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

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 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

Patient characteristics.

<p>Patient characteristics.</p

Possible Role of Minor H Antigens in the Persistence of Donor Chimerism after Stem Cell Transplantation; Relevance for Sustained Leukemia Remission

<div><p>Persistent complete donor chimerism is an important clinical indicator for remissions of hematological malignancies after HLA-matched allogeneic stem cell transplantation (SCT). However, the mechanisms mediating the persistence of complete donor chimerism are poorly understood. The frequent coincidence of complete donor chimerism with graft-versus-leukemia effects and graft-versus-host disease suggests that immune responses against minor histocompatibility antigens (mHags) are playing an important role in suppressing the host hematopoiesis after allogeneic SCT. Here, we investigated a possible relationship between donor immune responses against the hematopoiesis-restricted mHag HA-1 and the long-term kinetics of host hematopoietic chimerism in a cohort of 10 patients after allogeneic HLA-matched, HA-1 mismatched SCT. Functional HA-1 specific CTLs (HA-1 CTLs) were detectable in 6/10 patients lysing host-type hematopoietic cells in vitro. Presence of HA-1 CTLs in the peripheral blood coincided with low host hematopoiesis levels quantified by highly sensitive mHag specific PCR. Additionally, co-incubation of host type CD34⁺ cells with HA-1 CTLs isolated after allogeneic SCT prevented progenitor and cobblestone area forming cell growth in vitro and human hematopoietic engraftment in immunodeficient mice. Conversely, absence or loss of HA-1 CTLs mostly coincided with high host hematopoiesis levels and/or relapse. In summary, in this first study, presence of HA-1 CTLs paralleled low host hematopoiesis levels. This coincidence might be supported by the capacity of HA-1 CTLs isolated after allogeneic SCT to specifically eliminate host type hematopoietic stem/progenitor cells. Additional studies involving multiple mismatched mHags in more patients are required to confirm this novel characteristic of mHag CTLs as factor for the persistence of complete donor chimerism and leukemia remission after allogeneic SCT.</p></div

Cytotoxicity of isolated HA-1 CTLs.

<p>HA-1 CTL clones were isolated from peripheral blood of patients 1–6 at multiple time-points and expanded in vitro. Median 13 (range: 8–25) HA-1 CTL clones per patient were tested in a 4h ⁵¹Cr-release assay for HA-1 specific recognition of target cells in vitro. Left upper corner: percentage of HA-1 CTL clones lysing HA-1^H naturally expressing EBV LCLs; x-axis: HA-1^H naturally expressing EBV LCLs, HA-1^RR EBV LCLs loaded with HA-1^H peptide, HA-1^RR EBV LCLs, E/T ratio: back: 10:1, white: 1:1; y-axis: % specific lysis.</p

Patient characteristics.

<p><b>Abbreviations</b>: AML, acute myeloid leukemia; sAML, secondary AML; ALL, acute lymphoblastic leukemia; PMF, primary myelofibrosis; CLL: chronic lymphoblastic leukemia; SAA, severe aplastic anemia; Sib: matched sibling donor; PR: partial remission; CR: complete remission; PD: progressive disease; Dupriez score for the prognosis of PMF [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119595#pone.0119595.ref029" target="_blank">29</a>]; Bu, Busulphan; Cy, Cyclophosphamide; Flu, Fludarabin; TBI, Total Body Irradiation; VP-16, Etoposide; the FLAMSA protocol consisted of Fludarabine, Cytosine-Arabinoside and Amsacrine; D, donor; P, patient; M, male; F, female; MMF, Mycophenolate Mofetil; MTX, Methotrexate; CsA, Cyclosporine A; Pred, prednisolone; ATG, Anti-Thymocyte Globulin; Thy, Thymoglobuline; Cam, Campath; PBSC, peripheral blood stem cells; BM, bone marrow; MOF: multi organ failure; aG: acute GvHD graded according Glucksberg score [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119595#pone.0119595.ref030" target="_blank">30</a>]; cG: chronic GvHD graded according Seattle classification [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119595#pone.0119595.ref031" target="_blank">31</a>];</p><p>* lower detection limit of the used HA-1^H based chimerism assay: 0.01%</p><p>Patient characteristics.</p

HA-1^H specific elimination of human umbilical cord blood and bone marrow derived hematopoietic progenitor/stem cells by HA-1 CTLs studied in vitro.

<p>(A-F) Umbilical cord blood (UCB) derived CD34+ and bone marrow (BM) derived CD34⁺ and CD133⁺ cells from HLA-A2/HA-1^H or HLA-A2/HA-1^RR donors were incubated with medium, an irradiated control CMV CTL clone, an HA-1 CTL clone or an anti-HLA-A2 specific CTL clone overnight. Data are mean +/- Standard deviation of independent experiments with 3 HA-1^H and 2 HA-1^RR donors, respectively (*p<0,05; **p<0.01, ns = not significant using a two-tailed Student’s t-test). The results of the medium control were set as 100%. (A, D) % viable CD34⁺ or CD133⁺ cells (duplicate sample per donor) compared to the medium control directly after over-night co-incubation with CTLs. (B, E) % colony forming cells (single sample per donor) compared to the medium control as determined after 14 days; CFU-macrophage (CFU-M; striped bar), CFU-Granulocyte (CFU-G; black bar), blast forming unit (BFU-E; white bar). (C, F) % of cobblestone area positive wells (4–5 samples per donor) in a CAFC assay compared to the medium control as determined after 5 weeks. (G-H) An HA-1 CTL clone was titrated to BM derived CD34+ cells (white bars), UCB derived CD34+ cells (grey bars) and to BM derived CD133⁺ cells (black bars) from HLA-A2/HA-1^H donors and co-incubated over night. x-axis: HA-1 CTL to CD34 or CD133 cell ratio. Y-axis: % viable CD34⁺ or CD133+ cells (duplicate sample per donor) compared to the medium control (G) and % of cobblestone area positive wells in a CAFC assay (4–5 samples per donor) compared to the medium control as determined after 5 weeks (H).</p