harvested from mice and Mice collected in cold PBS until processing. Tumors were then excised Eight- to 12-week-old female WT C57BL/6J and WT BALB/c and finely minced, followed by enzymatic digestion for 30 to 40 mice were purchased from Janvier (Le Genest-St-Isle, France). minutes at 37C in PBS containing 1 mg/mL collagenase type VI C57BL/6J IL17A-deficient (Il17a / ), TCRd-deficient (Tcrd / ), (Sigma-Aldrich) 1 mg/mL DNase type I (Roche) and 100 ng/mL and IFNAR1-deficient (Ifnar1 / ) mice were bred in house at the Hyaluronidase (Sigma-Aldrich). Tumors were smashed and Pasteur institute of Lille. Mice were bred under pathogen-free homogenates were filtered and collected. Cells were centrifuged

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at 1,200 rpm at 4C for 10 minutes and pellets washed in PBS 2% models of breast cancer namely AT-3 (C57BL/6) and 4T1.2 FCS for direct surface marker staining or complete IMDM for (BALB/c). To this end, these tumor cell lines were implanted into intracellular staining. For intracellular IL17A detection, cells were the right flank of either Il17a / (AT-3) or IL17A–depleted incubated in complete IMDM containing Golgi Plug/Golgi Stop (4T1.2) mice. Compared with controls, a significant delay in (BD Biosciences) in presence of PMA (100 ng/mL) and ionomycin tumor growth was observed in absence or upon neutralization (1 mg/mL) for 4 hours at 37C before antibody staining. Cells were of IL17A (Fig. 1A; Supplementary Fig. S1). In addition, a decrease washed and incubated with the different antibodies for 30 min- number of spontaneous lung metastasis was numbered in AT-3 utes in PBS 2% FCS. For intracellular IL17A detection, cells were tumor-bearing Il17a / mice (Fig. 1B). Of interest, IL17A protein then washed with PBS and fixed using IC Fixation Buffer was mainly found at early-stage (day 14) of tumor development (eBioscience, CliniSciences). Fixed cells were then permeabilized in the primary tumor that may indicate a contribution for innate in permeabilization buffer (eBioscience), according to the man- components in IL17A production (Fig. 1C). To identify the ufacturer's instructions. Cells were finally stained with anti-17A cellular sources of IL17A within the tumor, infiltrating leukocytes antibody. Cells were analyzed on a LSR Fortessa (BD Biosciences). from AT3 tumor-bearing mice were analyzed 14 days after inoc- FACS analyses were performed using the FlowJo software. ulation. IL17A production by lymphocyte subsets (Supplemen- tary Fig. S2A) was analyzed by intracellular cytokine staining after Cell sorting and in vitro assay ex vivo stimulation with PMA/ionomycin. We observed that Lungs were harvested from naive mice and cell suspensions intratumoral innate-like gdT cells (43 %) rather than convention- þ were prepared as previously described (10). To purify gdT cells, al CD4 T cells (10 %) or NKT cells (9 %) represented the major lung mononuclear cells were labeled with anti-CD45, anti-CD3, contributors in IL17A production (Fig. 1D; Supplementary Fig. and anti-TCRd antibodies (conjugated as previously indicated). S2B). Remaining IL17A producers comprised other T-cell subsets Cells were sorted using an ARIA cell sorter (BD Biosciences). A (15 %) and non–T-cell populations (14 %; Supplementary Fig. total of 5 103 gdT cells were then stimulated for 20 hours with a S2B). Moreover, intrinsic capacity of IL17A production as well as þ combination of mouse recombinant IL1b/IL23 (1 ng/mL for each intensity of fluorescence in IL17A cells suggested that gdT cells cytokine) with or without mouse recombinant IFNb (20 ng/mL) had a higher intrinsic capacity to produce this cytokine compared in IMDM 10 % FCS media. Supernatants were collected for IL17A with the other intratumoral IL17A–producing cells, including þ detection by ELISA. NKT and CD4 T cells (Supplementary Fig. S2C and S2D). Interestingly, a similar tendency was observed in the 4T1.2 model RNA, cDNA, and real-time quantitative PCR 14 days post-inoculation (Supplementary Fig. S3A), which further fi RNA was extracted and purified using the NucleoSpin RNA con rmed gdT cells as a major source of IL17A in the breast tumor Extraction Kit (Macherey-Nagel) following the manufacturer's microenvironment (Supplementary Fig. S3B and S3C). Consis- instructions. The cDNA was synthesized from total RNA using tent with this, we detected a marked decrease in IL17A proteins – fi Tcrd/ the High Capacity RNA-to-cDNA Kit (Thermofisher Scientific). within the tumors of gdT-cell de cient ( ) mice (Fig. 1E). Gene expression was determined on the QuantStudio 12K Flex To assess the contribution of gdT17 cells in tumor outgrowth, we Tcrd/ Real-Time PCR System (Life Technologies) using specific primers monitored AT-3 tumor progression in controls versus 0 0 0 fi for Ifnb (5 -CAGGTGGATCCTCCACGCT-3 and 5 -CATTCAGCT- mice. Interestingly, gdT-cell de ciency was accompanied with a 0 Ifna4 0 reduced tumor progression (Fig. 1F). However, the number of GCTCCAGGAGC-3 ), (5 -TGATGAGCTACTACTGGTCAGC- 0 0 0 0 Tcrd / 3 and 5 -GATCTCTTAGCACAAGGATGGC-3 ), Il7 (5 -GCGGAC- lung metastases in mice was similar to control mice (Fig. GATCACTCCTTCTG-30 and 50-AGCCCCACATATTTGAAATTCCA- 1G). Altogether, these results suggest that IL17A mainly produced 30), and Gapdh (50-GCAAAGTGGAGATTGTTGCCA-30 and 50- by gdT cells has protumoral functions in breast cancer models. GCCTTGACTGTGCCGTTGA-30) and QuantiTect SYBR Green PCR Master Mix (Qiagen) according to the manufacturer's instructions. Type I IFN signaling restrains accumulation and activity of fi Gene expression was normalized to Gapdh. tumor-in ltrating gdT17 cells The factors that positively regulate gdT17 cell activity and proliferation have been extensively studied (3, 39). Conversely, Statistical analysis those curbing gdT17-cell functions in cancer are far less under- Data are presented as means SEM and are cumulative of two stood. Type I IFNs are endowed with potent immostimulatory to four independent experiments. Unpaired T test or one-way functions that confer them clinically relevant antitumor effects ANOVA followed by Bonferroni's post T test were used for statis- (25). The capacity of type I IFNs to constrain gdT17-cell activity tical analysis when two groups or multiple groups were analyzed, during viral and bacterial infections has recently been proposed respectively. When data did not follow a Gaussian distribution, (28–30). Interestingly, we observed high expression of Ifnb and Mann–Whitney U test or Kruskal–Wallis followed by Dunn's post Ifna4 transcripts in tumors at 14 days post-tumor inoculation T test were used for statistical analysis when two groups or multiple (Fig. 2A). To evaluate the putative role of type I IFNs on gdT-cell groups were analyzed, respectively. We used two-way ANOVA functions, AT-3 tumor cells were inoculated in mice deficient for when multiple parameters were studied. Statistical significance IFNAR1. We observed a significant increase in the frequency of was set at , P < 0.05; , P < 0.005; and , P < 0.001. tumor-infiltrating gdT cells in these animals compared to WT controls (Fig. 2B). Meanwhile, frequencies of NKT cells and þ Results conventional CD4 T cells were unchanged or decreased in this gdT cells are a prime source of protumoral IL17A in breast setting (Fig. 2B). Of great interest, accumulating intratumoral gdT cancer models cells from IFNAR1-deficient mice was mainly associated with To investigate the role of the pro-inflammatory cytokine IL17A a IL17A producer profile (Fig. 2C). However, these cells had a in tumor progression, we used two transplantable syngeneic comparable intrinsic ability to produce IL17A (Supplementary

