Application Of Live Cell Dye CFSE In Study On Proliferation And Cytokine Secretion Of Human γδT Cells

Objectives: 1. To develop the methodology combining live cell dye ,CFDA-SE(CFSE) labeling and flow cytometry to characterize lymphocyte proliferation. 2. To study the proliferation kinetics of human T lymphocyte subpopulations in response to different stimuli (including the Mtb-Ag) with live cell dye, CFSE labeling and flow cytometry. 3. After stimulated with Mtb-Ag, to identify the relationship between the secretion of IL-2 and IFN-γ by y8T cells and the proliferation kinetics.Methods: 1. PBMC isolated from normal subjects were labeled with CFSE, and then stimulated with PHA, CD3mAb, or Mtb-Ag, followed cultivation in IL-2 containing medium for 5 to 14 days. The phenotypes of proliferated lymphocytes were determined by staining with fluorescent labeled mAb to CD molecules or TCR by flow cytometry. The dynamics of proliferation of activated T cell subpopulations were analyzed with the ModFit software.2.PBMC stimulated with Mycobacterium tuberculosis antigen (Mtb-Ag) for four days,then labeled with CFSE and cultivated for anthother 72 hours under the conditions of 5%CO₂,37℃ . The obtained sample was futher stimulated with PMA, Ionomycin, and the secretion from interior to exterior of cytokine was inhibited by monesin. The secretionof IL-2and IFN-γ was monitored by flow cytometry.Results: 1. PHA and CD3mAb mainly activate the total T cells. After activation for five days, the population of CD₃+T cells is over 90% in PBMC. CD₄+T and CD₈+T cells exhibit apparent differentiation, and CD₈+T has greater reproduction than CD₄+T cells. Based on the analysis by ModFit software, the first four generations of CD₄+T outweigh CD₈+T cells, while the fifth and sixth generations have significantly lower populations than CD₈+T cells. 2.Mtb-Ag mostly promotes the proliferation of γδT cells. After...