Regulatory T cells (Treg) are critical mediators of immunosuppression in established tumors, although little is known about their role in restraining immunosurveillance during tumorigenesis. Here, we employ an inducible autochthonous model of melanoma to investigate the earliest Treg and CD8 effector T-cell responses during oncogene-driven tumorigenesis. Induction of oncogenic BRAFV600E and loss of Pten in melanocytes led to localized accumulation of FoxP3+ Tregs, but not CD8 T cells, within 1 week of detectable increases in melanocyte differentiation antigen expression. Melanoma tumorigenesis elicited early expansion of shared tumor/self-antigen–specific, thymically derived Tregs in draining lymph nodes, and induced their subsequent recruitment to sites of tumorigenesis in the skin. Lymph node egress of tumor-activated Tregs was required for their C-C chemokine receptor 4 (Ccr4)–dependent homing to nascent tumor sites. Notably, BRAFV600E signaling controlled expression of Ccr4-cognate chemokines and governed recruitment of Tregs to tumor-induced skin sites. BRAFV600E expression alone in melanocytes resulted in nevus formation and associated Treg recruitment, indicating that BRAFV600E signaling is sufficient to recruit Tregs. Treg depletion liberated immunosurveillance, evidenced by CD8 T-cell responses against the tumor/self-antigen gp100, which was concurrent with the formation of microscopic neoplasia. These studies establish a novel role for BRAFV600E as a tumor cell–intrinsic mediator of immune evasion and underscore the critical early role of Treg-mediated suppression during autochthonous tumorigenesis.

Significance: This work provides new insights into the mechanisms by which oncogenic pathways impact immune regulation in the nascent tumor microenvironment. Cancer Res; 78(17); 5038–49. ©2018 AACR.

Immune cells constantly survey host tissues for aberrant cells to limit neoplastic emergence. This cancer immunosurveillance is predicated on the ability of effector CD8 T cells to adequately detect and respond to tumor-associated antigens. Although effector responses spontaneously arise against highly immunogenic tumors, leading to elimination or equilibrium (1), poorly immunogenic tumors can instead evade immunosurveillance by inducing tolerance (2). FoxP3+ regulatory T cells (Treg) mediate this tolerance by suppressing effector responses against cancer (3). Hence, the balance of Tregs and CD8 T cells in the host is critical to effective immunosurveillance (4, 5). Accordingly, high intratumoral Treg:CD8 ratios portend weak antitumor responses in preclinical models (6) and poor outcomes in solid malignancies (7, 8). Although Treg:CD8 ratios have been well characterized in the microenvironments of established tumors, less is known about these T-cell responses during early tumorigenesis.

Generating and maintaining functional antitumor T-cell responses requires choreographed immune events, a process termed the “cancer immunity cycle” (9). Tregs are known to impede this process in draining lymph nodes (DLN), as they suppress CD8 T cells following the implantation of poorly immunogenic tumors (2). In transplantable models of ovarian cancer, Tregs preferentially migrate to tumor sites, restrain antitumor immunity, and, in turn, allow tumor growth (10). However, the utility of transplantable models remains limited, as they fail to mimic the autochthonous aspects of human malignancies. In an autochthonous model of pancreatic ductal adenocarcinoma, Tregs have been shown to accumulate during the preinvasive and invasive stages of the disease (11). However, the factors governing their early accumulation during tumorigenesis remain unexplored.

Oncogene-driven transgenic (Tg) mouse models offer a basis for studying host T-cell responses in settings that recapitulate the autochthonous nature of human cancers. The Braf/Pten melanoma model harbors inducible, melanocyte-restricted loss of the tumor suppressor gene Pten, and expression of the oncogenic MAPK kinase variant BRAFV600E, a common melanoma driver mutation (12, 13). We previously demonstrated that inhibiting BRAFV600E in established Braf/Pten tumors induces the selective apoptosis of intratumoral FoxP3+ Tregs and promotes CD8 effector T-cell responses (14). Moreover, we found that inhibiting BRAFV600E decreases infiltrating myeloid-derived suppressor cells (MDSC) and downregulates cytokines associated with MDSC recruitment (15). Other groups have similarly described a link between BRAFV600E and extrinsic mechanisms of immunosuppression (16, 17). However, it is unclear whether the initial activation of BRAFV600E signaling is sufficient to trigger Treg recruitment to the nascent tumor microenvironment.

Little is known about the antigen (Ag) specificity or origins of the earliest Treg responders during tumorigenesis. The Treg compartment in established cancers is known to include thymic Tregs (tTreg) and their peripherally induced counterparts (pTregs; ref. 18). Thymic Tregs arise through high-avidity interactions with self-epitopes and constitutively express FoxP3 (19). In an autochthonous mouse model of prostate cancer, such tumor/self-Ag–specific Tregs were identified and shown to require thymic Aire expression (20). Similarly, Tregs with high avidity for tumor-expressed self-Ags have been identified in patients with melanoma and pancreatic cancers (21, 22). On the other hand, pTregs develop from CD4+FoxP3 T cells and thus predominantly recognize non-self, tumor-specific antigens. Conversion of pTregs was shown to occur in transplantable models of kidney adenocarcinoma and colon carcinoma (23, 24). Moreover, Tregs recognizing tumor-derived viral epitopes have been identified in patients with human papillomavirus–associated cervical cancer (25). In contrast to virally and chemically derived tumors, autochthonous oncogene-driven tumors exhibit a low mutational burden and harbor no predicted neoepitopes (26), suggesting that tTregs may dominate the earliest responses during tumorigenesis. However, this has yet to be explored.

In these studies, we employ the inducible Braf/Pten mouse melanoma model to investigate the dynamics of early Treg responses during autochthonous tumorigenesis. By employing melanoma/melanocyte Ag-specific Tregs and CD8 T cells, we investigate the competing processes of tolerance and immunosurveillance during tumor emergence. We report that tumorigenesis induces the priming and recruitment of thymic Tregs at the expense of CD8 T cells, and further elucidate a requirement for oncogenic BRAFV600E as a driver of Treg trafficking.

Mice and tumor/nevus induction

These studies were approved by the Institutional Animal Care and Use Committee at Dartmouth College (Hanover, NH). Mice were maintained in pathogen-free conditions. Tyr::CreER+/−BrafCA/+Ptenfl/fl (Braf/Pten) mice were kindly provided by Marcus Bosenberg (Yale, New Haven, CT), and bred in-house onto a C57BL/6 background, with >98% purity confirmed by congenic testing (DartMouse). To generate nevus-inducible mice, BrafCA/CAPtenlox/lox were backcrossed to C57BL/6 to generate BrafCA/CAPtenWT/WT, which were then crossed with Tyr::CreER+ to generate Tyr::CreER+BrafCA/+ (BRAFV600E) mice. For induction of Braf/Pten tumors and BRAFV600E nevi, 0.5 mg of 4-hydroxy-tamoxifen (4-HT; Sigma) in DMSO was topically applied over approximately 1 cm2 on the shaved right flanks of 3-week-old mice. C57BL/6, Foxp3DTR, Rag1−/−, and Cd8−/− mice were obtained from The Jackson Laboratory, or Charles River Breeding Laboratories. Pmel-1 mice (referred to as pmel), which express a TCR specific for H-2Db–restricted gp10025-33, were a gift from Nicholas Restifo (NCI, Rockville, MD) and were crossed onto a Thy1.1+ background (Thy1.1+ C57BL/6 mice were from The Jackson Laboratory). Thy1.1+Rag1−/−Foxp3DTRTyrpB-w (TRP-1) mice express a TCR specific for I-Ab–restricted TRP-1113-125 (27) and were a gift from Paul Antony (University of Maryland, Baltimore, MD). OT-II mice were obtained from The Jackson Laboratory and bred in-house, to homozygosity, with CD45.1 congenic mice (NCI B6-Ly5.1/Cr mice; Charles River Laboratories). All animal studies were performed independently at least twice to generate a conclusion.

Gene expression analysis and protein detection

Total RNA was isolated using RNeasy Kit (Qiagen), and cDNA was amplified from 2 μg RNA using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Expression of melanocyte differentiation antigens was measured by real-time qRT-PCR using prevalidated gene-specific primers (Life Technologies) for Tyrp1 (Mm00453201_m1), gp100 (Mm00498996_m1), Tyr (Mm00495817_m1), Ccl17 (Mm01244826_g1), Ccl2 (Mm00441242_m1), Ccl22 (Mm00436439_m1), gapdh (Mm99999915_g1), and TaqMan master mix (Life Technologies) on a StepOne Plus Real-Time PCR System (Applied Biosystems). Gene expression was expressed as 2−(ΔCT), where |${\rm{\Delta }}{C_{\rm{T}}}{\rm{ = }}{C_{\rm{T}}}_{_{{\rm{gene\, of\, interest}}}} {\rm{- }}{C_{\rm{T}}}_{_{{\rm{Gapdh}}}}$|⁠. Fold changes were calculated as 2−(ΔCTreference sample – ΔCTtested sample).