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Figure 1. gdT cells are a major source of protumoral IL17A in AT-3 breast cancer model. A and B, A total of 5 105 AT-3 cells was injected subcutaneously into C57BL/6 WT or Il17a/ mice. A, Primary tumor growth was monitored every 2 to 3 days and areas under the curve were compared. Curves shown are representative of one experiment out of two (5–6 mice/group). Statistics were performed on cumulative data from two independent experiments and results are shown as means SEM of 10 to 12 mice/group of n ¼ 2. B, At end-point, lungs were harvested, sectioned, and metastases determined. One representative section of each group is shown on the left. Individuals and means SEM are shown on the right (n ¼ 2). C and D, A total of 5 105 AT-3 cells were injected subcutaneously into C57BL/6 WT mice. C, At time indicated, tumors were harvested and homogenates were tested for IL17A protein detection by ELISA. Results shown are the means SEM of 6 to 8 mice/group of n ¼ 2. D, After 14 days, tumors were collected. Means of cell subset proportions in overall live IL17-producing intratumoral leukocytes (IL17Aþ CD45þ þ þ þ þ cells) cells are represented on a pie chart. Leukocyte populations (CD45 ) are gated as follows: conventional CD4 T cells (CD4 tetramer CD1d/PBS57 TCRb ), NKT (tetramer CD1d/PBS57þ TCRbþ), and gdT (CD3þ TCRdþ). Results shown are the means of 15 mice/group of n ¼ 3. E–G, A total of 5 105 AT-3 cells were injected subcutaneously into C57BL/6 WT or Tcrd/ mice. E, After 14 days, tumors were collected and homogenates were tested for IL17A protein detection by ELISA. Results shown are the individuals and means SEM of 13 mice/group of n ¼ 2. F, Primary tumor growth was monitored every 2 to 3 days and areas under the curve were compared. Curves shown are representative of one experiment out of three (5–6 mice/group). Statistics were performed on cumulative data from two independent experiments and results are shown as means SEM of 17 mice/group of n ¼ 3. G, At end-point, lungs were harvested, sectioned, and metastases determined. Individuals and means SEM are shown (n ¼ 2; 13–14 mice/group); ns, not signiﬁcant; , P < 0.05; , P < 0.01; , P < 0.001.

