4A and B). Similar expression profiles of IFN-γ, IL-2 and TNF-α were observed when

comparing Ki67+ and OG dilution (Fig. 4C and D). To test the reproducibility of the Ki67 proliferation assay, we performed 5 proliferation assays per donor on whole blood AZD6244 molecular weight from 3 healthy adult volunteers. Intra-assay coefficient of variation (CV) values for PPD-specific Ki67+ CD4+ T cells were between 2% and 3%, and for Ki67+ CD8+ T cells, which were present at lower frequencies than Ki67+ CD4+ T cells, between 10 and 16%. Even lower CV values were observed for PHA-stimulated blood, which induced the highest frequencies of Ki67+ T cells (Table 1). These results indicate that the Ki67 proliferation assay generates highly reproducible findings. To establish if Ki67

can be used to measure vaccine-specific T cell proliferation, we determined Ki67 expression in T cells before and 11–13 days after tetanus toxoid (TT) re-immunisation of healthy, 18 month old infants. This post-vaccination time point was selected because it coincides with the peak TT-specific CD4+ T cell response in healthy adults (Cellerai et al., 2007). The frequency of proliferating, Ki67+ CD4+ T cells observed pre-vaccination, following in vitro incubation of whole blood with TT, was low (median, 0.15%). After vaccination, TT-specific CD4+ T cell proliferation increased markedly (median, 3.77%, Fig. 5A and B). To control for possible non-specific up-regulation of Ki67 after TT vaccination in vitro, we also quantified BCG-specific T cell proliferation

pre- and post-vaccination. Doxorubicin mouse Frequencies of BCG-specific Ki67+ CD4+ T cells were not different before and after TT vaccination ( Fig. 5A, C and D). These data suggest that in vivo T cell turnover does not interfere with the specificity of the Ki67 proliferation assay. This assay is therefore specific for the detection of antigen-specific T cell proliferation in vitro. Proliferation is a commonly measured indicator of T cell function. We assessed intracellular old Ki67 expression as a marker of in vitro proliferation in whole blood or PBMC-based assays. We show that the Ki67 assay provides an alternative approach to measuring antigen-driven T cell proliferation, and found that results obtained were very similar to those generated by commonly used proliferation assay systems. The development of fluorescent dyes and tracking markers has enabled combined analysis of antigen-specific T cell proliferation, phenotyping and cytokine expression by flow cytometry (Johannisson and Festin, 1995, Mehta and Maino, 1997, Lyons and Doherty, 2004 and Wallace et al., 2008). To date, whole blood BrdU and PBMC dye dilution assays have been the preferred flow cytometry based methods to assess lymphocyte proliferation. In comparison, Ki67 expression identified approximately double the frequency of proliferating CD4+ T cells detected by BrdU incorporation.