Human umbilical cord blood (UCB) represents a unique resource for hematopoietic stem cell transplantation for children and patients lacking suitable donors. UCB harbors a diverse set of leukocytes such as professional APCs, including monocytes, that could act as a novel source for cellular therapies. However, the immunological properties of UCB monocytes and monocyte-derived dendritic cells (MoDCs) are not fully characterized. In this study, we characterized the phenotype and functions of UCB-MoDCs to gauge their potential for future applications. UCB exhibited higher frequencies of platelets and lymphocytes as well as lower frequencies of neutrophils in comparison with adult whole blood. Leukocyte subset evaluation revealed significantly lower frequencies of granulocytes, NK cells, and CD14+CD16− monocytes. Surface marker evaluation revealed significantly lower rates of costimulatory molecules CD80 and CD83 while chemokine receptors CCR7 and CXCR4, as well as markers for Ag presentation, were similarly expressed. UCB-MoDCs were sensitive to TLR1–9 stimulation and presented quantitative differences in the release of proinflammatory cytokines. UCB-MoDCs presented functional CCR7-, CXCR4-, and CCR5-associated migratory behavior as well as adequate receptor- and micropinocytosis-mediated Ag uptake. When cocultured with allogeneic T lymphocytes, UCB-MoDCs induced weak CD4+ T lymphocyte proliferation, CD71 expression, and release of IFN-γ and IL-2. Taken together, UCB-MoDCs present potentially advantageous properties for future medical applications.

Background Antithrombin (AT) is an important anticoagulant in hemostasis. We describe here the characterization of a novel AT mutation associated with clinically relevant thrombosis. A pair of sisters with confirmed type I AT protein deficiency was genetically analyzed on suspicion of an inherited SERPINC1 mutation. A frameshift mutation, c.1247dupC, was identified and the effect of this mutation was examined on the cellular and molecular level. Methods Plasmids for the expression of wild-type (WT) and mutated SERPINC1 coding sequence (CDS) fused to green fluorescent protein ( GFP ) or hemagglutinin ( HA ) tag were transfected into HEK293T cells. Subcellular localization and secretion of the respective fusion proteins were analyzed by confocal laser scanning microscopy and Western blot. Results The c.1247dupC mutation results in a frameshift in the CDS of the SERPINC1 gene and a subsequently altered amino acid sequence (p.Ser417LysfsTer48). This alteration affects the C-terminus of the AT antigen and results in impaired secretion as confirmed by GFP- and HA-tagged mutant AT analyzed in HEK293T cells. Conclusion The p.Ser417LysfsTer48 mutation leads to impaired secretion, thus resulting in a quantitative AT deficiency. This is in line with the type I AT deficiency observed in the patients.

Topic: 11. Bone marrow failure syndromes incl. PNH - Biology & Translational Research Background: Aplastic anemia is characterized by bone marrow failure and multi-lineage cytopenia. T cells have been considered key players in immune-mediated aplastic anemia since immunosuppressive therapy with anti-thymocyte-globulin and cyclosporin A is an effective first-line treatment. However, phenotypes and target structures of dominant bone marrow T cell clones are poorly characterized. Aims: We identify immune-phenotypes and target antigens of dominant T cell clones in acquired aplastic anemia bone marrow at the single cell level. Methods: We applied multi-parameter flow cytometry single cell index sorting with subsequent targeted RNA sequencing of T-cell receptor (TCR) αβ and cytokine/transcription factor genes of approx. 8000 T cells from 15 patients with acquired aplastic anemia. Twenty-seven dominant CD8+ T cell clones from eight patients were re-expressed in reporter-T cells to determine reactivity against autologous in vitro expanded hematopoietic progenitor cells and viral epitopes. Results: While none of the 27 re-expressed T cell receptors was activated by CD34- bone marrow, three of them (out of two patients) were activated by magnetically enriched and in vitro expanded CD34+ bone marrow. These progenitor-reactive clones showed a CCR7-CD45RA+CD57+CD28-TBX21+ phenotype, which is indicative of chronic antigen exposure (e.g. viral antigens), and were persistent over time (follow up between five and twenty-four month). Therefore, we tested all 27 TCRs against viral peptide pools and found five of them recognizing epitopes derived from cytomegalovirus (CMV) and one from Epstein-Barr virus (EBV). Interestingly two TCRs recognized both, expanded hematopoietic progenitor cells and viral epitopes. For example, TCR 11A5 was HLA-A*02:01 restricted, recognized expanded progenitor cells, and an epitope derived from the EBV latent membrane protein 1 (LMP1). In vitro testing of an LMP1-minigene did not lead to correct processing/presentation and activation of TCR 11A5 suggesting that additional factors must have contributed to the expansion of T cell clone 11A5 in vivo. Summary/Conclusion: We show for the first time that dominant T cell clones in aplastic anemia bone marrow can be reactive with hematopoietic progenitor cells and epitopes derived from persistent viral infections. Our data suggest a role for chronic viral infections in aplastic anemia pathogenesis as drivers of progenitor cell-directed T cell immune responses. LH and FB contributed equally. Keywords: TCR, Bone marrow failure, Aplastic anemia, Antigen-specific T cells

