1. Novel application of Ki67 to quantify antigen-specific in vitro lymphoproliferation
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Gloria Khomba, Andreia Soares, Willem A. Hanekom, Lynnette Stone, Esme Janse van Rensburg, Gail Jacobs, Jane Hughes, Charlene Barnard, Thomas J. Scriba, Wendy Mavakla, Brian Abel, Bernadette Pienaar, Marwou de Kock, and Lerisa Govender
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Adult ,CD4-Positive T-Lymphocytes ,Male ,T cell ,Immunology ,T cells ,Biology ,CD8-Positive T-Lymphocytes ,TT, tetanus toxoid ,Clinical immunology ,Sensitivity and Specificity ,Flow cytometry ,03 medical and health sciences ,Interleukin 21 ,0302 clinical medicine ,Antigen ,medicine ,Tetanus Toxoid ,Cytotoxic T cell ,Immunology and Allergy ,Humans ,PPD, purified protein derivative ,030304 developmental biology ,Cell Proliferation ,0303 health sciences ,Antigens, Bacterial ,medicine.diagnostic_test ,Cell growth ,Vaccination ,Infant ,T lymphocyte ,Flow Cytometry ,Molecular biology ,3. Good health ,medicine.anatomical_structure ,Ki-67 Antigen ,Gene Expression Regulation ,OG, Oregon Green ,Cellular proliferation ,Cytokines ,Female ,Immunologic Memory ,Ki67 ,Vaccine ,CD8 ,030215 immunology ,Research Paper - Abstract
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.
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