DSB repair is affected by the type of sample preparation.

<p>Repair by NHEJ (A) or SSA (B) was measured in lymphocytes from different individuals (LYM2-6). LYM2 was only analyzed after freezing. LYM3-6 were analyzed in both fresh and frozen samples, each time starting with the cells recovered from an equivalent volume of blood (∼2.5 ml). In addition, LYM 5–6 were analyzed in samples that were first hetastarch-prepared and then depleted for CD15⁺ cells. Results indicate an enhanced repair in fresh cells that were hetastarch-prepared that is strongly associated with the presence of CD15⁺ cells in the environment of the studied lymphocytes and most of that difference is gone after freezing, at least for NHEJ repair. Mean and standard deviation of 3–4 measurements on 1–2 experimental days are shown.</p

Granulocytes Affect Double-Strand Break Repair Assays in Primary Human Lymphocytes

<div><p>Patients who develop therapy-related myelodysplasia/acute myeloid leukemia after autologous-hematopoietic stem cell (aHCT) transplant show lower expression levels of DNA repair genes in their pre-aHCT CD34⁺ cells. To investigate whether this leads to functional differences in DNA repair abilities measurable in patients, we adapted two plasmid-based host-cell reactivation assays for use in primary lymphocytes. Prior to applying these assays to patients who underwent aHCT, we wanted first to verify whether sample preparation affected repair measurements, as patient samples were simply depleted of erythrocytes (with hetastarch) prior to freezing, which is not the classical way to prepare lymphocytes prior to DNA repair experiments (with a density gradient). We show here that lymphocytes from healthy donors freshly prepared with hetastarch show systematically a higher level of double-strand break repair as compared to when prepared with a density gradient, but that most of this difference disappears after samples were frozen. Several observations points to granulocytes as the source for this effect of sample preparation on repair: 1) removal of granulocytes makes the effect disappear, 2) DSB repair measurements for the same individual correlate to the percentage of granulocytes in the sample and 3) nucleofection in presence of granulocytes increases the level of reactive oxygen species (ROS) in neighboring lymphocytes in a dose-dependent manner (R² of 0.95). These results indicate that co-purified granulocytes, possibly through the release of ROS at time of transfection, can lead to an enhanced repair in lymphocytes that obfuscates any evaluation of inter individual differences in repair as measured by host-cell reactivation. As a result, hetastarch-prepared samples are likely unsuitable for the assessment of DSB repair in primary cells with that type of assay. Granulocyte contamination that exists after a density gradient preparation, although much more limited, could have similar effects, but might be circumvented by freezing cells prior to analysis.</p></div

Different types of sample preparation evaluated.

<p>(A) Schematic representation of the different types of preparation protocols performed prior to repair measurements. Cells were either prepared on a density gradient (1) or incubated with hetastarch (2 and 3). When indicated for LYM5 and 6, hetastarch-preparation was followed by the depletion of CD15⁺ cells (3) (B) Cell composition of samples after purification depended on the preparation protocol that was used and whether cells were fresh or frozen. One distinctive feature of hetastarch-prepared samples is that the vast majority of cells recovered in fresh samples are granulocytes (GRA in red), whereas lymphocytes (LYM in blue) are the main cell type after density gradient preparation and/or after the cells were frozen. The last major cell type in samples are monocytes (MON).</p

Host cell reactivation DSB repair assays.

<p>Test plasmids are digested <i>in vitro</i> with XhoI and ApaI, leading to a DSB with a 3' and a 5' overhang. Digested plasmids are transfected in cells and express tdTomato as a transfection marker and EYFP when DSB repair occurred. A functional EYFP gene is indicated in yellow. (A) pSF-tdTomato-END with a double-strand break leads to EYFP expression only after NHEJ repair restores the physical link between the gene and its CMV promoter. (B) pSF-tdTomato-HOM contains two incomplete copies of EYFP (one with the 5' and the other with the 3' part of the gene with a 355 bp overlap (grey boxes) between the two copies) resulting in a non functional gene. Repair of the DBS break in the HOM construct leads to a functional EYFP only if repair is done by SSA, any repair by NHEJ remaining undetected.</p

Granulocytes increase ROS in neighboring lymphocytes after nucleofection.