Fig. S4A), which interestingly suggested that type I IFNs might antitumoral effects of type I IFNs, Ifnar1 / mice displayed a primarly influence the accumulation of these cells. Besides, con- slightly but significant faster progression of AT-3 tumor growth comitant analyses on the other IL17A-producing cells showed that (Fig. 2F). Altogether, these data indicate that type I IFNs control þ type I IFN signalling blockade restrained IL17A NKT cells but did the accumulation and functions of gdT17 cells in breast cancer. þ not interfere with the ability of CD4 T cells to express this cytokine (Supplementary Fig. S4B). In line with their TH17-like Type I IFNs preferentially exert its functions on tumor- phenotype, tumor-infiltrating gdT cells from Ifnar1-deficient mice infiltrating CD27 CD3bright gdT17 cells exhibited a pronounced activated/memory phenotype as judged Because gdT17 cells are composed of various subsets, we by CD69 and CD44 stainings (Fig. 2D). This increase was par- investigated in details whether or not type I IFNs could control ralleled with the detection of higher levels of IL17A protein in specific gdT17 cell subsets in the tumor microenvironment. In line the tumors of Ifnar1 / mice (Fig. 2E). Consistent with the with Fig. 2B, gdT cells in tumors of Ifnar1 / mice were mainly

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Figure 2. Type I IFN signaling negatively modulates IL17A–producing gdT cells. A, A total of 5 105 AT-3 cells was injected subcutaneously into C57BL/6 WT mice. At time indicated, tumors were collected and levels of transcripts for Ifnb and Ifna4 were determined by qRT-PCR. Means SEM of two independent experiments are shown (12 mice/group; n ¼ 2). B–F, A total of 5 105 AT-3 cells was injected subcutaneously into C57BL/6 WT or Ifnar1/ mice. B–E, After 14 days, tumors were collected and TILs prepared. B, Individuals and means SEM of the frequency of gdT, NKT, and CD4þ T cells in TILs were compared between þ þ þ groups (15 mice/group; n ¼ 3). C, IL17A-producing intratumoral gdT cells (CD45 CD3 TCRd ) are represented. A representative dot plot for each genotype þ is shown at the top (concatenate for 6 mice/group). Individuals and means SEM of the percentage of IL17A gdT within the gdT-cell population (bottom left) and total lymphocyte (bottom right) populations of 11 to 12 mice/group of n ¼ 2 are shown. D, Expression of CD69 and CD44 markers on intratumoral gdT cells was compared between groups. Means SEM of 11 to 12 mice/group of n ¼ 2 are shown. E, Tumors were harvested and homogenates were tested for IL17A protein detection by ELISA. Results shown are the individuals and means SEM of 12 mice/group of n ¼ 2. F, Primary tumor growth was monitored every 2 to 3 days and areas under the curve were compared. Curves shown are representative of one experiment out of 3 (6 mice/group). Statistics were performed on cumulative data from two independent experiments and results are shown as means SEM of 18 mice/group of n ¼ 3; , P < 0.05; , P < 0.01.

CD27 (Fig. 3A), a phenotype associated with preferential ability Ifnar1-deficient mice (Supplementary Fig. S5B) despite the fact þ to produce IL17A (5). that Vg4 T cells have important IL17A–producing capacities Compared with control AT-3 tumor-bearing mice, we observed (40). CD3bright gdT cells accounted for the majority of IL17A a significant increase of CD3bright gdT cells within the tumors of production within the gdT-cell compartment in both control and Ifnar1 / mice (Fig. 3B; Supplementary Fig. S5A) to become the Ifnar1-deficient mice (Fig. 3D). Collectively, these data support a main gdT cell subset (13.7 % vs. 58.8 %). This was parralleled with preferential role for type I IFNs in the negative control of protu- a reduced proportion of the CD3dim gdT-cell population (Fig. 3B; moral CD3bright gdT cells in breast cancer. þ þ Supplementary Fig. S5A), which include Vg1 and Vg4 T cells (Fig. 3C). In total TILs, the effect of type I IFN signaling blockade Type I IFN signaling inhibits accumulation of the IL17-driving on CD3bright gdT cells was even more striking (Supplementary cytokine IL7 in the tumor Fig. S5B). In the meantime, although their proportion within the To investigate the mechanisms involved in the regulatory gdT-cell compartment was decreased (Fig. 3C), the frequency of functions of type I IFNs on gdT17 cells, we first evaluated the þ þ both Vg1 and Vg4 T-cell subsets in total TILs was unaffected in possible direct effect of IFNb on IL17A production by gdT cells. As

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at both mRNA and protein levels, within the tumors of Ifnar1 / mice compared with their control counterparts (Fig. 4C). Since certain tumor cells have been previously shown to produce IL7 (41), we tested the ability of AT-3 in culture to secrete IL7. However, in these conditions, we failed to detect a signiﬁcant increase of IL7 production by the tumor cell line between 4 and 24 hours culture time points (Supplementary Fig. S6A). Furthermore, in vivo neutralization of IL7 in WT mice signiﬁ- cantly reduced the levels of IL17A within the tumors (Fig. 4D). In addition, IL7 neutralization led to a marked tumor growth deceleration in both AT-3– and 4T1.2-bearing mice, thus demonstrating the protumoral activity of this cytokine (Fig. 4E; Supplemen- tary Fig. S6B). In line, the number of spontaneous lung metastases was reduced in anti-IL7 mAb-treated mice (Fig. 4F). Thus, type I IFNs impair the expression of the pro-IL17A cyokine IL7 to favor tumor growth.

