Abstract
Flow cytometry-, ELISA-, and ELISpot-based in vitro assays have played important roles in assessing the frequencies and functional competence of antigen-specific T cells in the setting of infectious disease and cancer. Such methods have helped in the development of antigen-specific vaccines for human disease prevention/treatment and have also served as a foundation for the monitoring of patients’ immune responsiveness based on antigen-induced T cell expression of effector molecules (such as cytokines, chemokines, or proteins associated with cytolysis) as a consequence of therapeutic intervention.
The following method outlines a protocol employing quantitative real-time PCR (qRT-PCR) with SYBR® green technology to examine antigen-specific CD8+ T cell responses based on their rapid up-regulation of IFN-γ mRNA transcription following in vitro stimulation with peptide (antigen)-loaded, autologous peripheral blood mononuclear cells (PBMCs). The advantages of the current qRT-PCR approach over protein-based detection methods include the sensitivity to distinguish resident CD8+ T cell responses against multiple antigens without the need to artificially pre-expand T cell numbers ex vivo, as is commonly required for the latter in vitro assay systems. Following qRT-PCR setup and run, the level of human IFN-γ transcript is normalized to CD8 transcript expression level, with data reported as the relative fold change in this index versus a patient-matched PBMC sample stimulated with a negative control peptide (e.g., HIV NEF).
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Acknowledgments
This work was supported by NIH R01 grant CA140375 (W.J.S.) and a Postdoctoral Fellowship (PF-11-151-01-LIB) from the American Cancer Society (D.B.L.). The authors declare no conflicts of interest.
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Lowe, D.B., Taylor, J.L., Storkus, W.J. (2014). Monitoring Antigen-Specific T Cell Responses Using Real-Time PCR. In: Ranieri, E. (eds) Cytotoxic T-Cells. Methods in Molecular Biology, vol 1186. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1158-5_5
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DOI: https://doi.org/10.1007/978-1-4939-1158-5_5
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