<p>Frozen cells samples (LYM6) that were depleted in CD15⁺ cells either before freezing (CD15 in black) of after thawing (hetastarch-prepared LYM6 in white) were analyzed for the presence of ROS after or not a mock nucleofection (cells resuspended in nucleofection solution and electroporated without DNA). Each sample contains ∼135,000 lymphocytes. When indicated, a certain fraction F (or multiples thereof) of the CD15⁺ cells recovered from the MACS depletion column were added back to the recovered lymphocytes (F≈50,000 granulocytes). (<b>A</b>) After an overnight culture, untransfected cells, regardless of how the sample was prepared, showed a subpopulation of cells with higher level of ROS measured as a % Cy5 positive cells. (<b>B</b>) When a mock transfection step was added or after treatment with the NAC antioxidant, this population of Cy5⁺ cells tended to disappear (left Y axis). However, a <i>de novo</i> formation of ROS was detected in the whole lymphocyte population one hour after nucleofection in a manner dependant of the amount (F, 2F or 3F) of CD15⁺ cells added back to the sample. As the whole population of lymphocytes shifted towards higher level of Cy5 intensity, the levels of ROS in mock transfected samples were better measured using the median Cy5 intensity in lymphocytes (right Y axis). This Cy5 median intensity was strictly correlated to the estimated number of GRA added back to the mix.</p

Statistical analysis of the effect of sample preparation on repair measurements.

<p>Variation associated with the individual or the sample preparation was investigated using ANOVA.</p><p>**** p<0.0001.</p><p>ns: not significant.</p

Reproducibility of the host cell reactivation assays.

<p>A) Average transfection efficiency (tdTomato⁺ cells) for each of the constructs to measure NHEJ (END_LIN and END_DSB) or SSA (HOM_LIN and HOM_DSB). (B) Individual measurements of the RRE by NHEJ or SSA in GM01953 lymphoblastoid cell line and in LYM1 primary lymphocytes. Data represent 8–10 measurements for each type of repair performed on 3–6 experimental days over a period of 3 months. Mean of measurements and standard deviation are indicated.</p

Plasmids used for host cell reactivation assays.

(A) pSF-tdTomato-END (for NHEJ, left) and pSF-tdTomato-HOM (for SSA, right) plasmids used for DSB repair assays. XhoI+ApaI double-digestion (= DSB) generated in vitro is repaired by either NHEJ or SSA after transfection in cells, thereby restoring EYFP expression. The common sequence between the two overlapping halves of EYFP in the SSA template (“YF” in grey) is 350bp long. Repair is measured by the percentage of EYFP+ cells among tdTomato+ cells. (B) pM1-Luc plasmid (left) and pRL-CMV (right) plasmids. Oxidative (8-oxoG) or UV damage (pyrimidine dimers) generated in vitro on pM1-Luc plasmid impairs expression of firefly luciferase (FL) that is then restored by BER or NER, respectively, once transfected in cells. Repair is measured by the FL activity after normalization to renilla luciferase (RL) activity (transfection control) and in comparison to the undamaged FL template (100% expression) in the same cells.</p

Characteristics of aHCT lymphoma patients selected for DRC analysis.

<p>Characteristics of aHCT lymphoma patients selected for DRC analysis.</p

Splitstree of <i>Penicillium roqueforti</i> strains.

<p>Reticulation indicates likely occurrence of recombination. The colors correspond to those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0171387#pone.0171387.g001" target="_blank">Fig 1</a> of the clusters identified using Structure. The name of the clusters indicate whether they possess the <i>Wally</i> and <i>CheesyTer</i> genomic islands (W+C+) or not (W-C-) and whether they have been collected from cheeses, belonging or not to the Roquefort protected designation of origin (PDO), or from other environments, such as silage or spoiled food.</p