Human bone marrow samples show highly individual Ig-Repertoires.

<p>(a) Schematic overview of human VDJ-amplicons. Amplicon libraries for each Ig class contain framework (FR2, 3) and complementary determining regions (CDR1, 2, 3). (b) In a set of 4000 IgG-sequences per bone marrow sample, clones with 95% CDR3 sequence identity and identical VJ-usage were clustered as clonotypes. Frequencies of clonotypes are indicated by color code; clonotypes comprising more than 5% of the repertoire are highlighted in blue and numbers indicate their frequencies; grey color indicates absence of a given clonotype. Clontypes were sorted according to their frequency in healthy donor (HD) 1 and the 100 most frequent clonotypes of HD1 and their abundance in HD2-4 were displayed as heatmap. Similarity of repertoires was expressed as Morisita-Horn index (MHI), comparing 4000 clustered CDR3 sequences of HD1-4. Symbols represent pairwise comparisons. (c) The IgG-repertoire of HD4 (I) was re-investigated as independent V_H1DJ-amplificate (II) or de-novo cDNA plus V_H1DJ-amplificate (III). Repertoire-similarity of I to its technical replicates II and III is illustrated by heatmap and MHI, evaluating 2500 clustered sequences of each sample.</p

Repertoire diversity is reduced after allogeneic HSCT.

<p>(a) The Exponent Shannon (e^Shannon) indices were calculated based on 4000 clustered IgA and IgG repertoires of four healthy donors (HD1-4). Symbols indicate individual samples, horizontal lines indicate the mean. (b) Diversity (e^Shannon) of IgG and IgA repertoires pre- and post-HSCT was compared for eleven AML-patients. Exponent Shannon indices were calculated for each sample based on 2000 (patient 9) or 4000 (all other patients) clustered IgG and IgA sequences. Matched samples (pre- and post-transplantation) are connected by lines, numbers indicate the different patients. Statistical analysis was performed using paired T-test with Wilcoxon signed rank test, ** p< 0.01. Patient 11 (grey lines and symbols) was not considered for statistical analysis.</p

Anti-EGFR-Based Therapy in Recurrent or Metastatic HNSCC – What Difference Does it Make?

Patients with R/M HNSCC treated with palliative first-line therapy at Hannover Medical School between October 2005 and December 2016 have been included to show changes in survival following broad utilization of cetuximab. Treatment periods were defined from 10/2005 to 12/2008 (Period A) and 01/2009 to 12/2016. Overall survival did not improve over time. However, in subgroup analysis cetuximab utilized at any time vs. never showed a significant improve of overall survival (11.3 vs. 6.3 months, HR: 0.55, 95%-CI: 0.4–0.8, p = 0.04). Therefore, this study supports the application of cetuximab in this real-world population.</p

Switch Ig repertoires after HSCT comprise a spectrum of low and highly mutated clones.

<p>Numbers of somatic hypermutations in bone marrow switch IgA and IgG repertoires. Lines represent individual samples. Evaluation was based on all available sequences. Diagrams in (a) show results of four healthy donor repertoires (HD1-4), compared to (b) patient 1 and 6 evaluated separately for IgG (left column) and IgA (right column).</p

Clinical characteristics of AML-patients investigated for repertoire analysis before and after allogeneic HSCT.

<p>Clinical characteristics of AML-patients investigated for repertoire analysis before and after allogeneic HSCT.</p

Ig repertoire analysis reveals clonal sharing between isotypes and persistence of clonotypes after HSCT.

<p>(a) IgA, IgG and IgE repertoires sequences were clustered (4000 sequences; 95% CDR3 sequence identity; same VJ-usage), and sorted according to the 100 most abundant clonotypes present in the IgA (left panel), IgG (middle panel), or IgE (right panel) repertoire and plotted as heatmap (see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0168096#pone.0168096.g001" target="_blank">Fig 1</a>). Comparison of repertoire similarity of IgA, IgG and IgE repertoires of HD1 based on the analysis of 4000 sequences each. (b, c) IgG sequences of eleven patients pre- and post-HSCT (patient 1: 2000 sequences; other patients: 4000 sequences) were assigned to clonotypes. (b) Heatmaps show CDR3-overlap of the 100 most abundant IgG clonotypes of patients 1 and 5 pre- and post-HSCT. Repertoire similarity is described as Morisita-Horn index (MHI). (c) IgG-repertoire and (d) IgA-repertoire similarity quantified as MHI. Symbols represent comparisons of patient 1-11(X) pre- versus post-HSCT (X_pre vs. X_post) or versus itself as control (X_pre vs. X_pre). In addition, repertoires of patients 1 and 5 pre-HSCT were compared to all other patients pre HSCT (X₁ or X₅ vs.Y_pre). Pairwise match of all pre-HSCT or post-HSCT IgG repertoires investigated results in a mean MHI of 0.0001±0.0005 for all pre- and 0.0001±0.0004 and for all post-HSCT samples (mean±SD). Pairwise match of all pre-HSCT or post-HSCT IgA repertoires investigated results in MHI of 0.00007±0.00033 for all pre- and 0.00002±0.00011 for all post-HSCT samples (mean±SD). Statistical analysis was performed using Kruskal-Wallis test with Dunn’s multiple comparison post hoc test, * p<0.05.</p

Predefined human endpoints.