IL7 depletion decreases the accumulation of tumor-infiltrating gdT17 cells We next examined the potential impact of IL7 depletion on gdT17-cell homeostasis in the tumor microenvironment. Indeed, IL7 was recently shown to be a critical cytokine in the regulation and expansion of gdT17 cells (19). In the presence of IL7- neutralizing mAb, we noted a marked decrease in the percentages þ of IL17A tumor-infiltrating gdT cells in both AT-3– and 4T1.2- bearing mice (Fig. 5A; Supplementary Fig. S7A). Interestingly, IL7 depletion concomitantly caused reduction in the proportion of both CD27 and CD3bright gdT cells that clearly demonstrate the importance of IL7 in regulating gdT-cell subsets with great ability to secrete IL17A (Fig. 5A; Supplementary Fig. S7B). As previously shown in peripheral organs (Supplementary Fig. S8A; ref. 9), intratumoral CD3bright gdT cells expressed higher levels of IL7 receptor a (IL7Ra/CD127) at the cell surface compared with their CD3dim counterpart (Fig. 5B). It is also important to mention that type I IFN signalling blockade resulted in a increase expression of gdT-cell subsets to a similar extent (Fig. 5C). Nevertheless, this effect was likely to be indirect because incubation of purified CD3dim or CD3bright gdT cells with recombinant IFNb did not directly modulate CD127 expression (Supplementary Fig. S8B) whereas the positive control did (Supplementary Fig. S8C). Figure 3. Finally, we aimed to investigate the potential role of IL7 in Type I IFN signaling controls the accumulation of the IL17A-producing the type I IFN signaling-mediated control of the tumor- CD27 CD3bright gdT cell subset. A–C, A total of 5 105 AT-3 cells was fi bright / in ltrating IL17A-producing CD3 gdT-cell subset. Striking- injected subcutaneously into C57BL/6 WT or Ifnar1 mice. A, Individuals and fi ly, IL7 neutralization signi cantly decreased the percentages of means SEM of the frequency of CD27 gdT cells within the gdT-cell þ bright Ifnar1/ population were compared between groups (10–12 mice/group; n ¼ 2). IL17A as well as CD3 gdTcellsin mice (Fig. 5D). B, Comparison of intratumoral CD3dim versus CD3bright gdT cells. One Taken together, the type I IFN/IL7 axis exerts a critical role in the representative plot of 15 to 16 mice for each genotype is shown (n ¼ 3). regulation of intratumoral CD3bright gdT17-cell functions, an C, Proportions of gdT-cell subsets based on Vg expression within total gdT-cell effect that leads to the restrainement of tumor outgrowth. compartment are shown for each genotype as means (15–16 mice/group; n ¼ 3). D, Capacity of intratumoral gdT-cell subset to produce IL17A based on the CD3 phenotype in each genotype. Individuals and means SEM of subsets Discussion for each genotype is shown. n.s., not significant; , P < 0.01; , P < 0.001. Understanding the fine-tuning of immune responses in cancer is a useful goal to propose new prognosis biomarkers as well as shown in Fig. 4A, in vitro IFNb treatment failed to lower IL17A putative new therapeutic targets. In the present study, we inves- synthesis by purified gdT cells in response to IL1b and IL23, tigated the cytokine network responsible for the regulation of suggesting an indirect mode of action. tumor-infiltrating IL17A-producing gdT cells using two syngeneic We next turned to analyze the impact of type I IFN signaling models of breast cancer. Interestingly, we showed that protumoral blockade on the production of well-known gdT17-driving cyto- gdT17 cells, described herein as a major and early source of IL17A kines in the tumor tissue. While the levels of IL1b and IL23 in the tumor microenvironment during cancer progression, were proteins were not affected by type I IFN signaling disruption negatively regulated by type I IFN signaling. In addition, type I (Fig. 4B), we observed a significant increase in IL7 concentration, IFNs specifically control CD3bright gdT cells, a subset that displays

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Figure 4. Type I IFN signaling inhibits protumoral IL7. A, Purified gdT cells from the lungs of naive WT mice were stimulated in presence or not of recombinant IFNb (1,000 U/mL) and tested for IL17A production. Mean SEM of pooled data from three independent experiments is shown. B and C, A total of 5 105 AT-3 cells were injected subcutaneously into C57BL/6 WT or Ifnar1/ mice. B and C, After 14 days, tumors were harvested and homogenates were tested for the detection of transcripts or cytokines by qRT-PCR or ELISA, respectively. Results shown for IL1b and IL23 (B)andIL7(C) are individuals and means SEM of 10 to 12 mice/group of n ¼ 2 for ELISA and 16 mice/group of n ¼ 3 for qRT-PCR. D–F, A total of 5 105 AT-3 cells was injected subcutaneously into C57BL/6 WT mice and treated with an anti-IL7 (150 mg/mouse) or isotype control at day 0. D, After 14 days, tumors were collected and homogenates were tested for IL17A protein. Individuals and means SEM of 10 to 12 mice/group of n ¼ 2 are shown. E, Primary tumor growth was monitored every 2 to 3 days and areas under the curve were compared. Curves shown are representative of one experiment out of two (5–6 mice/group). Statistics were performed on cumulative data from two independent experiments and results are shown as means SEM of 10 to 12 mice/group of n ¼ 2. F, At end-point, lungs were harvested, sectioned, and metastases were numbered. Individuals and means SEM of one experiment out of two are shown in the right (8 mice/group); n.s., not significant; , P < 0.05; , P < 0.01. remarkable IL17A production capacities. Moreover, type I IFN previously shown by other groups, we demonstrated the main exerted their regulatory functions via an indirect mechanism source of IL17A in early stages of tumor progression to be gdT- involving inhibition of IL7, which was further identified as a cell–dependent in terms of both cell percentage and signal inten- critical cytokine for the accumulation of CD3bright gdT17 cells sity (14, 16). Surprisingly, even if gdT-cell deficiency led to a in tumors. significant reduction in tumor outgrowth, the number of lung Several studies have started to highlight the protumoral activ- metastasis at end-point was unchanged in comparison to WT AT-3 ities of IL17A-producing gdT cells (i.e., tumor burden on primary tumor-bearing mice. Since the gdT cell compartment comprises sites as well as metastasis and/or angiogenesis) in rodents and subsets endowed with either anti- or pro-tumoral functions (3), humans (3). Our observations confirmed previous investigations our observation might be explained by the absence of the pro- on both breast and ovarian cancer models showing deceleration tective activity of some tumorocidal gdT cells. In this context, the of tumor growth and/or decreased lung metastasis burden use of more specific animal models such as the Vg4/Vg6 / mice upon both deficiency and neutralisation of IL17A (14, 16). As that are deficient for the two major subsets of gdT17 cells (42) will

