Human CD8+ CD57- T_EMRA cells: Too young to be called "old"

<div><p>End-stage differentiation of antigen-specific T-cells may precede loss of immune responses against e.g. viral infections after allogeneic stem cell transplantation (SCT). Antigen-specific CD8+ T-cells detected by HLA/peptide multimers largely comprise CD45RA-/CCR7- effector memory (T_EM) and CD45RA+/CCR7- T_EMRA subsets. A majority of terminally differentiated T-cells is considered to be part of the heterogeneous T_EMRA subset. The senescence marker CD57 has been functionally described in memory T-cells mainly composed of central memory (T_CM) and T_EM cells. However, its role specifically in T_EMRA cells remained undefined. Here, we investigated the relevance of CD57 to separate human CD8+ T_EMRA cells into functionally distinct subsets. CD57- CD8+ T_EMRA cells isolated from healthy donors had considerably longer telomeres and showed significantly more BrdU uptake and IFN-γ release upon stimulation compared to the CD57+ counterpart. Cytomegalovirus (CMV) specific T-cells isolated from patients after allogeneic SCT were purified into CD57+ and CD57- T_EMRA subsets. CMV specific CD57- T_EMRA cells had longer telomeres and a considerably higher CMV peptide sensitivity in BrdU uptake and IFN-γ release assays compared to CD57+ T_EMRA cells. In contrast, CD57+ and CD57- T_EMRA cells showed comparable peptide specific cytotoxicity. Finally, CD57- CD8+ T_EMRA cells partially changed phenotypically into T_EM cells and gained CD57 expression, while CD57+ CD8+ T_EMRA cells hardly changed phenotypically and showed considerable cell death after in vitro stimulation. To the best of our knowledge, these data show for the first time that CD57 separates CD8+ T_EMRA cells into a terminally differentiated CD57+ population and a so far functionally undescribed “young” CD57- T_EMRA subset with high proliferative capacity and differentiation plasticity.</p></div

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

Distribution of CD57+ cells in CD8+ T cell subsets.

<p>(A-B) Gating strategy for the assessment of the CD57 distribution in subsets of (A) overall CD8+ T cells and (B) CMV specific CD8+ T cells. (A) Shown is one representative example of the CD57 distribution within CD8+ T_N, T_CM, T_EM and T_EMRA cells of 6 healthy individuals. (B) Shown is one representative example of the CD57 distribution within CD8+ CMV tetramer+ T_EM and T_EMRA cells of 10 patients after allogeneic SCT.</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

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

Phenotypic and functional characterization of CMV tetramer+ cells.

<p><b>(A)</b> CD8+ CMV tetramer+ T cells were FACS sorted from the peripheral blood of 3 patients after allogeneic SCT and in vitro expanded on autologous feeder cells. Depicted is the T_EM and T_EMRA subset distribution within CD8+ CMV HLA/tetramer+ T cells (left) and CD57+ distribution within CD8+ CMV HLA/tetramer+ T_EMRA cells (right) in the peripheral blood compared to after in vitro expansion of FACS sorted CD8+ CMV tetramer+ T cells. Y-axis: % subset distribution within CD8+ CMV HLA/tetramer+ T cells and CD8+ CMV HLA/tetramer+ T_EMRA cells. Error bars indicate standard deviation. (B) Sorting strategy for viable in vitro expanded CD8+ CMV HLA/tetramer+ CD8+ T cells for CD45RA and CD57 allowing functional analysis. (C) Absolute telomere length directly after sorting. (D) BrdU uptake 4 days after stimulation with CD14+ monocytes loaded with increasing concentrations of the relevant HLA/CMV peptide. (E) INF-γ release in the supernatant from the BrdU uptake assay. (F) Specific lysis of CFSE labelled PHA blasts loaded with increasing concentrations of the relevant HLA/CMV peptide. Significance was calculated using Mann-Whitney-U test. * indicates p<0.05, ** indicates p<0.01, ns indicates not significant.</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