Ccl22 Diverts T Regulatory Cells and Controls the Growth of Melanoma Jared Klarquist1, Kristen Tobin2, Peyman Farhangi Oskuei1, Steven W

Published OnlineFirst September 12, 2016; DOI: 10.1158/0008-5472.CAN-16-0618 Cancer Microenvironment and Immunology Research

Ccl22 Diverts T Regulatory Cells and Controls the Growth of Melanoma Jared Klarquist1, Kristen Tobin2, Peyman Farhangi Oskuei1, Steven W. Henning1, Manuel F. Fernandez1, Emilia R. Dellacecca1, Flor C. Navarro1, Jonathan M. Eby1, Shilpak Chatterjee3, Shikhar Mehrotra3, Joseph I. Clark1,2, and I. Caroline Le Poole1,4

Abstract

T regulatory cells (Treg) avert autoimmunity, but their the clinic. In assessing this hypothesis, we observed a marked increased levels in melanoma confer a poor prognosis. To explore increase in skin Treg in mice vaccinated with Ccl22, with repetitive the basis for Treg accumulation in melanoma, we evaluated vaccination sufficient to limit Treg accumulation and melanoma chemokine expression in patients. A 5-fold increase was docu- growth in the lungs of animals challenged by tumor cell injection, mented in the Treg chemoattractants CCL22 and CCL1 in mel- whether using a prevention or treatment protocol design. The anoma-affected skin versus unaffected skin, as accompanied by observed change in Treg accumulation in this setting could not be þ infiltrating FoxP3 T cells. In parallel, there was an approximately explained by Treg conversion. Overall, our findings offered a two-fold enhancement in expression of CCR4 in circulating Treg preclinical proof of concept for the potential use of CCL22 but not T effector cells. We hypothesized that redirecting Treg delivered by local injection as a strategy to enhance the efficacious away from tumors might suppress autoimmune side effects response to immune checkpoint therapy while suppressing its caused by immune checkpoint therapeutics now used widely in autoimmune side effects. Cancer Res; 76(21); 1–11. 2016 AACR.

Introduction may be particularly important (7). Elevated expression of CCL22 is observed in diverse tumor types (8, 9). Simultaneous- T regulatory cells (Treg) suppress the function of dendritic ly elevated expression of receptors for CCL22 and other che- cells (DC), B cells, and T cells in a contact-mediated fashion (1). mokines helps to explain an overabundance of Treg (10). The When tumors develop, the expansion of Treg interfering with intratumoral vasculature will likewise affect Treg extravasation successful immune surveillance is a natural extension of this and tumor infiltration (11). process (2). The abundance of peripherally induced Treg cor- To reduce Treg abundance and support antitumor immuno- relates with enhanced tumor growth (3). New tumor types therapy, patients can be pretreated with antibodies. In practice, where progression is correlated with Treg infiltration continue this challenge has primarily been met by antibodies to the IL2 to be identified (4), and an even greater cohort of patients receptor CD25, yet such treatment also depletes activated effector will face Treg-mediated immunosuppression in the future. T cells. Limiting Treg activation through an immune checkpoint Thus, a solid understanding of intratumoral Treg accumulation blockade can also be of help (12). Finally, depletion using is important. antibodies to CCR4 may be useful. This concept is being explored An increased abundance of Treg within tumors can result using mogamulizumab infusion in a clinical trial (13). from enhanced proliferation, for example, when Tregs recog- Systemic depletion of Treg can be associated with autoim- nize antigen (3). Also, the tumor can instruct infiltrating T cells munity (14). In fact, underrepresentation of Treg is common in to convert into Treg, for example, through thioredoxin expres- autoimmune conditions (15). A direct link between local sion (2). The plasticity of Th17 may play a role, giving rise to underrepresentation of Treg and autoimmunity can be made Treg by conversion in the presence of intratumoral IL10 (5). in tissue-specific conditions, such as vitiligo (16). The abun- Another mechanism supporting Treg abundance is chemoat- dance of Treg is reduced, apparently in response to limited traction (6). Among chemokines that can attract Treg, CCL22 numbers of CCL22-producing cells, including macrophages (17). In models of vitiligo, Treg depletion can induce depig- mentationevenintheabsenceofIFNg, highlighting their 1 Oncology Research Institute, Loyola University Chicago, Maywood, relative importance (18). Illinois. 2Department of Medicine, Loyola University Chicago, May- wood, Illinois. 3Department of Surgery/Hollings Cancer Center, Med- As tumor immunotherapeutic approaches meet with increas- ical University of South Carolina, Charleston, South Carolina. 4Depart- ed success, the development of autoimmunity takes center stage. ments of Pathology, Microbiology and Immunology, Loyola University This highlights the need for the development of precise Treg Chicago, Maywood, Illinois. depletion strategies. We recently demonstrated that Treg can Corresponding Author: I. Caroline Le Poole, Loyola University Chicago, 2160 be drawn to the skin by local Ccl22 overexpression (19). To South 1st Avenue, Maywood, IL 60153. Phone: 708-327-2032; Fax: 708-327- simultaneously address the accumulation of Treg in melanoma 3238; E-mail: [email protected] and their shortfall in autoimmunity, we postulate that cutane- doi: 10.1158/0008-5472.CAN-16-0618 ous overexpression of CCL22 can divert Treg away from tumors 2016 American Association for Cancer Research. and into the skin, as highlighted in Fig. 1.

