Discussion
CD26 was determined as one of co-stimulator for T cell activation [18], and the co-stimulatory effect of CD26 for T cell activation could be mediated by the interaction of CD26 with the ecto-adenosine deaminase (ADA), tyrosine phosphatase CD45, CARMA1 or caveolin-1 [4, 19]. In the present work, antigens of lymphocytes were stimulated by using an immobilized anti-CD3 mAb (OKT3, IgG2a) to further investigate the role of CD26 in T cell differentiation [20]. Three days after stimulation, the activation of lymphocytes was determined by the enhanced expression of lymphocyte activation markers CD69, CD71, CD25 or CD26 (Figure 1A and B). CD69 is one of the earliest cell surface antigens expressed by T cells following activation. It acts as a co-stimulatory molecular and surface marker for T cell activation and proliferation [21]. CD71 (the transferrin receptor) and CD25 (the IL-2 receptor) are other two molecular surface markers of T cell activation and proliferation [22]. The significant increase in the expression of CD69, CD71, and CD25 indicates that most of the lymphocytes are activated after stimulation. In addition, CD26 expression was also significantly upregulated after stimulation(Figure 1A) suggesting an association of CD26 to the activation of T lymphocytes, which is consistent with previous studies [23]. After stimulation, the co-expression level of CD26 with CD4+ or CD8+ was increased markedly(Figure 2) , which indicates that the expression of CD26 is related not only to the activation of CD4+ cells, but also to a certain extent, the activation of CD8 cells. A previous study reported that a unique pattern of CD26-high expression was identified on influenza-specific CD8+ T cells but not on CD8+ T cells specific for cytomegalovirus, Epstein Barr virus or HIV, which suggested that high CD26-expression may be a characteristic of long-term memory cells [24]. A later study indicated that CD26+CD8+ cells belong to the early effector memory T cell subsets. The CD26 mediated co-stimulation of CD8+ cells provokes effector function via granzyme B, tumor necrosis factor-α, IFN-γ and Fas ligand [25]. The role of CD26 in the differentiation and function of CD8+ cells needs further investigation.
Thereafter the proliferation of lymphocytes was analyzed after stimulation. It was found that in comparison to lymphocytes without stimulation (PBS control), which did not proliferate, the antigen stimulated lymphocytes proliferated up to five generations(Figure 1C, D) . Further analysis showed that after stimulation, the percentage of CD4+ cells in total HBLs was increased significantly while the percentages of CD8+did not change (Figure 2) . The up-regulated percentage of CD4+ cells suggests that the immobilized anti-CD3 mAb triggered mainly the proliferation of CD4+ lymphocytes [26]. It was found that the percentage of CD4+cells in the CD26high group was significantly higher than in the CD26low group, and most of the CD4 cells were co-expressed with CD26 (Figure 4A, B) . Previously, Ohnuma et al. have reported that CD26 was thought to be mostly expressed by memory T helper cells, and its expression was preferential on CD4+ cells and associated with T cell activation as a costimulatory molecule [4, 5]. Blockade of CD26-mediated T cell co-stimulation with soluble caveolin-1 induced anergy in CD4+ cells [27]. Besides studies on the involvement of CD26 in the activation and proliferation of CD4+ T cells in vitro , in vivoinvestigation using CD26 knockout mice presented a decreased percentage of CD4+ cells [10]. CD4+ cells are T helper cells and they can secret different cytokines upon T cell activation and these cytokines play a crucial role in the activation and/or proliferation of other effector cells, such as B cells, cytotoxic T cells and macrophages. The higher percentage of CD4+cells in CD26high group and CD26 high expression in activated CD4+ cells observed in the present work further confirms that CD26 expression is involved not only in the activation, but also in the proliferation and in further bioprocesses and functions of CD4+ cells.
After activation, CD4+ cells proliferate and differentiate into different subpopulations. Th1 and Th2 are the two main and earliest defined subpopulations of T helper cells [28]. Th1 cells can potentially produce large amounts of IFN-γ and IL-2 cytokines while Th2 effector cells are characterized by the production of IL-4 and IL-13 [29]. In present work, after cell sorting of CD26-expressing cells, the percentages of cells secreting each of Th1-typical cytokines IFN-γ and IL-2 in the CD26high group were significantly higher than in the CD26low group(Figure 5A, C) . Moreover, most of cells secreting IFN-γ or IL-2 were co-expressing CD26 (Figure 6) . In a previous study, the up-regulation of CD26 expression on CD4+ cell surfaces was identified to be related to the production of Th1 cytokines [4]. It was reported that the solid-phase immobilized anti-CD26 mAb had a comitogenic effect by inducing CD4+ lymphocytes proliferation and enhancing IL-2 production in conjunction with submitogenic doses of anti-CD3 [30]. The inhibitor of DPPIV/CD26 enzyme activity has been suggested to be able to reduce the production of IL-2, IL-6 and IFN-γ of human and mouse T cells under mitogen stimulation [8]. Supporting these findings, the results of the present work showed that the expression of CD26 is associated with the differentiation of Th1 cells. Th1 is an important subset of T helper cells. The positive relation between the activation of CD4+ cells and CD26 expression (Figure 4A, B)benefits the differentiation of CD4+ cells into a Th1 subset.
