Peptide microarrays for the profiling of cytotoxic T-lymphocyte activity using minimum numbers of cells |
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Authors: | Antje Hoff Ana-Cristina Bagû Thomas André Günter Roth Karl-Heinz Wiesmüller Brigitte Gückel Roland Brock |
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Institution: | 1. Department of Molecular Biology, Interfaculty Institute for Cell Biology, University of Tübingen, Auf der Morgenstelle 15, 72076, Tübingen, Germany 5. Trinity Centre for Health Sciences, Institute for Molecular Medicine, Trinity College Dublin, St. James Street, Dublin 8, Ireland 2. Department of Gynecology and Obstetrics, University Hospital Tübingen, Calwerstra?e 7, 72076, Tübingen, Germany 6. Bachem AG, Hauptstrasse 144, 4416, Bubendorf, Switzerland 7. Department of Microsystems Engineering (IMTEK), University of Freiburg, Georges-Koehler-Allee 106, 79110, Freiburg, Germany 3. EMC Microcollections GmbH, Sindelfinger Strasse 3, 72070, Tübingen, Germany 4. Department of Biochemistry, Nijmegen Centre for Molecular Life Sciences, Radboud University Nijmegen Medical Centre, PO Box 9101, 6500 HB, Nijmegen, The Netherlands
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Abstract: | The identification of epitopes that elicit cytotoxic T-lymphocyte activity is a prerequisite for the development of cancer-specific
immunotherapies. However, especially the parallel characterization of several epitopes is limited by the availability of T
cells. Microarrays have enabled an unprecedented miniaturization and parallelization in biological assays. Here, we developed
peptide microarrays for the detection of CTL activity. MHC class I-binding peptide epitopes were pipetted onto polymer-coated
glass slides. Target cells, loaded with the cell-impermeant dye calcein, were incubated on these arrays, followed by incubation
with antigen-expanded CTLs. Cytotoxic activity was detected by release of calcein and detachment of target cells. With only
200,000 cells per microarray, CTLs could be detected at a frequency of 0.5% corresponding to 1,000 antigen-specific T cells.
Target cells and CTLs only settled on peptide spots enabling a clear separation of individual epitopes. Even though no physical
boundaries were present between the individual spots, peptide loading only occurred locally and cytolytic activity was confined
to the spots carrying the specific epitope. The peptide microarrays provide a robust platform that implements the whole process
from antigen presentation to the detection of CTL activity in a miniaturized format. The method surpasses all established
methods in the minimum numbers of cells required. With antigen uptake occurring on the microarray, further applications are
foreseen in the testing of antigen precursors that require uptake and processing prior to presentation. |
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