The next generation of candidate HIV vaccines include replicating vectors selected for tropism to mucosal sites, where an efficacious T cell response will be required to limit T cell replication and HIV associated CD4 T cell loss. To fully assess immunogenicity of such candidates, there is a need to develop robust quality controlled analysis of gut derived HIV specific CD8+ T-cell responses. Despite obvious challenges in obtaining sufficient amounts of tissue, the highly compartmentalised nature of the mucosal immune responses, requires the assessment of CD8 T cells isolated directly from local tissue before any conclusions regarding the induction of mucosal responses are made. Here we describe the optimisation and subsequent qualification of a qualitative and quantitative polychromatic flow cytometry assay to assess antigen specific CD8+ T cells isolated from the gut, using samples from HIV positive and negative volunteers. Internal quality controls monitored over time, combined with the use of quality gating and standard operating procedures were used to demonstrate the generation of robust and reliable data.
Anjarwalla, I.; Ashraf, A.; Bergin, P.; Cheeseman, H.; Clark, L.; Cox, M.C.; Dally, L.; Gazzard, B.; Gill, D.K.; Gilmour, J.; Gomez, R.; Gotch, F.; Hayes, P.; Higgs, C.; Kaltsidis, H.; Kelleher, P.; Kopycinski, J.; Spentzou, A. Measuring human T cell responses in blood and gut samples using qualified methods suitable for evaluation of HIV vaccine candidates in clinical trials. Journal of Immunological Methods (2011) 370 (1-2) 43-54. [DOI: 10.1016/j.jim.2011.05.007]