Differential DNA Damage Response of Peripheral Blood Lymphocyte Populations

Abstract

DNA damage occurs constantly in every cell triggered by endogenous processes of replication and metabolism, and external influences such as ionizing radiation and intercalating chemicals. Large sets of proteins are involved in sensing, stabilizing and repairing this damage including control of cell cycle and proliferation. Some of these factors are phosphorylated upon activation and can be used as biomarkers of DNA damage response (DDR) by flow and mass cytometry. Differential survival rates of lymphocyte subsets in response to DNA damage are well established, characterizing NK cells as most resistant and B cells as most sensitive to DNA damage. We investigated DDR to low dose gamma radiation (2Gy) in peripheral blood lymphocytes of 26 healthy donors and 3 patients with ataxia telangiectasia (AT) using mass cytometry. γH2AX, p-CHK2, p-ATM and p53 were analyzed as specific DDR biomarkers for functional readouts of DNA repair efficiency in combination with cell cycle and T, B and NK cell populations characterized by 20 surface markers. We identified significant differences in DDR among lymphocyte populations in healthy individuals. Whereas CD56⁺CD16⁺ NK cells showed a strong γH2AX response to low dose ionizing radiation, a reduced response rate could be observed in CD19⁺CD20⁺ B cells that was associated with reduced survival. Interestingly, γH2AX induction level correlated inversely with ATM-dependent p-CHK2 and p53 responses. Differential DDR could be further noticed in naïve compared to memory T and B cell subsets, characterized by reduced γH2AX, but increased p53 induction in naïve T cells. In contrast, DDR was abrogated in all lymphocyte populations of AT patients. Our results demonstrate differential DDR capacities in lymphocyte subsets that depend on maturation and correlate inversely with DNA damage-related survival. Importantly, DDR analysis of peripheral blood cells for diagnostic purposes should be stratified to lymphocyte subsets.

Keywords: DNA damage response; ataxia telangiectasia; cell cycle; mass cytometry; peripheral blood lymphocyte subsets.

Figure 1

DNA damage response to ionizing radiation differs among T, B, and NK lymphocytes. Peripheral blood mononuclear cells (PBMCs) from 26 healthy donors were irradiated with 2Gy and fixed at indicated time points. Surface markers of lymphocyte subsets and intranuclear DDR biomarkers were analyzed by mass cytometry. Induction of γH2AX (A), p-ATM (B), p-CHK2 (C), and p53 (D) were calculated in CD45⁺ lymphocytes, CD45⁺CD3⁺ T cells, CD45⁺CD56^dimCD16⁺ NK cells and CD45⁺CD19⁺CD20⁺ B cells based on mean fluorescence intensities normalized on unirradiated samples. Bars represent mean values; error bars represent standard deviations. Significance is shown for NK and B lymphocytes in comparison to T cells (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 2

Lymphocyte subsets show differential survival response rates to ionizing radiation. PBMCs obtained from 26 healthy donors were irradiated with 2Gy and fixed at indicated time points. Cell counts of viable (cisplatin^-) T, NK, and B lymphocytes (A), naïve and memory CD4⁺ and CD8⁺ T cell subsets (B), CD56^brightCD16^-, CD56^brightCD16⁺, CD56^dimCD16⁺ NK cell subsets (C), and naïve and memory B cell populations (D) were compared at each time point following radiation. Statistical significance was calculated for each lymphocyte population using Turkey’s multiple comparison test and is shown for unirradiated lymphocytes vs. lymphocytes 24h after radiation (ns, not significant, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 3

DDR in naïve lymphocyte subsets is differential to mature memory populations. PBMCs obtained from 26 healthy donors were irradiated with 2Gy and fixed at indicated time points. Surface markers of lymphocyte subsets and intranuclear DDR biomarkers were assessed by mass cytometry. Fold inductions of γH2AX, p-ATM, p-CHK2, and p53 were calculated in CD45⁺ lymphocyte subsets, based on mean fluorescence intensities normalized on unirradiated samples. T cell subsets were characterized as CD3⁺, CD3⁺CD4⁺, CD3⁺CD8⁺, CD45RA⁺CCR7⁺ (naïve CD4/CD8), CD45RO⁺CCR7^+/- (central and effector memory CD4/CD8) (A). NK lymphocyte subsets were defined as CD3^-CD56^brightCD16^-, CD3^-CD56^brightCD16⁺, CD3^-CD56^dimCD16⁺ (B), which were further stratified to CD57 expression on CD56^brightCD16⁺ and CD56^dimCD16⁺ subsets. CD3^- B lymphocytes were characterized as CD19⁺CD20⁺, CD27^-IgD⁺(naïve B), CD27⁺ (memory B), CD27⁺IgM⁺ (unswitched memory B) CD27⁺IgM^-IgGκ⁺ (class switched memory B IgGκ), CD27⁺IgM^-IgGΛ⁺ (class switched memory B IgGΛ), CD27⁺IgM⁺IgD⁺ (Marginal Zone (MZ)-like B), IgM⁺⁺CD38⁺⁺ (transitional B), CD27⁺IgM⁺IgD^- (IgM only B), CD27^-IgM^-IgD^- (atypical memory B), CD19⁺CD20^-CD27⁺CD38⁺IgM⁺ (unswitched plasmablasts), CD19⁺CD20^-CD27⁺CD38⁺IgM^- (class switched plasmablasts), and CD21^lowCD38^low B cells. (C) Bars represent mean values of fold induction; error bars represent standard deviations. Significance is shown for naïve T and B cell subsets compared to memory subsets, and immature CD56^brightCD16^- to mature CD56^brightCD16⁺ and CD56^dimCD16⁺ NK lymphocytes (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 4

