Foxp3 + Regulatory T Cells Associated With CCL17/CCR4 Expression in Carcinomas of Dogs

Abstract

Regulatory T cells (Tregs) can be targeted in cancer immunotherapy. A previous study has shown that the chemokine CCL17 and the receptor CCR4 play a role in Treg recruitment in canine urothelial carcinoma. Here, we describe the association of tumor-infiltrating Tregs with CCL17/CCR4 expression in dogs with other carcinomas. In this study, we investigated 23 dogs with mammary carcinoma, 14 dogs with oral squamous cell carcinoma, 16 dogs with pulmonary adenocarcinoma, and 8 healthy control dogs. Immunohistochemistry showed that Foxp3⁺ Tregs and CCR4⁺ cells were increased in the tumor tissues of mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma, when compared with the healthy tissues. The number of CCR4⁺ cells was associated with that of Foxp3⁺ Tregs. Double immunofluorescence labeling confirmed that most tumor-infiltrating Foxp3⁺ Tregs expressed CCR4. In vitro, canine carcinoma cell lines expressed CCL17 mRNA. Quantitative RT-PCR (reverse transcriptase-polymerase chain reaction) showed that CCL17 mRNA expression in canine carcinomas was increased approximately 10- to 25-fold relative to that of healthy tissues. These results suggest that the CCL17/CCR4 axis may drive Treg recruitment in a variety of canine carcinomas. CCR4 blockade may be a potential therapeutic option for tumor eradication through Treg depletion.

Keywords

antitumor immunity cancer immunology chemokine dogs regulatory T lymphocytes

Regulatory T cells (Tregs) are a subset of CD4⁺ helper T lymphocytes expressing the IL-2 receptor alpha chain (CD25) and transcription factor forkhead box P3 (Foxp3). The function of Tregs is to suppress activity of effector immune cells. Tregs play a role not only in suppression of immune response and inflammation but also in tumor progression, presumably by inhibiting antitumor immunity. In humans, Treg infiltration has been reported in certain tumors and has been related to progression and prognosis.^5,24 High tumor-infiltrating Tregs are associated with poor prognosis in patients with ovarian,^3,29 breast,^1,9 lung,³⁶ gastric,²⁵ squamous cell,¹⁰ and hepatocellular carcinomas.^6,8 There is a growing interest in Treg infiltration in canine tumors; numerous studies have reported on the association of tumor-infiltrating Tregs with poor prognosis in various canine tumors, such as melanoma,^27,28 intestinal lymphoma,²¹ mammary carcinoma,² squamous cell carcinoma,²⁸ and pulmonary adenocarcinoma.²⁸

Evidence suggests that tumor-infiltrating Tregs are a potential target for cancer immunotherapy. In mice and humans, several molecules are candidates for Treg depletion or attenuation of their function, including CD25, CD15s, CD134, cytotoxic T-lymphocyte associated protein 4 (CTLA-4), glucocorticoid-induced TNF receptor (GITR), lymphocyte-activation gene 3 (LAG3), programmed cell death protein-1 (PD-1), and C-C chemokine receptor 4 (CCR4).^24,35 Anti-Treg therapy is under investigation for human malignant tumors, such as melanoma, adult T-cell leukemia/lymphoma, lung cancer, and esophageal cancer.^14,16,34,38 The mechanisms of Treg infiltration and the therapeutic efficacy of its depletion in canine tumors remain unclear.

CCR4 is a receptor for C-C chemokines CCL17 and CCL22, both of which are produced by mature dendritic cells.^12,13 It has been shown that CCR4 is preferentially expressed on type 2 helper T (Th2) cells or Tregs in both mice and humans.⁴³ A recent study by our group has demonstrated that Tregs infiltrate tumor tissues through the interaction between tumor-producing CCL17 and CCR4 on Tregs, and CCR4 blockade exerts an antitumor effect in dogs with urothelial carcinoma.¹⁹ However, little is known about the role of CCR4 in Treg infiltration in other canine tumors.

In the present study, we investigated the number of Foxp3⁺ Tregs and CCR4⁺ cells, and the gene expression of ligands for CCR4, in dogs with mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma, to elucidate the therapeutic potential of anti-CCR4 treatment for these canine carcinomas.