Flow cytometry

Skin samples were harvested, minced, and digested for 45 minutes at 37°C in 2 mL HBSS containing 7 mg/mL collagenase D (Roche) and 200 μg/mL DNase I (Roche), using magnetic bar stirring at 300 RPM. Tissue fragments were mechanically dissociated through a 40-μm nylon mesh filter. Samples were washed in RPMI1640 media containing 10% FBS and 2 mmol/L EDTA. Tumor-draining inguinal lymph nodes were mechanically dissociated. For surface molecule staining, samples were incubated with anti-CD16/32 (2.4G2; Bio X Cell) and stained with antibodies (BioLegend unless indicated) against CD45-APC-Cy7 (30-F11), CD11b-PCP (M1/70), CD4-APC (RM4-5), CD3-BrV420, -BrV510 (17A2), CD44-PE-Cy7 (IM7), CD62L-BrV510 (MEL-14), CD8-PE-Cy7, -BrV510 (53-6.7), Ccr4-BrV421 (2G12), Thy1.1-PE (30-H12), and FoxP3-FITC (FJK-16s; eBioscience) on ice for 30 minutes in PBS with 0.5% BSA and 2 mmol/L EDTA. For FoxP3 staining, cells were fixed and permeabilized using the Transcription Factor Staining Buffer Set (eBioscience). Cells were acquired on MacsQuant 10 Analyzer (Miltenyi Biotec), and analysis was performed using FlowJo 9.8.1 (TreeStar). To determine absolute cell number per gram of tissue, the total number of cells was multiplied by a correction factor for the acquired tumor fraction and normalized for tissue weight.

Adoptive cell transfer

Pmel cells were isolated from pooled lymph node and spleen of naïve pmel mice. Negative selection using anti–CD44-PE (IM7; BioLegend) and MACS anti-PE magnetic beads (Miltenyi Biotec) was performed, followed by positive selection using MACS anti-CD8 magnetics beads and validation of >90% purity. TRP-1 and OT-II T cells were isolated from pooled lymph nodes and spleens of naïve TRP-1 and OT-II mice, respectively. When indicated, 3 doses of daily 25 μg/kg diphtheria toxin (DT) were administered to deplete FoxP3+ Tregs prior to CD4+ T-cell isolation from TRP-1 mice. Positive selection using MACS anti-CD4 magnetic beads was performed, with >80% purity. Isolated lymphocytes were transferred retro-orbitally at a concentration of 1 × 105 cells/mouse (pmel) or 2 × 105 cells/mouse (TRP-1 or OT-II) on the indicated days. To assay in vivo migration, single-cell suspensions of bulk lymphocytes were obtained from inguinal DLNs of induced mice (26–28 days postinduction) or from naïve lymph nodes of wild-type (WT) counterparts, and 3 × 107 cells were transferred to Rag1−/− mice bearing induced skin grafts. When indicated, lymphocytes were incubated for 1.5 hours at 37°C in the presence or absence of 20 ng/mL pertussis toxin (PTx).

Skin grafting

Mice were anesthetized intraperitoneally with 90 mg/kg ketamine and 10 mg/kg xylazine. Tail skin pieces (∼5 × 5 mm) from 3-week-old Braf/Pten or WT donor mice were dorsally grafted with surgical sutures onto syngeneic recipients, which were allowed to recover for 7 days before 4-HT induction. Tumor growth was assessed by measuring skin thickness using a dial caliper.

In vivo depletions

For Treg depletion using DT (Sigma), Foxp3DTR mice bearing induced skin grafts were injected intraperitoneally with 25 μg/kg DT in PBS every 2 days. For antibody-mediated depletion, CD4-targeting (300 μL; clone GK.15) or CD8-targeting antibody (500 μL; clone 2.43) was administered intraperitoneally on indicated days. Both antibodies (∼1 mg/mL) were produced as bioreactor supernatant from hybridomas purchased from the ATCC, and each lot was confirmed to deplete >95% of target cells.

Drug treatments

For FTY720 treatments, 2-amino-2-(2[4-octylphenyl]ethyl)-1,3-propanediol hydrochloride (Cayman Chemical) was dissolved in sterile saline and 1 mg/kg was injected intraperitoneally daily between day 10 and 26. For BRAFi treatment, Braf/Pten mice were given a dose of 100 mg/kg PLX4032 (Selleckchem) by oral gavage 24 hours prior to adoptive transfer on day 19 or qRT-PCR on day 26. PLX4032 was compounded in aqueous vehicle (0.5% hydroxylpropyl cellulose; Sigma) on the day of treatment. When indicated, transferred mice were fed PLX4720-containing diet ad libitum to maintain inhibition from day 19 to 21. PLX4720 (provided by Plexxikon Inc. under a Materials Transfer Agreement) was compounded in AIN-76A rodent diet (417 mg PLX4720/kg) by Research Diets, Inc.

Statistical analyses

Statistical differences in normally distributed datasets were analyzed using unpaired Student two-tailed t test when comparing two groups, or one-way ANOVA with Bonferroni posttest when comparing three distinct cohorts. When Shapiro–Wilk test indicated non-Gaussian distributions, statistical differences were assessed using Mann–Whitney test (two groups), or Kruskal–Wallis with Dunn posttest (three groups). Differences in kinetics of skin thickness were determined using two-way ANOVA with Bonferroni posttest. Mice were randomized when assigned to treatment groups. Statistical analyses were performed using Prism 5 software (GraphPad), and differences were considered significant if P ≤ 0.05.

Autochthonous melanoma tumorigenesis induces priming and recruitment of self-Ag–specific thymic Tregs

To assess the earliest T-cell responses during autochthonous tumorigenesis, a tamoxifen-inducible melanoma model driven by BRAFV600E expression and Pten loss (referred to as the Braf/Pten model) was employed. We previously showed that dermal injection of tamoxifen leads to palpable tumor formation in this model by day 28 (14). However, topical tamoxifen led to more restrained tumor growth. Three weeks following topical induction, macroscopic hyperpigmented focal lesions developed, coalescing into palpable lesions by day 31 (Fig. 1A). Microscopically, hyperplastic foci could be detected in proximity to hair follicles as early as 16 days postinduction, with larger pigmented neoplasia evident throughout the dermis by day 26 (Fig. 1B). Accordingly, significant increases in expression of melanocyte differentiation Ags tyrosinase-related protein 1 (Tyrp1), glycoprotein 100 (gp100; Pmel), and tyrosinase (Tyr) were detected in the skin as early as 21 days postinduction (Fig. 1C).

Figure 1.

FoxP3+ Tregs preferentially accumulate in nascent Braf/Pten tumors. A, Representative skin from Braf/Pten mice on indicated days following 4-OHT application, with WT skin as a control. B, Representative hematoxylin and eosin staining of WT and Braf/Pten skin showing hyperplasia on day 16 and invasive neoplasia (demarcated in yellow) day 26 postinduction. C, qRT-PCR of melanocyte differentiation antigens in tumor-induced (Braf/Pten) versus WT skin over time (n = 3–10 mice/group). D–F, Induced Braf/Pten and WT skin analyzed for the frequency and absolute numbers of CD45+ cells, gated on live cells (D), CD3+ T cells gated on CD45+ cells (E), CD4+FoxP3+ Tregs gated on CD3+ cells (F), and CD8+ T cells gated on CD3+ cells (G), with representative plots showing differences 26 days postinduction. H, Ratio of intratumoral FoxP3+ Tregs to CD8 T cells over time. D–H, Data in each panel were pooled from two independent experiments, each with n ≥ 3 mice/group; symbols and error bars, means ± SEM, with *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, nonsignificant. Absence of error bar indicates SEM less than area represented by symbol. Analyzed by ANOVA (Bonferroni post hoc; C,E,F, and H) and Kruskal–Wallis (Dunn post hoc; D and G).

Figure 1.

FoxP3+ Tregs preferentially accumulate in nascent Braf/Pten tumors. A, Representative skin from Braf/Pten mice on indicated days following 4-OHT application, with WT skin as a control. B, Representative hematoxylin and eosin staining of WT and Braf/Pten skin showing hyperplasia on day 16 and invasive neoplasia (demarcated in yellow) day 26 postinduction. C, qRT-PCR of melanocyte differentiation antigens in tumor-induced (Braf/Pten) versus WT skin over time (n = 3–10 mice/group). D–F, Induced Braf/Pten and WT skin analyzed for the frequency and absolute numbers of CD45+ cells, gated on live cells (D), CD3+ T cells gated on CD45+ cells (E), CD4+FoxP3+ Tregs gated on CD3+ cells (F), and CD8+ T cells gated on CD3+ cells (G), with representative plots showing differences 26 days postinduction. H, Ratio of intratumoral FoxP3+ Tregs to CD8 T cells over time. D–H, Data in each panel were pooled from two independent experiments, each with n ≥ 3 mice/group; symbols and error bars, means ± SEM, with *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, nonsignificant. Absence of error bar indicates SEM less than area represented by symbol. Analyzed by ANOVA (Bonferroni post hoc; C,E,F, and H) and Kruskal–Wallis (Dunn post hoc; D and G).