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Figure 5. IL7 induces accumulation of IL17- producing CD27CD3bright gdT cells. A, A total of 5 105 AT-3 cells was injected subcutaneously into C57BL/6 WT mice and treated with an anti-IL7 (150 mg/mouse) or isotype control at day 0. Individuals and means SEM of the percentage of IL17þ, CD27, and CD3bright subset within the gdT-cell compartment of 10 to 12 mice per group of n ¼ 2 are shown. B, A total of 5 105 AT-3 cells was injected subcutaneously into C57BL/6 WT mice. At day 14, tumors were harvested and expression of IL7Ra (CD127) on infiltrating CD3dim and CD3bright gdT-cell subsets was analyzed. Means SEM of CD127 expression (mean fluorescence intensity, MFI) for each subset from 15 mice of n ¼ 3 are shown. C, A total of 5 105 AT- 3 cells was injected subcutaneously into C57BL/6 WT or Ifnar1/ mice. TILs are prepared from d14 tumors. Modulation of CD127 levels (MFI) on intratumoral CD3dim and CD3bright gdT subsets from WT versus Ifnar1/ mice was evaluated. Means SEM from 6 mice/genotype are shown. D, A total of 5 105 AT-3 cells was injected subcutaneously into C57BL/6 WT or Ifnar1/ mice and treated with an anti-IL7 (150 mg/mouse) or isotype control at day 0. Results show individuals and means SEM of the percentage of intratumoral þ IL17 gdT cells (left) and CD3bright gdT cells of 6 to 7 mice/group of n ¼ 2; n.s., not significant; , P < 0.05; , P < 0.01.

be worth undertaking. Little is known about the importance of type I IFN-mediated control of tumor-infiltrating gdT17 cells in tumor-infiltrating NKT cells in the production of IL17A. Interest- breast cancer. ingly, we observed substantial IL17A expression in NKT cells We next investigated whether type I IFN could directly inhibit although proportions appeared to be lower than gdT cells at IL17A production by gdT cells or whether it requires inter- the early stages of tumor progression. In any case, the putative mediates. Our data indicate that recombinant IFNb does role of IL17A–producing NKT cells should be considered in not directly modulate IL17A secretion by gdT cells in response future studies. to both IL1b and IL23. Interestingly, one report showed that We demonstrated in the present study that type I IFN signaling type I IFN could downregulate both IL1b and IL23 production impedes the accumulation of gdT17A cells in the tumor micro- in human DCs in vitro as well as subsequent Th17 response environment. Although our observations are in line with previous (43). However, we observed that IFNAR1 deficiency in mice investigations indicating the pivotal role of type I IFN in medi- did not impair the secretion of both IL1b and/or IL23 in the ating negative regulation of IL17A-producing gdT cells upon tumor microenvironment, strongly indicating the involvement infections (28–30), to our knowledge, this is the first time that of another mechanism. It is well known that secretion of type I a similar mechanism is defined in the context of cancer. We herein IFNs by antigen-presenting cells leads to sequential cellular extended those previous obervations by demonstrating that type I signaling events leading to the release of immunoregulatory IFNs preferentially exert their negative control on the CD3bright cytokines such as IL27 and IL10 (44). Cao and collegues CD27 gdT-cell subset. We recently described this population to reported inhibition of IL17A response in gdTcellsbytypeI express the canonical Vg6Vd1 TCR in multiple lymphoid or non- IFNs through an IL27-dependent mechanism during post-influ- lymphoid tissues (9, 10). However, our investigations on other enza pneumococcal infection (29). Accordingly, we can not þ þ populations, including both Vg1 and Vg4 showed that exclude in our model the possibility for IL27 to act as an those gdT-cell subsets were unlikely to be controlled by type I intermediate of type I IFN-mediated impairment of gdT17A IFN. These findings add another substantial level of regulation in enrichment in tumors. The same hypothesis could be

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Regulation of Protumoral gdT17 Cells in Breast Cancer