Materials and Methods

Canine Tumor Samples

For immunohistochemistry and immunofluorescence analyses, we obtained archival formalin-fixed, paraffin-embedded tumor tissues that were surgically removed from 53 dogs at the Veterinary Medical Center of the University of Tokyo. The following diagnoses were confirmed by histopathology: mammary simple carcinoma (n = 23), oral squamous cell carcinoma (n = 14), and pulmonary adenocarcinoma (n = 16). Lymph node involvement and lung metastasis were assessed by palpation, ultrasound, and radiographic examinations, respectively. In this study, lymph node involvement was observed in 4/23 dogs with mammary carcinoma, 4/14 dogs with squamous cell carcinoma, and 2/16 dogs with pulmonary adenocarcinoma. There were no cases with identified lung metastasis. No dogs received chemotherapy or radiation therapy before surgery. Normal tissues (mammary gland, oral mucosa, and lung) were obtained from 8 healthy beagles via necropsy for other experiments and used as controls.

For gene expression analysis, fresh tumor tissues from 15 dogs with mammary carcinoma, 10 dogs with oral squamous cell carcinoma, and 12 dogs with pulmonary adenocarcinoma were immediately submerged in RNAlater (Thermo Fisher Scientific, Waltham, MA) and stored at −20°C. Archival frozen normal tissues (mammary gland, oral mucosa, and lung) collected from 5 healthy beagles euthanized for another experimental purpose were used as a control. All procedures in this study were conducted in accordance with the guidelines of the Animal Care Committee of the Graduate School of Agricultural and Life Sciences, the University of Tokyo.

Immunohistochemistry

The expression of Foxp3 and CCR4 was examined by immunohistochemistry.¹⁹ Heat-induced antigen retrieval was performed by autoclaving the sections for 10 minutes at 121°C in 10 mM sodium citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation with REAL Peroxidase-Blocking Solution (DAKO, Glostrup, Denmark) at room temperature for 10 minutes. Sections were blocked with 5% skim milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 60 minutes, and then incubated with primary antibodies, rat anti-Foxp3 (1:400 dilution, clone FJK-16s, eBioscience, San Diego, CA), or mouse anti-CCR4 (1:100 dilution, clone 1G1, BD Biosciences, Franklin Lakes, NJ) at 4°C overnight. The cross-reactivity of these primary antibodies with canine Foxp3 and CCR4 was confirmed in previous studies.^20,23 Secondary antibodies were applied as follows: samples were incubated with EnVision polymer reagent anti-mouse (Dako) at room temperature for 45 minutes, or biotin-labeled anti-rat IgG (Vector Laboratories, Burlingame, CA) at 37°C for 30 minutes followed by HRP-labeled streptavidin (Dako) at room temperature for 30 minutes. For Foxp3 and CCR4, mesenteric lymph nodes were used as positive controls. Incubation of sections with TBST only served as negative controls. The reaction products were visualized with 3,3′-diaminobenzidine. Cells with clear lymphocyte morphology, distinct nuclear immunolabeling for Foxp3, or membranous immunolabeling for CCR4 were evaluated as positive. Foxp3⁺ and CCR4⁺ cells were manually counted in 10 representative fields (400× magnification: 0.238 mm² per field) with the most intense positivity and >50% of tumor cells using ImageJ software.^28,30

For double immunofluorescence labeling, antigen retrieval, endogenous peroxidase blocking, and milk blocking were performed as described above. Primary antibodies, rat anti-Foxp3 (1:100 dilution, clone FJK-16s, eBioscience) and mouse anti-CCR4 (1:100 dilution, clone 1G1, BD Biosciences), were applied at 4°C overnight. Immunofluorescence was performed using secondary antibodies, namely, Alexa Fluor 594 goat anti-rat IgG (1:500, Invitrogen, Carlsbad, CA) and Alexa Fluor 488 donkey anti-mouse IgG (1:500, Invitrogen). Images were captured using a fluorescence microscope (BZ-X800; Keyence, Osaka, Japan).

Cell Culture

A total of 7 canine carcinoma cell lines were used.^22,39,44 The cell lines were derived from mammary adenocarcinoma (CIPp, CIPm, and CTBp), squamous cell carcinoma (POCO, SYRUP, and MILK), and pulmonary adenocarcinoma (GEN). CIPp, CIPm, CTBp, and GEN cells were grown in RPMI-1640 (Sigma, St. Louis, MO), supplemented with 10% FBS (Thermo Fisher Scientific) and 100 μg/ml penicillin and streptomycin (Sigma) at 37°C under a humidified atmosphere with 5% CO₂. POCO, SYRUP, and MILK cells were grown in DMEM (Thermo Fisher Scientific, Waltham, MA), supplemented with 10% FBS (Thermo Fisher Scientific) and 100 μg/ml penicillin and streptomycin (Sigma) at 37°C under a humidified atmosphere with 5% CO₂. Two nontumor cell lines, established from normal lip epithelium COPK⁴¹ and keratinocytes CPEK,^33,41 were purchased from CELLnTEC Advanced Cell Systems (Bern, Switzerland). COPK and CPEK cells were maintained in CnT-09 medium (CELLnTEC Advanced Cell Systems) at 37°C under 5% CO₂.

