T Helper Type 2-Biased Natural Killer Cell Phenotype in Patients with Pemphigus Vulgaris Hayato Takahashi, Masayuki Amagai, Akiko Tanikawa, Shigeaki.

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T Helper Type 2-Biased Natural Killer Cell Phenotype in Patients with Pemphigus Vulgaris Hayato Takahashi, Masayuki Amagai, Akiko Tanikawa, Shigeaki Suzuki, Yasuo Ikeda, Takeji Nishikawa, Yutaka Kawakami, Masataka Kuwana Journal of Investigative Dermatology Volume 127, Issue 2, Pages 324-330 (February 2007) DOI: 10.1038/sj.jid.5700527 Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Analysis of NK cells in the peripheral blood from PV patients, ITP patients, MG patients, and healthy controls, by flow cytometry. (a) Representative density plot analysis for evaluating the expression of CD56 and CD3 in a PV patient and healthy control. The cells in the lymphocyte fraction, gated on forward and side scatter, were evaluated. The numbers indicate the proportions of cells in the corresponding quadrants in the lower part. The upper left portion (CD56+CD3−) corresponds to NK cells. (b) The proportion of CD56+CD3− NK cells in the circulating lymphocytes of 22PV patients (two with active disease), 11 ITP patients, 10MG patients, and 28 healthy controls. (c) The absolute number of CD56+CD3− NK cells in 1ml of peripheral blood from 12PV patients (one with active disease) and 13 healthy controls. (d) The proportion of CD69+-activated NK cells in the total NK cells of 15PV patients (one with active disease), 11 ITP patients, 10MG patients, and 18 healthy controls. Comparisons were made by the Mann–Whitney U-test. Horizontal bars represent the mean values. Journal of Investigative Dermatology 2007 127, 324-330DOI: (10.1038/sj.jid.5700527) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Relative mRNA expression levels of IL-10, IL-12Rβ2, perforin, and granzyme B in the NK cells from PV patients with active disease (aPV), PV patients in remission (rPV), and healthy controls. Total RNA was prepared from sorted CD56+CD3− cells and subjected to Taqman® quantitative PCR. Individual mRNA expression levels were normalized to the mRNA expression level of GAPDH. Comparison was made by the Mann–Whitney U-test. Horizontal bars represent the mean values. Journal of Investigative Dermatology 2007 127, 324-330DOI: (10.1038/sj.jid.5700527) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 The degree of Stat4 phosphorylation in IL-12-stimulated NK cells in PV patients and healthy controls. (a) Sorted NK cells from five PV patients (one with active disease) and five healthy controls were stimulated with IL-12 in vitro, and subjected to immunoblot analysis to detect phosphorylated Stat4 (pStat4) and total Stat4. (b) The degree of Stat4 phosphorylation in five PV patients and five healthy controls. Comparison was made by the Mann–Whitney U-test. Horizontal bars represent the mean values. Journal of Investigative Dermatology 2007 127, 324-330DOI: (10.1038/sj.jid.5700527) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 IL-10 production by NK cells from PV patients and healthy controls, upon in vitro stimulation with a combination of IL-2 and IL-12. NK cells freshly isolated from seven PV patients and nine healthy controls were cultured with IL-2 and IL-12 for 3 days, and the culture supernatants were subjected to ELISA for measurement of IL-10. Comparison was made by the Mann–Whitney U-test. Horizontal bars represent the mean values. Journal of Investigative Dermatology 2007 127, 324-330DOI: (10.1038/sj.jid.5700527) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 IL-5 expression in NK cells from PV patients according to the disease activity. (a) IL-5 and GAPDH expression by RT-PCR in sorted NK cells from four PV patients in remission, a PV patient with active disease, and four healthy controls. (b) IL-5 mRNA expression levels determined using Taqman® quantitative PCR in six PV patients with active disease (aPV), 13PV patients in remission (rPV), and 11 healthy controls. Each mRNA expression level was normalized to the GAPDH mRNA expression level. A relative mRNA expression level lower than 0 means the value was below the detectable limit. Comparison was made by the Mann–Whitney U-test. Horizontal bars represent the mean values. Journal of Investigative Dermatology 2007 127, 324-330DOI: (10.1038/sj.jid.5700527) Copyright © 2007 The Society for Investigative Dermatology, Inc Terms and Conditions