An updated view into the cell cycle kinetics of human T lymphocytes and the impact of irradiation

Abstract

Even though a detailed understanding of the proliferative characteristics of T lymphocytes is imperative in many research fields, prior studies have never reached a consensus on these characteristics, and on the corresponding cell cycle kinetics specifically. In this study, the general proliferative response of human T lymphocytes to phytohaemagglutinin (PHA) stimulation was characterized using a carboxyfluorescein succinimidyl ester-based flow cytometric assay. We were able to determine when PHA-stimulated T lymphocytes complete their first division, the proportion of cells that initiate proliferation, the subsequent division rate of the cells, and the impact of irradiation on these proliferative properties. Next, we accurately visualized the cell cycle progression of dividing T lymphocytes cultured in whole blood using an adapted 5-ethynyl-2'-deoxyuridine pulse-chase method. Furthermore, through multiple downstream analysis methods, we were able to make an estimation of the corresponding cell cycle kinetics. We also visualized the impact of X-rays on the progression of the cells through the cell cycle. Our results showed dose-dependent G2 arrest after exposure to irradiation, and a corresponding delay in G1 phase-entry of the cells. In conclusion, utilizing various flow cytometric assays, we provided valuable information on T lymphocyte proliferation characteristics starting from first division to fully dividing cells.

Figure 1

Schematic overview of the EdU pulse-chasing method for tracking T lymphocytes cultured in whole blood. (1) Whole blood is cultured and lymphocyte cell division is stimulated using PHA. (2) After 96 h of culture, the proliferating cells are pulse-labeled with 10 µM EdU for 30 min; and (3) during pulse-labeling, the cells are irradiated in vitro with 2 or 4 Gy of X-rays. (4) EdU pulse-chasing is performed by culturing the cells from 0 up to 25 h and chasing them over time. (5) Following red blood cell lysis, the lymphocytes are fixed and then stained using the Click-iT EdU imaging kit. (6) The samples are measured using flow cytometry and analyzed using FlowJo software.

Figure 2

Proliferation analysis of CFSE-labeled T lymphocytes after PHA stimulus. T lymphocytes were exposed to 0, 1, and 2 Gy of 220 kV X-rays and cell proliferation was examined at 0, 24, 48, 72, and 96 h. (a) Representative example of CFSE fluorescence profiles of live non-irradiated T lymphocytes. The open black histograms show stimulated CFSE-labeled cells. The solid grey histograms show unstimulated CFSE-labeled control samples. The open grey histograms show the autofluorescence of stimulated control samples, not labeled with CFSE. (b) The precursor frequency (PF) of CFSE-labeled cells for each irradiation dose, at various time points. Individual datapoints of 6 independent experiments are shown, with the mean ± standard deviation. No statistically significant differences were found (p > 0.05) between irradiation doses. (c) Representative example of CFSE fluorescence profiles of live irradiated T lymphocytes visualized as described for (a).

Figure 3

EdU pulse-chasing of T lymphocytes in whole blood cultures allows for the assessment of the cell cycle progression. T lymphocyte proliferation was stimulated with PHA for 96 h before EdU pulse-labeling. (a) Bivariate distributions of non-irradiated EdU pulse-labeled T lymphocytes showing DNA content (x-axis) and EdU incorporation (y-axis). The green population shows the EdU-positive T lymphocytes. The red population shows the EdU-negative cells. The black arrows indicate the progression of the cells through the cell cycle, over time. The bivariate profiles of one donor are displayed here. Other donors show similar distributions. (b) Bivariate profiles of T lymphocytes exposed to 2 Gy of X-rays, visualized as described for (a). (c) Bivariate profiles of T lymphocytes exposed to 4 Gy of X-rays, visualized as described for (a).

Figure 4

Estimation of the S and G2/M phase duration through the quantification of non-irradiated EdU-labeled cells in G2/M phase over time. Datapoints show the mean percentage of non-irradiated EdU-positive cells in the G2/M phase across four independent experiments, for each time point. Error bars show the standard deviation of the mean. A cubic fit was applied, on which the full-width-at-half-maximum was calculated, as shown by the red arrow. The blue arrow indicates the G2/M phase duration.

Figure 5

Progression of EdU-labeled T lymphocytes through both the G1 and the G2/M phase over time. (a) Datapoints show the mean percentage of EdU-positive cells for each irradiated dose in the G1 phase, for all time points. The grey shading shows the standard deviation of three independent experiments. The blue and red arrow indicate the cell cycle delay for 2 and 4 Gy, respectively. (b) The mean percentage of EdU-positive cells in the G2/M phase is shown for each irradiation dose, for each time point. The grey shading shows the standard deviation of three independent experiments.

Figure 6

G2 arrest of EdU-labeled T lymphocytes after exposure to X-ray irradiation over time. The bar plots show the mean percentage of EdU-positive cells still residing in G2 arrest for non-irradiated and irradiated conditions, over multiple time points. Error bars show the standard deviation of the mean of three independent experiments.