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Figure 1. Study concept: CCL22 to divert Treg and support antitumor responses in melanoma. A, melanoma cells commonly metastasize to the lung, generating surface lesions detectable as pigmented spots. B, in human melanoma, Tregs are detectable by nuclear FoxP3 expression and membrane CD3 staining, resulting in double-stained cells (arrows) that can be enumerated as a fraction of total T cells. C and D, vaccination using plasmid DNA encoding Ccl22 is applied to ventral murine skin (C) to provide proof of concept for diverting Tregs away from the circulation and toward skin (D).

Here, we describe the abundance of relevant chemokines in our liquid nitrogen inventory by tissue number until use. Tissue human melanoma by immunostaining. We also evaluated che- samples were embedded in optimal cutting temperature (OCT) mokine receptor expression levels in Treg from patients and compound (Sakura Finetek), snap frozen in liquid nitrogen, and controls, and compared this with expression by CTL. Looking to stored at 80C. Eight micrometer frozen sections were cut, fixed Ccl22-mediated chemoattraction of Treg to the skin and its in acetone at 4C, and stored at 20C until use. For mouse success toward mitigating autoimmune depigmentation, we experiments approved by the Loyola Institutional Animal Care applied our strategy in a mouse model of metastatic melanoma and Use Committee (IACUC), 6-week-old C57BL/6, Pmel-1 using B16 tumor cells. We measured the consequences of cuta- (gp100 reactive, TCR transgenic mice), and Foxp3-eGFP (Treg neous Ccl22 overexpression for tumor growth and intratumoral reporter) mice were purchased from The Jackson Laboratories Treg abundance, and addressed the possibility that cells may be under IACUC approval at Loyola University Chicago and bred in converted to Treg on site or preferentially migrate toward Ccl22. house. Skin and pulmonary tissues were harvested at euthanasia We propose that cutaneous Ccl22 overexpression can support and stored at 80C. antitumor responses while suppressing autoimmune side effects of immunotherapy. Immunostaining of human and mouse tissues Eight mm human tissue cryosections were preincubated with 10% normal human AB serum (Gemini Bio-Products). Materials and Methods When staining mouse tissues, sections were fixed in 10% neu- Human and mouse subjects tral buffered formalin. Sections were subsequently treated with in situ Melanoma and metastatic melanoma surgical samples SuperBlock (ScyTek Laboratories) and with 0.3% H2O2 to inac- not required for pathologic examination and 30-mL blood sam- tivate endogenous peroxidase. When detecting nuclear FoxP3 in ples from metastatic melanoma patients were obtained after mouse tissues, antigen retrieval was pursued after incubation informed consent under Institutional Review Board (IRB)– with the primary antibody by using a sequence of 0.05% Tween approved protocols according to the declaration of Helsinki. (Sigma Life Sciences), 1% Triton-X100 (Fischer Scientific), and Metastatic tissue samples were obtained from spine, lung, lymph 0.05% Tween, each in TBS. Primary antibodies include clones node, or brain, whereas melanoma in situ samples were skin- Ta99 to TRP-1 (Covance), polyclonal rabbit antiserum to mouse derived. In concordance with the approved protocols, no further and human FoxP3 (ThermoFisher) or MF-14PE rat monoclonal patient information was shared with investigators. Normal to mouse FoxP3 (Biolegend), 145-2C11bio, FITC or PE to mouse human skin was obtained as otherwise discarded fresh surgical CD3e (BD Biosciences) or HIT3AFITC to human CD3 (BD Bios- tissue, and normal human buffy coats were from LifeSource. No ciences), PK138Alexa Fluor 488 to mouse NK1.1 (BioLegend), 8P16- personal identifiers were provided to investigators, and studies N4-L21 to human CCL1 (Enzo Life Sciences), 57203 to human were approved by the IRB. Incoming blood samples were given a CCL22 (R&D Systems), and MAB4391 to mouse Ccl22 (R&D tissue sample number before subjecting the samples to Ficol Systems), TG6/CCR4 to human CCR4 (BioLegend), and 191704 density gradient centrifugation to enrich the lymphocytes. Result- to human CCR8 (R&D Systems). For immunofluorescent staining peripheral blood mononuclear cells (PBMC) were stored in ings, slides were coverslipped with anti-fade reagent with DAPI

