Figure legends
Figure 1. The activation and proliferation of T lymphocyte after stimulation. After 72 h of the stimulation with immobilized anti-CD3 mAb, the activation of lymphocytes was determined by the measurement of the expression of T lymphocyte activation markers (CD69, CD71, CD25, CD26). The proliferation of lymphocytes was analysed by CFSE assay. PBS treated cells were used as controls. (A ) Percentages of CD26+-HPBLs in control group and stimulated group. (B ) Percentages of CD69+-, CD71+- and CD25+-HPBLs in control group and stimulated group. Data represented mean value ± SD from a minimum 5 independent experiments with at least 5 healthy donor HPBLs samples, and each experiment was repeated more than 3 times.P -values were calculated with a Chi-square test. (C ) Histogram of the proliferated generations of lymphocytes after stimulation by immobilized anti-CD3 mAb (anti-CD3) or only PBS as control (PBS) for three days. The shaded histogram represents the original generation (0g) of PBS control group at day 3. The hollow histogram indicates the increased 5 generations (1g, 2g, 3g, 4g and 5g) of the stimulated group three days after stimulation. (D ) The dot plots show the proliferated generations of lymphocytes analyzed by flow cytometry.
Figure 2. Percentages of CD4+ and CD8+ cells, and cells co-expressing each of these surface markers with CD26 after stimulation. (A ) Percentages of CD4+ and CD4+CD26+ cells in control group and stimulated group. (C ) Percentages of CD8+ and CD8+CD26+ cells in control group and stimulated group. Data represented mean value ± SD from five independent experiments with five healthy donor HPBLs samples, and each experiment was repeated more than three times. P -values were calculated with a Chi-square test. The dot plots show one typical experiment for the analysis of percentages of CD4+ and CD4+CD26+ cells (B ) and percentages of CD8+ and CD8+CD26+ cells (D ) by flow cytometry.
Figure 3. Separation of CD26low and CD26high lymphocytes by magnetic cell sorting (MACS).(A ) Procedure of separation of CD26low and CD26high lymphocytes by MACS. After stimulation for three days, lymphocytes were labeled with the mouse anti-human CD26 mAb (anti-CD26 mAb350 prepared in our own laboratory) for 1 h at 4°C. Following the two washing steps, magnetic MicroBeads labeled with anti-mouse IgG were added to the cells and incubated further for 15 min at 4°C. After a washing step, cells were loaded into the column which was pre-placed in the magnetic field of a suitable MACS Separator (Miltenyi Biotec, Germany). The unlabeled cells were collected after flow-through with two times wash processes. The labeled CD26+ cells were bound to column, and then flushed out after removing the column from the separator by help of a plunger. (B ) Analysis of CD26 expression in CD26 high-expressing (CD26high) group and CD26 low-expressing (CD26low) group by flow cytometry. Data represented mean value ± SD from a minimum five independent experiments with at least five healthy donor HPBLs samples. (C ) The dot plots show one typical experiment for analysis of CD26 expression.
Figure 4. Percentages of CD4+, CD8+, CD4+CD26+and CD8+CD26+ cells in CD26low and CD26high groups.(A ) Percentages of CD4+ and CD4+CD26+ cells in CD26low and CD26high group. (C ) Percentages of CD8+ and CD8+CD26+ cells in CD26low and CD26high group. Data represented mean value ± SD from seven independent experiments with seven healthy donor HPBLs samples, and each experiment was repeated more than three times. Dot plots show the percentages of CD4+, CD4+CD26+cells (B ), CD8+, and CD8+CD26+ cells (D ) in CD26low and CD26high group.
Figure 5. Percentage of cells secreting different cytokines in the CD26low and the CD26highgroups. After separation, the cells in the CD26lowgroup and the CD26high group were labeled with different monoclonal antibodies against-cytokines or surface markers at 4°C for 30 min and then measured by flow cytometry. (A ) The percentages of cells secreting Th1- and Th2-typical cytokines in CD26low and CD26high group. (B ) The percentages of cells secreting Th17-typical cytokines or expressing Th17-typical biomarkers in CD26low and CD26high group. (C ) Overlay histograms demonstrate the relative expression of cells secreting different cytokines in CD26low and CD26highgroups. The black line indicates the values of CD26lowgroup cells while the color lines indicated the values of CD26high group cells. (D ) The percentages of cells expressing Tregs-typical biomarkers in CD26lowand CD26high group. Data represented mean value ± SD from a minimum of five independent experiments with at least five healthy donor HPBLs samples, and each experiment was repeated more than three times.
Figure 6. Co-expression of CD26 with each of Th1- or Th17-typical cytokines or surface markers in the cells of CD26low and CD26high groups.Lymphocytes were harvested at 72 h after stimulation and were double-stained with the FITC-conjugated anti-CD26 mAb and PE-conjugated anti-IL-2, anti-IFN-γ, anti-IL-17, anti-IL-6, anti-IL-22, or anti-IL-23R mAb. (A ) Percentages of cells co-expressing CD26 with each of Th1-typical cytokines (IL-2, or IFN-γ), Th17-typical cytokines (IL-6, IL-17, IL-22) or Th17-typical surface marker (IL-23R) in the CD26low and CD26high groups. Data represented mean value ± SD from a minimum five independent experiments with at least five healthy donor HPBLs samples, and each experiment was repeated more than three times. (B ) Co-expression of CD26 with Th1- or Th17-typical biomarkers was observed by fluorescence microscopy. Images were made at 600× magnifications. Co-expressing of CD26 with IL-2, IFN-γ, IL-17, IL-22, or IL-23R in some lymphocytes indicated by the merged images.