Novel application of Ki67 to quantify antigen-specific in vitro lymphoproliferation - PubMed (original) (raw)

. 2010 Oct 31;362(1-2):43-50.

doi: 10.1016/j.jim.2010.08.007. Epub 2010 Aug 25.

Lerisa Govender, Jane Hughes, Wendy Mavakla, Marwou de Kock, Charlene Barnard, Bernadette Pienaar, Esme Janse van Rensburg, Gail Jacobs, Gloria Khomba, Lynnette Stone, Brian Abel, Thomas J Scriba, Willem A Hanekom

Affiliations

Novel application of Ki67 to quantify antigen-specific in vitro lymphoproliferation

Andreia Soares et al. J Immunol Methods. 2010.

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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Figures

Fig. 1

Fig. 1

Ki67 as a specific marker of in vitro lymphoproliferation. Whole blood from healthy donors was incubated with the indicated antigens and Ki67 expression quantified on a daily basis over 6 days. (A) Representative example showing the frequencies of Ki67 expression by CD4+ T cells after incubation of whole blood with medium only (unstim.), PPD or αCD3/αCD28 over 6 days. Dotplots were gated on live, CD3+ CD4+ lymphocytes. Ki67+ CD4+ T cell frequencies after (B) PPD stimulation or (C) αCD3/αCD28 stimulation in 4 donors. Data are expressed as a percentage of the maximum response. The frequency of Ki67+ CD4+ T cells is indicated in each plot. (D) Frequencies of Ki67 expressing CD4+ T cells in whole blood from 15 donors after 6-day culture with medium only (unstim.) or PPD. (E) Frequencies of Ki67+ CD4+ T cells in PBMC from 14 donors. Differences were calculated using the Wilcoxon matched pairs test.

Fig. 2

Fig. 2

Comparison of the Ki67 proliferation assay with the BrdU and Oregon Green proliferation assays. (A) Representative dotplots showing Ki67 versus BrdU expression by CD4+ T cells in whole blood. Dotplots are gated on live, CD3+ CD8− lymphocytes. Frequencies of (B) PPD- and (C) TB10.4-specific CD4+ T cell proliferation as detected by Ki67 expression or BrdU incorporation (n = 15). CD4+ T cells are defined as CD3+ CD8− T cells (see Data analysis). (D) Representative dotplots showing Ki67 and dye dilution of Oregon Green by CD4+ T cells in PBMC. Dotplots are gated on live, CD3+ CD8− lymphocytes. Frequencies of (E) PPD- and (F) TB10.4-specific CD4+ T cell proliferation as detected by Ki67 expression or dye dilution of Oregon Green (OGlow) in 14 donors. CD4+ T cells are defined as CD3+ CD8− T cells (see Data analysis). Differences were calculated using the Wilcoxon matched pairs test.

Fig. 3

Fig. 3

Correlations between Ki67+ CD4+ T cell expression and BrdU incorporation or dye dilution of Oregon Green (OGlow). Whole blood was incubated with (A) PPD or (C) TB10.4 for 6 days (n = 15). PBMC were incubated with (B) PPD or (D) TB10.4 for 6 days (n = 14). Correlations were calculated using a Spearman's rank correlation coefficient.

Fig. 4

Fig. 4

Cytokine expression profiles of proliferating CD4+ T cells. Whole blood or PBMC were cultured for 6 days with no antigen or PPD. On day 6, cells were restimulated with PMA and ionomycin for 4 h in the presence of Brefeldin A to detect cytokine expression by proliferating T cells. Representative dotplots of the cytokine expression profiles of (A) Ki67+ or BrdU+ CD4+ T cells and (C) Ki67+ or OGlow CD4+ T cells. (B) Proportions of BrdU+, Ki67+ or Ki67+ BrdU− CD4+ T cells expressing IFN-γ, IL-2 or TNF-α (n = 15). (D) Proportions of Ki67+ or OGlow CD4+ T cells expressing IFN-γ, IL-2 or TNF-α (n = 14).

Fig. 5

Fig. 5

Monitoring of vaccine-induced T cell proliferation. (A) Dotplots showing Ki67 expression by CD4+ T cells from a representative 18 month old toddler before (pre-TT) and after TT vaccination (post-TT). Dotplots are gated on live, CD3+ lymphocytes. Values in each dotplot represent the frequency of Ki67+ T cells within the CD3+ CD8− T cell population. Frequencies of (B) TT-specific and (C) BCG-specific CD4+ T cells pre- and post-TT vaccination in 11 toddlers. CD4+ T cells are defined as CD3+ CD8− T cells (see Data analysis). (D) Relative increase in TT-specific or BCG-specific CD4+ T cells pre- and post-TT. The lines represent the medians. (E) Dotplots depicting frequencies of Ki67+ CD4+ T cells in whole blood directly ex vivo or after culture in the absence of antigen (unstim.) for 6 days. Values in each dotplot represent the frequency of Ki67+ T cells within the CD3+ CD8− T cell population. (F) Frequencies of Ki67+ CD4+ T cells directly ex vivo or after culture for 6 days with medium (n = 11). CD4+ T cells are defined as CD3+ CD8− T cells (see Data analysis). Differences were calculated using the Wilcoxon matched pairs test.

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