High throughput functional microdissection of pathogen-specific T-cell immunity using antigen and lymphocyte arrays.
The analysis of the human T-cell response specific for relevant pathogens is useful for diagnostic purposes and for research. Several methods enumerate antigen specific T-cells and measure their functions. Since screening of numerous antigens from pathogens is often needed to evaluate immunocompetence, lymphocytes, labor and cost are limiting factors. To examine pathogen-specific T-cell immunity, we have miniaturized the analysis of T-cell responses using an array approach in 384- and 1536-well plates with as few as 10 x 10(3) PBMC per well instead of the 500 x 10(3) PBMC used for current assays. Secreted cytokines were detected in the same wells used for lymphocyte cultures. The method can detect about ten CMV specific T-cells diluted into 50 x 10(3) PBMC (0.02%), and can quantify secreted cytokines. The microarray approach allowed evaluation of T-cell immunity in children with a sensitivity higher than current methods. When applied to CMV epitope mapping, the data obtained with conventional methods were confirmed. The assay could be automated, allowing high throughput processing. The assay provides quantitative information on cytokines induced by antigen stimulation and can be applied in a simplified format as a field test to monitor T-cell immunity in vaccine trials or in veterinary medicine.
Cellular Immunology Unit, Advanced Biotechnology Center, Largo Benzi 10, 16132 Genoa, Italy. lipira@email.it
Giuseppina
G
Li Pira
Federico
F
Ivaldi
Laura
L
Bottone
Fabrizio
F
Manca
D000946:Antigens, Fungal / Q000276:immunology; D000953:Antigens, Protozoan / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology*; D018929:Cell Culture Techniques; D002460:Cell Line; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D018984:Epitopes, T-Lymphocyte / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping; D013601:T-Lymphocytes / Q000276:immunology* / Q000382:microbiology / Q000821:virology*; D046888:Tissue Array Analysis
D000946:Antigens, Fungal; D000953:Antigens, Protozoan; D000956:Antigens, Viral; D016207:Cytokines; D018984:Epitopes, T-Lymphocyte