Interestingly, the percentages of cells secreting Th2-typical cytokines IL-4 or IL-13 were not only very low (<5%) in CD26low and CD26high groups, but they also didn´t present any difference between the both kinds of cell groups (Figure 5A) . As one of the main subpopulations of T helper cells, Th2 subset is often recognized as an opposite of Th1 cells since Th2 cytokines may suppress the activity and proliferation of Th1 cells during immune responses [31]. Our results indicate that CD26 expression is not related to the differentiation of CD4+ cells into the Th2 subset after antigen stimulation.
Besides Th1 and Th2 subsets, Th17 and Tregs are other two important subsets of T helper subpopulations. Th17 is a more recently identified subset of CD4+ cells [11], which is distinct from classic Th1 and Th2 subsets [32]. These cells originate from naive CD4+ precursor cells mainly in the presence of TGF-β and IL-6, and their differentiation requires IL-23 [32]. As a novel member of CD4+ T subset, it is important to clarify the role of CD26 in the differentiation and function of Th17 cells. After cell sorting, the percentage of cells secreting Th17 typical cytokines (IL-17 and IL-22), or expressing Th17 molecular markers (IL-23R, CD161, CD196) were found to be significantly higher in the CD26high group than in the CD26lowgroup (Figure 5B) . Moreover, most of cells secreting IL-17 and IL-22 or expressing IL-23R, CD161 and CD196 were co-expressed with CD26(Figure 6) . This indicates an involvement of CD26 in the differentiation of CD4+ cells into Th17 subset. A previous study showed that Th17 cells express a high level of CD26, and the phenotypic analysis of Th17 cells could be identified by the CD26 expression [15]. Th17 cells play an important role in preventing the pathogen invasion through secreting pro-inflammatory cytokines. Clinical research found that CD26 was related to some diseases which involved the immune response initiated by Th17 cells through inducing chronic inflammation or autoimmunity, like rheumatoid arthritis and multiple sclerosis [33].
Recently, it has been reported that inhibition of the enzyme activity of CD26 by sitagliptin reduced the proliferation and Th1/Th17 differentiation of human lymphocytesin vitro [34], and the deficiency of CD26 or the inhibition of DPPIV enzyme activity induced the reduction of IL-17 expression and increased the allograft acceptance in vivo [35, 36]. We have also shown recently that CD26-deficiency resulted in a delayed allogeneic skin graft rejection after allogeneic skin transplantation. The concentrations of serum IgG, including its subclasses IgG1 and IgG2a, were significantly reduced in CD26–/– mice during graft rejection. The secretion levels of the cytokines IFN-γ, IL-2, IL-6, IL-4, and IL-13 were significantly reduced whereas the level of the cytokine IL-10 was increased in the serum of CD26–/– mice compared to CD26+/+mice. Additionally, the concentration of IL-17 in serum and the percentage of cells secreting IL-17 in mouse peripheral blood lymphocytes (MPBLs) were both significantly lower while the percentage of regulatory T cells (Tregs) was significantly higher in MPBLs of CD26–/– mice than in those of CD26+/+ mice [17]. In line with the results of these in vivo experiments, the results of the present in vitro study confirm that the expression of CD26 is not only highly correlated to the differentiation of Th1 and Th7, but also plays an important role in the differentiation and function of Th1 and Th7. It is precisely because CD26 plays an indispensable role in the differentiation and function of Th1- and Th17-lymphocytes, which results in a lack of effective Th1 and Th17 cells when CD26 is absent under relevant pathological conditions. The present study provides more insight into the role of CD26 for the function of Th17 cells and related diseases and will support future research in this field.
It is reported that CD26 can be used as a negative selection marker for Tregs [37]. In the present study, the percentages of Tregs were very low in CD26high and CD26low groups, and no significant difference was found between the two groups(Figure 5D) , indicating that the expression of CD26 is not necessary for the differentiation of Tregs after immobilized anti-CD3 mAb stimulation.
In conclusion, CD26 is not only an activation marker for T lymphocytes, but its expression is closely related to the subsequent proliferation, differentiation, and functions of T lymphocytes. Considering that the balance between Th1 and Th2 and the balance between Th17 and Tregs play a prominent role in immune responses, our results in this study demonstrated that the high expression of CD26 is beneficial to the differentiation of T lymphocytes into Th1 and Th17 subpopulations after antigen stimulation, indicating a crucial role of CD26 in regulating the immune response to the inflammation and autoimmune reactions. The correlation of CD26 with the differentiation balance between Th1 and Th2 and between Th17 and Tregs observed in this study provides more insights into the role of CD26 in related diseases. The important role of CD26 in immune regulation suggests that it would become a therapeutic target for related diseases [38] .