TY - JOUR
T1 - Three-color flow cytometry detection of virus-specific CD4+ and CD8+ T cells in the cat
AU - de Groot - Mijnes, J.D.F.
AU - van der Most, R.G.
AU - van Dun, J.M.
AU - te Lintelo, E.G.
AU - Schuurman, N.M.P.
AU - Egberink, H.F.
AU - de Groot, R.J.
PY - 2004
Y1 - 2004
N2 - We describe a three-color flow cytometry assay for the detection of virus-specific CD4+ and CD8+ T cells in the cat. The assay is based upon detection of intracellular TNFα using the cross-reactive mAb 6401.1111, raised against the human cytokine. Allophycocyanin-conjugated mAb 6401.1111 specifically stained feline TNFα-producing murine cells and also Staphylococcus aureus Enterotoxin B-stimulated feline T cells, thus providing formal evidence for cross-reactivity. By using the anti-TNFα mAb in combination with PE- and FITC-conjugated mAbs against feline CD4 and CD8, respectively, antiviral CD4+ and CD8+ T cells could be identified in the peripheral blood and in the spleens of feline infectious peritonitis virus-infected cats. Moreover, feline calicivirus (FCV)-specific CD4+ T cells were detected in the spleens of FCV-vaccinated cats. As antigen-presenting cells (APCs), we used immortalized autologous fibroblast cell lines, PBMC or splenocytes. A straightforward protocol, in which splenocyte preparations served both as APCs and effector cells, consistently yielded best results. The assay will permit further studies of the cellular immune responses in cats during natural and experimental viral infections. It will contribute to vaccine development against feline viruses by facilitating the identification of T cell antigens and epitopes, and by allowing the quantitative detection of virus-specific T cells after vaccination. Furthermore, the assay will add to the value of those systems in which viral infections of the cat serve as models for human disease.
AB - We describe a three-color flow cytometry assay for the detection of virus-specific CD4+ and CD8+ T cells in the cat. The assay is based upon detection of intracellular TNFα using the cross-reactive mAb 6401.1111, raised against the human cytokine. Allophycocyanin-conjugated mAb 6401.1111 specifically stained feline TNFα-producing murine cells and also Staphylococcus aureus Enterotoxin B-stimulated feline T cells, thus providing formal evidence for cross-reactivity. By using the anti-TNFα mAb in combination with PE- and FITC-conjugated mAbs against feline CD4 and CD8, respectively, antiviral CD4+ and CD8+ T cells could be identified in the peripheral blood and in the spleens of feline infectious peritonitis virus-infected cats. Moreover, feline calicivirus (FCV)-specific CD4+ T cells were detected in the spleens of FCV-vaccinated cats. As antigen-presenting cells (APCs), we used immortalized autologous fibroblast cell lines, PBMC or splenocytes. A straightforward protocol, in which splenocyte preparations served both as APCs and effector cells, consistently yielded best results. The assay will permit further studies of the cellular immune responses in cats during natural and experimental viral infections. It will contribute to vaccine development against feline viruses by facilitating the identification of T cell antigens and epitopes, and by allowing the quantitative detection of virus-specific T cells after vaccination. Furthermore, the assay will add to the value of those systems in which viral infections of the cat serve as models for human disease.
KW - Diergeneeskunde (DGNK)
U2 - 10.1016/j.jim.2003.10.019
DO - 10.1016/j.jim.2003.10.019
M3 - Article
SN - 0022-1759
VL - 285
SP - 41
EP - 54
JO - Journal of Immunological Methods
JF - Journal of Immunological Methods
ER -