NEW APPROACH TO ANALYZING THE T LYMPHOCYTE CELL CYCLE USING FLOW CYTOMETRY

Abstract

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 determination of not just the fraction of dividing cells and the proliferation rate, but also the cell cycle phases, would substantially enhance our understanding of the causes and mechanisms behind impaired T lymphocyte division.

Aim. This study aimed to validate a novel approach for analyzing the cell cycle of T lymphocytes at different maturational stages via flow cytometry.

Materials and methods. The study utilized peripheral blood samples collected from healthy individuals. Peripheral blood mononuclear cells were isolated through density-gradient centrifugation, stimulated with phytohemagglutinin, and cultivated for 4 days under standard conditions (37^oC, 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. 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.

Results. The proposed technique successfully distinguished T lymphocytes in the 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, the numbers of CD4+ and CD8+ T lymphocytes progressing into active cell cycle phases were quantified, along with their phase-specific distributions. Differences in proliferation dynamics were also observed among naive CD4+ and CD8+ T lymphocytes, central memory, effector memory, and terminal effector subsets.

Conclusion. The proposed method enables comprehensive evaluation of the cell cycle progression in CD4+ and CD8+ T lymphocytes across distinct maturation stages through flow cytometry.