formulated in the case of IL10, although, to our knowlegde, the axis involved in the negative control of protumoral gdT17 cells in existence of a type I IFN/IL10 regulatory axis on gdT17A cells solid tumors. Those findings could also be potentially relevant in has never been reported. the context of human breast cancer regarding that elevated Numerous studies have reported the critical role of IL7 for expression of IL7 is considered usually as a poor prognosis for the differentiation and regulation of IL17A-producing gdT cells tumor progression in patients (21–23). Considering the pivotal (19, 45–47). Especially, administration of recombinant IL7 role of IL17A in regulating tumor growth, angiogenesis, and þ tended to increase the frequency of Vg6 cells in a model of metastasis during breast cancer in the mouse model, our study ovarian cancer (16). However, we highlighted here, using a suggests that the implementation of novel combined strategies to neutralizing antibody, the protumoral role of host IL7 on breast both specifically activate type I IFN and/or neutralize IL7 activities cancer tumor growth as well as in the accumulation of CD3bright could benefit the field of immunotherapy research against solid gdT17-cell population in the tumor bed. It is likely that the tumors. This could pave the way to the design of new curative preferential effect of IL7 on CD3bright gdT cells can be simply approaches that could increase the immunotherapeutic arsenal explained by the high level of IL7Ra surface expression on this for oncologists. very subset compared with CD3dim gdT cells. Besides its role in controling IL7 secretion within the tumor microenvironment, Disclosure of Potential Conflicts of Interest type I IFNs also exert regulatory functions on IL7Ra expression No potential conflicts of interest were disclosed. on CD3bright gdT cells. This may also play a part in the mechanism defined in the present study. Authors' Contributions Importantly, our results contrast with one previous report Conception and design: E.C. Patin, F. Trottein, C. Paget showing the role of type I IFNs in the induction of IL7 during Development of methodology: E.C. Patin, C. Paget viral hepatitis (47). Several reasons could explain this apparent Acquisition of data (provided animals, acquired and managed patients, discrepancy between the two models such as differences in exper- provided facilities, etc.): E.C. Patin, D. Soulard, S. Fleury, D. Dombrowicz, imental systems, in particular the nature and location of inflam- C. Paget Analysis and interpretation of data (e.g., statistical analysis, biostatistics, mation. Solid tumors are usually associated with a strong immu- computational analysis): E.C. Patin, D. Soulard, F. Trottein, C. Paget nosuppressive environment (48) whereas virus infection leads to Writing, review, and/or revision of the manuscript: E.C. Patin, M. Hassane, a generally IFN type I–mediated protective innate response. Then, D. Dombrowicz, C. Faveeuw, F. Trottein, C. Paget variation in cellular populations as well as inflammatory status Administrative, technical, or material support (i.e., reporting or organizing could subsequently lead to distinctive signaling mechanisms data, constructing databases): E.C. Patin, D. Soulard, M. Hassane, C. Faveeuw involved in the regulation of IL7 production. In addition, the Study supervision: F. Trottein, C. Paget cellular source of IL7 could represent another important factor of modulation. Acknowledgments fi Keratinocytes, stroma, and epithelium cells have been This work was supported by the recurrent annual nancial support from INSERM. E.C. Patin was supported by a post-doctoral fellowship from described as major cellular sources for IL7, although its produc- the French Institute of Cancer (INCa). C. Paget, S. Fleury, D. Dombrowicz, tion was also reported in dendritic cells (49, 50). Interestingly, IL7 and C. Faveeuw were supported by INSERM. F. Trottein was supported by CNRS. expression was observed in tumor cells of epithelial origin (41), M. Hassane was the recipient of a doctoral fellowship from the AZM Founda- which raised the question whether breast cancer cell lines could tion. We thank the Pasteur Lille animal facility for excellent mouse husbanding. spontaneously produce IL7. Nevertheless, our in vitro studies We also thank the BICeL flow cytometry core facility for technical assistance. This suggested no detectable secretion of IL7 from AT-3 cells, although work was supported by the "Institut National du Cancer" (INCa, PLBIO14-155; recipient: C. Paget). we cannot exclude the ability of those cells to produce this cytokine upon specific conditions in culture or in the appropriate environment in vivo. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked To conclude, the development of immunotherapeutic tools to advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate fi ght solid tumors has been proved to be challenging. Then, a this fact. better understanding of the complex regulatory mechanisms involved in the immunosurveillance inside the tumor microen- Received May 18, 2017; revised September 6, 2017; accepted October 20, vironment is required. We identified an important type I IFN/IL7 2017; published OnlineFirst October 25, 2017.

References 1. Fridman WH, Galon J, Pages F, Tartour E, Sautes-Fridman C, Kroemer G. interleukin 17-producing gammadelta T cell subsets. Nat Immunol 2009; Prognostic and predictive impact of intra- and peritumoral immune 10:427–36. inﬁltrates. Cancer Res 2011;71:5601–5. 6. Bonneville M, O'Brien RL, Born WK. Gammadelta T cell effector functions: 2. Gajewski TF, Schreiber H, Fu YX. Innate and adaptive immune cells in the a blend of innate programming and acquired plasticity. Nat Rev Immunol tumor microenvironment. Nat Immunol 2013;14:1014–22. 2010;10:467–78. 3. Silva-Santos B, Serre K, Norell H. gammadelta T cells in cancer. Nat Rev 7. O'Brien RL, Roark CL, Born WK. IL-17-producing gammadelta T cells. Eur J Immunol 2015;15:683–91. Immunol 2009;39:662–6. 4. Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, et al. 8. Haas JD, Ravens S, Duber S, Sandrock I, Oberdorfer L, Kashani E, et al. The orphan nuclear receptor RORgammat directs the differentiation pro- Development of interleukin-17-producing gammadelta T cells is restricted gram of proinﬂammatory IL-17þ T helper cells. Cell 2006;126: to a functional embryonic wave. Immunity 2012;37:48–59. 1121–33. 9. Paget C, Chow MT, Gherardin NA, Beavis PA, Uldrich AP, Duret H, et al. 5. Ribot JC, deBarros A, Pang DJ, Neves JF, Peperzak V, Roberts SJ, et al. CD27 CD3bright signals on gammadelta T cells identify IL-17A-producing is a thymic determinant of the balance between interferon-gamma- and Vgamma6Vdelta1þ T cells. Immunol Cell Biol 2015;93:198–212.

www.aacrjournals.org Cancer Res; 78(1) January 1, 2018 203

Patin et al.