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(Life Technologies, Fischer Scientific). For immunoenzymatic events was performed using the FACSCanto II (BD Biosciences) stains, primary antibodies were detected using biotinylated and Flowjo analysis software (TreeStar). A population enriched þ rabbit anti-mouse Ig (Dakopatts) combined with horseradish for CD8 effector T cells was selected as CD4 T cells among peroxidase–labeled streptavidin (Dakopatts) or goat anti-rabbit presorted T cells. Ig (Dakopatts). For TUNEL stainings, an Apoptag kit (Millipore) was used. Unfixed tissues were incubated with Ta99 Ab to TRP-1 Gene gun vaccination prior to formalin fixation and incubation with terminal deox- Ccl22 DNA or empty vector–coated microparticles were intro- ynucleotidyl transferase enzyme. Alkaline phosphatase–labeled duced into mouse skin using a Helios Gene gun (Bio-Rad). One polyclonal goat anti-mouse IgG (Dakopatts) was combined with micrometer gold microparticles (Alfa Aesar) were coated with peroxidase-labeled antidioxigenin. Alkaline phosphatase was mouse Ccl22-encoding cDNA expressed under a CMV promoter detected using FAST Blue BB substrate (Sigma-Aldrich). Amino- in pcDNA3.1 (19). DNA was precipitated onto gold particles in ethylcarbazole (AEC) was used as a peroxidase substrate (Sigma- the presence of spermidine (Sigma-Aldrich) and used to coat Aldrich). Some slides were counterstained in Harris hematoxylin Tefzel tubing (Bio-Rad). Mice were Naired (Church & Dwight) (Sigma Aldrich) before coverslipping in glycergel (Dakopatts). prior to vaccination under isoflurane anesthesia (E-Z Systems) Stainings were imaged using an Olympus Scope BX53 with a with 4.8 mg DNA. Mice were vaccinated before tumor challenge Retiga 4000R 4.2 Mpixel monochrome camera (QImaging), every 6 days, or after tumor challenge every 5 days. Prior findings followed by Adobe Photoshop–supported (Adobe Systems indicated that continued treatment was necessary to maintain Incorporated) quantification of stained cells. In skin tissues, elevated levels of cutaneous Treg (19). stained cells were analyzed within a dermal area to a depth of 3x epidermal height without reference to tumor location. Measuring tumor growth In prophylactic experiments, mice were challenged with 105 Treg conversion and migration analysis B16F10 mouse melanoma cells by retro-orbital injection after 6 To measure conversion of T cells into Treg in vitro,we vaccinations at 6-day intervals. In therapeutic experiments, vacci- exposed mouse splenocytes from a FoxP3-eGFP reporter mouse nations were performed at 5-day intervals starting 3 days after 6 to cytokines. Briefly, 10 splenocytes were incubated in 24-well tumor challenge. CRL-6475 mouse melanoma cells were plates (Corning Inc. Life Sciences), in X-VIVO 20 media obtained from the ATCC providing standardized authentication (Cambrex) in the presence of mouse Ccl22 (5, 50, or 500 and quality testing, expanded in DMEM with 10% FBS and ng/mL), human TGFb (0.2,2,or20ng/mL),ormediaalone antibiotic/antimycotic (all from Thermo Fisher Scientific) and in triplicate wells. After 18 hours, samples were individually passaged twice before storing stock vials in LN2 upon confluence analyzed for expression of CD3, CD25, and FoxP3 expression þ þ within 3 weeks