Predefined human endpoints.</p

Analysis of miR-125b function in granulocytes under steady phase.

(a) Chemotaxis of BM-derived granulocytes. Total bone marrow cells were harvested from BM chimeras 8 to 10 weeks post-transplantation. Granulocytes (> 90%) were enriched by using Percoll gradient cell separation. Chemotaxis of granulocytes was measured by using transfilter migration assays. Cell migration (5 x 104) was measured in the absence and presence of chemotactic agent, C5a (25 ng/ml). Cells migrated to the lower chamber were collected after 5 hours of incubation for FACS analysis. Data is displayed as fold induction calculated by dividing the number of migrating cells in the treated groups by the number of migrating cells in the untreated groups. Bar diagrams represent mean ± s.e.m. n = 8 for miR-125b over-expressing granulocytes (n = 8) and for control granulocytes (n = 5) and results were pooled from five independent experiments. (b) Survival of BM-derived granulocytes. Percoll-purified granulocytes (5 x 105) from BM chimeras were stimulated with PMA (10 ng/ml), TNF-α (50 ng/ml) or LPS (1 μg/ml) for 18 hours. Cell viability was determined by microscope cell counting using Trypan blue. Bar diagrams represent mean ± s.e.m. of absolute viable cell number. Data are pooled from three independent experiments. *Pt-test (two-tailed). (c) Reactive oxygen species (ROS) production in BM-derived granulocytes. Granulocytes harvested from BM chimeras were treated with manganese chloride (MnCl2) for 30 minutes. ROS production was detected by using CellROX oxidative stress reagents (Deep Red) and granulocyte fluorescence was measured by flow cytometry. Data (mean ± s.e.m.) is presented as frequency of ROS-positive cells. Results are pooled from three independent experiments (n = 4). *Pt-test (two-tailed).</p

Human Regulatory T Cells of G-CSF Mobilized Allogeneic Stem Cell Donors Qualify for Clinical Application

<div><p>Recent clinical studies demonstrate the high potency of regulatory T cells (Tregs) to control graft-versus-host disease in hematopoietic stem cell transplantation (SCT). However, the adoptive transfer of Tregs is limited by their low frequency in unstimulated donors and considerable concerns that G-CSF induced SC mobilization might have negative effects on the stability and function of Tregs. The isolation of Tregs from the G-CSF mobilized SC grafts would extend this novel strategy for tolerance induction to the unrelated setting and simplify global clinical application. We characterized CD4⁺CD25^highCD127⁻ Tregs from SC donors before and after G-CSF mobilization for their phenotype, function, and stability. After G-CSF application the Treg cell yield increased significantly. Donor Tregs retained their cytokine profile, phenotypic characteristics and <em>in vitro</em> expansion capacity after SC mobilization. Most importantly, <em>in vivo</em> G-CSF stimulated Tregs remained highly suppressive on the proliferation of effector T cells, also after <em>in vitro</em> expansion, and displayed a stable phenotype in epigenetic studies. The surface expression of CXCR3 is transiently reduced. However, donor-derived Tregs maintain their migratory properties after G-CSF stimulation. Therefore, the adoptive transfer of Tregs from G-CSF mobilized SC donors seems to be a feasible and safe strategy for clinical application in allogeneic SCT.</p> </div

Phenotypic analysis of <i>in vitro</i> expanded donor Tregs before and after SC mobilization.

<p>PBMCs were isolated before (n = 25) and after (n = 21) G-CSF mobilization. FACS isolated CD4⁺CD25^highCD127⁻Foxp3⁺ donor Tregs were expanded <i>ex vivo</i> for 14 days. FACS analysis was performed before (white bars) and after (grey bars) SC mobilization as well as after <i>in vitro</i> expansion of donor Tregs isolated before (white with black stripes) and after (grey with white stripes) SC mobilization. Expression values were calculated as percentage of CD4⁺CD25^highCD127⁻Foxp3⁺ Tregs and their mean values are presented as bar graphs with SD as error bars.</p