10. Hassane M, Demon D, Soulard D, Fontaine J, Keller LE, Patin EC, et al. 31. Pirhonen J, Siren J, Julkunen I, Matikainen S. alpha regulates Toll-like Neutrophilic NLRP3 inflammasome-dependent IL-1beta secretion regu- receptor-mediated IL-27 gene expression in human macrophages. lates the gammadeltaT17 cell response in respiratory bacterial infections. J Leukocyte Biol 2007;82:1185–92. Mucosal Immunol 2017;10:1056–68. 32. Hunter CA, Kastelein R. Interleukin-27: balancing protective and patho- 11. Ma S, Cheng Q, Cai Y, Gong H, Wu Y, Yu X, et al. IL-17A produced by logical immunity. Immunity 2012;37:960–9. gammadelta T cells promotes tumor growth in hepatocellular carcinoma. 33. Molle C, Goldman M, Goriely S. Critical role of the IFN-stimulated gene Cancer Res 2014;74:1969–82. factor 3 complex in TLR-mediated IL-27p28 gene expression revealing a 12. Hao J, Dong S, Xia S, He W, Jia H, Zhang S, et al. Regulatory role of two-step activation process. J Immunol 2010;184:1784–92. Vgamma1 gammadelta T cells in tumor immunity through IL-4 produc- 34. Patin EC, Jones AV, Thompson A, Clement M, Liao CT, Griffiths JS, et al. tion. J Immunol 2011;187:4979–86. IL-27 induced by select candida spp. via TLR7/NOD2 Signaling and 13. Carmi Y, Rinott G, Dotan S, Elkabets M, Rider P, Voronov E, et al. IFN-beta production inhibits fungal clearance. J Immunol 2016;197: Microenvironment-derived IL-1 and IL-17 interact in the control of lung 208–21. metastasis. J Immunol 2011;186:3462–71. 35. Nagai T, Devergne O, Mueller TF, Perkins DL, van Seventer JM, van Seventer 14. Coffelt SB, Kersten K, Doornebal CW, Weiden J, Vrijland K, Hau CS, GA. Timing of IFN-beta exposure during human dendritic cell maturation et al. IL-17-producing gammadelta T cells and neutrophils conspire to and naive Th cell stimulation has contrasting effects on Th1 subset promote breast cancer metastasis. Nature 2015;522:345–8. generation: a role for IFN-beta-mediated regulation of IL-12 family cyto- 15. Wakita D, Sumida K, Iwakura Y, Nishikawa H, Ohkuri T, Chamoto K, et al. kines and IL-18 in naive Th cell differentiation. J Immunol 2003; Tumor-infiltrating IL-17-producing gammadelta T cells support the pro- 171:5233–43. gression of tumor by promoting angiogenesis. Eur J Immunol 2010;40: 36. Remoli ME, Gafa V, Giacomini E, Severa M, Lande R, Coccia EM. IFN-beta 1927–37. modulates the response to TLR stimulation in human DC: involvement of 16. Rei M, Goncalves-Sousa N, Lanca T, Thompson RG, Mensurado S, Balkwill IFN regulatory factor-1 (IRF-1) in IL-27 gene expression. Eur J Immunol FR, et al. Murine CD27(-) Vgamma6(þ) gammadelta T cells producing IL- 2007;37:3499–508. 17A promote ovarian cancer growth via mobilization of protumor small 37. Beavis PA, Divisekera U, Paget C, Chow MT, John LB, Devaud C, et al. peritoneal macrophages. Proc Nat Acad Sci U S A 2014;111:E3562–70. Blockade of A2A receptors potently suppresses the metastasis of CD73þ 17. Wu P, Wu D, Ni C, Ye J, Chen W, Hu G, et al. gammadeltaT17 cells promote tumors. Proc Nat Acad Sci U S A 2013;110:14711–6. the accumulation and expansion of myeloid-derived suppressor cells in 38. Stewart TJ, Liewehr DJ, Steinberg SM, Greeneltch KM, Abrams SI. Modu- human colorectal cancer. Immunity 2014;40:785–800. lating the expression of IFN regulatory factor 8 alters the protumorigenic 18. Ma C, Zhang Q, Ye J, Wang F, Zhang Y, Wevers E, et al. Tumor-infiltrating behavior of CD11bþGr-1þ myeloid cells. J Immunol 2009;183:117–28. gammadelta T lymphocytes predict clinical outcome in human breast 39. Sutton CE, Mielke LA, Mills KH. IL-17-producing gammadelta T cells and cancer. J Immunol 2012;189:5029–36. innate lymphoid cells. Eur J Immunol 2012;42:2221–31. 19. Michel ML, Pang DJ, Haque SF, Potocnik AJ, Pennington DJ, Hayday AC. 40. Roark CL, French JD, Taylor MA, Bendele AM, Born WK, O'Brien RL. Interleukin 7 (IL-7) selectively promotes mouse and human IL-17-pro- Exacerbation of collagen-induced arthritis by oligoclonal, IL-17-producing ducing gammadelta cells. Proc Nat Acad Sci U S A 2012;109:17549–54. gamma delta T cells. J Immunol 2007;179:5576–83. 20. Webster KE, Kim HO, Kyparissoudis K, Corpuz TM, Pinget GV, Uldrich AP, 41. Roato I, Caldo D, Godio L, D'Amico L, Giannoni P, Morello E, et al. Bone et al. IL-17-producing NKT cells depend exclusively on IL-7 for homeostasis invading NSCLC cells produce IL-7: mice model and human histologic and survival. Mucosal Immunol 2014;7:1058–67. data. BMC Cancer 2010;10:12. 