Prothrombotic hereditary risk factors for cerebral vein thrombosis (CVT) are of clinical interest to better understand the underlying pathophysiology and stratify patients for the risk of recurrence. This study explores prothrombotic risk factors in CVT patients. An initial screening in patients of the outpatient clinic of the Department of Transfusion Medicine and Hemostaseology of the University Hospital Erlangen, Germany, revealed 183 patients with a history of CVT. An initial screening identified a number of common prothrombic risk factors, including Factor V Leiden (rs6025) and Prothrombin G20210A (rs1799963). All patients without relevant findings (58 individuals) were invited to participate in a subsequent genetic analysis of 55 relevant genes using next-generation sequencing (NGS). Three intron variants (ADAMTS13: rs28446901, FN1: rs56380797, rs35343655) were identified to occur with a significantly higher frequency in the CVT patient cohort compared to the general European population. Furthermore, the combined prevalence of at least two of four potentially prothrombic variants (FGA (rs6050), F13A1 (rs5985), ITGB3 (rs5918), and PROCR (rs867186)) was significantly higher in the CVT subjects. The possible impact of the identified variants on CVT is discussed.

The members of a Caucasian family were genetically analyzed on suspicion of hereditary protein S deficiency. A novel mutation, c.1904T>C, associated with severe quantitative protein S deficiency was found. The novel PROS1 mutation was identified by sequencing of the PROS1 gene coding sequence. The identified c.1904T>C point mutation results in p.Phe635Ser amino acid exchange, which is located in the Laminin G-like 2 domain of protein S. Computational analysis indicates that this amino acid exchange affects the correct folding of the protein S antigen. Furthermore, this mutation is located in a region of the Laminin G-like 2 domain where changes in the amino acid sequence often result in decreased secretion. We postulate that the novel p.Phe635Ser mutation might lead to an incorrect folding, and thus, to a strongly impaired secretion of this protein S variant. We named this novel variant protein.

