miR-625-3p expression is regulated during CD8+ T cell reconstitution in vivo.

<p>(A) Viable CD8+ T cells were quantified (grey triangles) and isolated by FACS sorting from peripheral blood samples collected on days 25 (n = 22), 45 (n = 53), 90 (n = 45) and 150 (n = 17) after allogeneic SCT. Subsequently, the relative miR-625-3p expression (black circles) was determined. Left Y-axis: Mean relative miR-625-3p expression calculated by miR-625-3p [RQ]/mean RNU48/U6 snRNA [RQ]. RQ: relative quantity. Right Y-axis: Mean CD8+ T cell count /μl at the time of blood collection (±5 days). Error bars indicate standard error of mean. All measurements were performed in duplicates. Statistical comparisons was calculated in comparison with the miR-625-3p expression in CD8+ T cells of healthy donors by Mann-Whitney U test; *indicates p<0.05. (B) Correlation analysis between relative miR-625-3p expression in patient derived CD8+ T cells (black circles) and the exact time of collection after allogeneic SCT is shown. The relationship between collection time and miR-625-3p expression in CD8+ T cells was analyzed by Spearman correlation coefficient (R²) analysis.</p

miR-625-3p upregulation is a late event after T cell activation.

<p>(A-D) CD8+ T cells isolated from PBMCs of 4 healthy donors were stimulated with PHA + 120 IU/ml IL-2 and (A) CD69, CD25 and CD79 surface expressions, (B) IFN-γ in the supernatant, (C) BrdU uptake and (D) intracellular miR-625-3p expression were measured after 0h, 6h, 1, 3, 5 and 7 days. Y-axis: (A) Mean fluorescence intensity (MFI), (B) Relative uptake of BrdU calculated as [absorbance of sample—absorbance of unstimulated control]/absorbance of unstimulated control, (C) IFN-γ levels in pg/ml, (D) Relative fold change in miR-625-3p expression calculated by 2^-ΔΔct method using RNU48 and U6 snRNA as reference genes. (A-C) Statistical analysis was calculated by paired t-test; *indicates p<0.05, **indicates p<0.01. (D) Fold change above dotted line indicates significant fold change value >2. Data are representative of three independent experiments. All measurements were performed in duplicates.</p

Patient characteristics.

<p>Patient characteristics.</p

miR-625-3p in CD8+ T cells in patients with GvHD.

<p>(A) Viable CD3+CD8+ T cells were isolated by FACS sorting from peripheral blood samples and miR-625-3p expression was determined. (B) Relative miR-625-3p expression in CD8+ T cells isolated from 137 peripheral blood samples of 74 patients collected on days 25, 45, 90 and 150 after allogeneic SCT (triangles) compared to healthy donors (n = 9, circles). (C) Mean CD8+ T cell count / μl at the time of blood collection (left) and relative miR-625-3p expression in CD8+ T cells (right) isolated from peripheral blood samples collected on day 45 (±5 days) after allogenic SCT from patients without (n = 20, circles) and with (n = 13, triangles) severe GvHD (grade II-IV). (D) Mean CD8+ T cell count / μl at the time of blood collection (left) and relative miR-625-3p expression in CD8+ T cells (right) of 13 patients with severe GvHD (grade II-IV) determined in the last sample before GvHD onset (circles) and in a sample during GvHD (triangles). Relative miR-625-3p expression in individual patient samples calculated by miR-625-3p [RQ]/mean RNU48/U6 snRNA [RQ]. RQ: relative quantity. Median is shown as a bar. All measurements were performed in duplicates. Statistical comparisons between independent samples were performed by Mann-Whitney U test (B-C) and between dependent samples by Wilcoxon matched-pairs signed rank test (D). * indicates p<0.05.</p

miR-625-3p upregulation in CD8+ T cells after stimulation.

<p>(A-B) CD8+ T cells were isolated from PBMCs of 3 healthy donors by MACS negative selection and stimulated with CD2/CD3/CD28 beads, PHA + IL-2, PHA alone, IL-2 or IL-7 + IL-15. BrdU uptake (A) and miR-625-3p expression (B) were measured 5 days after stimulation. (C-D) Established HLA-A2 restricted CMV and HA-1 specific CD8+ T-cell clones were stimulated with CD14+ monocytes loaded with CMV pp65 NLV or HA-1 peptide, respectively, in the presence of IL-2. BrdU uptake (C) and miR-625-3p expression (D) were measured 5 days after stimulation. Y-axis: (A, C) Relative uptake of BrdU was calculated as [absorbance of sample—absorbance of unstimulated control] / absorbance of unstimulated control; Statistical analysis for BrdU uptake was calculated by (A) one way ANOVA followed by Dunnett’s multiple comparison test for overall CD8+ T cells and (C) Unpaired t-test for antigen-specific cells. **indicates p<0.01, *** indicates p<0.001. (B, D) relative fold change in miR-625-3p expression was calculated by 2^-ΔΔct method using RNU48 and U6 snRNA as reference genes. Fold change above dotted line indicates significant fold change value >2. Data are representative of three independent experiments. All measurements were performed in duplicates.</p

miR-625-3p expression in CD8+ T cells in relation to proliferation.

<p>(A-F) CD8+ T cells isolated from PBMCs of 4 healthy donors were stimulated with PHA and 80 IU/ml IL-2 was repeatedly added every second day (A-C) or only on day 2 (D-F). BrdU uptake (A, D), miR-625-3p expression (B, E) and viable cell counts determined by trypan blue staining (C, F) were measured on different days until day 20 (A-C) or 17 (D-F) after stimulation. (G, H) CD8+ T cells isolated from PBMCs of 3 healthy donors were stimulated with CD2/CD3/CD28 beads in the presence or absence of rapamycin (RapaB). BrdU uptake (G) and miR-625-3p expression (H) were measured on day 5 after stimulation. X axis: (A-F) days post SCT. Y-axis: (A,D,G) Relative uptake of BrdU was calculated as [absorbance of sample—absorbance of unstimulated control] / absorbance of unstimulated control. (B,E,H) Relative fold change in miR-625-3p expression was calculated by 2^-ΔΔct method using RNU48 and U6 snRNA as reference genes. All measurements were performed in duplicates. Fold change above dotted line indicates significant fold change value >2. Statistical comparisons were performed by paired t-test; *indicates p<0.05.</p