Valerie Künzi, Patrick A

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Recombinant Measles Virus Induces Cytolysis of Cutaneous T-Cell Lymphoma In Vitro and In Vivo Valerie Künzi, Patrick A. Oberholzer, Lucie Heinzerling, Reinhard Dummer, Hussein Y. Naim Journal of Investigative Dermatology Volume 126, Issue 11, Pages 2525-2532 (November 2006) DOI: 10.1038/sj.jid.5700529 Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Quantification of CD46 and CD150 expression by FACS analysis. Surface expression of MV receptors by CTCL cell lines was analyzed by means of anti-CD46 or anti-CD150 (signaling lymphocytic activation molecule) antibodies and a secondary antibody conjugated to FITC. All three lymphoma cell lines, MyLa2059, SeAx, and HUT78, expressed (a) CD46 and (b) CD150 at relatively high levels. Open histograms represent staining of CD46 or CD150. Filled histograms represent the isotype control. Numbers indicate the percentage of gated positive cells. Journal of Investigative Dermatology 2006 126, 2525-2532DOI: (10.1038/sj.jid.5700529) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Infection of T-lymphoma cell lines by rMV. Cells were infected with rMeGFPV at an MOI of 0.2PFU/cell. Virus replication and cell death was visualized by microscopy (UV for GFP and phase contrast for total cell culture). The change of cell morphology (vesiculation and disintegration) is a typical indication of lysis as shown for SeAx and MyLa2059 cells (arrows). Bar=200μm. Journal of Investigative Dermatology 2006 126, 2525-2532DOI: (10.1038/sj.jid.5700529) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 CTCL cells are infected and lysed by rMV. (a) Determination of infectious virus dose in tumor cell culture. MyLa2059 cells were infected by MV expressing the eGFP at MOI 0.01, 0.1, 1, 5, and 10PFU/cell. The percentage of infected cells (in triplicates) was assessed daily by counting cells expressing GFP, and the average of triplicates was blotted. The experiment was terminated at a 100% cell infection. (b) Time course of infection and death of MyLa2059 cells infected with rMeGFPV at an MOI of 5PFU/cell. Arrows indicate cellular debris and aggregates as a result of cellular burst. Control non-infected cells are shown in the third panel. Original magnification × 10. (c) MyLa2059 cells were infected with rMeGFPV at an MOI of 5PFU/cell. On day 1–4 and on day 7, cells and medium were harvested. Virus replication within cells was analyzed by SDS–PAGE using an antibody mix directed against MV N, P, and H. The total amount of virus shed into the medium was determined by plaque assays on Vero cells. Values are means of duplicates (Ctrl, control non-infected cells; dpi, days post infection; pfu, plaque forming unit), bar=100μm. Journal of Investigative Dermatology 2006 126, 2525-2532DOI: (10.1038/sj.jid.5700529) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 I.t. injections of CTCL xenografts with rMV results in significant retardation of tumor growth in nude mice. CTCL grafts in outbreed ICR nu/nu mice were left to grow to an average size of 0.2cm3 before start of treatment with recombinant MV (rMeGFPV). Mice were injected with rMeGFPV i.t. (3.0 × 104PFU/dose) or i.p. (5.0 × 104PFU/dose) on days 1, 2, 3, 5, 7, 9, 11, 13, and 15. Control mice were injected on the same days with NaCl i.t., i.p., or left without treatment. (a) Significant tumor growth inhibition after i.t. treatment (n=6; rhombs) as compared to i.p. treatment (n=6; squares) and the untreated controls (n=6; triangles). Means of tumor sizes in cm3 (±SD) during the course of the treatment (arrows indicate injections). (b) Regression of tumor grafts in nude mice during i.t. treatment shown in (a). Tumor development was documented by photography. Typical regression of tumor growth is shown. (c) Immunoreactivity for MV NP protein is demonstrated by peroxidase staining. Treated tumor shows an intensively positive stained cluster of lymphocytes in the central part. Bar=50μm. Journal of Investigative Dermatology 2006 126, 2525-2532DOI: (10.1038/sj.jid.5700529) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions