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Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides


Cytotoxic CD8+ T cells recognize the antigenic peptides presented by class I major histocompatibility complex (MHC) molecules. These T cells have key roles in infectious diseases, autoimmunity and tumor immunology, but there is currently no unbiased method for the reliable identification of their target antigens. This is because of the low affinities of antigen-specific T cell receptors (TCR) to their target MHC-peptide complexes, the polyspecificity of these TCRs and the requirement that these TCRs recognize protein antigens that have been processed by antigen-presenting cells (APCs). Here we describe a technology for the unbiased identification of the antigenic peptides presented by MHC class I molecules. The technology uses plasmid-encoded combinatorial peptide libraries and a single-cell detection system. We validated this approach using a well-characterized influenza-virus–specific TCR, MHC and peptide combination. Single APCs carrying antigenic peptides can be detected among several million APCs that carry irrelevant peptides. The identified peptide sequences showed a converging pattern of mimotopes that revealed the parent influenza antigen. This technique should be generally applicable to the identification of disease-relevant T cell antigens.

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Figure 1: Schematic overview of the experimental strategy.
Figure 2: Activation of TCR-transfected T hybridoma cells.
Figure 3: Activation of 58-JM22-CD8-sGFP cells by library-transfected COS-7 cells and the identification of mimotopes of flu(58–66).

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We thank E. Meinl and W.E.F. Klinkert for comments on the manuscript, D. Hackl and M. Ackmann for help with synthesizing the plasmid pcDNA-NFAT-sGFP, I. Bartholomäus and M. Mues for advice regarding fluorescence microscopy, I. Eiglmeier for expert technical assistance and K. Ogston for editing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (grant SFB 571 A1).

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Authors and Affiliations



K.S. designed and performed most of the experiments and contributed to the writing of the manuscript, J.M. contributed to cloning experiments, N.K. contributed to microscopy experiments, H.W. and R.H. supervised and supported the study and contributed to the writing of the manuscript, and K.D. conceived of, designed and supervised the research and wrote the manuscript.

Corresponding author

Correspondence to Klaus Dornmair.

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Competing interests

The Ludwig-Maximilians-University, Munich, Germany, has filed European and International patent applications describing the technology used in this study (K.D., K.S. and R.H.).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–4, Supplementary Table 1 and Supplementary Methods (PDF 6560 kb)

Supplementary Video 1

Activation of a 58-JM22-CD8-sGFP cell. Time-lapse fluorescence microscopy of a coculture of 58-JM22-CD8-sGFP cells and COS-7 cells. (AVI 1060 kb)

Supplementary Video 2

Isolation of an antigen-expressing cell. We show the isolation of an activated green fluorescent 58-JM22-CD8-sGFP cell together with a subjacent antigen-expressing COS-7 cell. (AVI 1171 kb)

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Siewert, K., Malotka, J., Kawakami, N. et al. Unbiased identification of target antigens of CD8+ T cells with combinatorial libraries coding for short peptides. Nat Med 18, 824–828 (2012).

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