Part of the lymphocytes were activated and proliferated, and the expression of CD26 was upregulated after antigen stimulation
Three days after stimulation by immobilized anti-CD3 mAb, the activation of HPBLs was determined by the measurement of expression of different lymphocyte activation markers (CD69, CD25, CD71, and CD26). In comparison to non-activated control cells, the percentage of CD26+ HPBLs was significantly increased after stimulation by 85% (33±8% vs. 61±14% of total HPBLs,p <0.001) (Figure 1A) , while the percentages of CD69+ and CD71+ cells were 6-fold and 5-fold compared to control cells (54.29± 20.87% vs.9.07±7.28%, p <0.01; 30.6±14% vs. 5.8±2.46%,p <0.05), respectively, and the percentage of CD25+ HPBLs was 68% higher than the value in the control group (17.65±6.58% vs. 10.49±9.41%) (Figure 1B) . These results indicate that a substantial part of the HPBLs was activated after stimulation with immobilized anti-CD3 mAb.
To determine the proliferated new generations of lymphocytes after stimulation, the CFSE assay was used. As shown in Figure 1C andD , at day three after stimulation, the stimulated group (hollow black histogram) showed five additional peaks that represent five increased generations of HPBLs whereas the PBS control group (shaded red histogram) showed only one peak remaining in the original position, indicating that no new generation was generated. These results provide evidence that the lymphocytes proliferated and increased by up to five new generations after stimulation compared to the lymphocytes of the PBS control group that had not proliferated within three days.