File S1 - Impaired Functionality of Antiviral T Cells in G-CSF Mobilized Stem Cell Donors: Implications for the Selection of CTL Donor
Supporting Information files. Figure S1 in File S1. Effects of sample storage time and temperature. Multimer analysis is usually performed on whole blood samples stored at room temperature within 24 h. As apheresis and graft processing took longer, some samples were analyzed later. Thus, we evaluated the influence of storage time and temperature on the reliability of the results. Nine patient samples were analyzed consecutively to detect changes in CD3, mNeg (non-specific background) and total percentages of mCMV_pp65_A02 and/or mCMV_pp65_B07 over time. Representative examples are shown. Storage time is plotted on the X-axis, and percentages of total CD3 (A), mCMV_pp65_A02 (B) and mCMV_pp65_B07 (C) on the Y-axis. Staining results for fresh samples (unstored) are depicted as a reference line (baseline; dotted horizontal line), and those for samples stored at room temperature (RT) and 4°C as filled circles (black line) and white circles (dashed line), respectively. Changes in CD3 and specific multimer percentages over time were less pronounced at 4°C in all sample types. Background staining did not change significantly at 24 h or 48 h (data not shown). After 48 h of storage at 4°C, a mean of 93% (mCMV_pp65_A02, n = 4) and 96% (mCMV_pp65_B07, n = 5) of the frequency in the fresh sample (baseline) was obtained. After 72 h, the variation from baseline was more pronounced, even in samples stored at 4°C. For example, in the 5 samples analyzed with mCMV_pp65_A02, only 82% and 30% (mean) of the multimer-positive population detected in fresh material was detected after 72 h of storage at 4°C or room temperature, respectively (Table S2 in File S1). Therefore, donor samples analyzed after more than 48 h (n = 2) were excluded from further analysis. As storage at 4°C led to less deviation from baseline (percentage change), it is preferable. Figure S2 in File S1. Gating strategy. (A) Gating strategy for one-platform quantification of percentage and total numbers of CD3CD8-double positive T cells. From left to right: A gate is set on the fluorescent beads for calculation of absolute cell numbers. Flow Count over time is recorded in order to detect changes in the flow rate. The 3rd plot from the left shows gating on lymphocytes for exclusion of debris and other mononuclear cells. CD4CD8-double-positive cells are excluded. The percentage of CD3+CD4+ and CD3+CD8+ T cells is determined in the lymphocytes (beads and CD4+CD8+ cells excluded) and the absolute number per µL blood can be calculated. For A and G samples, beads were added to confirm constant flow rates and signal intensity. (B) Gating strategy for quantification of multimer-positive T cells. From top to bottom, staining in all 3 sample types is shown. The percentage of multimer positive cells is given as percentage of CD3+CD8+ T cells. FSc – forward scatter, SSc – side scatter, FITC - fluorescein isothiocyanate, PE - Phycoerythrin, PCy7 – Phycoerythrin-Cyanin 7, WBM – whole blood mobilized, A – material from apharesis filter, G – material from graft quality control, Lymph – lymphocyte gate. For multimer abbreviations, please refer to Table S1 in File S1. Figure S3 in File S1. In vitro application of G-CSF reduces IFN-γ production in PBMCs. The optimal G-CSF concentration for stimulation was determined by treating PBMCs with different concentrations (5 to 50 ng/ml) of G-CSF for one week and subsequent stimulation with anti-CD3 monoclonal antibodies on day 7. IFN-γ production was measured by intracellular staining and flow cytometry. Results of four independent experiments are shown and expressed as mean ± SD. Asterisks indicate significant differences (**p<0.01). The IFN-γ production in untreated PBMCs (controls) was used as the reference value (100%). The percentage of IFN-γ-expressing cells decreased after G-CSF. A strong effect was achieved using 10 ng/ml G-CSF. At this dose, only 38.73% of IFN-γ-producing cells were retrieved on day 7. Because there was no significant difference after treatment with higher concentrations of G-CSF, 10 ng/ml G-CSF was used in further experiments. Figure S4 in File S1. Staining with specific multimers is not significantly influenced by sample type. (A) Unspecific background staining (mNeg): The background detected (mNeg) is shown for each donor sample and the sample type is indicated on the X-axis. Groups were compared by Kruskal-Wallis analysis followed by Dunn's multiple comparison. (B) Exemplary analysis of mCMV_pp65_A02 staining in different samples (Kruskal-Wallis test followed by Dunn's multiple comparison) in CMV-seropositive donors. (C–E) Blots C to E show the comparison of detection of multimer positive cells in the mobilized samples (WBM, A, G) to freshly isolated whole blood (WB). Percentages detected in the mobilized samples only differed slightly from those in WB samples. Sample sources: (A) apheresis tubing set, (G) graft, (WB) whole blood, (WBM) mobilized whole blood on day of apheresis. Figure S5 in File S1. Multimer staining examples. Examples for multimer staining in the different sample types (WBM – whole blood mobilized, A – material from apharesis filter, G – material from graft quality control) in 5 donors are depicted. The total sum of multimer positive CD3+CD8+ T cells after substraction of background (mNeg) for CMV (ΣmCMV) and EBV (ΣmEBV) is given. These values are also reported in Figure 1A and 1B. Plots show CD8-FITC signal versus multimer-PE signal gated on CD3+CD8+ T cells. Figure S6 in File S1. Multimer staining with mCMV_pp65_A02 (tetramer) and mCMV_IE1_A02 (pentamer). Staining examples in CMV-seropositive (CMV+, upper row) and CMV-seronegative (CMV-, lower row) donors are given, for each donor, all 3 samples types are shown (WBM – whole blood mobilized, A – material from apharesis filter, G – material from graft quality control). Staining with mCMV_pp65_A02 (tetramer) gives clear populations in CMV+ donors and low background in CMV-donors. In contrast, staining with mCMV_IE1_A02 (pentamer) results in less defined populations, furthermore positive staining results are found in CMV-donors (e.g. D#059, D#072, D#101). Figure S7 in File S1. Functional assays for in vitro G-CSF-treated pCMV_pp65_A02-stimulated cells on day 7. (A) IFN-γ ELISpot reveals a decrease in IFN-γ secretion after G-CSF treatment. Unstimulated cells served as control. Means of spots per well (spw) are indicated. (B) Granzyme B ELISpot shows a reduction in granzyme B secretion in a target cell dependent manner. Unloaded target cells served as a control. Means of spw are indicated. (C) Representative staining results from the CD107a degranulation assay show a reduced degranulation activity of CD8+ T cells after G-CSF treatment. Figure S8 in File S1. Follow up in R+/D+ patients. (A–C) Number of CMV-CTLs detected in the patient and donor before HSCT and post-HSCT follow-up in patients UPN2104 (A), UPN2131 (B) and UPN2145 (C). CMV reactivation led to expansion of CMV-CTL in UPN2145 (C). Figure S9 in File S1. Overview of CMV-serostatus of recipients and donors at Hannover Medical School (MHH). Number of CMV-seropositive patients with seropositive donors (R+D+; dotted bars) and seronegative donors (R+D−; striped bars) per year. Table S1 in File S1. Overview of multimers and antigens used. Table S2 in File S1. Variation of total multimer-positive population from baseline (fresh samples) after 24, 48 and 72 hoursof storage at 4°C or room temperature (RT). Table S3 in File S1. Donors with no, low and high percentages of multimer-positive T cells. Table S4 in File S1. Sequence alignment for the A02-restricted IE-1 epitope VLAELVKQI.
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