Immunosuppression induced by apoptosis of mixed lymphocyte culture is associated with p53

Abstract

A lymphocyte inhibition model was created using a co-culture of donor and host lymphocytes, resulting in apoptosis of the latter, and subsequently, inducing immune tolerance. This method may be used to resolve the immune rejection problem prior to organ transplantation. Using mixed lymphocyte culture (MLC) and/or addition of anti-anti-IL-2 neutralizing monoclonal antibody, we successfully developed a lymphocyte inhibition model in vitro. In this model, the apoptosis of recipient lymphocytes co-cultured with donor lymphocytes was observed by Wright-Giemsa stain, electron microscopy imaging and flow cytometry. The growth and proliferation of mixed lymphocytes were detected by XTT and BrdU assays. Cell viability was determined using CCK-8 cell viability assay. The activity of the recipient lymphocytes was very high when stimulated by antigens alone [PMLC+D2 (Bm) group] but markedly lowered by anti-anti-IL-2 neutralizing monoclonal antibody [PMLC+D2 (Bm)+anti‑IL-2 group]. The suppression of recipient lymphocyte activity was due to apoptosis mediated by p53 and caspase-3, and the optimal ratio of donor and recipient lymphocytes for apoptosis was explored. With the exception of the control group, the ratio of apoptotic cells was highest in the PMLC+D2 (Bm)+anti-IL-2 group and lowest in the PMLC+D2 (Bm) group. Blockade of IL-2 with anti-IL-2 neutralizing antibody resulted in an increased number of apoptotic lymphocytes in our experiment, which suggested that IL-2 inhibits the apoptosis of lymphocytes. These data suggest that IL-2 is involved in MLC-induced apoptosis of recipient lymphocytes, and that apoptosis may be associated with p53 and caspase-3 pathways.