However, an

unexpected advantage of the Ca2+ restoration was seen as the antigen-specific T cell proliferation was elevated especially in CD4+ T cells, but also in CD8α+ T cells. As both cell separation and wash in our analysis were carried out within 3–4 h after blood sampling, our findings are in good agreement with those of Bull et al. [17] who found that antigen-specific responses to HIV antigen even after cryopreservation was very sensitive to time after blood sampling but not to the type of anticoagulant. Chickens of the MHC haplotypes B13 and B130 (B21 like) were vaccinated against selleck inhibitor NDV. Forty-nine days after the first vaccination, the antigen-specific proliferation was measured. Within chickens of each haplotype, the percentage of proliferated CD8α+ T cells in unstimulated cells varied greatly, while this was not the case for CD4+ T cells. This contrasts the results shown in Fig. 3, as both B13 and B130 chickens in that case varied a lot in CD4+ T cell proliferation also. The discrepancy Selleckchem GS-1101 may be explained by the more uniform set-up in experiment 2. This confirms an NDV vaccine–induced cellular immune response as it was earlier demonstrated in chickens either cyclo-phosphamide-treated or bursectomized [4, 8, 29]. In those cases, it was also shown that the cellular response offered protection although the virus persisted for

a longer time in bursectomized and vaccinated chickens than was the case in control-vaccinated chickens. The fold increase in unstimulated to stimulated cells (SI) was calculated, and for the CD4+ T cells, the MHC difference was significant as the SI in B13 chickens was larger than that in the B130 chickens. The same tendency was seen for CD8α+ T cells although it was not significant. This

is in concordance with an earlier description of the B13 haplotype showing a larger mitogen-induced lymphocyte proliferation activity than the B21 haplotype [30]. In conclusion, we have established and optimized an assay for testing NDV-specific T cell Benzatropine proliferation in chickens upon vaccination. We concluded that it was an advantage to use EDTA as an anticoagulant if the CIS was used simultaneously as serum additive to the culture medium. Ca2+ restoration immediately after cell separation on Ficoll enhanced antigen-specific proliferation especially in CD4+ T cells. The levels of antigen-specific proliferation in chickens vaccinated at least 1 year prior to testing indicated a possible dependence on the MHC haplotype of the chicken. This was finally confirmed as two chicken lines that differed in MHC were subsequently compared. The authors acknowledge financial support from the Danish Poultry Council and from Aarhus University, Denmark. Hanne Svenstrup and Lene R. Dal are thanked for their technical assistance and Karin V. Østergaard for critical review of the manuscript.