Gene Expression Analysis

We extracted total RNA from each canine cell line using the RNAspin Mini RNA Isolation Kit (GE Healthcare, Buckinghamshire, England). Reverse transcription (RT) was accomplished using a ReverTra Ace (Toyobo, Osaka, Japan). PCR (polymerase chain reaction) was performed over 35 cycles using Taq polymerase (AmpliTaq Gold; Thermo Fisher Scientific) under a 3-step thermal cycling process of 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 30 seconds. Table 1 lists the primer pair sequences. Amplicons were separated by electrophoresis and stained by ethidium bromide for visualization and confirmation of the amplicon size.

Table 1.

Primer Pair Sequences Used for RT-PCR.

Primer Set	GenBank Accession Number		Primer Sequence (5′–3′)
CCL17	NM_001003051	Forward	GGC TGA CAA GGT GGT ACA AGA CTT C
CCL17	NM_001003051	Reverse	CAG ATG GAC TTG CCT TGG ACA G
CCL22	XM_003433778	Forward	TAT GGT GCC AAC GTG GAA GA
CCL22	XM_003433778	Reverse	GAT CTC CCG ATC CTT GAC AGT TAG
RPL13A	AJ388525	Forward	GCC GGA AGG TTG TAG TCG T
RPL13A	AJ388525	Reverse	GGA GGA AGG CCA GGT AAT TC
RPL32	XM_848016	Forward	TGG TTA CAG GAG CAA CAA GAA A
RPL32	XM_848016	Reverse	GCA CAT CAG CAG CAC TTC A

Abbreviations: RT-PCR, reverse transcriptase polymerase chain reaction; RPL13A, ribosomal protein L13a; RPL32, ribosomal protein L32.

For quantitative RT-PCR, we extracted the total RNA from tumor and normal tissues using the RNAspin Mini RNA Isolation Kit (GE Healthcare). Two-step real-time RT-qPCR (StepOnePlus; Applied Biosystems, Waltham, MA) was performed using THUNDERBIRD SYBR qPCR Mix (Toyobo) under the amplification conditions of 95°C for 10 seconds, followed by 40 cycles of PCR (95°C for 5 seconds and 60°C for 30 seconds). RPL13A and RPL32 were used as references.²⁶ Table 1 lists the primer pair sequences. All primer sets exhibited >95% efficiency, which was determined by using a 10-fold dilution series from representative cDNA samples. The nuclease-free water was used as a negative control. Cycle threshold (Ct) values that indicate points of threshold intersects with the amplification curve of PCR were determined using StepOne Plus v2.3 software (Applied Biosystems). All samples were examined in duplicate, and the mean ΔCt was calculated. Each mRNA expression level (relative quantity) was calculated using 2^-ΔCt, resulting in the evaluation of carcinoma tissues as the n-fold difference from the mean value of normal tissues after normalizing based on the reference genes.

Statistical Analysis

We used JMP Pro version 14.2.0 (SAS Institute, Cary, NC) for statistical analyses. Intergroup comparisons were assessed using the Mann-Whitney U test. The Kruskal-Wallis test, followed by the Dunn test, was used for multiple comparisons. Correlation between 2 variables was assessed using Spearman’s rank correlation coefficient. Statistical significance was defined as P < .05.

Results

Foxp3⁺ Tregs and CCR4⁺ Cells Infiltrate Into Canine Carcinoma Tissues

We visualized Foxp3 and CCR4 with immunohistochemistry and examined the localization and number of tumor-infiltrating Tregs and CCR4⁺ cells. We observed only a few Foxp3⁺ Tregs in the normal tissues (Figs. 1–3), whereas Foxp3⁺ Tregs were densely present in mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma (Figs. 4–6). Tumor-infiltrating CCR4⁺ cells were also increased in these carcinomas but not in the normal tissues (Figs. 7–12). Both Foxp3⁺ Tregs and CCR4⁺ cells were observed mainly in the peritumoral stroma, and some of them were present in the intratumoral area. Compared to normal tissues, Foxp3⁺ Tregs and CCR4⁺ cells were more frequent in dogs with mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma (Figs. 13 –21). The density of Foxp3⁺ Tregs was positively correlated to the number of CCR4⁺ cells (Figs. 13 –21).

