Establishment of a method for culturing cynomolgus T lymphocytes induced by human CD3Ab / 中国肿瘤生物治疗杂志

ABSTRACT

@#[Abstract] Objective: To establish a method for in vitro isolation and culture of T lymphocytes from peripheral blood of cynomolgus monkeys that induced by human CD3 antibody based on the foundation of protein homology of CD3 from human, cynomolgus monkey and porcine. Methods: The amino acid sequences of human, cynomolgus monkeys and porcine CD3 proteins were obtained from NCBI, and the sequence, homology and phylogenetic tree were analyzed by DNAMAN software. Western blotting was used to detect the expression of CD3 protein on T cell membranes from the three species. PBMCs of healthy cynomolgus were isolated and divided into three groups group A was stimulated with anti-human CD3Ab alone, group B was stimulated with IL-2 alone, and group C was costimulated with human CD3Ab and IL-2. Cell morphology and growth status were observed under inverted microscope and the cell growth curve was plotted. Cell viability was detected by trypan blue staining and the expressions of CD3, CD4 and CD8 on T cell surface were detected by flow cytometry. Results: The homology of the amino acid sequence of human CD3 protein to cynomolgus monkey and porcine were 86.9% and 65.6% respectively. The expression levels of CD3 protein on cynomolgus and porcine T cell membrane were 79% and 17% contrast to human, respectively. Cells of group A did not proliferate. Proliferation, viability and CD3 expression [(93.8±3.6)% vs (70.3±4.7)%, P<0.01] in T cells of group C were significantly higher than those in group B. Growth curve of T cells in group C showed an S-shape, which is consistent with Logistic growth curve. T cells in group C exhibited high purity and expressed high level CD3; moreover, the CD8+T cell took a high proportion. Conclusion: The membrane of T lymphocytes from peripheral blood of cynomolgus can express CD3 protein that highly homological to human. Co-stimulation of human CD3Ab, IL-2 and 1% PHAcan induce the proliferation and differentiation of T lymphocytes of cynomolgus, and obtain T lymphocytes with good growth status, high proliferation ability and high purity.