Close modal

Measurable infiltration of T cells into skin was not detected until 26 days postinduction, with both proportions and absolute numbers of CD45+ leukocytes increased in comparison with WT skin (Fig. 1D). Total CD3+ lymphocytes, although slightly decreased by total proportion of CD45+ cells, were still substantially increased by absolute number in tumor-induced skin (Fig. 1E). Within this CD3+ population, FoxP3+ Treg cells were significantly increased by proportion, resulting in a log-fold increase in absolute numbers of Tregs in the skin between days 21 and 26 (Fig. 1F). In contrast, CD8 T-cell proportions were slightly decreased in tumor-induced skin (Fig. 1G). However, this did not translate to a drop in overall numbers of CD8 T cells (Fig. 1G), consistent with the quantitative increase in FoxP3+ Tregs. These dynamics resulted in a 6-fold increase in the Treg:CD8 ratio between days 21 and 26 (Fig. 1H). Thus FoxP3+ Tregs, but not CD8 T cells, accumulated in nascent melanomas within days following detectable increases in tumor Ag expression.

Consistent with the paradigm that T cells are primed in lymph nodes prior to their recruitment to peripheral sites, elevated proportions of Tregs were also detected in the DLNs of tumor-induced mice compared with naïve counterparts (Fig. 2A). Furthermore, Treg proportions and numbers were elevated in tumor-DLNs compared with non-DLNs from tumor-induced mice (Supplementary Fig. S1A). A significant increase in the Treg:CD8 ratio also became evident in DLNs by day 26 (Fig. 2B) as a result of an 8-fold increase in Treg numbers compared with a more modest (4-fold) increase in CD8 T cells (Supplementary Fig. S1B). Moreover, Treg populations in DLNs were enriched for phenotypically activated/effector (CD44hiCD62Llow) and central memory (CD44hiCD62Lhi) subsets (Fig. 2C), and were significantly more suppressive ex vivo relative to Tregs derived from naïve lymph nodes (Fig. 2D). To further assess whether tumor Ag-specific Tregs were primed during tumorigenesis, CD4+ T cells specific for the melanocyte-expressed differentiation antigen Tyrp1 (TRP-1 cells) were harvested from naïve TCR Tg mice (on a Tyrp-1KO background; ref. 27) and adoptively transferred into WT and Braf/Pten mice 16 days postinduction (Fig. 2E). Ten days later, significantly larger populations of CD4+FoxP3+ TRP-1 cells were detected in DLNs of tumor-induced Braf/Pten mice compared with WT counterparts (Fig. 2F), and compared with non-DLNs within the same host (Supplementary Fig. S2A), indicating tumor-driven Treg expansion. Whereas the transferred population exhibited a predominantly naïve phenotype, TRP-1 Tregs in induced skin-DLNs acquired an overwhelmingly CD44hiCD62Llow/hi effector/memory phenotype (Fig. 2G), as evidence of Ag-experience. A similar experiment involving tumor Ag-irrelevant OT-II Tg T cells indicated no difference between OT-II Treg accumulation in tumor-DLNs versus naïve lymph nodes, further confirming the tumor/self-Ag specificity of the response (Supplementary Fig. S2B). These data illustrate that melanoma tumorigenesis induces early priming and expansion of tumor/self-Ag–specific Tregs in draining lymph nodes.

Figure 2.

Tumor/self-antigen–specific thymic Tregs proliferate and function in draining lymph nodes during tumorigenesis. Lymph nodes draining 4-HT–induced skin were analyzed in Braf/Pten and WT mice. A–C, Frequency of CD4+FoxP3+ Tregs gated on CD3+ cells (A), ratio of FoxP3+ Treg to CD8+ T cells over time (B), and frequency of T-cell subsets 26 days postinduction, gated on CD4+FoxP3+ Tregs (C). D, CD4+CD25+ Tregs from pooled Braf/Pten DLNs versus WT-naïve lymph nodes (n = 4–5 mice/group) were assayed for suppression of CellTrace-Violet–labeled CD8 T-cell proliferation ex vivo (see Supplementary Materials and Methods). Representative flow plots depict 1:1 Treg:CD8 ratio. Dashed lines represent percentage of divided cells in the absence of Tregs (top) or absence of stimulation (bottom); symbols represent mean of 3 wells ± SD. E, Schematic for FI depicting the adoptive transfer of Thy1.1+CD4+ TRP-1 T cells into WT versus Braf/Pten mice on day 16 and analysis 10 days later. F, Absolute numbers of TRP-1 Tregs 26 days postinduction. G, Frequency of TRP-1 Tregs with indicated phenotypes on the day of transfer (input) and 26 days postinduction. H, Effect of DT administration on the input population of CD4+ TRP-1 cells. I, Number of TRP-1 Tregs on day 26 (gated on CD3+ cells) in induced Braf/Pten mice that received a transfer of intact or Treg-depleted TRP-1 cells. Data were pooled from A–C, F, G, and I or are representative of two independent experiments (D), each with n ≥ 3 mice/group; *, P < 0.05; **, P < 0.01; ***, P < 0.001, analyzed by ANOVA (Tukey post hoc, A–D) and t test (F and I). Symbols and error bars, means ± SEM (AD, and G); symbols represent individual mice and horizontal lines depict means (F and I). Absence of error bar indicates SEM less than area represented by symbol.

Figure 2.

Tumor/self-antigen–specific thymic Tregs proliferate and function in draining lymph nodes during tumorigenesis. Lymph nodes draining 4-HT–induced skin were analyzed in Braf/Pten and WT mice. A–C, Frequency of CD4+FoxP3+ Tregs gated on CD3+ cells (A), ratio of FoxP3+ Treg to CD8+ T cells over time (B), and frequency of T-cell subsets 26 days postinduction, gated on CD4+FoxP3+ Tregs (C). D, CD4+CD25+ Tregs from pooled Braf/Pten DLNs versus WT-naïve lymph nodes (n = 4–5 mice/group) were assayed for suppression of CellTrace-Violet–labeled CD8 T-cell proliferation ex vivo (see Supplementary Materials and Methods). Representative flow plots depict 1:1 Treg:CD8 ratio. Dashed lines represent percentage of divided cells in the absence of Tregs (top) or absence of stimulation (bottom); symbols represent mean of 3 wells ± SD. E, Schematic for FI depicting the adoptive transfer of Thy1.1+CD4+ TRP-1 T cells into WT versus Braf/Pten mice on day 16 and analysis 10 days later. F, Absolute numbers of TRP-1 Tregs 26 days postinduction. G, Frequency of TRP-1 Tregs with indicated phenotypes on the day of transfer (input) and 26 days postinduction. H, Effect of DT administration on the input population of CD4+ TRP-1 cells. I, Number of TRP-1 Tregs on day 26 (gated on CD3+ cells) in induced Braf/Pten mice that received a transfer of intact or Treg-depleted TRP-1 cells. Data were pooled from A–C, F, G, and I or are representative of two independent experiments (D), each with n ≥ 3 mice/group; *, P < 0.05; **, P < 0.01; ***, P < 0.001, analyzed by ANOVA (Tukey post hoc, A–D) and t test (F and I). Symbols and error bars, means ± SEM (AD, and G); symbols represent individual mice and horizontal lines depict means (F and I). Absence of error bar indicates SEM less than area represented by symbol.

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To determine the extent to which thymically derived Tregs contributed to this early response during tumorigenesis, we predepleted FoxP3+ thymic Tregs in the input TRP-1 T-cell population by DT treatment prior to adoptive transfer on day 16 (Fig. 2H; TRP-1 Tg mice also expressed the diphtheria toxin receptor under the control of the Foxp3 promoter; ref. 27). Ten days posttransfer, mice that received FoxP3-depleted TRP-1 T cells generated an order of magnitude fewer Ag-specific Tregs in DLNs, as compared with mice transferred with an intact FoxP3+ compartment (Fig. 2I). Thus, the priming of thymic Tregs, as opposed to the conversion of CD4+FoxP3 T cells, dominates the early Ag-specific Treg response to tumorigenesis in DLNs.

To determine whether thymic Tregs also constituted the earliest population recruited to nascent tumor sites, the same experiment was performed, and skin was analyzed on day 26. Following DT depletion of thymic Tregs, accumulation of FoxP3+ cells was drastically reduced in induced skin (Fig. 3A), indicating that nascent tumor-infiltrating Tregs also derived from the thymic subset. To determine whether these Tregs originated from populations primed in DLNs, the sphingosine-1-phosphate analogue FTY720 was used to block T-cell egress from lymph nodes. Indeed, upon treatment with FTY720, the number of transferred TRP-1–specific Tregs was significantly decreased in skin (Fig. 3B). A similar decrease was observed in the polyclonal Treg population (Fig. 3C). Thus, optimal Treg accumulation in nascent tumors required thymic Treg egress from lymph nodes.

Figure 3.

