Novel application of Ki67 to quantify antigen-specific in vitro lymphoproliferation.
Antigen-specific proliferation is a critical function of memory T cells that is often utilised to measure vaccine immunogenicity and T cell function. We proposed that measurement of intracellular expression of the nuclear protein, Ki67, could reliably assess specific T cell proliferation in vitro. Ki67 was expressed in CD4+ and CD8+ T cells that had undergone in vitro proliferation after 6-day culture of human whole blood or PBMC with antigens. T cells cultured with no antigen did not express Ki67. When compared to current flow cytometry based proliferation assays, Ki67 detected proliferating cells with greater sensitivity than BrdU incorporation, whereas its sensitivity was similar to dye dilution of Oregon Green (OG), a CFSE derivative. Overall, the magnitude and cytokine expression profile of proliferating T cells detected by Ki67 expression correlated strongly with T cells detected with BrdU or OG. The intra-assay variability of Ki67 proliferation was 2-3% for CD4+ T cells, and 10-16% for CD8+ T cells. Finally, we demonstrate that the Ki67 assay detects tetanus toxoid-specific CD4+ T cell proliferation after infant vaccination with tetanus toxoid (TT). Overall our data suggest that intracellular Ki67 expression provides a specific, quantitative and reproducible measure of antigen-specific T cell proliferation in vitro.
Soares
Andreia
A
South African Tuberculosis Vaccine Initiative, Institute of Infectious Diseases and Molecular Medicine and School of Child and Adolescent Health, University of Cape Town, Cape Town, South Africa.
Govender
Lerisa
L
Hughes
Jane
J
Mavakla
Wendy
W
de Kock
Marwou
M
Barnard
Charlene
C
Pienaar
Bernadette
B
Janse van Rensburg
Esme
E
Jacobs
Gail
G
Khomba
Gloria
G
Stone
Lynnette
L
Abel
Brian
B
Scriba
Thomas J
TJ
Hanekom
Willem A
WA
D000328:Adult; D000942:Antigens, Bacterial / Q000276:immunology / Q000494:pharmacology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D049109:Cell Proliferation; D016207:Cytokines / Q000096:biosynthesis / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000379:methods; D005786:Gene Expression Regulation / Q000187:drug effects / Q000276:immunology; D006801:Humans; D007156:Immunologic Memory / Q000502:physiology; D007223:Infant; D019394:Ki-67 Antigen / Q000096:biosynthesis* / Q000276:immunology; D008297:Male; D012680:Sensitivity and Specificity; D013745:Tetanus Toxoid / Q000276:immunology / Q000494:pharmacology*; D014611:Vaccination
D000942:Antigens, Bacterial; D016207:Cytokines; D019394:Ki-67 Antigen; D013745:Tetanus Toxoid
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