Tehran University of Medical Sciences Iranian Journal of Allergy, Asthma and Immunology 1735-1502 20 3 2021 06 06 364 375 Mahdieh Motiee Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Ahmad Zavaran Hosseini Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Sara Soudi Department of Immunology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran Seyed Mehdi Hassanzadeh Vaccine Production Unit, Research and Production Complex, Pasteur Institute of Iran, Karaj, Iran 2020 11 29 2021 01 16 T-lymphocytes have critical functions in the immune responses against viral and intracellular bacterial infections as well as cancers. Antigen (Ag)-specific T-lymphocyte clones enriched and expanded in vitro are valuable tools in the study of immune responses in animal models and adoptive T-cell therapy of patients with cancer or infection. We described a method for inducing, enriching, and replicating Ag-specific poly-clonal T-cells from BALB/c mice infected with live Bacillus Calmette Guérin (BCG) bacterium. During a 7-8 days procedure, T-lymphocytes were purified from immune cells of lymph nodes stimulated with immunodominant Ag of BCG, TB10.4, and expanded by interleukin -2 cytokine. We evaluated the effect of Ag doses (1, 10, and 100 μg/mL) and exposure method of Ag presenting cells (APCs) to T-cells, on T-cells’ proliferation, viability, and Interferon-gamma (IFN-γ) secretion at 2, 5, and 7 days after Ag stimulation. Increasing Ag concentration increased the average cell division, but at the highest dose of Ag (100 μg/mL), T-cell viability is decreased. Only clones induced by 10 μg/mL Ag produced a desirable amount of IFN-γ. Incubation of Ag and APCs, 24 h before T-lymphocytes addition, increased the proliferation and viability of cells. T cells are in a more favorable condition around day 5 of Ag stimulation in terms of proliferation and survival, and it is the desired time for T cell restimulation. For optimal preparation of specific T-cells for adoptive cell transfer, optimization of Ag dose, the order of APCs and T-cells exposure with Ag, and the duration of initial Ag stimulation, as well as the time for restimulation, is essential. https://ijaai.tums.ac.ir/index.php/ijaai/article/view/3055 https://ijaai.tums.ac.ir/index.php/ijaai/article/download/3055/1707