DYNAMICS OF PROLIFERATIVE RESPONSE AND SUBSET COMPOSITION OF CD4+ T LYMPHOCYTES DURING SHORT-TERM AND LONG-TERM CULTIVATION WITH PHYTOHEMAGGLUTININ

Assessment of the proliferative activity of CD4+ T lymphocytes is an important tool for characterizing the functional status of adaptive immunity. The use of flow cytometry with the CFSE dye allows for the tracking of cell division kinetics. However, data on the heterogeneity of the proliferative response to T-cell mitogens, such as phytohemagglutinin (PHA), remain limited. In particular, the phenomenon of ultra-fast division of a small fraction of stimulated T cells in vitro is poorly understood. The aim of this study was to investigate the dynamics of the proliferative response and to analyze changes in the subset composition (naive, central memory, and effector memory cells) of CD4+ T lymphocytes during short-term (1–3 days) and longer-term (6 days) cultivation with PHA in vitro. Peripheral blood mononuclear cells from relatively healthy volunteers were stained with CFSE and cultured in the presence of PHA (15 µg/ml) for 1, 3, and 6 days. Analysis of proliferation and the subset composition of CD4+ T cells (CCR7+CD45RO– – naive; CCR7+CD45RO+ – central memory T cells; CCR7–CD45RO+ – effector memory T cells) was performed using multicolor flow cytometry. Cells that had completed more than five mitotic cycles (CFSE-negative) were examined in detail. It was found that after 3 days of culture with PHA, the relative number of CD4+ T cells that had entered division was 68.8%. Of these, 21.4% belonged to a subset that had completely lost the CFSE label, i.e., had completed more than five mitotic cycles. Analysis of the CFSE-negative fraction on day 3 showed that the composition of rapidly proliferating cells was diverse (naive – 20.8%, central memory T cells – 67.9%, effector memory T cells – 10.7%). By day 6, 84.5% of CD4+ T cells had completed one or more division cycles. The proportion of extensively divided lymphocytes was 34.3%. In contrast to the CFSE-negative cells formed during short-term culture, by day 6 the fraction of extensively divided cells consisted almost exclusively of memory cells (central memory T cells – 72.8%, effector memory T cells – 25.1%). This indicates either phenotypic conversion during division or a selective advantage of memory cells upon prolonged stimulation. Thus, it was demonstrated that short-term cultivation with PHA reveals a fraction of CD4+ T lymphocytes with ultra-fast proliferation that is not associated with the dominance of any single subset. Ignoring the phenomenon of ultra-fast division of a subset of cells in short-term experiments may lead to skewed conclusions regarding T-cell functional activity.