Multiple myeloma is a malignancy of monoclonal plasma cells accumulating in the bone marrow. The critical influence of tumor-infiltrating T cells on disease control and therapeutic responses has been shown in a variety of malignancies, however, the role of multiple myeloma bone marrow-infiltrating T cells is incompletely understood. Although it has been shown that multiple myeloma neo-antigen-specific T cells can be expanded in vitro, little is known about functions and specificities of clonally expanded multiple myeloma-infiltrating bone marrow T cells. Here we asked at the single cell level whether clonally expanded T cells i) were detectable in multiple myeloma bone marrow and peripheral blood, ii) showed characteristic immune phenotypes, and iii) recognized antigens selectively presented on multiple myeloma cells. A total of 6,744 single bone marrow T cells from 13 treatment-naïve patients were index-sorted and sequenced using our methodologies for determination of paired T cell receptor (TCR) αβ sequences along with immune phenotype, transcription factor and cytokine expression. Clonal T cell expansion occurred predominantly within the CD8+ compartment. Phenotypes of clonally expanded T cells were distinctive of cytolytic effector differentiation and significantly different from non-expanded CD8+ T cells. Less than 25% of expanded CD8+ T cell clones expressed the immune checkpoint molecules programmed death-1 (PD-1), cytotoxic T lymphocyte antigen-4 (CTLA-4), or T cell immunoglobulin and mucin-domain containing-3 (TIM-3), while B and T lymphocyte attenuator (BTLA) was expressed on more than half of the expanded clones. Clonal T cell expansion did not correlate with neo-antigen load as determined by whole exome and RNA sequencing of purified multiple myeloma cells. Furthermore, peripheral blood TCRβ repertoire sequencing from five selected patients with substantial bone marrow T cell expansion identified 90% of expanded bone marrow T cell clones overlapping with peripheral blood. To determine whether clonally expanded bone marrow T cells recognized antigens selectively presented on multiple myeloma cells, 71 dominant TCRs from five selected patients with substantial clonal T cell expansion were re-expressed in 58α-β- T-hybridoma reporter T cells and co-incubated with CD38-enriched multiple myeloma cells from the same patients. Only one of these TCRs recognized antigens selectively presented on multiple myeloma cells and this TCR was not neo-antigen-specific. Hypothesizing that the target antigen was a non-mutated self-antigen, we could show that this TCR also recognized the plasma cell leukemia cell line U-266 in an HLA-A*02:01-restricted manner. In summary, clonally expanded T cells in multiple myeloma bone marrow of newly diagnosed patients show cytolytic effector differentiation. In the majority of patients, clonally expanded bone marrow T cells do not recognize antigens presented on multiple myeloma cells and are not neo-antigen-specific. Our findings are relevant for the design of future therapeutics and clinical trials. The identified TCR, which recognizes a multiple myeloma antigen shared with U-266 in an HLA-A*02:01-restricted manner, could be a promising candidate for T cell therapy. Disclosures Bullinger: Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Hexal: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Menarini: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees.

A 36-year-old female (Wo) delivered a full-term girl in the 40th week of gestation with no signs of bleeding. Her neonatal platelet count was 29 G/L (dropping to 20 G/L), and fetal-neonatal alloimmune thrombocytopenia (FNAIT) was suspected. In the serological work-up, no anti-platelet antibodies were detectable using PAK Lx (Immucor Medizinische Diagnostik). Genetic testing did not reveal incompatibility for HPA-1, -2, -3, -4, -5, -6, -9 and -15. Two years later, Mrs. Wo gave birth to a white European full-term boy in the 39th week of gestation, again with no signs of bleeding. The neonatal platelet count was 31 G/L. Platelet antibody testing in the monoclonal antibody of platelet specific antigens (MAIPA) assay using random donor platelets was negative. In contrast, a MAIPA cross-match analysis between maternal serum and paternal platelets showed positive reactions with αIIbβ3, indicating an alloimmunization against a lowfrequency antigen. On Day 1, the newborn was transfused with random donor platelets. The platelet count rose to 170 G/L, remained above 50 G/L without further transfusions, and was in normal range at Day 28.

HLA‐A*01:234 was identified by next‐generation sequencing and confirmed by Sanger sequencing.

HLA‐C*01:136 identified by next generation sequencing and confirmed by Sanger sequencing.

BACKGROUND:For cryopreservation of stem cells, a cryoprotective agent is essential. Dimethyl sulfoxide is commonly used, but has deleterious effects on cell vitality in the warmth and for the recipients of stem cells. The aim of the study was to reduce DMSO and find one cryoprotective solution suitable for stem cells of different origin. Materials: Small volumes of both stem cell apheresis products and cord blood derived stem cells were frozen using six different cryoprotective solutions. Suitability of these solutions was tested by comparing cell vitalities and recovery rates of CD45 and CD34 positive cells and colony forming unit recovery rates.RESULTS:No single cryoprotective solution being significantly superior regarding all cell qualities and recovery rates could be identified. However, mixing approximately 5% DMSO with hydroxyethyl starch with a molecular weight of 450,000 Dalton showed better results for most qualities examined than DMSO alone, especially when looking at cord blood derived stem cells.CONCLUSIONS:There might not be an all-in-one cryoprotective solution suitable for every purpose regarding the cryopreservation of stem cell concentrates produced from different cell sources. However, when trying to reduce the DMSO amount used, hydroxyethyl starch of a molecular weight of 450,000 Dalton is a suitable option.

