Functional MHC Class II Is Upregulated in Neurofibromin-Deficient Schwann Cells David E. Reuss, Jana Mucha, Nikola Holtkamp, Ute Müller, Hans-Peter Berlien,

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Functional MHC Class II Is Upregulated in Neurofibromin-Deficient Schwann Cells David E. Reuss, Jana Mucha, Nikola Holtkamp, Ute Müller, Hans-Peter Berlien, Victor-Felix Mautner, Volker Ehemann, Michael Platten, Klaus Scheffzek, Andreas von Deimling Journal of Investigative Dermatology Volume 133, Issue 5, Pages 1372-1375 (May 2013) DOI: 10.1038/jid.2012.488 Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Upregulation of major histocompatibility complex II (MHCII) in neurofibromin-deficient Schwann cells (SCs) in vitro and in tumor tissue. (a) Immunoblots of normal human SC culture (SC, +/+) and cultures enriched for NF1+/- and -/- SCs from three neurofibromas (ID52, ID98, and ID64). (b) Immunohistochemistry of neurofibroma shows MHCII expression in a subpopulation of tumor cells (left panel: 100-fold magnification, right panel: 400-fold magnification; brown areas correspond to antibody binding, nuclei are blue; bar=10μm). (c) Immunofluorescence of neurofibroma demonstrating inverse expression of neurofibromin (red) and MHCII (green) in SC (S100B, blue). Bar=10μm. (d), Real-time PCR for class II transactivator (CIITA) and HLA-DRα of a NF1+/-/ -/- pair (ID52) along with +/+ SC. (e) Immunoblots of NF1+/+ and three different NF1-/- neurofibroma SC cultures showing strong overexpression of HLA-DR in NF1-/- SCs. (f) Immunoblots of NF1-/- SC after small interfering RNA (siRNA)–mediated knockdown of CIITA demonstrating decreased levels of MHCII. Data represent six independent experiments with two different cultures. Journal of Investigative Dermatology 2013 133, 1372-1375DOI: (10.1038/jid.2012.488) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Anti-HLA-DR antibody L243 induces apoptosis and G2 arrest in NF1-/- SCs. (a) Cell growth upon incubation with L234 (HLA-DR, major histocompatibility complex II (MHCII)) or W6/32 (HLA-A,B,C, MHCI) antibody of three primary NF1-/- SC cultures and normal human SC (+/+). Within 72hours cell numbers increased 2- to 3-fold in untreated cultures (control); (b) cell growth of NF1-/- SCs (ID34) upon incubation with L234 or IgG within 24hours. (c) Induction of apoptosis was evaluated by flow cytometry determination of terminal transferase dUTP nick-end labeling (TUNEL) staining (2.5hours). (d) Cell cycle analysis of NF1-/- SCs treated with L243 or IgG control antibody (2.5hours). (e) Small interfering RNA (siRNA)–mediated knockdown of class II transactivator (CIITA) rescues NF1-/- SC from L243-induced apoptosis and G2 arrest. Results are expressed as mean±SD of three independent experiments (n=3); *P<0.05, **P<0.01 Student’s t-test. NS, nonsignificant. Journal of Investigative Dermatology 2013 133, 1372-1375DOI: (10.1038/jid.2012.488) Copyright © 2013 The Society for Investigative Dermatology, Inc Terms and Conditions