Thymic Tregs home from draining lymph nodes to nascent tumor sites in skin. A, Absolute number of Thy1.1+Foxp3+ TRP-1 Tregs in induced skin of Braf/Pten mice (day 26) that received adoptive transfer of intact versus DT-depleted TRP-1 T cells on day 16. B and C, Absolute numbers of FoxP3+ Tregs in Braf/Pten versus WT skin 26 days postinduction ± FTY720 treatment. B, Numbers of TRP-1–specific Tregs; intact TRP-1 T cells were transferred 10 days prior. C, Endogenous Foxp3+ Tregs. D, Braf/Pten tail skin was grafted onto Rag−/− versus WT hosts, and numbers of FoxP3+ Tregs were quantified in skin grafts 21 days postinduction. E, Schematic depicting Treg recruitment assay used in FH; Rag−/− mice bearing Braf/Pten skin grafts received adoptive transfer (A.T.) of T cells on day 19, and Tregs were assessed in grafts 48 hours later (day 21). F, Absolute numbers of Tregs in skin grafts following adoptive transfer of T cells derived from tumor DLNs versus naïve lymph nodes. G, Proportions (left) and absolute numbers (right) of Tregs in induced skin grafts versus adjacent skin and DLNs. H, Treg:CD8 ratios in induced skin grafts versus adjacent skin. Data in each panel were pooled from two independent experiments, each with n ≥ 3 mice/group (AG) or n ≥ 2 (F), with *, P < 0.05; **, P < 0.01; ***, P < 0.001, analyzed by Mann–Whitney test (A), Kruskal–Wallis (Dunn post hoc; B), ANOVA (Tukey post hoc; C and H), and t test (D,F, and G). Symbols represent individual mice, horizontal lines depict means (A–G), and symbols and error bars, means ± SEM (H).

Figure 3.

Thymic Tregs home from draining lymph nodes to nascent tumor sites in skin. A, Absolute number of Thy1.1+Foxp3+ TRP-1 Tregs in induced skin of Braf/Pten mice (day 26) that received adoptive transfer of intact versus DT-depleted TRP-1 T cells on day 16. B and C, Absolute numbers of FoxP3+ Tregs in Braf/Pten versus WT skin 26 days postinduction ± FTY720 treatment. B, Numbers of TRP-1–specific Tregs; intact TRP-1 T cells were transferred 10 days prior. C, Endogenous Foxp3+ Tregs. D, Braf/Pten tail skin was grafted onto Rag−/− versus WT hosts, and numbers of FoxP3+ Tregs were quantified in skin grafts 21 days postinduction. E, Schematic depicting Treg recruitment assay used in FH; Rag−/− mice bearing Braf/Pten skin grafts received adoptive transfer (A.T.) of T cells on day 19, and Tregs were assessed in grafts 48 hours later (day 21). F, Absolute numbers of Tregs in skin grafts following adoptive transfer of T cells derived from tumor DLNs versus naïve lymph nodes. G, Proportions (left) and absolute numbers (right) of Tregs in induced skin grafts versus adjacent skin and DLNs. H, Treg:CD8 ratios in induced skin grafts versus adjacent skin. Data in each panel were pooled from two independent experiments, each with n ≥ 3 mice/group (AG) or n ≥ 2 (F), with *, P < 0.05; **, P < 0.01; ***, P < 0.001, analyzed by Mann–Whitney test (A), Kruskal–Wallis (Dunn post hoc; B), ANOVA (Tukey post hoc; C and H), and t test (D,F, and G). Symbols represent individual mice, horizontal lines depict means (A–G), and symbols and error bars, means ± SEM (H).

Close modal

As FTY720 treatment did not completely eliminate Treg accumulation in induced skin, we separately assessed whether skin-resident Tregs proliferated in situ during tumorigenesis. Skin from Braf/Pten mice was grafted onto T cell–deficient (Rag−/−) mice (Supplementary Fig. S3A). As grafting necessitated the use of melanocyte-rich tail skin, tumor appearance and Treg infiltration was accelerated in this model, occurring on day 21 (Supplementary Fig. S3B–S3D). When tumors were induced in Braf/Pten skin grafts on Rag−/− hosts, Tregs were virtually absent, indicating a lack of contribution from skin-resident Tregs (Fig. 3D). Separately, when Braf/Pten skin was grafted onto congenically distinct (Thy1.1+) mice, Tregs on day 21 overwhelmingly expressed the congenic marker of the host (Supplementary Fig. S3E), further supporting their origination from the lymphoid compartment.

To formally demonstrate that migration mediates the accumulation of tumor-experienced Tregs in the skin, T cells from either naïve lymph nodes or tumor-DLNs were transferred into Rag−/− recipients 19 days following induction of Braf/Pten skin grafts, and infiltration was assessed 48 hours later (Fig. 3E). Whereas the input cell population from tumor-bearing donors had a slightly elevated Treg proportion as compared with naïve donors (Supplementary Fig. S4A), this difference was amplified in vivo with tumor-experienced Tregs migrating in 4-fold greater numbers to tumor-induced skin (Fig. 3F). More detailed analysis of DLN-derived Treg migration indicated preferential trafficking to induced skin grafts, as compared with adjacent host skin or lymph nodes (Fig. 3G). This was in contrast to the CD8 T cells from the transferred, tumor-experienced DLN population, which predominantly trafficked to lymph nodes, resulting in a high Treg:CD8 ratio in skin grafts (Fig. 3H). Taken together, these data illustrate that melanoma tumorigenesis induces the priming and recruitment of Tregs, but not CD8 T cells, and suggest a dominant role for Ag-specific thymic Tregs at this early stage.

BRAFV600E signaling governs chemokine-driven recruitment of Tregs during tumorigenesis

The mechanisms of early thymic Treg recruitment to sites of tumorigenesis remained to be elucidated. To determine whether chemotaxis was involved, tumor-experienced T cells were treated ex vivo with PTx to block chemokine receptor signaling, prior to adoptive transfer as in Fig. 3E. PTx had negligible effects on the viability of T cells in the input population (Supplementary Fig. S4B). However, consistent with a role for chemokine receptors, PTx-pretreatment significantly decreased the number of Tregs migrating to induced skin (Fig. 4A). As reports have implicated C-C chemokine receptor 4 (Ccr4) in the accumulation of Tregs in established tumors (10, 28), Ccr4 expression was assessed in Tregs from induced-skin DLNs. Compared with Tregs from naïve lymph nodes, approximately twice as many Tregs from DLN expressed Ccr4 (Fig. 4B). Similarly, a majority of TRP-1–specific Tregs expressed Ccr4 following tumor Ag encounter in DLNs (Fig. 4C). Moreover, the expression of three Ccr4-cognate chemokines, Ccl17, Ccl2, and Ccl22, increased in Braf/Pten skin within the 21- to 26-day window following tumor induction (Fig. 4D; Supplementary Fig. S5A). To determine whether these chemokines were required for Treg accumulation during tumorigenesis, mice were treated with a combination of Ccl17, Ccl2, and Ccl22 neutralizing mAbs throughout the day 21 to 26 Treg recruitment window. Chemokine neutralization significantly reduced Treg accumulation by proportion of CD3+ T cells; however, absolute numbers of Tregs were only modestly decreased (Supplementary Fig. S5B). To more definitively test whether Ccr4 was required for Treg accumulation during tumorigenesis, the Ccr4 antagonist C-021 was administered to tumor-induced mice throughout days 21 to 26. FoxP3+ Treg numbers were significantly decreased in induced skin of antagonist-treated mice (Fig. 4E), confirming a role for Ccr4 in Treg recruitment.

Figure 4.

The Ccr4 chemotactic axis controls Treg homing during melanoma tumorigenesis. A, According to schematic in Fig. 3E, mice were grafted with Braf/Pten skin and T cells were transferred following ex vivo treatment with vehicle (Veh) or pertussis toxin (PTx). CD4+FoxP3+ Tregs were enumerated in grafts 48 hours posttransfer. B, Naïve lymph nodes (LN) versus LNs that drain day 26–induced skin from Braf/Pten mice (DLN) were analyzed for Ccr4 on endogenous CD4+Foxp3+ Tregs. C, Thy1.1+ TRP-1 T cells were transferred into Braf/Pten mice as in Fig. 3E, and Ccr4 was assessed on Thy1.1+Foxp3+ TRP-1 Tregs on the day of transfer (input) versus 10 days later in DLNs. D, Quantitative RT-PCR analysis of Ccr4 cognate ligands over time in Braf/Pten versus WT skin. E, Tregs were enumerated in the skin of WT versus Braf/Pten mice 26 days postinduction, with or without daily administration of Ccr4 antagonist (C-021) beginning on day 21. AE, Data in each panel were pooled from two independent experiments, each with n ≥ 3 mice/group (“input” group in C represents two in vitro measurements), with **, P < 0.01; ***, P < 0.001; NS, nonsignificant, analyzed by t test (A), or ANOVA (Bonferroni post hoc; BE). Symbols represent individual mice, and horizontal lines depict means (A–C, and E); symbols and error bars, means ± SEM (D).

Figure 4.