Background and objectives Monocytes can be cultured into dendritic cells with addition of autologous plasma, which is highly prone to platelet contamination due to the apheresis process. Since platelets affect the maturation process of monocytes into dendritic cells and might even lead to a diminished harvest of dendritic cells, it is very important to reduce the platelet contamination. A new collection device (Spectra Optia) was analyzed, compared to two established devices (COM.TEC, Cobe Spectra) and evaluated regarding the potential generation of source plasma. Materials and methods Concurrent plasma collected during leukapheresis was analyzed for residual cell contamination in a prospective study with the new Spectra Optia apheresis device (n = 24) and was compared with COM.TEC and Cobe Spectra data (retrospective analysis, n = 72). Donor pre-donation counts of platelets were analyzed for their predictive value of contaminating PLTs in plasma harvests. Results The newest apheresis device showed the lowest residual platelet count of the collected concurrent plasma (median 3.50 × 109/l) independent of pre-donation counts. The other two devices and sets had a higher platelet contamination. The contamination of the plasma with leukocytes was very low (only 2.0% were higher than 0.5 × 109/l). Conclusions This study showed a significant reduction of platelet contamination of the concurrent plasma collected with the new Spectra Optia device. This plasma product with low residual platelets and leukocytes might also be used as plasma for fractionation.

We have recently shown that memory B cells from murine CMV immune donor animals adoptively transferred into immunodeficient mice were highly effective in protecting from a viral infection indicating a therapeutic potential of virus specific memory B cells. These preclinical data provided evidence that a cell-based strategy supporting the humoral immune response might be effective in a clinical setting of immunodeficiency after allogeneic hematopoietic stem cell transplantation. As adoptive transfer of B cells has not been used before in a clinical setting it was necessary to establish a technology for the generation of good manufacturing practice (GMP)-grade B cell products.

BACKGROUND Quantification of CD34+ cells in peripheral blood stem cell apheresis is normally performed by single platform flow cytometric measurements according to the ISHAGE protocol. Peripheral blood stem cell concentrates (PBSC) produced by apheresis normally contain many T cells. Those T cells can be used for production of donor lymphocyte infusion doses, if abundant amounts of CD34+ cells have been collected. Therefore, it is of interest to know both the CD3+ and the CD34+ cell count of allogeneic PBSC. This is the first study comparing the performance of a modified ISHAGE protocol allowing additional quantification of CD3+ cells on two different flow cytometers, the FACSCalibur and the FACSVerse, respectively. METHODS CD45+ and CD34+ cell concentrations were measured using a standard and a modified ISHAGE protocol including CD3+ cell quantification on both machines. All cell concentrations were measured using a Trucount bead based stem cell enumeration kit. The FACSVerse machine can additionally be equipped with a sample volume sensor allowing cell quantification without using beads. The samples analysed were taken from granulocyte-colony-stimulating factor mobilized peripheral blood stem cell apheresis procedures (pre- and post-apheresis, and apheresis concentrate). RESULTS There were no significant differences in cell concentrations measured by the standard and modified ISHAGE protocol, regardless of which machine had been used when using bead quantification. No significant differences between the results of the two flow cytometers using the modified ISHAGE protocol were observed. Pearson´s correlation was always > 0.96, and regression coefficients were higher than 0.93. The only significant differences were observed between bead quantification and volume sensor quantification on the FACSVerse machine. CONCLUSIONS The modified ISHAGE protocol can effectively be used on both flow cytometers tested, especially if bead quantification is used.

BACKGROUND:We present a multivariate test system using flow cytometry after in-vitro lymphocyte stimulation using 5 mitogens and 7 antigens to describe in-vitro immunofunction. METHODS:The present work is a crucial step towards establishing a simple, CFSE-based, multivariate test system that can describe the dynamics of stimulus-induced lymphocyte proliferation with considerably more precision than is possible with the radionucleotide method using 3H-thymidine. Using multicolour flow cytometry, our method allows additional phenotyping of the proliferating cells and quantifies the proliferation behaviour by precisely resolving daughter generations and determining the precursor frequency. CONCLUSIONS: Taking the calculated apoptosis parameters into account not only provides additional information about the stimulus-specific response behaviour but also improves the validity of the commonly used proliferation indices. Not only can we confirm previous findings that healthy people have marked differences in a multivariate test system in terms of the individual in-vitro reactivity to various stimuli but also substantiate that the response pattern of an individual is remarkably constant. In follow-up studies we can show for the first time that the results of immunofunctional testing do not change over a period of at least 6 months and appear to be an inherent characteristic of the individual and thus possibly have a genetic basis.

