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.

Manual Scoring with Shortened 48 h Cytokinesis-Block Micronucleus Assay Feasible for Triage in the Event of a Mass-Casualty Radiation Accident

“…As stated in EPR-Biodosimetry, Cyt-B should be preferably added at 24 h for ''biological dosimetry to ensure only first division cells are captured'' (13). As supported in Duthoo et al's study (53) of PHAstimulated non-irradiated and irradiated T cell cycles, the estimated total cell division time was 16.6 h in nonirradiated cells. First-cycle divisions were completed in 48-72 h and unaffected by irradiation of up to 2 Gy after PHA stimulation.…”

“…2 and 4, though no strong conclusions can be drawn due to a low number of donors analyzed. In Rodrigues et al's study, a much lower MN frequency in 48 h (24 h at Cyt-B) than 72 h (24 h at Cyt-B) WB cultures in automatically scored cells with imaging flow cytometry (22), which could be due to damaged cells dividing slower than healthy cells (53). In Ko ¨ksal et al's study comparing 48 h (24 h at Cyt-B) and 72 h (44 h at Cyt-B) CBMN assay, a lower MN frequency was also seen in 48 than 72 h WB cultures in manually scored cells (52).…”

Neoadjuvant radiotherapy in ER+, HER2+, and triple-negative -specific breast cancer based humanized tumor mice enhances anti-PD-L1 treatment efficacy

“…Alongside the objective to preserve manipulated by mainly vital cells single dose irradiation instead of fractionated scheme was later on applied for in-vivo studies in order to avoid inhibitory effects of irradiation on infiltrating lymphocytes. Irradiation induced negative effects have been reported, for example, by a study in which T cell propagation and activity is reduced upon irradiation with 4 Gy (or higher doses) ( 33 ).…”

Methods for Inferring Cell Cycle Parameters Using Thymidine Analogues

“…In both procedures, a marker is given according to two schedules where both interjection intervals and marker doses are similar. As an example of the “short procedure”, see Figure 1 in [ 45 ], Figure 4 in [ 33 ], Figure 8a–b in [ 46 ], and Figure 4 in [ 47 ]. For the “long procedure”, see Figure 3 in [ 48 ], Figures 2–4 in [ 49 ], Figure 4 in [ 50 ], Figure 8 in [ 51 ], and Figure 4 in [ 34 ].…”