TIM-3 Is Expressed in Melanoma Cells and Is Upregulated in TGF-Beta Stimulated Mast Cells Zoltan Wiener, Barbara Kohalmi, Peter Pocza, Judit Jeager,

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TIM-3 Is Expressed in Melanoma Cells and Is Upregulated in TGF-Beta Stimulated Mast Cells Zoltan Wiener, Barbara Kohalmi, Peter Pocza, Judit Jeager, Gergely Tolgyesi, Sara Toth, Eva Gorbe, Zoltan Papp, Andras Falus Journal of Investigative Dermatology Volume 127, Issue 4, Pages 906-914 (April 2007) DOI: 10.1038/sj.jid.5700616 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Cord blood-derived mast cells magnetically isolated after 5-week culture expressed TGF-βRI and TGF-βRII and they responded to TGF-βI. (a) TGF-βRI and TGF-βRII expression in cultured human cord blood-derived mast cells (real-time PCR). (b) Flow cytometry with anti-human CD117 PE. Dotted line: isotype control; simple line: untreated mast cells; bold line: mast cells treated with 2ng/ml TGF-βI for 5 days. Journal of Investigative Dermatology 2007 127, 906-914DOI: (10.1038/sj.jid.5700616) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 The mast cell cultures contained only MC(T) cells. Both control and TGF-βI-treated (2ng/ml, 5 days after 5 weeks culturing) human mast cell cultures expressed (a) tryptase, but none of them were positive for (b) chymase (flow cytometry). The figure shows one representative out of the four repeated experiments. Dotted line: isotype control; simple line: untreated mast cells; bold line: treated mast cells. Journal of Investigative Dermatology 2007 127, 906-914DOI: (10.1038/sj.jid.5700616) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 TGF-βI upregulates TIM-3 in human mast cells. (a) Real-time PCR results for TIM-3 from human mast cells with TaqMan probes fitting to either exon6/7 (right, P=0.007) or exon 2/3 (left, P=0.025). Results were normalized to glyceraldehyde-3-phosphate dehydrogenase housekeeping (three paired samples are shown, the P-values were determined by paired t-test). (b) TIM-3 is overexpressed in human mast cells by TGF-βI (2ng/ml, 5 days) as detected by immunoblot (with tubulin housekeeping). C: control; T: TGF-βI-treated samples; 1,2, and 3: different mast cell preparates. (c) TIM-3 level is increased on the surface of human mast cells (flow cytometry) after TGF-βI treatment (2ng/ml, 5 days). Dotted line: isotype control; simple line: untreated, bold line: treated mast cell preparate. Journal of Investigative Dermatology 2007 127, 906-914DOI: (10.1038/sj.jid.5700616) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 TIM-3 is expressed both in melanoma-surrounding human mast cells and in melanoma cells. (a) Immunohistochemistry with anti-TIM3 antibody in melanoma tissue sections. Two representatives out of three different patients. The background staining of the control slides was negligible (see Materials and Methods). Single asterisks indicate melanoma cells. Double asterisk labels the area that is magnified in (b). Bar=20μm. (b) Asterisks mark mast cells in serial sections. Left side: immunohistochemistry with anti-TIM3 antibody (the magnification from a part of a). Right side: mast cells show metachromatic staining with toluidine blue. Bar=20μm. (c) WM35 and HT168-M1 melanoma cell lines express TIM-3 as detected by real-time PCR with TaqMan probes fitting to either exon6/7 or exon2/3 (mean+SEM, n=4, the WM35 data obtained by TaqMan assay fitting to exon 2/3 were taken as 100%). (d) Both WM35 and HT168-M1 melanoma cell lines were positive for cell surface TIM-3 (flow cytometric analysis). Simple line: isotype control, bold line: anti-TIM3. (e) Melanoma cell lines express TIM-3 at a higher level than HEMn-DP melanocytes (flow cytometry, simple line: melanocytes; bold line: WM35; dotted line: isotype control. With HT168-M1 similar results were obtained). Journal of Investigative Dermatology 2007 127, 906-914DOI: (10.1038/sj.jid.5700616) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Galectin-9 messenger RNA level is about 1,000-fold greater in human mast cells than in melanoma cell lines. Real-time PCR results were normalized to glyceraldehyde-3-phosphate dehydrogenase housekeeping (mean+SEM, n=4, Student's t-test: P<0.001 for both mast cell WM35 and mast cell HT108-M1 comparisons) and mast cell mean expression level was taken as 100%. Journal of Investigative Dermatology 2007 127, 906-914DOI: (10.1038/sj.jid.5700616) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions