The protocol for the animal study was approved by the ethics committee at Tohoku University Graduate School of Medicine for Animal Experimentation, Sendai, Japan (permit number: 2017MdLMO-216). The research complied with the Tohoku University Graduate School of Medicine's Animal Experimentation Ethics guidelines and policies. All surgeries were performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. The protocol for the human study was approved by the ethics committee at Tohoku University Graduate School of Medicine, Sendai, Japan (permit number: 2018-1-772). All patients provided written informed consent to participate.

C57BL/6 mice (5–8 weeks old) were purchased from Japan Shizuoka Laboratory Animal Center (Shizuoka, Japan) and housed in the animal facility at Tohoku University Graduate School of Medicine. The EL4 murine T-cell lymphoma cell line was obtained from American Type Culture Collection (Manassas, VA) and cultured in Dulbecco's minimal essential medium supplemented with 10% heat-inactivated fetal calf serum. All mice were bred under specific pathogen-free conditions at Tohoku University Graduate School of Medicine.

EL4 T-cell lymphoma cells (100 μl of 2 × 10⁶ cells/ml) were subcutaneously injected into female C57BL/6 mice (13). For quantitative (q)RT-PCR and enzyme-linked immunosorbent assay (ELISA), 0.15 mg bexarotene was intraperitoneally injected on day 12, and the tumor was harvested on day 14. For qRT-PCR, the whole tumor was frozen with liquid nitrogen, then crushed with a Cryo-Press (MICROTEC, Chiba, Japan), as described previously (14). Total RNA was extracted using ISOGEN (NIPPON GENE, Tokyo, Japan) according to the manufacturer's instructions. For the therapeutic experiments, we measured the size of established tumors with calipers (Mitsutoyo, Utsunomiya, Japan) and estimated tumor volume using the following formula: π/6 × length × width (14). Starting on day 6, we intraperitoneally injected 0.15 mg of bexarotene or 0.30 mg of anti-CCL22 antibodies (R&D Systems, Minneapolis, MN) on day 6 and day 12. Tumor-bearing animals were sacrificed when the tumor resulted in severe ulceration or reached a size of 1,000 mm³.

Total RNA was extracted using an RNeasy Micro kit (Qiagen, Courtaboeuf, France) in accordance with the manufacturer's instructions. RNA was eluted with 14 μl of RNase-free water. DNase I treatment (RNase-Free DNase Set; Qiagen) was performed to remove contaminating genomic DNA. Reverse transcription was performed with the SuperScript VILO cDNA Synthesis kit (Invitrogen, Carlsbad, CA). Amplification reactions were performed using an Mx 3000P Real-Time Quantitative PCR System (Stratagene, San Diego, CA). Relative mRNA expression levels were calculated for each gene and each time point after normalization against GAPDH using the ΔCt method or ΔΔCt method.

Data from 25 CTCL patients were collected from five clinical sites in Japan. Pathologists and dermatologists in each institute had diagnosed these patients with CTCL both clinically and pathologically. No patients who were administered bexarotene had received any systemic therapies previously. We have summarized the clinical information in Table 1.

The following antibodies were used for immunofluorescence (IF): mouse anti-human CD163 phycoerythrin-conjugated monoclonal antibody (R&D Systems), rabbit polyclonal anti-CCL22 antibody (Biorbyt, Cambridge, UK), rabbit polyclonal anti-CXCL5 antibody (Lifespan Bioscience, Seattle, WA), mouse anti-CXCL10 antibody (Lifespan Bioscience), Alexa Fluor 488-conjugated anti-mouse rat immunoglobulin (Ig)G (Abcam, Tokyo, Japan), and Alexa Fluor 488-conjugated anti-rabbit goat IgG (Abcam). We used the following antibodies for immunohistochemical staining: rabbit polyclonal antibodies for human CCL22 (R&D Systems) and human CXCL5 (LifeSpan Bioscience).

Each sample was processed for single staining of CCL22 and CXCL5, and developed with liquid permanent red (DAKO, Santa Clara, CA). Briefly, formalin-fixed, paraffin-embedded tissue samples were sectioned at 4 μm and deparaffinized. After protease treatment for antigen retrieval, sections were blocked with goat serum for 10 min, then exposed to primary antibodies at 4°C overnight. Sections were developed with 3-Amino-9-ethylcarbazole (AEC).

For cryosections, each sample was frozen in optimal cutting temperature embedding medium, and 6-μm sections were fixed with cold acetone for 10 min and blocked with IF buffer (PBS, 5% bovine serum albumin). Each section was therefore incubated with relevant antibodies. Slides were mounted in DAPI Fluoromount-G (Southern Biotech, Birmingham, AL) and examined using an LSM 700 microscope equipped with a SPOT digital camera (Zeiss, Oberkochen, Germany).

Secretion of CCL22 in each tumor was determined using ELISA kits (R&D Systems), according to the manufacturer's instructions. Serum levels of CCL22, CXCL5, and CXCL10 in each CTCL patient were measured according to the manufacturer's instructions. For patients treated with bexarotene, serum was obtained at 0 days and/or 4 weeks after bexarotene administration (300 mg/m²), then stored for ELISA analysis of serum levels of each chemokine. For peripheral blood monocytes (PBMo)-derived M2 macrophages, after the 7-day culture, supernatants were collected and secretion of CCL22, CXCL5, and sCSD163 was determined by ELISA kits (R&D Systems), according to the manufacturer's instructions.

Data from each donor were obtained as the mean of duplicate assays.