21. Schroten C, Dits NF, Steyerberg EW, Kranse R, van Leenders AG, Bangma 42. Okamoto Yoshida Y, Umemura M, Yahagi A, O'Brien RL, Ikuta K, CH, et al. The additional value of TGFbeta1 and IL-7 to predict the course of Kishihara K, et al. Essential role of IL-17A in the formation of a prostate cancer progression. Cancer Immunol Immunother 2012;61: mycobacterial infection-induced granuloma in the lung. J Immunol 905–10. 2010;184:4414–22. 22. Silva A, Laranjeira AB, Martins LR, Cardoso BA, Demengeot J, Yunes JA, 43. Zhang X, Jin J, Tang Y, Speer D, Sujkowska D, Markovic-Plese S. IFN-beta1a et al. IL-7 contributes to the progression of human T-cell acute lympho- inhibits the secretion of Th17-polarizing cytokines in human dendritic blastic leukemias. Cancer Res 2011;71:4780–9. cells via TLR7 up-regulation. J Immunol 2009;182:3928–36. 23. Al-Rawi MA, Rmali K, Watkins G, Mansel RE, Jiang WG. Aberrant expres- 44. MacDonald BA, Chakravarthy KV, Davidson BA, Mullan BA, Alluri R, sion of interleukin-7 (IL-7) and its signalling complex in human breast Hakansson AP, et al. Halothane modulates the type i interferon response cancer. Eur J Cancer 2004;40:494–502. to influenza and minimizes the risk of secondary bacterial pneumonia 24. McNab F, Mayer-Barber K, Sher A, Wack A, O'Garra A. Type I interferons in through maintenance of neutrophil recruitment in an animal model. infectious disease. Nat Rev Immunol 2015;15:87–103. Anesthesiology 2015;123:590–602. 25. Zitvogel L, Galluzzi L, Kepp O, Smyth MJ, Kroemer G. Type I interferons in 45. Corpuz TM, Stolp J, Kim HO, Pinget GV, Gray DH, Cho JH, et al. anticancer immunity. Nat Rev Immunol 2015;15:405–14. Differential responsiveness of innate-like IL-17- and IFN-gamma-Produc- 26. Tsai CY, Liong KH, Gunalan MG, Li N, Lim DS, Fisher DA, et al. Type I IFNs ing gammadelta T cells to homeostatic cytokines. J Immunol 2016; and IL-18 regulate the antiviral response of primary human gammadelta T 196:645–54. cells against dendritic cells infected with Dengue virus. J Immunol 46. Kasten KR, Prakash PS, Unsinger J, Goetzman HS, England LG, Cave CM, 2015;194:3890–900. et al. Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment 27. Devilder MC, Allain S, Dousset C, Bonneville M, Scotet E. Early triggering of through gamma delta T-cell IL-17 production in a murine model of sepsis. exclusive IFN-gamma responses of human Vgamma9Vdelta2 T cells by Infect Immun 2010;78:4714–22. TLR-activated myeloid and plasmacytoid dendritic cells. J Immunol 47. Hou L, Jie Z, Desai M, Liang Y, Soong L, Wang T, et al. Early IL-17 2009;183:3625–33. production by intrahepatic T cells is important for adaptive immune 28. Henry T, Kirimanjeswara GS, Ruby T, Jones JW, Peng K, Perret M, et al. Type responses in viral hepatitis. J Immunol 2013;190:621–9. I IFN signaling constrains IL-17A/F secretion by gammadelta T cells during 48. Beavis PA, Slaney CY, Kershaw MH, Neeson PJ, Darcy PK. Enhancing the bacterial infections. J Immunol 2010;184:3755–67. efficacy of adoptive cellular therapy by targeting tumor-induced immu- 29. Cao J, Wang D, Xu F, Gong Y, Wang H, Song Z, et al. Activation of IL-27 nosuppression. Immunotherapy 2015;7:499–512. signalling promotes development of postinfluenza pneumococcal pneu- 49. Gao J, Zhao L, Wan YY, Zhu B. Mechanism of action of IL-7 and its potential monia. EMBO Mol Med 2014;6:120–40. applications and limitations in cancer immunotherapy. Int J Mol Sci 30. Li W, Moltedo B, Moran TM. Type I interferon induction during influenza 2015;16:10267–80. virus infection increases susceptibility to secondary Streptococcus pneu- 50. Zarogoulidis P, Lampaki S, Yarmus L, Kioumis I, Pitsiou G, Katsikogiannis moniae infection by negative regulation of gammadelta T cells. J Virol N, et al. Interleukin-7 and interleukin-15 for cancer. J Cancer 2014;5: 2012;86:12304–12. 765–73.

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Type I IFN Receptor Signaling Controls IL7-Dependent Accumulation and Activity of Protumoral IL17A-Producing δγ T Cells in Breast Cancer

Emmanuel C. Patin, Daphnée Soulard, Sébastien Fleury, et al.

Cancer Res 2018;78:195-204. Published OnlineFirst October 25, 2017.

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