Increased Soluble CD226 in Sera of Patients with Cutaneous T-Cell Lymphoma Mediates Cytotoxic Activity against Tumor Cells via CD155  Naomi Takahashi, Makoto Sugaya, Hiraku Suga, Tomonori Oka, Makiko Kawaguchi, Tomomitsu Miyagaki, Hideki Fujita, Takashi Inozume, Shinichi Sato  Journal of Investigative Dermatology  Volume 137, Issue 8, Pages 1766-1773 (August 2017) DOI: 10.1016/j.jid.2017.03.025 Copyright © 2017 The Authors Terms and Conditions

Figure 1 Immunohistochemical staining for CD155 and TOX. (a) Representative pictures of CD155 staining in CTCL lesional skin (n = 23, 4 patients with patch MF, 5 patients with plaque MF, 4 patients with tumor MF, 4 patients with erythrodermic MF, and 6 patients with SS) and in healthy skin (n = 6). Scale bars = 100 μm. (b) CD155 and TOX staining in serial sections of CTCL lesional skin (upper panel, CD155; lower panel, TOX). Scale bar = 50 μm. Representative pictures are shown. (c) CD155-positive cells were counted per high power field (×400). Data are presented as mean ± standard deviation. ∗P < 0.05 between patch and tumor, †P < 0.05 between each stage of CTCL and normal skin. CTCL, cutaneous T-cell lymphoma; MF, mycosis fungoides; SS, Sézary syndrome. Journal of Investigative Dermatology 2017 137, 1766-1773DOI: (10.1016/j.jid.2017.03.025) Copyright © 2017 The Authors Terms and Conditions

Figure 2 CD155, CD226, and TIGIT mRNA expression in CTCL lesional skin. (a) Results of quantitative reverse transcriptase–PCR for CD155, CD226, and TIGIT using mRNA extracted from CTCL lesional skin (n = 19, 4 patients with MF, 3 patients with plaque MF, 5 patients with tumor MF, 3 patients with erythrodermic MF, and 4 patients with SS) and normal skin (n = 8). The measured values from individual patients are plotted as dots. ∗P < 0.05 and ∗∗P < 0.01. (b) Correlations between CD155 and CCL17, CD226, and TIGIT mRNA levels in CTCL lesional skin. CTCL, cutaneous T-cell lymphoma; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MF, mycosis fungoides; SS, Sézary syndrome. Journal of Investigative Dermatology 2017 137, 1766-1773DOI: (10.1016/j.jid.2017.03.025) Copyright © 2017 The Authors Terms and Conditions

Figure 3 Decreased membrane CD226 expression on NK and CD8+ cells in CTCL patients. (a) Peripheral blood mononuclear cells (PBMCs) from patients with CTCL (n = 18, 3 patients with patch MF, 7 patients with plaque MF, 6 patients with tumor MF, and 2 patients with erythrodermic MF) and healthy subjects (n = 11) were stained with phycoerythrin-conjugated anti-CD56 mAb or allophycocyanin–conjugated anti-CD8 mAb and FITC-conjugated anti-CD226 mAb. Numbers indicate the percentage of cells in the respective quadrants. (b, c) The frequencies of CD226-positive cells among (b) total PBMCs and (c) CD56+ or CD8+ T cells are shown. The measured values from individual patients are plotted as dots. ∗P < 0.05 and ∗∗P < 0.01. CTCL, cutaneous T-cell lymphoma; FSC, forward scatter; MF, mycosis fungoides; NK, natural killer; PBMC, peripheral blood mononuclear cell; SS, Sézary syndrome; SSC, side scatter. Journal of Investigative Dermatology 2017 137, 1766-1773DOI: (10.1016/j.jid.2017.03.025) Copyright © 2017 The Authors Terms and Conditions

Figure 4 Serum soluble CD226 levels in CTCL patients. (a) Serum soluble CD226 levels in CTCL patients (n = 42, 7 patients with patch MF, 12 patients with plaque MF, 12 patients with tumor MF, 3 patients with erythrodermic MF, and 8 patients with SS) and healthy control subjects (n = 20). (b) Soluble CD226 levels in CTCL patients (n = 8) before and after treatment. (c) A negative correlation between the frequency of CD226+ cells in CD56+ cells in peripheral blood and serum soluble CD226 levels in CTCL patients. (d) Correlations between serum soluble CD226 levels and soluble IL-2 receptor (sIL-2R), LDH, and CCL17 levels in CTCL patients. The measured values from individual patients are plotted as dots. ∗∗P < 0.01. CTCL, cutaneous T-cell lymphoma; LDH, lactate dehydrogenase; MF, mycosis fungoides; SS, Sézary syndrome. Journal of Investigative Dermatology 2017 137, 1766-1773DOI: (10.1016/j.jid.2017.03.025) Copyright © 2017 The Authors Terms and Conditions

Figure 5 Cytotoxic activity of recombinant CD226 against CD155-expressing CTCL cells. (a) CD155 surface expression on SeAX cells, MJ cells, Myla cells, and HH cells was analyzed by flow cytometry. (b) CD226 expression on KHYG-1 cells (NK cell line) was analyzed. The histograms show the overlay of CD155- or CD226-specific staining (gray color) and the isotype controls (dotted lines). Shown is a representative example of four independent experiments giving similar results. (c) Cytotoxicity assay was performed at an effector-to-target ratio of 5:1. Cells were co-cultured for 36 hours. (d) The frequencies of dead target CTCL cells (SeAX cells) expressing CD155 when they were cultured alone, with recombinant CD226, or with NK cells. (e) Cytotoxic effect of recombinant CD226 against CD155-expressing cells (MJ cells and Myla cells) and CD155-negative cells (HH cells). ∗P < 0.05 and ∗∗P < 0.01. (f) CD155 surface expression on tumor cells from SS patients and normal lymphocytes. The histogram shows the overlay of CD155-specific staining (gray color) and the isotype control (dotted lines). Shown is a representative example of five independent experiments giving similar results. (g) The frequencies of dead cells from SS patients when they were cultured with recombinant CD226. ∗P < 0.05. NK, natural killer; SS, Sézary syndrome. Journal of Investigative Dermatology 2017 137, 1766-1773DOI: (10.1016/j.jid.2017.03.025) Copyright © 2017 The Authors Terms and Conditions