Figures 1–6. Immunohistochemistry for Foxp3, dogs. Figures 1–3. Normal tissues. No Foxp3⁺ cells are observed in the normal mammary gland (Fig. 1), oral mucosa (Fig. 2), or lung (Fig. 3). Figures 4–6. Carcinomas. There is nuclear immunolabeling of Foxp3⁺ mononuclear cells, indicating the presence of regulatory T cells (Tregs), in mammary carcinoma (Fig. 4), squamous cell carcinoma (Fig. 5), and pulmonary adenocarcinoma (Fig. 6).

Figures 7–12. Immunohistochemistry for CCR4, dogs. Figures 7–9. Normal tissues. No CCR4⁺ cells are observed in the normal mammary gland (Fig. 7), oral mucosa (Fig. 8), or lung (Fig. 9). Figures 10–12. Carcinomas. CCR4⁺ mononuclear cells are present in mammary carcinoma (Fig. 10), squamous cell carcinoma (Fig. 11), and pulmonary adenocarcinoma (Fig. 12).

Figures 13–14. The number of tumor-infiltrating Foxp3⁺ Tregs (Fig. 13) and CCR4⁺ cells (Fig. 14) in normal mammary gland (n = 8) and canine mammary carcinoma (n = 23). Horizontal lines indicate the median number. ***P < 0.001. HPF, high-power field. Figure 15. The correlation between the number of Foxp3⁺ and CCR4⁺ cells in canine mammary carcinoma.

Figures 16–17. The number of tumor-infiltrating Foxp3⁺ Tregs (Fig. 16) and CCR4⁺ cells (Fig. 17) in normal oral mucosa (n = 8) and canine oral squamous cell carcinoma (n = 14). Figure 18. The correlation between the number of Foxp3⁺ and CCR4⁺ cells in canine oral squamous cell carcinoma.

Figures 19–20. The number of tumor-infiltrating Foxp3⁺ Tregs (Fig. 19) and CCR4⁺ cells (Fig. 20) in normal lung (n = 8) and canine pulmonary adenocarcinoma (n = 16). Figure 21. The correlation between the number of Foxp3⁺ and CCR4⁺ cells in canine pulmonary adenocarcinoma.

Tumor-Infiltrating Tregs Express CCR4 in Various Canine Carcinomas

To confirm the expression of CCR4 in tumor-infiltrating Tregs, we conducted double immunofluorescence labeling. Tumor-infiltrating Foxp3⁺ Tregs expressed CCR4 in mammary carcinoma (Fig. 22), squamous cell carcinoma (Fig. 23), and pulmonary adenocarcinoma (Fig. 24). In addition, Foxp3⁻ CCR4⁺ non-Treg cells were also present in the carcinoma tissues (Figs. 22–24). However, Foxp3⁺CCR4⁻ Tregs were only rarely observed in the carcinoma tissues (Figs. 22–24). The median proportions of Foxp3⁺CCR4⁺ cells, Foxp3^-CCR4⁺ cells, and Foxp3⁺CCR4⁻ cells were 28.4% (range, 20.1% to 44.3%), 69.5% (58.1% to 79.4%), and 1.2% (0% to 2.8%) in dogs with mammary carcinoma; 27.7% (20.6% to 39.1%), 72.4% (47.3% to 81.6%), and 0% (0% to 2.2%) in dogs with squamous cell carcinoma; and 34.1% (25.8% to 48.2%), 63.1% (43.1% to 74.2%), and 0% (0% to 3.5%) in dogs with pulmonary adenocarcinoma, respectively.

Figures 22–24. Representative images of immunofluorescence for (a) Foxp3 (red), (b) CCR4 (green), and (c) merged. Insets: Higher magnifications of stained cells. Figure 22. Canine mammary carcinoma. Figure 23. Canine squamous cell carcinoma. Figure 24. Canine pulmonary adenocarcinoma.

CCL17 mRNA Expression Is Elevated in Various Canine Carcinomas

To examine the mRNA expression of CCL17 and CCL22 in tumor cells, RT-qPCR was performed using canine carcinoma cell lines. We detected mRNA expression of CCL17, but not CCL22, in mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma cell lines (Fig. 25). Interestingly, CCL17 mRNA expression varied among cell lines of the same tumor type. CCL17 mRNA was detected in 2/3 mammary carcinoma cell lines, 2/3 squamous cell carcinoma cell lines, and 1/1 pulmonary adenocarcinoma cell line (Fig. 25). The normal canine squamous epithelial cell line COPK and the epidermal keratinocyte cell line CPEK did not express CCL17 or CCL22 (Fig. 25).

