Peptide microarrays for the profiling of cytotoxic T-lymphocyte activity using minimum numbers of cells.
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.
Hoff
Antje
A
Department of Molecular Biology, Interfaculty Institute for Cell Biology, University of Tübingen, Tübingen, Germany.
Bagû
Ana-Cristina
AC
André
Thomas
T
Roth
Günter
G
Wiesmüller
Karl-Heinz
KH
Gückel
Brigitte
B
Brock
Roland
R
D002452:Cell Count; D045744:Cell Line, Tumor; D003601:Cytotoxicity Tests, Immunologic; D018604:Epitope Mapping / Q000379:methods*; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D015234:HLA-A Antigens / Q000378:metabolism; D015789:HLA-A2 Antigen; D006801:Humans; D016219:Immunotherapy, Adoptive; D046228:Microarray Analysis; D008904:Miniaturization; D018396:Mucin-1 / Q000276:immunology / Q000378:metabolism; D009369:Neoplasms / Q000276:immunology / Q000473:pathology / Q000628:therapy*; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism* / Q000473:pathology
D005452:Fluoresceins; D005456:Fluorescent Dyes; D015234:HLA-A Antigens; C524866:HLA-A*02 antigen; D015789:HLA-A2 Antigen; C060192:MUC1 protein, human; D018396:Mucin-1; D010446:Peptide Fragments; C007740:fluorexon