ThromboLUX (TLX)‐Score was compared with hypotonic shock response (HSR) and extent of shape change (ESC) in 99 samples from 42 platelet concentrates. Tests were performed in parallel and duplicate. Mean values for TLX Score, HSR and ESC were 30·3 ± 3·8%, 69·0 ± 12·2% and 23·2 ± 4·9%, respectively. We found no significant correlation between TLX Score and HSR or ESC (r = −0·158, P = 0·118 and r = −115, P = 0·255, respectively), whereas HSR and ESC correlated significantly (r = 0·351, P < 0·001). As TLX Score did not show significant correlation with HSR and ESC, the value of TLX for platelet quality testing remains unclear. Studies comparing these parameters with transfusion outcome are needed.

Background and ObjectivesRegulatory T cells (Tregs) and other T‐cell subsets are of importance in the setting of autologous and allogeneic stem cell transplantations. We conducted a study to assess the content of peripheral blood stem cell concentrates and related apheresis parameters in the autologous and allogeneic setting.Material and MethodsWe characterized 53 donors, patients and peripheral blood stem cell concentrates (PBSC) regarding the content of CD45+ cells, lymphocytes, CD3+ cells, CD3+ CD4+ T cells, CD3+ CD4+ CD25+ T cells, CD3+ CD4+ CD25+ CD127low/negative Tregs and CD34+ cells and calculated cell yields, recruitment factors and collection efficiency for all cell types. We compared allogeneic data with autologous data.ResultsAutologous PBSC show significantly lower concentrations of T‐cell subsets compared to allogeneic PBSC (17 112/μl CD4+, 14 858/μl CD4+ CD25+ and 1579/μl CD3+ CD4+ CD25+ CD127low/negative Tregs in autologous compared to 65 539/μl CD4+, 44 208+/μl CD4+ CD25+ and 5040/μl CD3+ CD4+ CD25+ CD127low/negative Tregs in allogeneic PBSC, respectively), in contrast to CD34+ concentrations (5342/μl CD34+ in autologous compared to 2367/μl CD34+ in allogeneic PBSC, respectively). Accordantly, all T‐cell yields are lower in the autologous setting compared to allogeneic PBSC. However, recruitment factor and collection efficiency of all cell types are higher in autologous compared to allogeneic PBSC, but not all parameters differ significantly when groups are compared.ConclusionT‐cell subsets and especially Tregs are a substantial part of PBSC transplantation, as considerable recruitment during apheresis occurs. In large volume apheresis, the collection efficiency of Treg is comparable to that of CD34+ cells, while recruitment factors are even higher.

BackgroundThe only accepted way to avoid transfusion-associated graft-versus-host disease is irradiation of blood components. With respect to irradiation and associated storage time, different recommendations exist. We examined early irradiated (day +3) leucoreduced red blood cell units for irradiation-associated damages during storage.Study Design and MethodsWe studied 80 leucoreduced units from two manufacturers. All RBCs were stored in the additive solution saline-adenine-glucose-mannitol (SAG-M) and leucoreduced on collection day. Forty components were irradiated on day +3 with 30 Gy, 40 served as non-irradiated controls. Samples were drawn and analysed from these 80 units on days +3, +7, +14, +21, +28, +35 and +42.ResultsFrom day +14, there was a significant difference in the in vitro haemolysis rate between the non-irradiated and the irradiated components. Two of the irradiated units showed a haemolysis rate over the recommended limit of 08% on day +42. Potassium and lactate dehydrogenase activity increased faster in irradiated groups during storage.ConclusionsOur findings show that leucoreduced RBCs which are irradiated early after collection show an obvious radiation damage over storage period. Interestingly, on days +28, +35 and +42 the quality of RBCs which were irradiated on day +3 was nearly identical to the quality of RBCs which were irradiated on day +14 in a former series. Early irradiation does not cause more damage of RBCs during subsequent storage than irradiation on day +14. The maximum storage period of irradiated RBCs should remain restricted to 28 days from collection, however independently from the day of irradiation within this period.