Figure 25. CCL17 and CCL22 mRNA expression in canine carcinoma cell lines. RT-qPCR products were separated by electrophoresis and stained by ethidium bromide for visualization of the amplicons. CIPp, CIPm, and CTBp cells were derived from mammary carcinoma (MC); POCO, SYRUP, and MILK cells were from squamous cell carcinoma (SCC); GEN cells were from pulmonary adenocarcinoma (PA); COPK and CPEK cells were from nontumor epithelium (Non). RPL13A and RPL32 were used as internal controls.

Within samples of tumor tissue, mRNA expression of CCL17 in mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma was increased by approximately 8-, 21-, and 26-fold compared with normal control tissues, respectively (Fig. 26). There was no significant difference in CCL22 mRNA expression between normal and tumor tissues in each carcinoma type (Fig. 27). Tumoral CCL17 mRNA was positively correlated to the number of Foxp3⁺ Tregs in dogs with mammary carcinoma (Fig. 28), but not in dogs with squamous cell carcinoma (Fig. 29) or pulmonary adenocarcinoma (Fig. 30). Tumoral CCL17 mRNA was positively correlated to the number of CCR4⁺ cells in dogs with mammary carcinoma (Fig. 31), squamous cell carcinoma (Fig. 32), and pulmonary adenocarcinoma (Fig. 33). However, there was no association between CCL17 and tumor grade (data not shown). Tumoral CCL22 mRNA expression did not correlate to the number of Foxp3⁺ Tregs and CCR4⁺ cells (data not shown).

Figures 26–27. Expression of CCL17 (Fig. 26) and CCL22 (Fig. 27) mRNA in canine normal tissues (n = 5 each; mammary, oral mucosa, and lung), mammary carcinoma (n = 15), squamous cell carcinoma (n = 10), and pulmonary adenocarcinoma (n = 12). RPL13A and RPL32 were used as internal controls. Data are presented as mean ± SEM. *P < .05, **P < .01.

Figures 28–30. Correlation between the number of Foxp3⁺ Tregs and the level of CCL17 mRNA expression in tissue samples of canine mammary carcinoma (n = 15; Fig. 28), squamous cell carcinoma (n = 10; Fig. 29), and pulmonary adenocarcinoma (n = 12; Fig. 30). HPF, high-power field.

Figures 31–33. Correlation between the number of CCR4⁺ cells and the level of CCL17 mRNA expression in tissue samples of canine mammary carcinoma (n = 15; Fig. 31), squamous cell carcinoma (n = 10; Fig. 32), and pulmonary adenocarcinoma (n = 12; Fig. 33). HPF, high-power field.

Discussion

In this study, we observed an increased number of Foxp3⁺ Tregs expressing CCR4 in canine mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma. Furthermore, high mRNA expression of CCL17, but not CCL22, was evident in these carcinomas, suggesting that Tregs infiltrate the tumor tissue through the CCL17/CCR4 axis in a variety of canine carcinomas, including urothelial carcinoma.¹⁹ Previous studies have shown that high tumor-infiltrating Tregs are associated with poor prognosis in dogs with these carcinomas,^2,28 and our recent investigation has demonstrated that Treg-targeting therapy, using anti-CCR4 antibodies, prolongs survival with low incidence of clinically relevant toxicities in dogs with urothelial carcinoma.¹⁹ Thus, blocking CCR4 or tumor-producing CCL17 may be a rational approach that could be clinically tested as a potential treatment option for dogs with carcinomas.

Although numbers of tumor-infiltrating Tregs were increased in canine mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma, the degree of Treg infiltration varied from case to case. Likewise, the mRNA expression of the Treg-attracting chemokine CCL17 was also different between cell lines and clinical cases regardless of tumor grade. Tumoral CCL17 mRNA expression was positively correlated to tumor-infiltrating CCR4⁺ cells. Although not all CCR4⁺ cells were Foxp3⁺ Tregs, the very low number of Foxp3⁺CCR4⁻ cells suggested that most Tregs express CCR4 in the tumor tissues. These observations suggest that the degree of Treg infiltration might be influenced by the amount of tumor-producing CCL17.

The underlying mechanisms of differences in CCL17 expression remain uncertain. In human and canine keratinocytes, CCL17 production was induced by tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) through nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK).^7,15,32,40 Furthermore, serum levels of TNF-α and IFN-γ were increased in dogs with mammary carcinoma compared to healthy female dogs,¹⁸ indicating that TNF-α and/or IFN-γ in the tumor-microenvironment might regulate CCL17 production. Additional studies will be necessary to investigate the mechanisms of upregulated CCL17 expression in canine carcinomas.

