Comparison of proliferation and rapid cytokine induction assays for flow cytometric T-cell epitope mapping.
BACKGROUND
T-cell epitope mapping by flow cytometry based on rapid ex vivo peptide-specific cytokine induction in T cells is very efficient and time saving compared with traditional assays. We investigated whether the same epitopes could be identified by proliferation studies.


METHODS
An assay based on rapid interferon-gamma induction in T cells (6 h of ex vivo stimulation) was run in parallel with a proliferation assay based on the incremental loss of carboxy-fluorescein diacetate succinimidyl ester staining in proliferating cells. The proliferation assay was chosen because it can be evaluated by high-resolution modern multiparameter flow cytometry. In both cases, T cells were stimulated with the same cytomegalovirus-derived peptides. The peptides identified by the rapid induction of interferon-gamma were compared with those inducing T-cell proliferation.


RESULTS
Most epitopes were identified by proliferation and rapid cytokine induction methods; however, each method also identified epitopes that the other one did not. In general, rapid cytokine induction was associated with considerably less background noise, making epitope identification easier, and, owing to the short stimulation time necessary, several identification steps could be carried out on material stored in the incubator.


CONCLUSIONS
Even though most epitopes were identified by both approaches, the rapid cytokine induction method had major logistic advantages. However, it may be best to use both assays, particularly in situations in which the identification of epitopes may depend on prior clonal T-cell expansion.
Tesfa
Lydia
L
Institute for Medical Immunology, Universitätsklinikum Charité der Humboldt Universität zu Berlin, Berlin, Germany.
Volk
Hans-Dieter
HD
Kern
Florian
F
D000328:Adult; D000911:Antibodies, Monoclonal / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism; D018414:CD8-Positive T-Lymphocytes / Q000378:metabolism; D002455:Cell Division; D016207:Cytokines / Q000378:metabolism; D018604:Epitope Mapping / Q000379:methods*; D000939:Epitopes; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006680:HLA Antigens / Q000737:chemistry; D006801:Humans; D007074:Immunoglobulin G / Q000378:metabolism; D007371:Interferon-gamma / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D013997:Time Factors
D000911:Antibodies, Monoclonal; D016207:Cytokines; D000939:Epitopes; D006680:HLA Antigens; D007074:Immunoglobulin G; D010455:Peptides; D007371:Interferon-gamma