| Aplastic anemia (AA) is a unique autoimmune disease which is believed to be resulted from aberrant activated T cell-mediated suppression of hematopoiesis. The abnormal activation of T cell might be associated with aberrant expression and function of costimulatory molecules. Objective: The effect and mechanism of PD-1 on the aberrant regulation of T cells in AA was studied. The significance of PD-1 in AA patients administrated with tetramethylpyrazine (TMP) and the effect of TMP on proliferation of T cells and production of cytokines in vitro were observed. Method: 1). Two mouse anti-human PD-1 molecule mAbs (1F2 and 5F10) recognizing two different sites were used. 1F2 was applied as coating mAb and 5F10, labeled with biotin, was used as detecting mAb. A human soluble PD-1 (sPD-1) ELISA kit was established. The concentration of sPD-1 in sera of healthy volunteers, hematonosis and transplant patients were detected. 2). Flow cytometry was applied to observe the expression of PD-1 on CD4+ and CD8+ T cells from peripheral blood and bone marrow from AA patients. The sPD-1 ELISA kit was used to valuate the concentration of sPD-1 in sera of AA patients. Meanwhile, we observed the effect of sPD-1 on proliferation of T cells and production of IL-2 and IFN-γin vitro. Real-time PCR was applied to detect the expression of different PD-1 splice variants. 3). Two cases of clinical AA patients administrated with TMP for one month were observed in the study. The changes of expression of PD-1, CD4, CD8, CD25 on T cells and the concentration of sPD-1 in sera were observed before and after the therapy separately. MTT method was used to observe the effect of TMP on the proliferation of T cells from healthy volunteers and AA patients in vitro; Flow cytometry was applied to detect the effect of TMP on the expression of CD25 and PD-1 on CD4~+ and CD8~+ T cells; ELISA kits were also used to evaluate the concentration of IL-2, IFN-γand sPD-1. Result: 1). A human sPD-1 ELISA kit was successfully established and the normal value of sPD-1 in sera from healthy volunteers was about 1.103±0.240ng/ml. The concentration of sPD-1 in sera of AA patients was significantly increased compared with controls. And there were no significant differences between the values of sPD-1 in sera of hyperthyroidism patients and transplant patients compared with controls. 2). Results from flow cytometry showed that the expression of PD-1 on CD4+ and CD8+ T cells from peripheral blood and bone marrow of AA patients was low,but ELISA result indicated that level of sPD-1 in sera of AA patients was increased; results from Real-time PCR suggested that sPD-1 might be the production of PD-1Δex3 transcript and in the function assay, the result indicated that sPD-1 could up-regulate the proliferation of T cells and stimulate the production of IL-2 and IFN-γ. 3). In two cases of clinical therapy, we found that TMP could up-regulate the expression of CD4, and down-regulate the expression of CD8 and CD25, as well as the concentration of sPD-1 in sera. TMP could suppress the proliferation and activation of T cells in healthy voluntary and AA patients, and reduce the production of IL-2 and IFN-γin vitro. It could also suppress the expression of CD25 on CD4+ and CD8+ T cells, up-regulate the expression of PD-1 on T cells from AA patients, and reduce the level of sPD-1, but it had little effect on expression of PD-1 on T cells in normal. Conclusion: The aberrant activation of T cells in AA might be associated with the abnormal expression and function of PD-1 molecule. The mechanism might be that the high level of sPD-1, which might be a production of PD-1Δex3 transcript in sera of AA patients, could serve as a PD-L specific auto-antibody to block the PD-1/PD-L pathway, and up-regulate the proliferation, activation and cytokine production of T cells, then enhance the toxicity of T cells and inhibiting factor of IFN-γon hematopoietic cells, and therefore result in AA. The mechanism of TMP in AA patients might be that TMP could suppress the over-activated T cells. Its suppression effect might be associated with reducing the production of IL-2 and IFN-γ, inhibiting the expression of CD25 (IL-2R), and decreasing the level of sPD-1 in sera of AA patients. This study provides an experimental basis for further understanding the reasons, the treatment and disease progression of AA. It also provides a theoretical basis that PD-1 might be a novel target of immune interference of AA. Besides, it suggests that TMP might be potentially used to treat AA. |
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