Among the types of lymphocytes, CCR4 is preferentially expressed on Tregs and Th2 cells.⁴³ In this study, immunofluorescence showed that a large number of Foxp3⁻CCR4⁺ cells, as well as Foxp3⁺CCR4⁺ Tregs, were observed in tumor tissues. Previous studies has shown that peripheral CCR4⁺ lymphocytes exhibit a Th2 phenotype in dogs.^11,20 Therefore, CCR4⁺ non-Treg cells observed in tumor tissues might be Th2 cells. Th2 cells produce IL-4, IL-5, and IL-13 and are involved in humoral immunity and allergic diseases.⁴³ In the tumor-microenvironment, the role of Th2 cells is controversial.⁵ Several studies have demonstrated the association of tumor-infiltrating Th2 cells and Th2 cytokines with poor prognosis in human patients with ovarian,¹⁷ gastric,³⁷ and pancreatic cancers.⁴ However, other groups have reported that tumor-infiltrating Th2 cells are associated with favorable outcome in patients with breast cancer⁴² and Hodgkin’s lymphoma.³¹ Thus, it remains unknown whether Th2 cells exacerbate or protect neoplastic diseases. Further study is needed to characterize the phenotype and role of CCR4⁺ non-Treg cells in canine tumor tissues.

For precision medicine, biomarkers predicting Treg infiltration before anti-Treg treatment in each case are needed. In dogs with urothelial carcinoma, assessment of urinary CCL17, but not serum CCL17, concentrations can predict the response to CCR4 blockade therapy,¹⁹ which is likely due to the direct contact between tumor and urine. Unlike urothelial carcinoma, CCL17 derived from other tumors including mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma could not be detected in urine (data not shown); therefore, it is necessary to examine other detection methods for predicting Treg infiltration. A possible biomarker predicting anti-Treg therapy is peripheral blood Tregs. Our previous study showed that a reduction in the proportion of circulating CCR4⁺Foxp3⁺ Tregs correlates to the therapeutic response to anti-CCR4 treatment.¹⁹

One limitation of this study is that healthy control dogs were not well matched to the clinical cases by breed. As it was impossible to obtain normal tissues from various breeds matched to the carcinoma cases, all healthy control dogs were of the same breed (beagle). This limitation could lead to erroneous results regarding the expression of some genes that might differ from breed to breed.

In conclusion, we detected increased numbers of CCR4-expressing Tregs in various canine carcinoma tissues. Furthermore, we found that expression of CCL17, the ligand of CCR4, was elevated in these carcinomas in vitro and in vivo. These results suggest that the CCL17/CCR4 pathway may drive Treg recruitment, contributing to the immune-suppressive nature of the tumor-microenvironment in canine carcinomas. The present study paves the way for clinical trials of CCR4 blockade therapy for dogs with mammary carcinoma, squamous cell carcinoma, and pulmonary adenocarcinoma.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the JSPS KAKENHI Grant-in-Aid for Young Scientists (A) (Grant No. JP16H06208), the Grant-in-Aid for Scientific Research (A) (Grant No. JP19H00968), and the Anicom Capital Research Grant (EVOLVE).

ORCID iD

Shingo Maeda

References

Bates

Fox

Han

, et al. Quantification of regulatory T cells enables the identification of high-risk breast cancer patients and those at risk of late relapse. J Clin Oncol. 2006;24(34):5373–5380.

Carvalho

Pires

Prada

, et al. Intratumoral FoxP3 expression is associated with angiogenesis and prognosis in malignant canine mammary tumors. Vet Immunol Immunopathol. 2016;178:1–9.

Curiel

Coukos

Zou

, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med. 2004;10(9):942–949.

De Monte

Reni

Tassi

, et al. Intratumor T helper type 2 cell infiltrate correlates with cancer-associated fibroblast thymic stromal lymphopoietin production and reduced survival in pancreatic cancer. J Exp Med. 2011;208(3):469–478.

Fridman

Pages

Sautes-Fridman

, et al. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer. 2012;12(4):298–306.

Liu

, et al. Increased regulatory T cells correlate with CD8 T-cell impairment and poor survival in hepatocellular carcinoma patients. Gastroenterology. 2007;132(7):2328–2339.

Fujii-Maeda

Kajiwara

Ikizawa

, et al. Reciprocal regulation of thymus and activation-regulated chemokine/macrophage-derived chemokine production by interleukin (IL)-4/IL-13 and interferon-gamma in HaCaT keratinocytes is mediated by alternations in E-cadherin distribution. J Invest Dermatol. 2004;122(1):20–28.