of purchase. A stock vial was thawed and plated 16 by FACS to identify Treg. The percentage of CD25 ,FoxP3 þ months later and passaged once before being harvested for tumor Treg among CD3 T cells was graphed. For Transwell migration injections. Another aliquot plated 2 weeks later tested pathogen- 6 assays, 10 splenocytes from FoxP3-eGFP mice were added in free by Charles River Laboratories using a rodent agent 360 PCR triplicate to a 5-mm pore size Transwell insert (Costar) with the panel. B16 cells consistently showed >95% viability upon thaw. outer well containing chemokines Ccl1 or Ccl22 (R&D Sys- Tumor-challenged mice received continued vaccinations with tems) at the indicated concentrations. The Treg fraction was Ccl22 or empty vector DNA for 9 days (prophylactic experiments) measured as reporter gene expressing cells among total cells by or 18 days (therapeutic experiments) before euthanasia. Lungs FACS analysis of the top and bottom wells after 2 hours. were imaged and surface pulmonary metastases were enumerated as melanized growths per pulmonary surface area prior to snap Fluorocytometry of human PBMC freezing the OCT-embedded tissues (Tissue Tek). In therapeutic Approximately 3 million PBMCs were thawed for negative experiments, the percent area occupied by tumor calculated in þ magnetic T-cell sorting using a labeled CD3 enrichment anti- ImageJ (20) was used as the more accurate estimate of tumor body cocktail (StemCell Technologies). Monoclonal antibodies burden. Tissues were immunostained to confirm metastatic inva- used for FACS analysis and sorting of peripheral blood samples sion and to detect infiltrating Treg. include antibodies to human CD3 (clone F7.2.38; BD Bio- sciences); CD4FITC (clone RPA-T4; BD Biosciences); FoxP3PE Statistical comparisons (clone 206D; Biolegend); CD25PE-Cy7 (clone M-A251; BD Bio- When two groups were compared as in melanoma and control sciences) or CD25PE (clone 4E3; MACS Miltenyi Biotech); lymphocytes or Ccl22-treated and empty vector–treated mice, a t CD127APC-eFlour780 (clone eBioRDR5; eBioscience); test for groups of unequal variance was applied. In therapeutic CCR4PerCP-Cy5.5 (clone TG6/CCR4; Biolegend); CCR8APC (clone experiments where tumors were followed for 18 days and larger FAB1429A; R&D Systems); CLAbio (HECA452bio)with group sizes were included, trimmed means were used. For com- fi streptavidinPaci cOrange(Invitrogen). T lymphocytes were parisons among multiple groups, an ANOVA was used followed then first incubated in the presence of antibodies CLAbio, by the Dunnett multiple comparison test to compare treatment CCR4PerCP-Cy5.5, and CCR8APC, subsequently adding CD4FITC, samples with an untreated control group. CD25PE-Cy7, and CD127APC-eFlour780 to distinguish Tregs. fi Washed cells were incubated with streptavidinPaci cOrange, followed by permeabilization and intracellular staining Results with anti-FoxP3PE according to the manufacturer's protocol Treg chemokines are expressed within human melanoma (BioLegend). For gating purposes, an FMO (FoxP3 excluded) tumors and a fluorescence minus three controls (CCR4, CCR8, and CLA Immunohistology provided a first impression of Treg excluded) were used. Multicolor analysis of >200,000 acquired homing in melanoma. Representative serial sections are

