the effect of antigen dose and antigen presenting process on t cell stimulation: a method for enrichment of tb10.4 antigen-specific t-cell clones

T-lymphocytes have critical functions in the immune responses against viral and intracellularbacterial infections as well as cancers. antigen (ag)-specific t-lymphocyte clones enriched andexpanded in vitro are valuable tools in the study of immune responses in animal models andadoptive t-cell therapy of patients with cancer or infection.we described a method for inducing, enriching, and replicating ag-specific poly-clonal t-cellsfrom balb/c mice infected with live bacillus calmette guérin (bcg) bacterium. during a 7-8 daysprocedure, t-lymphocytes were purified from immune cells of lymph nodes stimulated withimmunodominant ag of bcg, tb10.4, and expanded by interleukin -2 cytokine. we evaluatedthe 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 at2, 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 adesirable 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 aroundday 5 of ag stimulation in terms of proliferation and survival, and it is the desired time for t cellrestimulation.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.