Effects Of ALA-PDT On GVHD And GVL Following Allo-BMT In Mice

Allogenic bone marrow transplantation (allo-BMT) has been an effective and available therapy in leukemia patients, however, graft-versus-host disease (GVHD), induced by the reaction of donor T cells to recipient histoincompatible antigens (HLA), is a serious complication of allo-BMT, resulting in considerable morbidity and mortality. GVHD occurs when transplanted donor-derived T cells recognize and react to histoincompatible recipient antigens and cells. Final consequences of the GVHD process are a wide variety of host tissue injuries in varying degrees of clinical severity. Depleting such reactive T lymphocytes has been a strategy to prevent GVHD following allo-BMT in acute lymphoblastic leukemia (ALL) patients. Nevertheless, depleting the T lymphocytes in the graft thoroughly increases the risk of tumor relapse; therefore, recent studies have been focusing on selectively depleting alloreactive T cells from allograft.Photodynamic therapy (PDT) is a newly emerging biological therapy for tumor, which has been widely used in solid tumor and dermatonosis but seldom practiced in hematologic diseases, especially in patients following bone marrow transplantation (BMT). The anti-tumor effects of PDT are based on higher affinity of photosensitizer and tumor tissues. When exerted specified wavelenghth of light, a series of photochemical reaction will occur and produces singlet oxygen and the other reactive oxygen species (ROS), which will induce tumor cells death in an apoptosis or necrosis manner.Alloreactive T lymphocytes in allograft that causes GVHD can aggregate photosensitizer like tumor cells, thus PDT can effectively deplete such T cells and increases achievement ratio of allo-BMT. However, another kind of T lymphocytes in allograft which induce graft-versus-leukemia (GVL) does not induce GVHD. Therefore, preserving these effective T cells will preserve GVL in allo-BMT patients in theory.Present study predisposed allograft with aminolevulinic acid (ALA) and aimed to clarify the beneficial effects of ALA-PDT on GVHD in allo-BMT mice. The major results are the following:1). Identified the optimal concentration of rhIL-2 and PHA in vitro Dissected the mouse spleen sterily and isolated lymphocytes using Ficoll-Hypaque density gradient centrifugation. Added rhIL-2 and PHA to the lymphocytes in culture for 5 days and MTT was used to measure OD value. The optimal concentration of PHA and rhIL-2 were 40μg/ml and 25 U/ml respectively.2). Identified the optimal concentration of mitomycin in vitro Added mitomycin to the lymphocytes of recipients to reach the final concentration (μg/ml) of 10, 25, 30, 40, 50 in culture for 5 days and MTT was used to measure OD value. The optimal concentration of mitomycin was 25μg/ml.3). Prepared one-way mixed lymphocytes and identified mixed cells ratio Mixed the recipient lymphocytes and donor lymphocytes according to 2:1, 1:1, 1:2 in culture. MTT was used to measure and identify optimal mixed cells ratio. The 2:1 ratio was the optimal concentration of mixed lymphocytes culture (MLC).4). Identified the optimal ALA concentration of ALA-PDT selectively depleting allogenic lymphocytes Added 1.5, 2.0, 2.5 mmol/L ALA to one-way mixed lymphocytes for 4 h and exerted specified wavelength light. Washed the cells and then co-cultured with recipient cells. MTT was used to identify the effects of one-way mixed lymphocytes on recipient cells. The lowest kill rate occured at 1.5 mmol/L. 5). ALA-PDT alleviated aGVHD in mice following allo-BMT by depleting activated T lymphocytes One-way mixed lymphocytes predisposed with ALA-PDT and 3×107/ml marrow cells from the donors (0.5 ml in volume) was infused into the recipients suffered from 8.5 Gy 60Co irridiation. The results shown that the general status, peripheral WBC and GVHD were better than all the other groups.6). ALA-PDT alleviated GVHD following allo-BMT and preserved GVL in ALL mice One-way mixed lymphocytes predisposed with ALA-PDT and marrow cells from the donors were infused into ALL mice. The body weight increase, haematogenesis recovery and survival time were better then all the other groups.In conclusion, our study demonstrated that ALA-PDT could selectively deplete allogenic T lymphocytes in allograft. Mixed lymphocytes predisposed by ALA-PDT plus marrow cells from donors transplantation could alleviate GVHD following allo-BMT and increased the survival rate. Therefore, ALA-PDT maybe a promising therapy for GVHD and preserve GVL following allo-BMT in mice.