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Figure 2. Treg chemoattractant cytokines are abundant in melanoma tumors. A–D, expression of melanoma marker TRP-1 in a spinal metastasis (A) is inversely related to FOXP3 expression detected just outside a melanoma lesion (B), whereas such FOXP3 expression colocalizes with CCR4 chemokine receptor expression (C) and faint CCL22 expression (D) detected in the same area. E and F, CCR8 chemokine receptor expression and TRP-1 expression are likewise readily mutually exclusive (E), whereas CCL1 expression again colocalizes with its receptor CCR8 in areas surrounding the metastasis (F). In metastatic lesions of the lung (G), lymph node (H), and brain (I), similar distributions of CCL22-expressing cells are observed. In J, we quantiﬁed CCL22 expression in three melanoma in situ samples compared with four unaffected adult skin samples, detecting 5-fold upregulated chemokine expression, whereas in K, an 8-fold upregulation of CCL1 expression was observed.

shown in Fig. 2. Immunostaining of a metastatic tumor in Fig. 2K) were observed in melanoma-affected skin, suggesting Fig. 2A illustrates tumor cells tightly packed in a central tumor that Tregs are preferentially attracted toward tumors. area. Just outside, sparsely differentiated tumor tissue dis- plays irregular TRP-1 staining, where Tregs are abundant as Enhanced expression of homing markers by Treg from þ the same area is comprised primarily of FoxP3 cells melanoma patients (Fig. 2B). Tregs express CCR4 (Fig. 2C), whereas CCL22 Besides enhanced expression of chemokines, accumulation expression is at times punctate (Fig. 2D). Expression of CCR8 of Treg in tumors can be driven by homing marker expression (Fig. 2E) and its ligand CCL1 (Fig. 2F) is found outside the on the surface of circulating Treg. We stained lymphocytes central tumor area (Fig. 2D). In metastatic tumors of the lung from blood samples of 5 melanoma patients and 5 healthy þ þ (Fig.2G),lymphnode(Fig.2H),orbrain(Fig.2I),asimilar controls, selecting Treg based on expression of CD4 ,CD25 , þ distribution of CCL22 expressing cells is found (arrows). CD127low,andFoxP3 amongsingletsasshowninFig.3A.The Thus, chemoattractant cytokines may support Treg homing resulting percentages of Treg among lymphocytes were not to the tumor. We quantified CCL22- and CCL1-expressing significantly different at 2.3 0.5% among healthy controls cells in four samples of melanoma in situ and superficial and 3.0 1% among patient samples. Expression of CCR4, spreading melanoma, compared with four samples of control CCR8, and CLA (cutaneous lymphocyte antigen) was evaluat- skin; the majority of unaffected tissues (3/4) originated from ed. Both the percent stained Treg (Fig. 3B) and the mean metastatic melanoma patients. An impressive 5-fold increase fluorescence intensity (Fig. 3C) among CCR4-expressing cells in the expression of CCL22 (Fig. 2J) and an average 45-fold were increased among melanoma patients (P < 0.05), with 16% increase in the number of CCL1-expressing cells (P < 0.05; more Tregs staining at a 1.9-fold increased intensity. Similarly,

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Figure 3. Circulating Tregs from melanoma patients overexpress chemokine receptors. A, Tregs were selected based on forward/side scatter and singlet selection, as CD25þCD127low,FOXP3-expressingCD4þ cells. For comparison, selection of FOXP3þ cells among CD4þ cells is shown without including CD127low. The percentages of CCR4þ (B), CCR8þ (C), and CLAþ (D) Tregs are compared between 5 melanoma patients and 5 healthy controls, indicating a signiﬁcant increase in the number of CCR4- and CCR8-expressing Tregs (P < 0.05 for each). When examining mean ﬂuorescence intensities for CCR4 expression (E), CCR8 expression (F), and CLA (G), these receptors are upregulated as well (P < 0.05 for CCR4 and <0.01 for CCR8). In H, it is clear that the population overexpressing CCR4 also incorporates most CLA expressing and the majority of CCR8-expressing Tregs.

1.5% more melanoma Tregs expressed CCR8 at a 1.6-fold lated by interfering with Treg homing if chemokine receptor increased intensity overall. By comparison, the expression of expression does not likewise increase on effector lymphocytes. CLA did not vary among patient and control Treg. These We thus evaluated expression of chemokine receptors on non- ﬁndings suggested that antitumor responses can be manipu- CD4 T cells (primarily CTL) in Fig. 4. Interestingly, these T cells

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targeting CCR8 and its interaction with CCL1 would not be fortuitous (not shown). However, expression of CCR4 is much greater among Treg compared with CTL, rendering the CCR4–CCL22 axis a valuable interaction to interfere with in melanoma. Enhanced expression of CLA on Treg compared þ with CD8 T cells suggests that blocking this receptor would also relatively favor CTL inﬁltration;however,targetingthe chemokine receptor CCR4 may be a better choice based on the 3.1-fold greater % expressing melanoma Tregs in Fig. 3 (P < 0.001). Thus, targeting the CCR4/CCL22 axis would be preferable for therapeutic applications.

