ELISPOT and intracellular cytokine staining: novel assays for quantifying T cell responses in the chicken.
The measurement of T cell responses in chickens, not only for quantitative aspects but also for the qualitative nature of the responses, becomes increasingly important. However, there are very few assays available to measure T cell function. Therefore, we have developed enzyme-linked immunosorbent spot assay (ELISPOT) and an intracellular cytokine staining (ICCS) assay. ELISPOT assay for the detection of chicken interferon-gamma (ChIFN-gamma) production was set up and shown to be reproducible for both polyclonal and antigen-specific stimuli such as Newcastle disease virus (NDV). However, the ELISPOT assay lacks the ability to identify individual cytokine-producing cells. Separation of CD4+ and CD8+ T cell populations gave additional information, but appeared to have the disadvantage of a loss of cell interactions during stimulation. In a further refinement, individual cells were identifiable by ICCS, which gives the possibility to characterize for multiple characteristics, such as cytokine production and phenotype of the cell. Using ICCS, ChIFN-gamma production was evaluated. Although cells were detected at only low frequencies, polyclonal stimulation of peripheral blood mononuclear cell (PBMC) or spleen cells resulted in a significant increase in ChIFN-gamma production by CD4+ and CD8+ cells.
Ariaans
M P
MP
Faculty of Veterinary Medicine, Department of Infectious Diseases and Immunology, Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands.
van de Haar
P M
PM
Lowenthal
J W
JW
van Eden
W
W
Hensen
E J
EJ
Vervelde
L
L
D000818:Animals; D002645:Chickens / Q000276:immunology*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D007371:Interferon-gamma / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology*; D042541:Intracellular Space / Q000276:immunology*; D008938:Mitosis; D013154:Spleen / Q000276:immunology; D013601:T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D014611:Vaccination
D007371:Interferon-gamma