[A flow cytometric assay for the expression of interferon gamma in T lymphocytes and its application in the study of EIAV-induced immune response].

This research paper presents a flow cytometry-based assay for detecting interferon gamma (IFN-gamma) expressed by T lymphocytes in horses infected with equine infectious anemia virus (EIAV). The study further demonstrates the specificity, stability, and reproducibility of this flow cytometry technique, making it a crucial tool for understanding the protective immunity induced by the EIAV vaccine.

Methodology

• In this study, the researchers prepared Peripheral blood mononuclear cells (PBMC) from horses that had been inoculated with either EIAV vaccine strain FDDV (fetal donkey dermal-attenuated virus vaccine), a virulent strain LV, or saline (as a healthy control group).
• The cells were then stimulated in vitro (in a controlled environment outside a living organism) with either FDDV or PMA + Inomycin. To block protein secretion, Brefeldin A was added to the cell culture.
• After stimulation, the cells were labeled with monoclonal antibodies that are specific for equine CD4+ and CD8+ surface markers, as well as an IFN-gamma-specific monoclonal antibody.
• Using flow cytometry, the researchers were able to detect CD4+ and CD8+ lymphocytes that were expressing IFN-gamma.

Results

• The results showed that the IFN-gamma positive cell population isolated from FDDV-immunized horses was 1.7 +/- 0.9% in CD4+ T cells and 6.1 +/- 1.2% in CD8+ T cells.
• In contrast, for the PBMC isolated from LV-infected horses, only 0.6 +/- 0.1% of IFN-gamma positive cells in the CD4+ subset and 2.4 +/- 0.9% of IFN-gamma positive cells in the CD8+ subset of T cells were detected.

Conclusion

• The research concluded that the multi-fluorescence cell flowmetry technique, which simultaneously detects both IFN-gamma expressing cells and different T lymphocyte subsets, is a specific, stable, and reproducible method.
• The authors emphasized that this technique is crucial for evaluating the antigen-specific response of IFN-gamma-producing lymphocytes and studying the protective immunity induced by the EIAV vaccine.