Cutaneous overexpression of Ccl22 diverts Treg Gene gun vaccination provides a mode of mechanical trans- fection of cutaneous cells. Marked overexpression of Ccl22 in vaccinated skin was previously demonstrated, and the same protocol was used here (19). At euthanasia, we found a trend toward a 2-fold increase in Ccl22-expressing cells in the spleen (n ¼ 6, P ¼ 0.07), yet the main site of overexpression was the skin, where the gold beads concentrated at the vaccination site. Ccl22 vaccination dramatically reduced vitiligo development in T-cell receptor transgenic Pmel-1 mice, where T cells respond to gp100 (19). Here, we tested whether Ccl22 may convert skin and tumor-infiltrating T cells into Treg, as reported for TGFb. Splenocytes from FoxP3-eGFP reported mice were exposed to Ccl22, TGFb, or medium for 18 hours before quantifying Tregs by FACS analysis. As shown in Fig. 5A–C, in example, FACS plots generated under each culture condition, and in Fig. 5D, where Tregs are quantified, exposure to Ccl22 did not induce a greater Treg compartment among splenocytes at any tested concentration, even at higher than physiologic (serum) concentrations (21). However, comparing TGFb treatment with untreated cells indicates a significant treatment effect by ANOVA (P ¼ 0.0002), and the % of Treg is significantly different from untreated samples (P < 0.05) at both 2 and 20 ng/mL using the Dunnett multiple comparison post- test.InTranswellmigrationassays(22),Tregsweresigni- ficantly enriched among total splenocytes migrating toward Ccl22 (Fig. 5E) but not toward Ccl1 (Fig. 5F). Taken together, these data sparked the concept that Treg can be diverted to the skin, providing a tumor microenvironment more conducive to tumor immune surveillance. Figure 4. CCR4 is not elevated on the surface of circulating CTL. If Tregs were to be A strategy to contain tumor growth rerouted based on chemokine receptor expression, selective upregulation on An important advantage of redirecting Treg over simply the Treg subset is critical. Thus, we probed CD4 ,FOXP3 T cells for preferential expression of CCR4 (A), noting similar expression among blocking Treg tumor homing by antibodies to deplete Treg is healthy and melanoma nonregulatory T cells that is less than half of what is that skin-migrating Treg can suppress autoimmunity. We observed on Tregs (see Fig. 3). B, CCR8 expression does not differ tumor-challenged Ccl22-vaccinatedmiceandcontinuedtotreat significantly between 5 patients and 5 controls, whereas mean fluorescence the animals by gene gun vaccination until dissection (Fig. 6A). intensities (MFI) are not reduced compared with Tregs. C, CLA expression Examples of pulmonary tumor growth imaged for an empty (MFI) is also the same among patients and controls. Overall however, vector–vaccinated mouse compared with the Ccl22-vaccinated expression is markedly (65%) higher among Treg (P < 0.05). Though Treg may be selectively targetable via CLA, the number of CCR4-expressing mouse are shown in Fig. 6B, revealing marked differences in patient Treg is markedly greater. number and size of the tumors. Quantifying pulmonary tumors, an advantage to cutaneous Ccl22 treatment is evident (Fig. 6C). We next performed therapeutic experiments where display no differences in CCR4 or in CLA expression, whereas a gene gun vaccination was initiated 3 days after tumor challenge trend toward increased CCR8 expression was observed among and repeated only twice before euthanasia (Fig. 6D). A remark- þ CTL. When comparing CCR8 lymphocytes among melanoma able reduction in tumor burden is illustrated in Fig. 6E and patients and controls, CCR8 was expressed not only by a 2-fold quantified as 39% reduction in area occupied by tumor (Fig. greater percentage of patient Treg (P < 0.05), but also among a 6F), representing a significant therapeutic effect. We demon- 2.5-fold greater percentage of patient CTL (P < 0.05), thus strated that cutaneous Ccl22 vaccination will suppress ongoing

