Fig. 1

CD4⁺ T-cell proliferative responses to 23 peptide pools. PBMCs were obtained from five BLV-infected cattle (S2, S4, S6, R1, and N1). CD4⁺ T-cells were then isolated and used as effector cells. PBMCs were pre-treated with mitomycin C (4 × 10⁵/50 μl; 50 μg/ml) for 1 h at 37 °C and then co-incubated with CD4⁺ T-cells (1 × 10⁵/50 μl) and different peptide pools (each pool contained five different peptides, each at 20 μM) for 113 h at 37 °C. Cell Counting Kit-8 was used to measure CD4⁺ T-cell proliferation. The absorbance of the test wells was compared with that of control wells that did not contain peptides. The Stimulation Index (SI) was calculated as follows: \( \mathrm{Stimulation}\ \mathrm{Index}\ \left(\mathrm{SI}\right)=\frac{\left[\mathrm{PBMC},\mathrm{CD}4,\mathrm{peptide}\right]-\left[\mathrm{Medium}\ \mathrm{only}\right]}{\left[\mathrm{PBMC},\mathrm{CD}4,\mathrm{DMSO}\right]-\left[\mathrm{Medium}\ \mathrm{only}\right]} \). The bars represent the mean ± standard deviation (SD) of triplicate wells. Asterisk and shade box bar mean the pool showed significantly higher value than DMSO (negative control) well (p < 0.01)