PBMC kept in growth medium were used to assess the background proliferation, while induction of the antigen-specific proliferation
see more was carried out by adding 1 or 1.5 doses of processed NDV antigen to PBMC. Figure 2 shows the effect of substituting heparin with EDTA and FBS with CIS on the proliferative capacity of CD4+ and CD8α+ T cells. In general, substitution of heparin with EDTA alone had no effect on unspecific proliferation. Substitution of FBS with CIS alone reduced unspecific proliferation in CD4+ cells, but at the same time the antigen-specific proliferation was also reduced considerably. The greatest effect was seen when both substitutions were made in that unspecific proliferation was reasonably low in both CD4+ and CD8α+ T cells while still maintaining a high antigen-specific proliferation. Using the EDTA/CIS combination, BI 6727 nmr the ability of NDV-vaccinated chickens of four different MHC haplotypes (B12, B13, B130 and B201) to perform antigen-specific T cell proliferation was measured.
Figure 3 clearly shows that large variations in recall proliferation exist not only between MHC haplotypes but also between individuals with identical MHC haplotype. CD4+ and CD8α+ T cells from B130 chickens respond intermediately or well to recall stimulation with NDV antigen. CD4+ and CD8α+ T cells from B12 chickens on the contrary respond very poorly. Interestingly, it seems that CD4+ cells from B13 chickens respond well whereas CD8α+ cells from the same chickens respond poorly, and the opposite is seen for the B201 chickens. During the assessment of the proliferative capacity in the NDV-vaccinated chickens of different MHC haplotypes in experiment 1, it was noticed that
CD8α+ T cells were undetectable in some chickens independent of the MHC haplotype. We realized that a known polymorphism in the CD8α gene probably existed in some of the chickens tested , and so the chickens with poorly detectable CD8α T cells were excluded from the data shown in Fig. 2. As a consequence, we decided to test three different Buspirone HCl monoclonal antibodies for the detection of CD8α+ T cells. As seen in Table 1, the CT8 antibody normally used failed to detect CD8α+ T cells in 8 out of 20 cases, and the EP72 antibody in 9 out of 20 cases. The 3-298 antibody, however, was capable of detecting the CD8α+ T cells in all cases. Examples of detection patterns are given in Fig. 4 with cells from three different chickens gated through a small lymphocyte gate on the FSC–SSC dot plot. As shown, the CT8 antibody is able to detect CD8α+ T cells in chicken nos. 2 and 13, and EP72 is able to detect the CD8α+ T cells in chicken no. 3 and partly in no. 1. Compared with these two, the 3-298 antibody was shown to be superior, in that it was able to detect CD8α+ T cells distinctly in all cases (Fig. 4 and Table 1).