The Ccr4 chemotactic axis controls Treg homing during melanoma tumorigenesis. A, According to schematic in Fig. 3E, mice were grafted with Braf/Pten skin and T cells were transferred following ex vivo treatment with vehicle (Veh) or pertussis toxin (PTx). CD4+FoxP3+ Tregs were enumerated in grafts 48 hours posttransfer. B, Naïve lymph nodes (LN) versus LNs that drain day 26–induced skin from Braf/Pten mice (DLN) were analyzed for Ccr4 on endogenous CD4+Foxp3+ Tregs. C, Thy1.1+ TRP-1 T cells were transferred into Braf/Pten mice as in Fig. 3E, and Ccr4 was assessed on Thy1.1+Foxp3+ TRP-1 Tregs on the day of transfer (input) versus 10 days later in DLNs. D, Quantitative RT-PCR analysis of Ccr4 cognate ligands over time in Braf/Pten versus WT skin. E, Tregs were enumerated in the skin of WT versus Braf/Pten mice 26 days postinduction, with or without daily administration of Ccr4 antagonist (C-021) beginning on day 21. AE, Data in each panel were pooled from two independent experiments, each with n ≥ 3 mice/group (“input” group in C represents two in vitro measurements), with **, P < 0.01; ***, P < 0.001; NS, nonsignificant, analyzed by t test (A), or ANOVA (Bonferroni post hoc; BE). Symbols represent individual mice, and horizontal lines depict means (A–C, and E); symbols and error bars, means ± SEM (D).

Close modal

We have previously reported that established Braf/Pten tumors express Ccl17 and Ccl2, and that these transcripts are downregulated following treatment with small-molecule BRAFV600E inhibitors (BRAFi; ref. 15). To assess whether Ccr4-cognate chemokines are also regulated during tumorigenesis, their expression was analyzed 26 days postinduction, with or without BRAFi administered 24 hours earlier. Indeed, BRAFi treatment rapidly decreased expression of all three chemokines in induced skin (Fig. 5A; Supplementary Fig. S5C). To separately determine whether BRAFV600E signaling regulates Treg migration to induced skin, T cells from DLNs were adoptively transferred into Rag−/− mice bearing induced Braf/Pten skin grafts (see Fig. 3E), with or without concurrent BRAFi treatment. Forty-eight hours posttransfer, both the proportions and absolute numbers of skin migrating Tregs were reduced in BRAFi-treated animals (Fig. 5B). In contrast, BRAFi had no effect on Treg accumulation in lymph nodes (Fig. 5C). Taken together, these data show that BRAFV600E signaling promotes the expression of Ccr4 cognate chemokines and the associated recruitment of Tregs to nascent tumor sites.

Figure 5.

Oncogenic BRAFV600E governs Treg recruitment during tumor emergence. A, qRT-PCR of Ccr4 cognate ligands in WT versus induced Braf/Pten skin, in mice gavaged with PLX4032 (BRAFi; +), or vehicle (–) 24 hours prior to analysis. B and C, According to schematic in Fig. 3E, mice were grafted with Braf/Pten tail skin and treated with vehicle or BRAFi 24 hours prior to adoptive transfer of total T cells derived from day 26 tumor-DLNs (see Materials and Methods). B, Proportions and absolute number of FoxP3+ Tregs in induced Braf/Pten grafts. C, Proportions of Tregs in DLN-induced grafts. D–F, WT versus BRAFV600E skin (nevus) was analyzed 31 days postinduction. Representative plots showing CD4+FoxP3+ Tregs (D) gated on CD3+ cells and CD8+ T cells (E) gated on CD3+ cells. F, Ratio of FoxP3+ Tregs to CD8 T cells. G, WT and nevus-induced mice received 2 × 105 Thy1.1+CD4+ TRP-1 T cells on day 21, and numbers of TRP-1 Tregs were analyzed in skin 10 days later. AG, Data in each panel were pooled from two independent experiments, each with n ≥ 4 mice/group (symbols represent individual mice; horizontal lines, means), with **, P < 0.01; ***, P < 0.001; and NS, nonsignificant, analyzed by ANOVA (Bonferroni post hoc; A), t test (B–F), or Mann–Whitney test (G).

Figure 5.

Oncogenic BRAFV600E governs Treg recruitment during tumor emergence. A, qRT-PCR of Ccr4 cognate ligands in WT versus induced Braf/Pten skin, in mice gavaged with PLX4032 (BRAFi; +), or vehicle (–) 24 hours prior to analysis. B and C, According to schematic in Fig. 3E, mice were grafted with Braf/Pten tail skin and treated with vehicle or BRAFi 24 hours prior to adoptive transfer of total T cells derived from day 26 tumor-DLNs (see Materials and Methods). B, Proportions and absolute number of FoxP3+ Tregs in induced Braf/Pten grafts. C, Proportions of Tregs in DLN-induced grafts. D–F, WT versus BRAFV600E skin (nevus) was analyzed 31 days postinduction. Representative plots showing CD4+FoxP3+ Tregs (D) gated on CD3+ cells and CD8+ T cells (E) gated on CD3+ cells. F, Ratio of FoxP3+ Tregs to CD8 T cells. G, WT and nevus-induced mice received 2 × 105 Thy1.1+CD4+ TRP-1 T cells on day 21, and numbers of TRP-1 Tregs were analyzed in skin 10 days later. AG, Data in each panel were pooled from two independent experiments, each with n ≥ 4 mice/group (symbols represent individual mice; horizontal lines, means), with **, P < 0.01; ***, P < 0.001; and NS, nonsignificant, analyzed by ANOVA (Bonferroni post hoc; A), t test (B–F), or Mann–Whitney test (G).

Close modal

To formally determine whether BRAFV600E signaling is sufficient to recruit Tregs, we generated mice expressing melanocyte-restricted tamoxifen-inducible BRAFV600E, but with normal Pten expression. Consistent with prior reports in similar models (29, 30), topical induction of BRAFV600E elicited hyperpigmented, benign melanocytic lesions in skin (Supplementary Fig. S6A). These nevus-like lesions expressed elevated levels of melanocyte Ags (Tyrp1, gp100, and Tyr) and Ccr4-cognate chemokines (Ccl17, Ccl2, and Ccl22) by both RNA and protein (Supplementary Fig. S6B–S6D). In nevus-induced skin on day 31, FoxP3+ Tregs were significantly increased by both proportion of CD3+ T cells, and by absolute number (Fig. 5D), indicating that BRAFV600E expression is sufficient to promote the accumulation of Tregs. In contrast, CD8 T-cell proportions decreased, while absolute numbers remained unchanged in nevi (Fig. 5E). This is consistent with an influx of CD3+ Tregs, and resulted in a 2-fold increase in the Treg:CD8 ratio (Fig. 5F). This change was less pronounced in nevi than in Braf/Pten skin (see Fig. 1H), albeit significant. Moreover, adoptively transferred TRP-1–specific Tregs accumulated in nevus-bearing skin (Fig. 5G), demonstrating an Ag-specific Treg response. These data collectively support the conclusion that BRAFV600E governs the recruitment of Tregs during autochthonous tumorigenesis.

Optimal tumorigenesis requires Treg suppression of CD8 T cell–mediated immunosurveillance

The tumor-promoting role of Tregs has previously been demonstrated in established cancers (3). However, it has remained unknown whether Tregs prevent early T cell–mediated immunosurveillance against autochthonous, poorly immunogenic cancers. To assess this, Braf/Pten skin was grafted onto Foxp3DTR mice, and Tregs were depleted by DT treatment beginning 17 days postinduction. The depletion of Tregs resulted in a 40-fold increase in CD8 T cells in the skin 26 days postinduction (Fig. 6A), indicating that CD8 T cells could readily infiltrate early tumor lesions if Tregs were absent. To separately address this question in a more high-throughput (i.e., non skin-grafted) setting, we selected anti-CD4 mAb as an established therapy to deplete Tregs without impairing CD25+ activated effector T cells (4). Consistent with data from Foxp3DTR mice, anti-CD4 treatment also resulted in robust CD8 T-cell accumulation in tumor-induced skin (Fig. 6B). These data collectively indicate that Tregs suppress CD8 T-cell accumulation, and further suggest that CD4 T-cell help is not absolutely required for CD8 T-cell infiltration during tumorigenesis.

Figure 6.

Optimal Braf/Pten tumorigenesis requires FoxP3+ Tregs. A,Foxp3DTR mice bearing day 17–induced Braf/Pten skin grafts received DT every 2 days, or were untreated, and absolute numbers of CD8 T cells were assessed in grafts 26 days postinduction. B, Absolute numbers of CD8 T cells in Braf/Pten skin 26 days postinduction, ± anti-CD4–depleting antibody (clone GK1.5) on days 16 and 22. C, Braf/Pten versus WT mice (treated as in B) received adoptive transfer of 1 × 105 Thy1.1+CD8+CD44 naïve pmel cells on day 12; proportions of pmel cells in DLN-induced skin (left) and absolute numbers in induced skin (right) were assessed 26 days postinduction. D and E, WT versus Braf/Pten mice received naïve pmel cells 14 days prior to analysis on indicated days (postinduction); transfer was followed by CD4 depletion on days 4 and 10; pmel cells were then enumerated in DLNs (D) or induced skin (E). F, Proportions of pmel cells in DLNs (left) and absolute numbers in induced skin (right) 31 days (WT and nevus BRAFV600E mice) postinduction (Braf/Pten mice were analyzed on day 26 as a positive control); mice were treated with anti-CD4 as in D. G, Thickness of induced Braf/Pten skin grafts on WT versus Foxp3DTR mice that received DT (white arrows) alone or concurrent with anti-CD8 mAb (gray arrows). Data in each panel were pooled from two independent experiments each with n ≥ 3 mice/group (B–G) or n ≥ 2 (F), with *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, nonsignificant, analyzed by t test (A, B, and D), Mann–Whitney test (E), ANOVA (Bonferroni post hoc; C, left; F, left; G), and Kruskal–Wallis (Dunn post hoc; C, right; F, right). Symbols represent individual mice (A–F), and symbols and error bars represent means ± SEM (G).

