Ex vivo detection and enumeration of human antigen-specific CD8+ T lymphocytes using antigen delivery by a recombinant vaccinia expression vector and intracellular cytokine staining.

We describe an intracellular cytokine staining, flow cytometry based technique for quantitative and qualitative assessment of CD8+ T lymphocyte-mediated antigen specific immune responses. This adaptation of the technique first described by Suni et al. [J. Immunol. Methods, 212 (1998) 89; Cytometry, 40 (2000) 60] utilises a recombinant vaccinia expression vector to deliver the test antigen to the MHC class I processing pathway. This allows the measurement of antigen-specific CD8+ T cell responses in human subjects, irrespective of HLA type, and un-restricted to responses directed against a single peptide epitope. This method offers an advantage over the ELISPOT methods because the cells producing cytokine in the assay can be readily phenotyped, and it permits the simultaneous analysis of multiple cytokines. Finally, this technique does not require the prior establishment of autologous transformed B cell lines from patients/subjects.