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Updated: Jun 9, 2026
A DNA/Ki67-Based Flow Cytometry Assay for Cell Cycle Analysis of Antigen-Specific CD8 T Cells in Vaccinated Mice
Published on: January 5, 2021
Novel application of Ki67 to quantify antigen-specific in vitro lymphoproliferation
Andreia Soares1, Lerisa Govender, Jane Hughes
1South 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.
Insights
Measuring intracellular Ki67 protein expression reliably assesses antigen-specific T cell proliferation in vitro. This method offers a sensitive, reproducible alternative for evaluating vaccine immunogenicity and T cell function.
Area of Science:
- Immunology
- Cell Biology
Background:
- Antigen-specific T cell proliferation is crucial for memory T cell function and vaccine immunogenicity.
- Current methods for measuring T cell proliferation have limitations in sensitivity and reproducibility.
Purpose of the Study:
- To evaluate the utility of intracellular Ki67 protein expression as a reliable measure of antigen-specific T cell proliferation in vitro.
- To compare the sensitivity and reproducibility of Ki67 detection with established proliferation assays.
Main Methods:
- Human whole blood or peripheral blood mononuclear cells (PBMCs) were cultured with specific antigens for 6 days.
- Intracellular Ki67 expression was measured using flow cytometry.
- Ki67 detection was compared with Bromodeoxyuridine (BrdU) incorporation and Oregon Green (OG) dye dilution assays.
Main Results:
- Ki67 was expressed in CD4+ and CD8+ T cells undergoing antigen-specific proliferation, but not in unstimulated cells.
- Ki67 assay demonstrated greater sensitivity than BrdU incorporation and comparable sensitivity to OG dye dilution.
- Ki67 detection showed strong correlation with BrdU and OG in terms of proliferation magnitude and cytokine profiles.
- Intra-assay variability for Ki67 was low (2-3% for CD4+, 10-16% for CD8+ T cells).
- The assay successfully detected tetanus toxoid-specific CD4+ T cell proliferation in infants post-vaccination.
Conclusions:
- Intracellular Ki67 expression is a specific, quantitative, and reproducible marker for in vitro antigen-specific T cell proliferation.
- The Ki67 assay provides a sensitive and reliable method for assessing T cell function and vaccine responses.
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.