Stimulation with IL-2 does not impact on DDR in lymphocyte subsets. PBMCs of healthy donors were cultured in RPMI media without (w/o) supplementation of IL-2 (A) and in presence of 100U/ml human IL-2 (B) for 48h. Subsequently, cells were irradiated with 2Gy and fixed at indicated time points. Shown are tSNE Plots demonstrating expression level of DDR markers γH2AX, p-ATM, p-CHK2 and p53 on all populations analyzed at indicated time points. Scale bars on the right-hand side of each panel indicate intensities of DDR markers. The bottom panel of (A, B), respectively, represent lymphocyte populations that are color coded by the legend underneath. This figure demonstrates one representative experiment of one donor out of six.

Figure 5

Differential DDR in lymphocyte subsets is independent from IL-2 stimulation. PBMCs of 6 healthy donors were cultured in RPMI media without and in presence of 100U/ml human IL-2 for 48h. Cells were irradiated with 2Gy and fixed at indicated time points. Mean fluorescence intensities (MFI) of DDR markers γH2AX, p-ATM, p-CHK2 and p53 are shown in T, NK and B lymphocyte subsets of unirradiated lymphocytes and 1h, 4h, 8h, 24h following IR with 2Gy (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 6

Stimulation with IL-2 impacts on cell cycle of lymphocyte subsets. PBMCs of six healthy donors were cultured in RPMI media without (w/o) supplementation of IL-2 and in presence of 100U/ml human IL-2 for 48h. Cells were irradiated with 2Gy, and 30min before fixation at indicated time points, IdU was added to cell culture to be incorporated in the DNA of replicating cells. Cell cycle was assessed by mass cytometry using ki67 and IdU to distinguish G0 (ki67^-IdU^-), G1 (ki67⁺IdU^-) and S (ki67⁺IdU⁺) cell cycle phases. Bars represent mean percentages of cell cycle phases G0 (grey), G1 (red), and S (blue) in T, NK and B lymphocyte subsets of unirradiated lymphocytes and 1h, 4h, 8h, 24h after IR with 2Gy. CD45⁺ lymphocytes are shown in each panel as internal control. Error bars represent standard deviations.

Figure 7

DDR to ultraviolet C radiation is differential in lymphocyte subsets. PBMCs of healthy donors were thawed and cultured in RPMI media supplemented with 100U/ml human IL-2 for 96h before ionizing radiation with 2Gy (A) and UVC radiation with 100mJ/m² (B). Cells were fixed unirradiated, and 1h, 4h, 8h, 24h after radiation. Shown are tSNE Plots demonstrating expression level of DDR markers γH2AX, p-ATM, p-CHK2 and p53 on all populations at indicated time points. Scale bars on the right-hand side of each panel indicate intensities of DDR markers. The bottom panel of (A, B), respectively, represent populations color coded by the legend underneath. Shown is one representative experiment obtained from one donor out of eight.

Figure 8

DDR of PBMCs to IR is differential to DDR induced by UVC. PBMCs obtained from 8 healthy donors were irradiated with 2Gy or 100mJ/m² UVC, respectively. Surface markers of lymphocyte subsets and intranuclear DDR biomarkers were analyzed by mass cytometry. Fold inductions of γH2AX, p-ATM, p-CHK2, and p53 were calculated in CD45⁺ lymphocyte subsets, based on mean fluorescence intensities normalized on unirradiated samples. Bars indicate mean values of fold induction; error bars represent standard deviations. Significance is shown for naïve T and B cell subsets compared to memory subsets, and immature CD56^brightCD16^- to mature CD56^dimCD16⁺ NK lymphocytes (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001).

Figure 9

DDR is diminished in all lymphocyte subsets of patients with ataxia telangiectasia. PBMCs obtained from healthy donors and 3 patients with ataxia telangiectasia (AT) were treated with 2Gy ionizing radiation and fixed after 1h, 4h, 8h and 24h. Fold inductions of γH2AX (A), p-ATM (B), p-CHK2 (C), and p53 (D) were calculated in CD45⁺, CD45⁺CD3⁺, CD45⁺CD3^-CD56^dimCD16^-, and CD45⁺CD3^-CD19⁺CD20⁺ lymphocyte subsets, based on mean fluorescence intensities normalized on unirradiated samples. Box plots indicate distribution of fold inductions obtained from 26 healthy donors and 3 AT patients; error bars represent standard deviations. Significance is shown for differences between controls and patients (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.001).