Figure 6.

Optimal Braf/Pten tumorigenesis requires FoxP3+ Tregs. A,Foxp3DTR mice bearing day 17–induced Braf/Pten skin grafts received DT every 2 days, or were untreated, and absolute numbers of CD8 T cells were assessed in grafts 26 days postinduction. B, Absolute numbers of CD8 T cells in Braf/Pten skin 26 days postinduction, ± anti-CD4–depleting antibody (clone GK1.5) on days 16 and 22. C, Braf/Pten versus WT mice (treated as in B) received adoptive transfer of 1 × 105 Thy1.1+CD8+CD44 naïve pmel cells on day 12; proportions of pmel cells in DLN-induced skin (left) and absolute numbers in induced skin (right) were assessed 26 days postinduction. D and E, WT versus Braf/Pten mice received naïve pmel cells 14 days prior to analysis on indicated days (postinduction); transfer was followed by CD4 depletion on days 4 and 10; pmel cells were then enumerated in DLNs (D) or induced skin (E). F, Proportions of pmel cells in DLNs (left) and absolute numbers in induced skin (right) 31 days (WT and nevus BRAFV600E mice) postinduction (Braf/Pten mice were analyzed on day 26 as a positive control); mice were treated with anti-CD4 as in D. G, Thickness of induced Braf/Pten skin grafts on WT versus Foxp3DTR mice that received DT (white arrows) alone or concurrent with anti-CD8 mAb (gray arrows). Data in each panel were pooled from two independent experiments each with n ≥ 3 mice/group (B–G) or n ≥ 2 (F), with *, P < 0.05; **, P < 0.01; ***, P < 0.001; NS, nonsignificant, analyzed by t test (A, B, and D), Mann–Whitney test (E), ANOVA (Bonferroni post hoc; C, left; F, left; G), and Kruskal–Wallis (Dunn post hoc; C, right; F, right). Symbols represent individual mice (A–F), and symbols and error bars represent means ± SEM (G).

Close modal

To determine whether early, Treg-restrained CD8 T-cell responses are tumor/self-Ag specific, mice received sentinel populations of congenically marked naïve CD8 T cells specific for gp10025-33 (pmel cells). Despite local tumorigenesis, pmel cells remained virtually undetectable in DLNs or early tumors on day 26 (Fig. 6C). However, Treg depletion with anti-CD4 promoted a significant accumulation of pmel cells in both locations in tumor-induced mice, but not in WT mice (Fig. 6C). To determine whether Tregs functionally prevent CD8 T-cell responses during incipient stages of tumorigenesis, earlier time points were analyzed. Indeed, in DLNs of Treg-depleted mice, pmel cells accumulated as early as 16 days postinduction (Fig. 6D), which was concurrent with the earliest microscopically detected melanocytic lesions (see Fig. 1B). However, pmel cells were not detected in induced skin at these earlier time points (Fig. 6E), suggesting that recruitment of CD8 T cells, like Tregs, is limited by local tumor-derived factors. Interestingly, Treg depletion did not elicit pmel cell priming or migration in response to benign melanocytic nevi (Fig. 6F), indicating that malignant transformation is required for CD8 T-cell responsiveness. Finally, to determine whether Treg-restrained immunosurveillance can suppress tumorigenesis, tumor formation was assessed in Foxp3DTR mice bearing Braf/Pten skin grafts. Indeed, the depletion of Tregs with DT resulted in marked control of induced skin thickening, an effect that was lost upon codepletion of CD8 T cells (Fig. 6G). Therefore, in this early autochthonous malignancy, Treg suppression potently restrains CD8 T cell–mediated immunosurveillance.

This work has uncovered the origins of Treg responses during autochthonous tumorigenesis and unveiled oncogenic BRAFV600E as a key driver of early Treg recruitment. We show that Treg recruitment requires the preactivation of Tregs in DLNs, and that self-Ag–specific thymic Tregs are involved in the process. This Treg response leads to the suppression of CD8 T cell–mediated immunosurveillance as early as microscopic neoplastic lesions develop. Thus, Treg-mediated immunosuppression potently counteracts cancer immunosurveillance even in settings of early autochthonous tumorigenesis.

Previous studies by ourselves (14) and others (16, 17, 31) have defined T-cell infiltrates in established BRAFV600E-driven melanoma tumors, yet little was known about T-cell responses during tumorigenesis. The current studies now identify Tregs as the dominant T-cell responders to early tumors. This observation is consistent with prior studies showing early preferential accumulation of Tregs in pancreatic ductal adenocarcinoma in patients (32) and in a Kras-driven pancreatic mouse model (11). The current finding that Ag-specific Tregs, but not CD8 T cells, are activated in early tumor-DLNs is consistent with previous findings that Tregs outpace CD8 T cells during early tumor growth (2). In accordance with the poor immunogenicity of autochthonous tumors and their lack of strong neoantigens, we observe a lack of endogenous CD8 T-cell responses during tumorigenesis, which is due to the presence of Tregs. In conjunction with published studies (2), our work supports the conclusion that Tregs are the earliest T-cell responders to neoplastic transformation.

Our work also underscores the participation of thymically derived, self-Ag–specific Tregs during melanoma emergence. Our demonstration that self-Ag–specific tTregs characterize the early response during tumorigenesis is consistent with reports showing that the TCR repertoires of tumor-infiltrating CD4+FoxP3+ and CD4+FoxP3 cells are largely distinct, and instead resembled their DLN counterparts (33). Our findings are also consistent with a previous report that Tregs in autochthonous prostate tumors are Aire dependent and recognize a tumor-expressed self-Ag (20). As autochthonous oncogene-driven tumors were shown to lack predicted neoepitopes (26), our findings lend credence to the notion that self-Ag–specific thymic Tregs dominate the T-cell responses elicited during early tumorigenesis.

The current in vivo migration studies illustrate that BRAFV600E is a critical tumor cell–intrinsic driver of Treg recruitment. This observation is consistent with reports by us and others showing that treatment with BRAFV600E inhibitors for >7 days decreases Treg prevalence in established Braf/Pten tumors (14–16). The current work underscores the rapidity with which Treg migration is inhibited in response to BRAFi. Importantly, this work also provides a new example in a growing body of research linking oncogenic signaling and immune regulation (34, 35). Previous reports have linked c-KIT signaling to IDO expression in gastrointestinal stromal cell tumors (36), Pten loss to innate anti–PD-1 resistance in melanomas (37), Myc to CD47, and PD-L1 expression in lymphomas (38), and β-catenin to reduced effector T-cell infiltration in melanomas (31). As BRAFV600E was previously shown to govern the accumulation of MDSCs in established tumors (14, 15), future studies should address whether BRAFV600E induction also promotes MDSC accumulation during tumorigenesis.

Our studies underscore the role of oncogene-driven chemotactic cues in Treg recruitment. The finding that FTY720 decreases Treg recruitment during tumorigenesis corroborates a previous report that S1PR1 deficiency reduced Treg accumulation in implanted tumors (39). We demonstrate that Treg recruitment to early melanoma sites requires Ccr4 signaling, consistent with the role of this axis in Treg trafficking to transplantable ovarian and melanoma tumors (10, 28). Our studies also indicate that tumor induction significantly increases expression of multiple Ccr4 cognate chemokines in the skin. This is consistent with previous findings that BRAFi results in the downregulation of Ccl2 and Ccl17 in established Braf/Pten tumors (15) and Ccl2 in transplantable tumors derived from Braf/Pten mice (40). Although it remains unclear which chemokine(s) mediate Treg migration, this relationship may be complex due to the functional redundancy of Ccl2, Ccl22, and Ccl17 (41). In addition, the inability of the Ccr4 antagonist to completely abrogate Treg migration suggests that additional chemokine receptors may be involved.

BRAFV600E mutations exist in 78% of human nevi (42) and persist in the course of melanoma's genetic evolution from precursor lesions (43). Yet, T-cell responses have remained poorly understood in relation to preneoplastic lesions. Our studies reveal that Tregs also preferentially accumulate in response to BRAFV600E-driven nevus formation. This recruitment was less pronounced than in induced Braf/Pten skin, which may relate to the modest induction of chemokines and melanocyte Ags, and underscores the role of additional oncogenic pathways (e.g., Pten) in the optimal recruitment of Tregs. Prior studies indicate that BRAFV600E nevus-like lesions are largely benign (29, 30) and exhibit senescence-like cell-cycle arrest typical of human nevi (44). However, these lesions progress to melanoma with a median latency of 12 months (29). Interestingly, FoxP3+ Tregs are prevalent in human atypical nevi and radial growth phase melanomas (45), suggesting a link between Tregs and the transition of melanocytic lesions to malignancy. One may speculate that nevus-associated Tregs could facilitate progression to melanoma through local immunosuppression.

