These authors contributed equally to this study.
Immunodominance and functional alterations of tumor-associated antigen-specific CD8+ T-cell responses in hepatocellular carcinoma
Article first published online: 20 FEB 2014
© 2014 The Authors. Hepatology published by Wiley on behalf of the American Association for the Study of Liver Diseases
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Volume 59, Issue 4, pages 1415–1426, April 2014
How to Cite
Flecken, T., Schmidt, N., Hild, S., Gostick, E., Drognitz, O., Zeiser, R., Schemmer, P., Bruns, H., Eiermann, T., Price, D. A., Blum, H. E., Neumann-Haefelin, C. and Thimme, R. (2014), Immunodominance and functional alterations of tumor-associated antigen-specific CD8+ T-cell responses in hepatocellular carcinoma. Hepatology, 59: 1415–1426. doi: 10.1002/hep.26731
See Editorial on Page 1232
Supported by the Deutsche Forschungsgemeinschaft (RT: Heisenberg Professorship TH-719/3-1), the European Union (EFRE, INTERREG IV Oberrhein), the Wellcome Trust (DAP), and the Excellence Initiative of the German Federal and State Governments (GSC-4, Spemann Graduate School).
Potential conflict of interest: Nothing to report.
- Issue published online: 24 MAR 2014
- Article first published online: 20 FEB 2014
- Accepted manuscript online: 3 SEP 2013 11:49AM EST
- Manuscript Accepted: 29 AUG 2013
- Manuscript Revised: 10 JUL 2013
- Manuscript Received: 7 MAR 2013
Additional Supporting Information may be found in the online version of this article.
Supplementary Figure 1 Correlation between TAA-specific CD8+ T-cell response breadth and patient survival. PFS data are shown for a total of 76 HCC patients after stratification based on the number of TAA targeted by CD8+ T-cells. The correlation between TAA and PFS was analyzed by logrank-test for trend.
Supplementary Figure 2 Fine-mapping of novel CD8+ T-cell epitopes. Antigen-unspecifically expanded CD8+ T-cells were either left unstimulated, stimulated with overlapping peptides or stimulated with exact epitopes. Production of IFN-γ is shown in representative dot plots. Each row represents one patient. Peptide sequences are indicated. The exact epitope in the overlapping peptide is underlined. Numbers indicate %IFN-γ+/CD8+ T-cells. (A) Epitope GPC-3519-528/HLA-A*03:01 within peptide GPC-3 65. (B) Epitope GPC-3281-289/HLA-A*02:01 within peptide GPC-3 36. (C) Epitope NY-ESO-1145-153/HLA-A*02:01 within peptide NY-ESO-1 18.
Supplementary Figure 3 Expression of cytotoxic mediators after antigen-specific expansion. After two weeks of antigen-specific expansion, TAA-specific CD8+ T-cell lines were analyzed for expression of cytotoxic mediators. Representative dot plots and histogram showing the tetramer-binding population and its expression of Granzyme B, Perforin, FasL and CD25 (black lines) compared to total CD8+ T-cells (grey) for one patient with a response to MAGE-A196-104 (A) and one patient with a response to NY-ESO-1157-165 (B). Numbers indicate %tetramer+/CD8+ T-cells. The mean fluorescence intensities (MFI) of tetramer+CD8+ T-cells are shown for Granzyme B (C), Perforin (D), FasL (E) and CD25 (F). Each dot represents one of seven TAA-specific CD8+ T-cell lines. Values were compared by paired t-test (C-E) or by Wilcoxon matched-pair test (F).
Supplementary Figure 4 Expression of inhibitory receptors after antigen-specific expansion. After two weeks of antigen-specific expansion, the expression of the inhibitory receptors programmed death-1 (PD-1) and T-cell immunoglobulin and mucin-3 (Tim-3) was analyzed on tetramer-binding CD8+ T-cells. Representative dot plots and histograms showing the tetramer-binding population and its expression of PD-1 and Tim-3 (black lines) compared to total CD8+ T-cells (grey) of one cell line specific for HLA-A*02/NY-ESO-1157-165 (A) and one specific for HLA-A*02/EBV BMLF-1280-288 (B) are shown. The percentages of PD-1+ (C) and Tim-3+ (D) among tetramer+CD8+ T-cells are shown for virus-specific and TAA-specific CD8+ T-cell lines. Comparisons were performed by Mann-Whitney U-test.
Supplementary Figure 5 Effect of inhibitory receptor blockade and cytokine supplementation on antigen-specific expansion. Antigen-specific expansion was essentially performed as described. At initiation of cultures either anti-PD-L1 mAb (A) or a mixture of IL-7 and IL-12 (B) was added. Black squares represent frequencies of tetramer-binding cells, white circles represent frequencies of IFN-γ producing cells. Each dot is representative of one cell line.
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