CD14⁺ monocytes were isolated from peripheral blood mononuclear cells from healthy donors using MACS beads (CD14 microbeads; Miltenyi Biotec, Sunnyvale, CA) according to the manufacturer's protocol. CD14⁺ monocytes (2 × 10⁵/ml) were cultured in complete medium containing 100 ng/ml of recombinant human M-CSF for 5 days, as previously reported (15, 16). On day 5, monocyte-derived macrophages were treated with recombinant human IL-4 (20 ng/ml) with or without bexarotene (10 ng/ml−1 μg/ml) for 48 h, and culture supernatant was harvested.

The surface expression of CD163 and arginase 1 on macrophages was analyzed by flow cytometry. Cell staining was conducted with PE-conjugated anti-CD163 (R & D system), FITC-conjugated anti-arginase 1 (R & D system), PE-conjugated isotype control Ab (BD Bioscience, Tokyo, Japan), or FITC-conjugated isotype control Ab (BD Bioscience). The cells were analyzed with a C6 flow cytometer (Acuri Cytometers Inc., Ann Arbor, MI).

For a single comparison of two groups, the Mann–Whitney U-test was used. The level of significance was set at p < 0.05.

Since the immunological microenvironment of CTCL resembles that of atopic dermatitis (16–18), and as tumor-associated macrophages (TAMs) have been reported to play a significant role in stimulating the developing tumor microenvironment by periostin and IL-4 in lesional skin of mycosis fungoides (MF) (16), we hypothesized that bexarotene might affect the immunological functions of TAMs in tumor sites of CTCL. To investigate the immunomodulatory effects of bexarotene on the tumor microenvironment in vivo, we used the mouse EL4 T-cell lymphoma model. First, we evaluated TAM-related chemokines, Th1/Th2-related cytokines and proinflammatory cytokines. Intraperitoneal administration of bexarotene significantly decreased expressions of CCL22, CXCL5, CXCL10, IL-4, and p19 mRNA in the tumor microenvironment (Figure 1A). No significant differences were seen in the expressions of CCL17, IL-17A, p35, or p40 mRNA.

Since bexarotene significantly decreased expression of CCL22 mRNA in the tumor microenvironment, we hypothesized that bexarotene, as well as anti-CCL22 antibody (Ab) could suppress the growth of EL-4 T cell lymphoma in vivo. We examined the therapeutic effects of bexarotene in vivo using the EL-4 murine T-cell lymphoma model. We treated EL-4 murine T-cell lymphoma (3–4 mm in diameter) on the backs of mice by intraperitoneal injection of bexarotene (0.15 mg/mouse) or anti-CCL22 Ab (0.30 mg/mouse) on days 6 and 12. For the control antibody, we used rat IgG (0.30 mg/mouse). Both bexarotene and anti-CCL22 Ab significantly suppressed the growth of EL-4 T-cell lymphoma (Figure 1B).

Since intraperitoneal injection of bexarotene decreased expression of CCL22, CXCL5, and CXCL10 mRNA in EL4 mouse T-cell lymphoma, we hypothesized that serum levels of CCL22, CXCL5, or CXCL10 might be associated with response in CTCL patients treated with bexarotene. We therefore first evaluated serum levels of CCL22, CXCL5, and CXCL10 in 9 patients with early CTCL and 16 patients with advanced CTCL (Table 1). Serum levels of CCL22 were significantly increased in advanced CTCL compared with early CTCL (Figure 2). In contrast, no significant difference in serum levels of CXCL5 and CXCL10 were seen between early and advanced CTCL (Figure 2). Next, we evaluated serum levels of CCL22, CXCL5, and CXCL10 before and after administration of oral bexarotene. All patients were administered bexarotene at 300 mg/m²/day. Serum levels of CCL22 were decreased in CTCL patients who responded to bexarotene therapy, but were increased in CTCL patients who showed progressive disease 4 weeks after starting bexarotene therapy (Figure 3A). The change ratio of serum CCL22 in CTCL patients who responded to bexarotene therapy was significantly lower than that in CTCL patients who showed progressive disease by 4 weeks after starting bexarotene therapy (Figure 3A). In contrast to serum CCL22, no differences were identified in the change ratios of serum CXCL5 (Figure 3B) or CXCL10 (Figure 3C) among responders and non-responders to bexarotene therapy.

Since oral intake of bexarotene decreased serum CCL22 in CTCL patients, we next investigated source cells of CCL22, CXCL5, and CXCL10 in the lesional skin of MF, as the largest subtype of CTCL. The number of CCL22-producing cells significantly increased in advanced stages compared to that in the early stage (Figure 4A). In contrast, the number of CXCL5-producing cells significantly decreased in parallel with MF stage (Figure 4A). CXCL10-producing cells were not detected in the lesional skin of MF (data not shown). IF staining revealed that CD163⁺ M2 macrophages mainly produced CCL22 (Figure 4B), but a few CD163⁺ M2 macrophages produced CXCL5 in the lesional skin of MF (Figure 4B).

Since CD163⁺ TAMs produced CCL22 in the lesional skin of MF, we hypothesized that bexarotene might decrease the production of CCL22 from CD163⁺ M2 macrophages. To test this, we evaluated the production of chemokines from CD163⁺ M2 macrophages using M2 macrophages generated from peripheral blood mononuclear cells (PBMCs) in healthy donors (15). Production of CCL22 was significantly decreased by bexarotene in a dose-dependent manner (Figure 5A). The purity of cultured CD163+ M2 macrophages is >90% as assessed by FACS analysis (Figure 5B).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.