Abstract: CD8+ cytolytic activity is traditionally measured by detecting the release of 51Cr after incubation of effector cells with HLA-matched, infected, radiolabeled targets. An alternative method to detect CD8+ activity is to measure the production of intracellular interferon gamma (IFNγ) after antigen-specific stimulation, either by ELISPOT or by flow cytometry. Studies were performed in 19 volunteers enrolled in a phase 1 trial of candidate canarypox HIV-1 vaccines that encoded multiple HIV-1 genes. The vaccines including vCP205 (Env, Gag, and protease), vCP1433 (Env, Gag, protease, and CTL epitope-rich regions of pol and nef) and vCP1452 (equivalent to vCP1433 with additional immunomodulatory genes of vaccinia). PBMCs were stimulated in vitro with vaccinia constructs encoding env and gag or a lacZ control, and the effectors were cultured for 12–14 days. EBV-transformed B cell lines were infected overnight with the vaccinia vectors, and then incubated with the effector cells for 4 h in the presence of monensin. CD8+ gene-specific activity was determined as a percentage of IFNγ cells in the CD3+CD8+CD45RO+ gate after subtracting both the isotype control and the lacZ control stimulation. CD4 memory IFNγ production was simultaneously determined in the CD3+CD8−CD45RO+ gate. Using these techniques in blinded studies, we found that CD8+ IFNγ activity could be measured in the majority of volunteers given four immunizations. Specifically, the responses to the gag gene were control — 0/2; vCP205 — 2/4; vCP1433 — 5/6; vCP1452 — 4/7. Most of the positive responses were detected after the fourth immunization. Flow cytometric techniques hold promise as a surrogate measure of CTL and for ease of phenotyping of the effector population.

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{{Explor lien
   |wiki=    Sante
   |area=    MersV1
   |flux=    France
   |étape=   Analysis
   |type=    RBID
   |clé=     ISTEX:65A64D2B74F2447A5B5798DCB3ED28295022CD77
   |texte=   Evaluation of canarypox-induced CD8+ responses following immunization by measuring the effector population IFNγ production
}}