English abstract
Measurement of lymphocyte functions is important clinically to assess the status of immunity and to evaluate the successfulness of immuno-therapies. These functions are usually assessed in vitro by measuring lymphocyte responsiveness to mitogens or antigens. The common measurements of activated lymphocytes are assessment of cellular proliferation, cytokine production and expression of membrane activation markers. Several methods have been developed and employed for such purposes due to their clinical relevance to many immunodeficiencies. The aim of the study was to utilize flow cytometry and Enzyme-linked immunosorbent assay (ELISA) in assessing T cell functions through expression of activation markers and cytokine production, respectively. Common activation markers; CD69, HLA-DR, and CD25 were analyzed in unstimulated and stimulated lymphocytes from ten healthy individuals (5 males and 5 females) after PHA-stimulation at different time intervals (0, 4, 24, and 48 hours). Production of IL-12 and IFN-y were assessed using ELISA in stimulated lymphocytes for 15 healthy individuals (7 males and 8 females) after 24 hours of stimulation. Flow cytometric analysis showed a general increase in the expression of the studied markers on CD3+ T cells after 24 hours of stimulation. Data showed a peak expression of CD69 after 24 hours; 44.25 + 20.86% in males, and 44.10 + 31.33% in females. Expression of HLA-DR and CD25 was higher after 48 hours; 17.63 + 10.79% HAL-DR, and 33.47 +16.01% CD25 in males. In females, the percentages were 16.04 + 11.83% HLA-DR, and 46.21 + 26.75% CD25. ELISA measurements showed a significant increase (P<0.05) in the production of IL-12 in males and females, but no significant increase in the production of IFN-y. Both flow cytometry and ELISA techniques showed no differences between males and females in the production of cytokines and the levels of activation marker expression. In conclusions, flow cytometry is an accurate and sensitive tool that allows the study of several activation responses in lymphocyte subsets. ELISA is a rapid and easy assay that provides measurement of total cytokine secretion in the cellular supernatant. Such methods for total secretion of cytokines do not allow for a direct correlation between cytokine producing T cell and surface marker expression. However, further studies may be needed and including higher sample size.
