Novel approach to cell cycle analysis of T lymphocytes using flow cytometry

Intensive proliferation of T lymphocytes is essential for the development of both natural and vaccine-induced immunity. As a result, impaired T cell proliferation could influence disease susceptibility and vaccination effectiveness. A methodology enabling definition of dividing cells fraction, proliferation rate, as well as the cell cycle phases, would substantially enhance our understanding of the causes and mechanisms inderlying impaired T lymphocyte division. The aim of our study was to validate a novel approach for analyzing cell cycle of T lymphocytes at different maturation stages via flow cytometry. The study was performed with peripheral blood samples collected from healthy individuals. Peripheral blood mononuclear cells were isolated by density-gradient centrifugation, stimulated with phytohemagglutinin, and cultivated for 4 days under standard conditions (37 °C, 5% CO2). Activated leukocytes were labeled with the LIVE/DEAD Fixable Violet Dead Cell Stain and antibodies specific for CD3 BV605, CD4 PE, CD8 BV510, CCR7 PE/Cy7, and CD45RO APC-eF780. The fixed and permeabilized cells were further stained with antibodies targeting pRb AF488 and pHH3 AF647, along with the DAPI nuclear stain. Flow cytometric analysis was conducted using a CytoFLEX S instrument. The proposed technique allowed successful distinction between T lymphocytes in G0, G1, S, G2, and M phases of the cell cycle. Moreover, the designed fluorescence panel permits concurrent detection of CD4+ and CD8+T cells at diverse maturation states. During the pilot study, we have quantified the numbers of CD4+ and CD8+T lymphocytes progressing into active cell cycle phases, along with their phase-specific distributions. The differences in proliferation dynamics were also observed for naive CD4+ and CD8+T lymphocytes, central memory, effector memory, and terminal effector subsets. The proposed method enables comprehensive evaluation of the cell cycle progression in CD4+ and CD8+T lymphocytes across distinct maturation stages by means of flow cytometry.