Although our studies do not detect Treg accumulation in tumor-induced skin prior to day 26, Tregs play a critical role in suppressing immunosurveillance prior to this day. Indeed, when Tregs were depleted, tumor/self-Ag–specific CD8 T cells accumulated in DLNs very early. Our analyses show that T cell–mediated immunosurveillance is compromised even before increased tumor/self-Ag expression is detectable in induced skin. Our failure to detect CD8 T-cell responses in Treg-intact mice is in contrast to a recent report that SV40 neoantigen-specific CD8 T cells proliferate in response to premalignant lesions of SV40-driven autochthonous liver cancer, and only subsequently do these antitumor T cells become dysfunctional (46). In our model, CD8 T-cell ignorance is Treg mediated from the outset, and only following Treg depletion are CD8 T cells capable of controlling tumor outgrowth. Interestingly, pmel cells remained ignorant in nevus-bearing animals even after Treg depletion, indicating that neoplastic transformation is required to break tolerance. The expansion of self-Ag–specific CD8 T cells in induced Braf/Pten mice corroborates our previous reports that pmel cells are primed following Treg depletion in mice bearing established B16 and Braf/Pten tumors (14, 47), while further demonstrating the highly sensitive nature of CD8 T cells during early microscopic tumor formation.

The current work is not without limitations. Analysis of the causal connection between Tregs and CD8 T cells required the use of skin grafts on Foxp3DTR mice, so it remains uncertain whether Tregs are the predominant tumor escape mechanism during autochthonous tumorigenesis. These studies are also limited to a single model, and it is not yet clear whether implications extend to all melanomas or other types of cancer. Finally, the mechanistic underpinnings of oncogene-driven chemokine production remain unclear. Oncogenic BRAFV600E increases the activity of transcriptional factors including NFκB and AP-1 (48, 49), which can promote chemokine expression (50). However, pathways linking BRAFV600E signaling and Ccr4-cognate chemokine expression will require further investigation.

In summary, this work illustrates an absence of effective cancer immunosurveillance in the presence of dominant early Treg suppression. By establishing a link between BRAF signaling and Treg responses, these studies highlight the inherently immunosuppressive nature of oncogene-driven cancers. Tregs remain a formidable obstacle to generating effective immunity against cancer. Their key requirement during early tumorigenesis further underscores the integral role of Tregs throughout cancer progression.

T.J. Curiel is a consultant/advisory board member for Agenus. No potential conflicts of interest were disclosed by the other authors.

Conception and design: T.B. Shabaneh, A. Boni, M.J. Turk

Development of methodology: T.B. Shabaneh, P. Zhang, M.J. Turk

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): T.B. Shabaneh, A.K. Molodtsov, S.M. Steinberg, P. Zhang, G.M. Torres

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): T.B. Shabaneh, A.K. Molodtsov, S.M. Steinberg, P. Zhang, T.J. Curiel, M.J. Turk

Writing, review, and/or revision of the manuscript: T.B. Shabaneh, T.J. Curiel, C.V. Angeles, M.J. Turk

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): T.B. Shabaneh, P. Zhang, G.A. Mohamed

