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Updated: Jul 29, 2026
Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination
Published on: April 29, 2015
Evaluation of canarypox-induced CD8(+) responses following immunization by measuring the effector population IFNgamma
T G Evans1, E G Kallas, M Campbell
1Infectious Diseases Unit, Suite 500, Department of Medicine, University of Rochester School of Medicine and Dentistry, PSSB 4150 V St., Sacramento, CA 95817, USA. tgevans@ucdavis.edu
Insights
Flow cytometry effectively measures CD8(+) interferon gamma (IFNγ) activity, a key indicator of cellular immune response to HIV-1 vaccines. This method shows promise for assessing vaccine efficacy and characterizing effector cells.
Area of Science:
- Immunology
- Vaccinology
- Molecular Biology
Background:
- Traditional CD8(+) cytolytic activity assays using 51Cr release are complex.
- Measuring intracellular interferon gamma (IFNγ) production via ELISPOT or flow cytometry offers an alternative for assessing CD8(+) T cell responses.
- Assessing immune responses to novel HIV-1 vaccine candidates is crucial for public health.
Purpose of the Study:
- To evaluate the utility of flow cytometry for measuring CD8(+) T cell-mediated IFNγ production in response to canarypox HIV-1 vaccines.
- To compare the sensitivity of flow cytometry with traditional methods for detecting cellular immune responses.
- To characterize the phenotype of effector cells involved in the vaccine-induced immune response.
Main Methods:
- Phase 1 clinical trial involving 19 volunteers receiving canarypox HIV-1 vaccines (vCP205, vCP1433, vCP1452).
- Peripheral blood mononuclear cells (PBMCs) were stimulated in vitro with vaccinia constructs encoding HIV-1 genes (env, gag) or a lacZ control.
- Intracellular IFNγ production in CD8(+) T cells was measured using flow cytometry after co-incubation with infected target cells.
Main Results:
- CD8(+) IFNγ activity was successfully measured in the majority of volunteers after four immunizations.
- Specific responses to the gag gene varied across vaccine groups, with higher frequencies observed in vCP1433 and vCP1452 recipients.
- Flow cytometry enabled simultaneous phenotyping of CD4 memory IFNγ production.
Conclusions:
- Flow cytometry is a viable and promising method for measuring CD8(+) T cell-specific IFNγ responses to HIV-1 vaccines.
- This technique facilitates the assessment of cellular immunity and the characterization of effector cell populations.
- The findings support the use of flow cytometry as a surrogate measure for cytotoxic T lymphocyte (CTL) activity in vaccine trials.
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 (IFNgamma) 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 IFNgamma cells in the CD3(+)CD8(+)CD45RO(+) gate after subtracting both the isotype control and the lacZ control stimulation. CD4 memory IFNgamma production was simultaneously determined in the CD3(+)CD8(-)CD45RO(+) gate. Using these techniques in blinded studies, we found that CD8(+) IFNgamma 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.
