Antigen-specific responses assessment for the evaluation of Bordetella pertussis T cell immunity in humans.
Measurement of antigen-specific T cell responses is an adjunctive parameter to evaluate protection induced by a previous Bordetella pertussis infection or vaccination. The assessment of T cell responses is technically complex and usually performed on fresh peripheral blood mononuclear cells (PBMC). The objective of this study was to identify simplified methods to assess pertussis specific T cell responses and verify if these assays could be performed using frozen/thawed (frozen) PBMC. Three read-outs to measure proliferation were compared: the fluorescent dye 5,6-carboxylfluorescein diacetate succinimidyl ester (CFSE) dilution test, the number of blast cells defined by physical parameters, and the incorporation of (3)H-thymidine. The results of pertussis-specific assays performed on fresh PBMC were compared to the results on frozen PBMC from the same donor. High concordance was obtained when the results of CFSE and blast read-outs were compared, an encouraging result since blast analysis allows the identification of proliferating cells and does not require any use of radioactive tracer as well as any staining. The results obtained using fresh and frozen PBMC from the same donor in the different T cell assays, including IFNγ and TNFα cytokine production, did not show significant differences, suggesting that a careful cryopreservation process of PBMC would not significantly influence T cell response evaluation. Adopting blast analysis and frozen PBMC, the possibility to test T cell responses is simplified and might be applied in population studies, providing for new instruments to better define correlates of protection still elusive in pertussis.
Department of Infectious, Parasitic and Immune-mediated Diseases, Istituto Superiore di Sanità, 00161 Rome, Italy.
Maria
M
Carollo
Raffaella
R
Palazzo
Manuela
M
Bianco
Kaatje
K
Smits
Françoise
F
Mascart
Clara Maria
CM
Ausiello
D000328:Adult; D000942:Antigens, Bacterial / Q000276:immunology*; D001886:Bordetella pertussis / Q000276:immunology*; D018929:Cell Culture Techniques; D049109:Cell Proliferation; D002478:Cells, Cultured; D015925:Cryopreservation; D005434:Flow Cytometry; D006801:Humans; D007371:Interferon-gamma / Q000276:immunology; D007963:Leukocytes, Mononuclear / Q000276:immunology; D008213:Lymphocyte Activation; D037342:Pertussis Toxin / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*; D013936:Thymidine / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
D000942:Antigens, Bacterial; D014409:Tumor Necrosis Factor-alpha; D007371:Interferon-gamma; D037342:Pertussis Toxin; D013936:Thymidine