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Figure 5. Ccl22 favors Treg migration but does not convert T cells to become Treg. To demonstrate that Ccl22 vaccination increases the fraction of skin Treg without converting T cells into Treg, we exposed three samples of T cells from a FoxP3 reporter mouse to Ccl22 or to TGFb overnight. Splenocytes were gated for Cd3þ T cells and exposed to Cd4 and Cd25 immunostaining. A, FoxP3þCd25þ Treg in a representative untreated sample. B and C, Treg abundance is increased in a representative sample of TGFb-treated cells (B), whereas exposure to Ccl22 does not induce a Treg phenotype in vitro (C). D, Tregs are quantified, demonstrating a significant increase in response to 2 and 20 ng/mL TGFb of up to 60%. E and F, in a representative Transwell migration assay among three performed, Treg abundance is increased among splenocytes migrating toward Ccl22 (E), whereas preferential Treg migration is not observed toward Ccl1 (F). autoimmunity accompanied by greater Treg accumulation in The reduced proportion of Treg apparently resulted in reduced the skin of TCR transgenic vitiligo mouse models (19). Here, we suppression and a 57% expansion of T cells overall as evaluated evaluated pulmonary Treg numbers in tissues from tumor- in 3 tumors/group (P < 0.01; Fig. 7H), whereas natural killer challengedmicetodeterminewhetherthesameprocessresults (NK)–cell abundance was not significantly affected (Fig. 7I). in reduced pulmonary immunosuppression by limiting Treg Treatment similarly revealed a trend toward 38% increased availability. In Fig. 7A–C, images of immunostained pulmo- apoptosis among tumor cells at 3 tissues/group (Fig. 7J), þ þ nary tissue are shown to represent FoxP3 ,CD3 Treg detec- suggesting that reduced tumor growth partially results from tion. A 33% greater pulmonary Treg to total T-cell ratio was impaired proliferation instead. Thus, cutaneous overexpression observed in untreated mice (Fig. 7D), with the majority of T of Ccl22 can divert Treg away from tumor metastases, suggest- cells expressing FoxP3 in untreated mice, whereas Tregs were in ing that equivalent treatment of human skin can be of imme- the minority in pulmonary tissue from Ccl22-vaccinated mice diate benefit to patients with melanoma metastases. þ (n ¼ 3, P < 0.05). In separate efforts to quantify FoxP3 cells immunoenzymatically, we found a 2.8-fold greater pulmonary Treg density in untreated mice (n ¼ 5, P < 0.01, not shown). Discussion Conversely, in the skin where Treg densities are generally We observed overexpression of Treg chemoattractant cyto- markedly lower than in pulmonary tissues, a 45% increase in kines in melanoma in situ tumors compared with unaffected the ratio of Treg among T cells was found among tumor-bearing human skin. Overexpression of CCL22 in melanoma has Ccl22-vaccinated over tumor-bearing control mice (Fig. 7E–G; n been associated with an accumulation of plasmacytoid DCs ¼ 3, P < 0.05), indicating that Tregs were redirected to the skin. (23). These studies also accentuate the contribution of the A 13.9-fold increase in absolute Treg densities following Ccl22 tumor microenvironment to immunosuppression in melano- vaccination was reported in the absence of tumor (19), yet here ma. Thus, another strategy to circumvent tumor immuno- we calculated Treg density as a fraction of total infiltrating T suppression could be to convert immunosuppressive pDCs cells as the more important parameter to reflect Treg function. into a phenotype more supportive of antitumor immunity.

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Figure 6. Tumor growth is inhibited by cutaneous Ccl22 overexpression. A, in a representative experiment of two performed, 5 mice/group were gene gun vaccinated with Ccl22-encoding or empty vector DNA as shown. B, at euthanasia, lung tissues were collected to identify tumors. C, significantly reduced pulmonary tumor counts were observed in Ccl22-vaccinated mice as quantified for 5 mice/group (P < 0.05). D, in therapeutic experiments, mice were tumor challenged 3 days before vaccination applied every 5 days as shown. E, examples of pulmonary tissues from the Ccl22 (n ¼ 7) and empty vector control (n ¼ 8) groups. F, trimmed means (highest and lowest values removed from each group) were used to calculate that significantly reduced portions of the lung surface were covered by tumor in Ccl22 mice (representative experiment of two performed).

Oversecretion of CCL1, meanwhile, can possibly be ascribed interest in rerouting Tregs that were sparse in vitiligo and to infiltrating gd-T cells and NK cells (24), which are more decided to address both deficiencies at once by overexpressing likely to support immune reactivity to tumors. Our studies CCL22. This concept is supported by cutaneous Ccl22 over- suggest that the contribution of CCL1 to chemoattraction expression suppressing vitiligo, accompanied by preferential of Treg is not very selective. Yet the contribution of the cutaneous Treg infiltration (19). Furthermore, that outcome is CLA–P-selectin axis in attracting Treg may be a positive one; supported by overexpression of CCR4 among melanoma CLA is overexpressed by skin-infiltrating T cells and favors skin patient Treg. over tumor homing (25). Gene gun vaccination has been applied to human subjects Overexpression of chemokine receptors including CXCR3 vaccinated against HPV (31). One advantage of DNA vaccination on T cells in tumor patients and intratumoral overexpression is that it allows for longer lasting expression of the gene product. of the associated chemokines is beneficial in the case of However, other methods of introducing CCL22 DNA may be tumor-reactive effector cells (26, 27). Interestingly, much more suitable, such as a microneedle, tattooing, or electropora- importance has been assigned to tumor overexpression of tion (32–33). The most effective means of vaccination likely Ccl2, an additional ligand for CCR4 (28). Perhaps the ambiv- depend on the encoded gene product. alent outcomes when promoting such interaction are due to Remarkably, cutaneous overexpression of CCL22 may super- an overarching support for Treg infiltration. We initially sede the effects of CCL22 naturally overexpressed in the tumor. reported that Tregs in blood from melanoma patients over- This leads us to believe that redirecting Treg may be of parti- express CCR4 (and CCR8) and may thus be preferentially en cular importance to the treatment of micrometastases, whereas route to the tumor (29). It was later shown that blocking larger tumors with greater CCL22 expression may require more CCR4 can prevent the immunosuppressive consequences of drastic treatment. Redirecting Treg might also be useful in other þ Treg infiltration to tumors (30). Meanwhile, we developed an cancers where CCR4 Tregs reportedly interfere with antitumor