Study supervision: T.B. Shabaneh, M.J. Turk

The authors thank W. Green, R. Noelle, Y. Huang, and K. Hvorecny for helpful discussions, technical guidance, and providing reagents. We thank M. Bosenberg, N. Restifo, R. Noelle, and P. Antony for providing mice. PLX4720 was provided by Plexxikon Inc. under a Materials Transfer Agreement. This work was supported by NIH R21CA209375-01 (NCI), NIH R01CA120777-06 (NCI), NIH R01 CA225028, the generous philanthropy of the Knights of the York Cross of Honour, and a grant from The Melanoma Research Alliance, to M.J. Turk. This work was also supported by NIH R01CA205965 to T.J. Curiel. C.V. Angeles was supported by NIH KL2TR991088. T.B. Shabaneh was supported by NIH T32-AI0073634. Some analyses were carried out in DartLab, a shared resource supported (http://dx.doi.org/10.13039/100000002) by NIH P30CA023108-36 and NIH P30GM103415-14.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Dunn
GP
,
Old
LJ
,
Schreiber
RD
. 
The three Es of cancer immunoediting
.
Annu Rev Immunol
2004
;
22
:
329
60
.
2.
Darrasse-Jèze
G
,
Bergot
A
,
Durgeau
A
,
Billiard
F
,
Salomon
BL
,
Cohen
JL
, et al
Tumor emergence is sensed by self-specific CD44hi memory Tregs that create a dominant tolerogenic environment for tumors in mice
.
J Clin Invest
2009
;
119
:
2648
62
.
3.
Bos
PD
,
Plitas
G
,
Rudra
D
,
Lee
SY
,
Rudensky
AY
. 
Transient regulatory T cell ablation deters oncogene-driven breast cancer and enhances radiotherapy
.
J Exp Med
2013
;
210
:
2435
66
.
4.
Turk
MJ
,
Guevara-Patiño
JA
,
Rizzuto
GA
,
Engelhorn
ME
,
Houghton
AN
. 
Concomitant tumor immunity to a poorly immunogenic melanoma is prevented by regulatory T cells
.
J Exp Med
2004
;
200
:
771
82
.
5.
Onizuka
S
,
Tawara
I
,
Shimizu
J
,
Sakaguchi
S
,
Fujita
T
,
Nakayama
E
. 
Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody
.
Cancer Res
1999
;
59
:
3128
33
.
6.
Quezada
SA
,
Peggs
KS
,
Curran
MA
,
Allison
JP
. 
CTLA4 blockade and GM-CSF combination immunotherapy alters the intratumor balance of effector and regulatory T cells
.
J Clin Invest
2006
;
116
:
1935
45
.
7.
DeLeeuw
RJ
,
Kost
SE
,
Kakal
JA
,
Nelson
BH
. 
The prognostic value of FoxP3+ tumor-infiltrating lymphocytes in cancer: a critical review of the literature
.
Clin Cancer Res
2012
;
18
:
3022
9
.
8.
Shang
B
,
Liu
Y
,
Jiang
S
,
Liu
Y
. 
Prognostic value of tumor-infiltrating FoxP3+ regulatory T cells in cancers: a systematic review and meta-analysis
.
Sci Rep
2015
;
5
:
15179
.
9.
Chen
DS
,
Mellman
I
. 
Oncology meets immunology: the cancer-immunity cycle
.
Immunity
2013
;
39
:
1
10
.
10.
Curiel
TJ
,
Coukos
G
,
Zou
L
,
Alvarez
X
,
Cheng
P
,
Mottram
P
, et al
Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival
.
Nat Med
2004
;
10
:
942
9
.
11.
Clark
CE
,
Hingorani
SR
,
Mick
R
,
Combs
C
,
Tuveson
DA
,
Vonderheide
RH
. 
Dynamics of the immune reaction to pancreatic cancer from inception to invasion
.
Cancer Res
2007
;
67
:
9518
27
.
12.
Dankort
D
,
Curley
DP
,
Cartlidge
RA
,
Nelson
B
,
Karnezis
AN
,
Damsky
WE
, et al
BrafV600E cooperates with Pten loss to induce metastatic melanoma
.
Nat Genet
2009
;
41
:
544
52
.
13.
Hodis
E
,
Watson
IR
,
Kryukov
GV
,
Arold
ST
,
Imielinski
M
,
Theurillat
JP
, et al
A landscape of driver mutations in melanoma
.
Cell
2012
;
150
:
251
63
.
14.
Steinberg
SM
,
Zhang
P
,
Malik
BT
,
Boni
A
,
Shabaneh
TB
,
Byrne
KT
, et al
BRAF inhibition alleviates immune suppression in murine autochthonous melanoma
.
Cancer Immunol Res
2014
;
2
:
1044
50
.
15.
Steinberg
SM
,
Shabaneh
TB
,
Zhang
P
,
Martyanov
V
,
Li
Z
,
Malik
BT
, et al
Myeloid cells that impair immunotherapy are restored in melanomas with acquired resistance to BRAF inhibitors
.
Cancer Res
2017
;
77
:
1599
610
.
16.
Hooijkaas
AI
,
Gadiot
J
,
Morrow
M
,
Stewart
R
,
Schumacher
TN
,
Blank
CU
. 
Selective BRAF inhibition decreases tumor-resident lymphocyte frequencies in a mouse model of human melanoma
.
Oncoimmunology
2012
;
1
:
609
17
.
17.
Ho
PC
,
Meeth
KM
,
Tsui
YC
,
Srivastava
B
,
Bosenberg
MW
,
Kaech
SM
. 
Immune-based antitumor effects of BRAF inhibitors rely on signaling by CD40L and IFNg
.
Cancer Res
2014
;
74
:
3205
17
.
18.
Adeegbe
DO
,
Nishikawa
H
. 
Natural and induced T regulatory cells in cancer
.
Front Immunol
2013
;
4
:
1
14
.
19.
Kieback
E
,
Hilgenberg
E
,
Stervbo
U
,
Lampropoulou
V
,
Shen
P
,
Bunse
M
, et al
Thymus-derived regulatory T cells are positively selected on natural self-antigen through cognate interactions of high functional avidity
.
Immunity
2016
;
44
:
1114
26
.
20.
Malchow
S
,
Leventhal
DS
,
Nishi
S
,
Fischer
BI
,
Shen
L
,
Paner
GP
, et al
Aire-dependent thymic development of tumor associated regulartory T cells
.
Science
2011
;
339
:
1219
24
.
21.
Amedei
A
,
Niccolai
E
,
Benagiano
M
,
Della Bella
C
,
Cianchi
F
,
Bechi
P
, et al
Ex vivo analysis of pancreatic cancer-infiltrating T lymphocytes reveals that ENO-specific Tregs accumulate in tumor tissue and inhibit Th1/Th17 effector cell functions
.
Cancer Immunol Immunother
2013
;
62
:
1249
60
.
22.
Wang
HY
,
Lee
DA
,
Peng
G
,
Guo
Z
,
Li
Y
,
Kiniwa
Y
, et al
Tumor-specific human CD4+regulatory T cells and their ligands: implications for immunotherapy
.
Immunity
2004
;
20
:
107
18
.
23.
Valzasina
B
,
Piconese
S
,
Guiducci
C
,
Colombo
MP
. 
Tumor-induced expansion of regulatory T cells by conversion of CD4+CD25- lymphocytes is thymus and proliferation independent
.
Cancer Res
2006
;
66
:
4488
95
.
24.
Liu
VC
,
Wong
LY
,
Jang
T
,
Shah
AH
,
Park
I
,
Yang
X
, et al
Tumor evasion of the immune system by converting CD4+CD25- T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGFb
.
J Immunol
2007
;
178
:
2883
92
.
25.
van der Burg
SH
,
Piersma
SJ
,
de Jong
A
,
van der Hulst
JM
,
Kwappenberg
KMC
,
van den Hende
M
, et al
Association of cervical cancer with the presence of CD4+ regulatory T cells specific for human papillomavirus antigens
.
Proc Natl Acad Sci U S A
2007
;
104
:
12087
92
.
26.
Evans
RA
,
Diamond
MS
,
Rech
AJ
,
Chao
T
,
Richardson
MW
,
Lin
JH
, et al
Lack of immunoediting in murine pancreatic cancer reversed with neoantigen
.
JCI Insight
2016
;
1
:
1
16
.
27.
Goding
SR
,
Wilson
KA
,
Xie
Y
,
Harris
KM
,
Baxi
A
,
Akpinarli
A
, et al
Restoring immune function of tumor-specific CD4+ T cells during recurrence of melanoma
.
J Immunol
2013
;
190
:
4899
909
.
28.
Spranger
S
,
Spaapen
RM
,
Zha
Y
,
Williams
J
,
Meng
Y
,
Ha
TT
, et al
Up-regulation of PD-L1, IDO, and Tregs in the melanoma tumor microenvironment is driven by CD8+ T cells
.
Sci Transl Med
2013
;
5
:
200ra116
.
29.
Dhomen
N
,
Reis-Filho
JS
,
da Rocha Dias
S
,
Hayward
R
,
Savage
K
,
Delmas
V
, et al
Oncogenic braf induces melanocyte senescence and melanoma in mice
.
Cancer Cell
2009
;
15
:
294
303
.
30.
Goel
VK
,
Ibrahim
N
,
Jiang
G
,
Singhal
M
,
Fee
S
,
Flotte
T
, et al
Melanocytic nevus-like hyperplasia and melanoma in transgenic BRAFV600E mice
.
Oncogene
2009
;
28
:
2289
98
.
31.
Spranger
S
,
Bao
R
,
Gajewski
TF
. 
Melanoma-intrinsic β-catenin signalling prevents anti-tumour immunity
.
Nature
2015
;
523
:
231
5
.
32.
Hiraoka
N
,
Onozato
K
,
Kosuge
T
,
Hirohashi
S
. 
Prevalence of FOXP3+ regulatory T cells increases during the progression of pancreatic ductal adenocarcinoma and its premalignant lesions
.
Clin Cancer Res
2006
;
12
:
5423
34
.
33.
Hindley
JP
,
Ferreira
C
,
Jones
E
,
Lauder
SN
,
Ladell
K
,
Wynn
KK
, et al
Analysis of the T-cell receptor repertoires of tumor-infiltrating conventional and regulatory T cells reveals no evidence for conversion in carcinogen-induced tumors
.
Cancer Res
2011
;
71
:
736
46
.
34.
Spranger
S
,
Gajewski
TF
. 
Impact of oncogenic pathways on evasion of antitumour immune responses
.
Nat Rev Cancer
2018
;
18
:
139
147
.
35.
Dias Carvalho
P
,
Guimarães
CF
,
Cardoso
AP
,
Mendonça
S
,
Costa
ÂM
,
Oliveira
MJ
, et al
KRAS oncogenic signaling extends beyond cancer cells to orchestrate the microenvironment
.
Cancer Res
2017
;
78
:
7
15
.
36.
Balachandran
VP
,
Cavnar
MJ
,
Zeng
S
,
Bamboat
ZM
,
Ocuin
LM
,
Obaid
H
, et al
Imatinib potentiates antitumor T cell responses in gastrointestinal stromal tumor through the inhibition of Ido
.
Nat Med
2011
;
17
:
1094
100
.
37.
Peng
W
,
Chen
JQ
,
Liu
C
,
Malu
S
,
Creasy
C
,
Tetzlaff
MT
, et al
Loss of PTEN promotes resistance to T cell–mediated immunotherapy
.
Cancer Discov
2016
;
6
:
202
16
.
38.
Casey
SC
,
Tong
L
,
Li
Y
,
Do
R
,
Walz
S
,
Fitzgerald
KN
, et al
MYC regulates the antitumor immune response through CD47 and PD-L1
.
Science
2016
;
352
:
227
31
.
39.
Priceman
SJ
,
Shen
S
,
Wang
L
,
Deng
J
,
Yue
C
,
Kujawski
M
, et al
S1PR1 is crucial for accumulation of regulatory T cells in tumors via STAT3
.
Cell Rep
2014
;
6
:
992
9
.
40.
Knight
DA
,
Ngiow
SF
,
Li
M
,
Parmenter
T
,
Mok
S
,
Cass
A
, et al
Host immunity contributes to the anti-melanoma activity of BRAF inhibitors
.
J Clin Invest
2013
;
123
:
1371
81
.
41.
Zlotnik
A
,
Yoshie
O
. 
The chemokine superfamily revisited
.
Immunity
2012
;
36
:
705
12
.
42.
Ross
AL
,
Sanchez
MI
,
Grichnik
JM
. 
Molecular nevogenesis: an update
.
Nevogenes Mech Clin Implic Nevus Dev
2012
;
2011
:
99
110
.
43.
Shain
AH
,
Yeh
I
,
Kovalyshyn
I
,
Sriharan
A
,
Talevich
E
,
Gagnon
A
, et al
The genetic evolution of melanoma from precursor lesions
.
N Engl J Med
2015
;
373
:
1926
36
.
44.
Michaloglou
C
,
Vredeveld
LCW
,
Soengas
MS
,
Denoyelle
C
,
Kuilman
T
,
Van Der Horst
CMAM
, et al
BRAFE600-associated senescence-like cell cycle arrest of human naevi
.
Nature
2005
;
436
:
720
4
.
45.
Mourmouras
V
,
Fimiani
M
,
Rubegni
P
,
Epistolato
MC
,
Malagnino
V
,
Cardone
C
, et al
Evaluation of tumour-infiltrating CD4+CD25+FOXP3+ regulatory T cells in human cutaneous benign and atypical naevi, melanomas and melanoma metastases
.
Br J Dermatol
2007
;
157
:
531
9
.
46.
Schietinger
A
,
Philip
M
,
Krisnawan
VE
,
Chiu
EY
,
Delrow
JJ
,
Basom
RS
, et al
Tumor-specific T cell dysfunction is a dynamic antigen-driven differentiation program initiated early during tumorigenesis
.
Immunity
2016
;
45
:
389
401
.
47.
Zhang
P
,
Côté
AL
,
De Vries
VC
,
Usherwood
EJ
,
Turk
MJ
. 
Induction of postsurgical tumor immunity and T-cell memory by a poorly immunogenic tumor
.
Cancer Res
2007
;
67
:
6468
76
.
48.
Maurus
K
,
Hufnagel
A
,
Geiger
F
,
Graf
S
,
Berking
C
,
Heinemann
A
, et al
The AP-1 transcription factor FOSL1 causes melanocyte reprogramming and transformation
.
Oncogene
2017
;
36
:
5110
21
.
49.
Liu
J
,
Suresh Kumar
KG
,
Yu
D
,
Molton
SA
,
McMahon
M
,
Herlyn
M
, et al
Oncogenic BRAF regulates beta-Trcp expression and NF-kappaB activity in human melanoma cells
.
Oncogene
2007
;
26
:
1954
8
.
50.
Singha
B
,
Gatla
HR
,
Vancurova
I
. 
Transcriptional regulation of chemokine expression in ovarian cancer
.
Biomolecules
2015
;
5
:
223
43
.