These authors contributed equally to this work.
Overcoming regulatory T-cell suppression by a lyophilized preparation of Streptococcus pyogenes
Version of Record online: 26 FEB 2013
© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
European Journal of Immunology
Volume 43, Issue 4, pages 989–1000, April 2013
How to Cite
Hirayama, M., Nishikawa, H., Nagata, Y., Tsuji, T., Kato, T., Kageyama, S., Ueda, S., Sugiyama, D., Hori, S., Sakaguchi, S., Ritter, G., Old, L. J., Gnjatic, S. and Shiku, H. (2013), Overcoming regulatory T-cell suppression by a lyophilized preparation of Streptococcus pyogenes. Eur. J. Immunol., 43: 989–1000. doi: 10.1002/eji.201242800
- Issue online: 16 APR 2013
- Version of Record online: 26 FEB 2013
- Accepted manuscript online: 22 FEB 2013 03:59AM EST
- Manuscript Accepted: 28 JAN 2013
- Manuscript Revised: 7 DEC 2012
- Manuscript Received: 6 JUL 2012
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Figure S1. (A) Preparation of NY-ESO-1 and 146HER2 proteins complexed with cholesteryl pullulan (CHP): Recombinant NY-ESO-1 and 146HER2 proteins for clinical use were prepared, and the nano-particles consisting of CHP and the NYESO-1 protein, and CHP and the HER2 complex were formulated. (B) Study design of the clinical trial. (C) Patient characteristics in this study.
Figure S2. (A) DCs were prepared from four healthy individuals as described in Materials and Methods. TNF-⟨ (100 ng/ml), LPS (1 mg/ml), or OK-432 (1 ìg/ml) was added in the culture of 1 × 105 immature DCs on day 6. After 48 h, supernatant was collected and cytokine production was analyzed with ELISA. (B) Summary of cytokine secretion in from four healthy individuals. These experiments were performed independently at least twice with similar results. Data are expressed as mean ± SD. *p < 0.05 and **p < 0.01 as compared to control.
Figure S3. (A) Fleshly isolated CD4+CD25- and CD4+CD25+ T cells were stimulated with anti-CD3 mAb (0.5 ìg / mL) and IL-12 receptor (IL-12R) ® chain expression was analyzed with flowcytometry. Bold line : CD4+CD25- T cells, Thin line: CD4+CD25+ T cells, filled grey : isotype control. (B) Expression level of IL-12R®2 after siRNA treatment was confirmed. 1 × 106 siRNA transfected or untreated CD4+CD25- T cells were cultured with 1 × 105 irradiated autologous CD4-depleted PBMCs and anti-CD3 mAb. Three days later, cells were harvested and RNA was extracted to confirm knockdown of IL-12R®2 expression by real-time RT-PCR. The relative differences in gene expression were calculated using threshold cycle (Ct) values that were normalized to those of TATA-box-binding protein gene, and compared with the relative Ct value of untreated CD4+CD25- T cells by the 2-ddCt. (C)) 1 × 104 CD4+CD25- T cells with/without siRNA treatment were cultured with 1 × 105 irradiated autologous CD4-depleted PBMCs and anti-CD3 mAb in the presence or absence of 5 × 103 CD4+CD25high Tregs with OK-432 (1 ìg / mL). Proliferation was evaluated as described in Materials and Methods. These experiments were performed independently at least twice with similar results. Data are expressed as mean ± SD.
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