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Figure 7. Cutaneous Ccl22 overexpression limits tumor-infiltrating Treg. Treg in formalin-fixed cryosections by immunofluorescent double staining. CD3 is shown in green and FoxP3 staining in red. DAPI counterstaining was used. Shown is a sequence representing FoxP3-stained (A) and CD3-stained (B)lung tissue as well as an overlay (C) in an untreated mouse, including DAPI counterstaining, and the number of Treg was quantified as a ratio to total infiltrating T cells in pulmonary tissue (D), revealing a marked reduction in Ccl22-vaccinated mice (n ¼ 3; , P < 0.05); representative experiment of two evaluated. The overall abundance of pulmonary T cells (E) and NK cells (F)wasnotaffected.G, example TUNEL stainings of lung tissue from empty vector– (top) and Ccl22 (bottom)-treated mice in a prophylactic experiment. H, the number of Treg is elevated in the skin of vaccinated mice. I, Tregs in a sample of Ccl22-vaccinated skin. responses, including colon carcinoma, lymphoma, and glio- CCL22-mediated enhancement of antitumor responses is blastoma (34–36). Support for our strategy is most convincing however not due to conversion, but rather to redirection of when investigating larger sample numbers, yet our materials existing regulatory T cells to the site of cutaneous overexpres- were limited. sion. The added benefitofsuppressingautoimmunitycom- The proposed cutaneous overexpression of CCL22 may monly associated with antimelanoma responses can be sig- have consequences that can negatively affect its application nificant (38). Few mechanisms of supporting antitumor in patients. We specifically investigated the possibility that responses will simultaneously allow for reducing autoim- exposure to CCL22 may convert T cells into Tregs, as reported mune side effects of therapy. Cutaneous overexpression of for TGFb (37), because we have proposed cutaneous over- CCL22 may truly separate autoimmunity from antitumor expression as a treatment modality for vitiligo (19). The immunity (39).

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Disclosure of Potential Conﬂicts of Interest Administrative, technical, or material support (i.e., reporting or organiz- No potential conﬂicts of interest were disclosed. ing data, constructing databases): S.W. Henning, J.M. Eby Study supervision: I.C. Le Poole Authors' Contributions Conception and design: S.W. Henning, F.C. Navarro, I.C. Le Poole Grant Support Development of methodology: F.C. Navarro, S. Chatterjee This study was supported by NIH/NCI RO1CA191317 and NIH/NIAMS- Acquisition of data (provided animals, acquired and managed pati- RO1AR057643 (I.C. Le Poole, Principal Investigator) and NIH/ ents, provided facilities, etc.): K. Tobin, P. Farhangi Oskuei, S.W. Henning, NHLBIT35HL120835 (P. Farhangi Oskuei; N.J. Zeleznik-Le Ph.D., Principal M.F. Fernandez, E.R. Dellacecca, J.M. Eby, J.I. Clark Investigator). Analysis and interpretation of data (e.g., statistical analysis, biostatistics, The costs of publication of this article were defrayed in part by the computational analysis): K. Tobin, P. Farhangi Oskuei, S.W. Henning, payment of page charges. This article must therefore be hereby marked advertisement M.F.Fernandez,E.R.Dellacecca,F.C.Navarro,J.M.Eby,S.Chatterjee, in accordance with 18 U.S.C. Section 1734 solely to indicate I.C. Le Poole this fact. Writing, review, and/or revision of the manuscript: K. Tobin, P. Farhangi Oskuei, S.W. Henning, M.F. Fernandez, E.R. Dellacecca, F.C. Navarro, J.M. Eby, Received March 1, 2016; revised August 25, 2016; accepted August 30, 2016; S. Mehrotra, J.I. Clark, I.C. Le Poole published OnlineFirst September 12, 2016.

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www.aacrjournals.org Cancer Res; 76(21) November 1, 2016 OF11

Ccl22 Diverts T Regulatory Cells and Controls the Growth of Melanoma

Jared Klarquist, Kristen Tobin, Peyman Farhangi Oskuei, et al.

Cancer Res Published OnlineFirst September 12, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-0618

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