Gao

Qiu

Fan

, et al. Intratumoral balance of regulatory and cytotoxic T cells is associated with prognosis of hepatocellular carcinoma after resection. J Clin Oncol. 2007;25(18):2586–2593.

Gobert

Treilleux

Bendriss-Vermare

, et al. Regulatory T cells recruited through CCL22/CCR4 are selectively activated in lymphoid infiltrates surrounding primary breast tumors and lead to an adverse clinical outcome. Cancer Res. 2009;69(5):2000–2009.

10.

Ihara

Sakurai

Horinaka

, et al. CD45RA(-)Foxp3(high) regulatory T cells have a negative impact on the clinical outcome of head and neck squamous cell carcinoma. Cancer Immunol Immunother. 2017;66(10):1275–1285.

11.

Iio

Motohashi

Kunisada

, et al. Preferential gene transcription of T helper 2 cytokines in peripheral CCR4(+) CD4(+) lymphocytes in dogs. Vet Dermatol. 2014;25(3):199–150.

12.

Imai

Baba

Nishimura

, et al. The T cell-directed CC chemokine TARC is a highly specific biological ligand for CC chemokine receptor 4. J Biol Chem. 1997;272(23):15036–15042.

13.

Imai

Chantry

Raport

, et al. Macrophage-derived chemokine is a functional ligand for the CC chemokine receptor 4. J Biol Chem. 1998;273(3):1764–1768.

14.

Ishida

Joh

Uike

, et al. Defucosylated anti-CCR4 monoclonal antibody (KW-0761) for relapsed adult T-cell leukemia-lymphoma: a multicenter phase II study. J Clin Oncol. 2012;30(8):837–842.

15.

Komine

Kakinuma

Kagami

, et al. Mechanism of thymus- and activation-regulated chemokine (TARC)/CCL17 production and its modulation by roxithromycin. J Invest Dermatol. 2005;125(3):491–498.

16.

Kurose

Ohue

Wada

, et al. Phase Ia study of FoxP3+ CD4 Treg depletion by infusion of a humanized anti-CCR4 antibody, KW-0761, in cancer patients. Clin Cancer Res. 2015;21(19):4327–4336.

17.

Kusuda

Shigemasa

Arihiro

, et al. Relative expression levels of Th1 and Th2 cytokine mRNA are independent prognostic factors in patients with ovarian cancer. Oncol Rep. 2005;13(6):1153–1158.

18.

Machado

Crivellenti

Bottari

, et al. Oxidative stress and inflammatory response biomarkers in dogs with mammary carcinoma. Pathol Res Pract. 2015;211(9):677–681.

19.

Maeda

Murakami

Inoue

, et al. CCR4 blockade depletes regulatory T cells and prolongs survival in a canine model of bladder cancer. Cancer Immunol Res. 2019;7(7):1175–1187.

20.

Maeda

Ohmori

Yasuda

, et al. Increase of CC chemokine receptor 4-positive cells in the peripheral CD4 cells in dogs with atopic dermatitis or experimentally sensitized to Japanese cedar pollen. Clin Exp Allergy. 2004;34(9):1467–1473.

21.

Maeda

Ohno

Fujiwara-Igarashi

, et al. Changes in Foxp3-positive regulatory T cell number in the intestine of dogs with idiopathic inflammatory bowel disease and intestinal lymphoma. Vet Pathol. 2016;53(1):102–112.

22.

Miyamoto

Kurita

Tani

, et al. Canine squamous cell carcinoma cell lines with high expression of survivin are sensitive to survivin inhibitor YM155. Vet J. 2018;240:31–36.

23.

Mizuno

Suzuki

Umeki

, et al. Crossreactivity of antibodies to canine CD25 and Foxp3 and identification of canine CD4+CD25+Foxp3+ cells in canine peripheral blood. J Vet Med Sci. 2009;71(12):1561–1568.

24.

Nishikawa

Sakaguchi

. Regulatory T cells in cancer immunotherapy. Curr Opin Immunol. 2014;27:1–7.

25.

Perrone

Ruffini

Catalano

, et al. Intratumoural FOXP3-positive regulatory T cells are associated with adverse prognosis in radically resected gastric cancer. Eur J Cancer. 2008;44(13):1875–1882.

26.

Peters

Peeters

Helps

, et al. Development and application of multiple internal reference (housekeeper) gene assays for accurate normalisation of canine gene expression studies. Vet Immunol Immunopathol. 2007;117(1–2):55–66.

