Langerhans Cells from Human Cutaneous Squamous Cell Carcinoma Induce Strong Type 1 Immunity Hideki Fujita, Mayte Suárez-Fariñas, Hiroshi Mitsui, Juana.

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Langerhans Cells from Human Cutaneous Squamous Cell Carcinoma Induce Strong Type 1 Immunity Hideki Fujita, Mayte Suárez-Fariñas, Hiroshi Mitsui, Juana Gonzalez, Mark J. Bluth, Shali Zhang, Diane Felsen, James G. Krueger, John A. Carucci Journal of Investigative Dermatology Volume 132, Issue 6, Pages 1645-1655 (June 2012) DOI: 10.1038/jid.2012.34 Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Flow cytometric analysis of maturation-associated molecules expressed on Langerhans cells (LCs) from squamous cell carcinoma (SCC) and peritumoral nonlesional (PTNL) skin. HLA-DR+CD207+ LCs in the epidermal cell suspension from SCC and PTNL skin were subjected to the analysis of cell surface expression of CD40, CD80, CD83, and CD86. Dashed lines indicate isotype controls. Mean fluorescence intensity value is shown in the upper right-hand corner of each histogram. Data are representative of three independent experiments. Journal of Investigative Dermatology 2012 132, 1645-1655DOI: (10.1038/jid.2012.34) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Langerhans cells (LCs) from squamous cell carcinoma (SCC) stimulate proliferation of IFN-γ-producing CD4+ and CD8+ T cells. (a) Carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled, allogeneic T cells were cultured with FACS-sorted LCs from SCC or peritumoral nonlesional (PTNL) skin. Percentages of proliferating cells within live CD3+CD4+ and CD3+CD8+ cells were determined by CFSE dilution. (b) Representative FACS histograms of CFSE dilution assay using LCs from SCC and PTNL skin. Numbers show percentages of proliferating (CFSElow) cells. (c) Frequencies of cells positive for intracellular IFN-γ, IL-4, IL-17, and IL-22 detected in proliferating CD4+ and CD8+ T-cell populations following LC coculture experiments. NS, not significant. (d) Dot plot analysis of IFN-γ, IL-4, IL-17, and IL-22 expression in proliferating CD4+ and CD8+ T cells stimulated by LCs from SCC versus PTNL skin. Numbers indicate percent gated cells. Journal of Investigative Dermatology 2012 132, 1645-1655DOI: (10.1038/jid.2012.34) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Tumor supernatant (TSN) enhances Langerhans cell (LC)-driven CD4+ and CD8+ T-cell proliferation, IFN-γ, IL-4, IL-17, and IL-22 production by CD4+cells, and IFN-γ production by CD8+ cells. (a) LCs from normal skin were cultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled allogeneic T cells in the presence or absence of 25% TSN. Percentages of proliferating cells within live CD3+CD4+ and CD3+CD8+ cells were determined by CSFE dilution. (b) FACS histograms of CFSE dilution assay using LCs and TSN. Numbers show percentages of proliferating cells. (c) The effect of TSN on production of IFN-γ, IL-4, IL-17, and IL-22 by CD4+ and CD8+ T cells. NS, not significant. (d) Dot plot analysis of IFN-γ, IL-4, IL-17, and IL-22 expression in proliferating CD4+ and CD8+ T cells stimulated by LCs in the presence or absence of TSN. Numbers show percent gated cells. Journal of Investigative Dermatology 2012 132, 1645-1655DOI: (10.1038/jid.2012.34) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Tumor supernatant (TSN) enhances proliferation and IFN-γ response by CD4+ and CD8+ T cells induced by in vitro–generated Langerhans cells (LCs), but suppresses those by monocyte-derived dendritic cells (mono-DCs). (a) CD1a+CD14- LC-type DCs derived from CD34+ hematopoietic progenitors, or mono-DCs, were cultured with carboxyfluorescein diacetate succinimidyl ester (CFSE)-labeled T cells in the presence or absence of 25% TSN. Percentages of proliferating cells were determined by CFSE dilution. (b) FACS histograms of CFSE dilution assay. Numbers show percentages of proliferating cells. (c) Effect of TSN on in vitro–generated LC- or DC-driven production of IFN-γ, IL-4, IL-17, or IL-22, by CD4+ and CD8+ T cells. NS, not significant. (d) Dot plot analysis of IFN-γ, IL-4, IL-17, and IL-22 expression in proliferating CD4+ and CD8+ T cells stimulated by LC-type DCs or mono-DCs in the presence or absence of TSN. Numbers show percent gated cells. Journal of Investigative Dermatology 2012 132, 1645-1655DOI: (10.1038/jid.2012.34) Copyright © 2012 The Society for Investigative Dermatology, Inc Terms and Conditions