27.

Porcellato

Brachelente

De Paolis

, et al. FoxP3 and IDO in canine melanocytic tumors. Vet Pathol. 2019;56(2):189–199.

28.

Sakai

Maeda

Yamada

, et al. Association of tumour-infiltrating regulatory T cells with adverse outcomes in dogs with malignant tumours. Vet Comp Oncol. 2018;16(3):330–336.

29.

Sato

Olson

Ahn

, et al. Intraepithelial CD8+ tumor-infiltrating lymphocytes and a high CD8+/regulatory T cell ratio are associated with favorable prognosis in ovarian cancer. Proc Natl Acad Sci U S A. 2005;102(51):18538–18543.

30.

Schneider

Rasband

Eliceiri

. NIH Image to ImageJ: 25 years of image analysis. Nat Methods. 2012;9(7):671–675.

31.

Schreck

Friebel

Buettner

, et al. Prognostic impact of tumour-infiltrating Th2 and regulatory T cells in classical Hodgkin lymphoma. Hematol Oncol. 2009;27(1):31–39.

32.

Shibata

Maeda

Kondo

, et al. Identification of the signaling pathway of TNF-alpha-induced CCL17/TARC transcription in a canine keratinocyte cell line. Vet Immunol Immunopathol. 2011;139(2–4):90–98.

33.

Shibata

Maeda

Tsuchida

, et al. Phenotypic analysis for a cell line of canine epidermal keratinocytes. J Vet Med Sci. 2008;70(8):853–855.

34.

Sugiyama

Nishikawa

Maeda

, et al. Anti-CCR4 mAb selectively depletes effector-type FoxP3+CD4+ regulatory T cells, evoking antitumor immune responses in humans. Proc Natl Acad Sci U S A. 2013;110(44):17945–17950.

35.

Tanaka

Sakaguchi

. Regulatory T cells in cancer immunotherapy. Cell Res. 2017;27(1):109–118.

36.

Tao

Mimura

Aoe

, et al. Prognostic potential of FOXP3 expression in non-small cell lung cancer cells combined with tumor-infiltrating regulatory T cells. Lung Cancer. 2012;75(1):95–101.

37.

Ubukata

Motohashi

Tabuchi

, et al. Evaluations of interferon-gamma/interleukin-4 ratio and neutrophil/lymphocyte ratio as prognostic indicators in gastric cancer patients. J Surg Oncol. 2010;102(7):742–747.

38.

Ueda

. Clinical application of anti-CCR4 monoclonal antibody. Oncology. 2015;89(suppl 1):16–21.

39.

Uyama

Nakagawa

Hong

, et al. Establishment of four pairs of canine mammary tumour cell lines derived from primary and metastatic origin and their E-cadherin expression. Vet Comp Oncol. 2006;4(2):104–113.

40.

Vestergaard

Bang

Gesser

, et al. A Th2 chemokine, TARC, produced by keratinocytes may recruit CLA+CCR4+ lymphocytes into lesional atopic dermatitis skin. J Invest Dermatol. 2000;115(4):640–646.

41.

Yagihara

Kinjo

Kobayashi

, et al. Expression of canine Kdap in normal, hyperplastic and neoplastic epidermis. Vet J. 2009;180(3):348–355.

42.

Yoon

Maresh

Shen

, et al. Higher levels of GATA3 predict better survival in women with breast cancer. Hum Pathol. 2010;41(12):1794–1801.

43.

Yoshie

Matsushima

. CCR4 and its ligands: from bench to bedside. Int Immunol. 2015;27(1):11–20.

44.

Yoshitake

Saeki

Watanabe

, et al. Molecular investigation of the direct anti-tumour effects of nonsteroidal anti-inflammatory drugs in a panel of canine cancer cell lines. Vet J. 2017;221:38–47.

Foxp3 + Regulatory T Cells Associated With CCL17/CCR4 Expression in Carcinomas of Dogs

Abstract

Keywords

Materials and Methods

Canine Tumor Samples

Immunohistochemistry

Cell Culture

Gene Expression Analysis

Statistical Analysis

Results

Foxp3+ Tregs and CCR4+ Cells Infiltrate Into Canine Carcinoma Tissues

Tumor-Infiltrating Tregs Express CCR4 in Various Canine Carcinomas

CCL17 mRNA Expression Is Elevated in Various Canine Carcinomas

Discussion

Footnotes

Declaration of Conflicting Interests

Funding

ORCID iD

References

Foxp3⁺ Tregs and CCR4⁺ Cells Infiltrate Into Canine Carcinoma Tissues