bluuebunny/crossref_metadata_2025_split huggingface dataset:
Peptide-Major Histocompatibility Complex Class I Tetramers with Enhanced Coreceptor Binding Properties Enable Visualization of Low Avidity Leukemia-Associated Antigen-Specific CD8+ T Cells.
Authors: Melenhorst J. Joseph , Scheinberg Phillip , Gostick Emma , Chattopadhyay Pratip K. , Lissina Anna , Cole David K. , Roederer Mario , Bornstein Ethan , Hensel Nancy F. , Douek Daniel C. , Sewell Andrew K. , Barrett A. John , Price David A. 
Published: October 2019

Abstract
There is substantial evidence to indicate that CD8+ T cells specific for epitopes derived from overexpressed antigens form part of the immune response to leukemia in patients both prior to and after stem cell transplantation (SCT) and following adoptive immunotherapy with donor leukocyte infusion. However, the immunobiology of these CD8+ T cell populations has proven difficult to study directly ex vivo due to technical obstacles that preclude the reliable detection of low frequency CD8+ T cells that bind antigen poorly. We engineered a mutation (Q115E) in the heavy chain a2 domain of HLA-A*0201 that moderately enhances monomeric CD8 binding without impacting the TCR binding platform. Combined flow cytometric studies and biophysical analysis of recombinant TCR/peptide-major histocompatibility complex class I (pMHCI) interactions showed that tetrameric complexes of these coreceptor-enhanced HLA-A*0201 molecules were more sensitive reagents for the detection of specific CD8+ T cells at the clonal level compared to corresponding tetramers produced with wildtype HLA-A*0201 complexes; thus, the threshold of detection was enhanced approximately twofold relative to the monomeric TCR/pMHCI cut-off (KD=80mM) observed with the wildtype tetramer. These novel reagents were incorporated in a polychromatic flow cytometric panel to detect CD8+ T cell populations specific for HLA-A*0201-restricted epitopes derived from Wilm’s tumor-1 (WT1) and proteinase 3 (PR1) in the bone marrow and peripheral blood of patients with acute myeloid leukemia before SCT. The HLA-A*0201-restricted CMV pp65-derived epitope NLVPMVATV, which induces high avidity cognate CD8+ T cell populations, was used as a control. Both wildtype and coreceptor-enhanced tetramers consistently identified populations of NLVPMVATV-specific CD8+ T cells that were identical in magnitude; molecular analysis of TCR usage in these populations after high resolution flow cytometric sorting confirmed the equivalent cognate binding properties of these reagents. In contrast, coreceptor-enhanced tetramers enabled the visualization and simultaneous immunophenotyping of CD8+ T cell populations specific for PR1 and WT1 in patients with AML that were not detectable with the corresponding wildtype reagent. These results provide the first validation of a novel approach to the physical detection of low avidity antigen-specific CD8+ T cell populations by flow cytometry; such coreceptor-enhanced tetrameric reagents are likely to be useful in a multitude of settings for the detection of tumor-specific and autoreactive CD8+ T cells.
Modulation of Tryptophan Catabolism by Acute Myeloid Leukemia Cells Acts as a General Mechanism of Immune Tolerance Via the Induction of T Regulatory Cells.
Authors: Curti Antonio , Salvestrini Valentina , Aluigi Michela , Trabanelli Sara , Ottaviani Emanuela , Durelli Ilaria , Horenstein Alberto L. , Fiore Francesca , Massaia Massimo , Piccioli Milena , Pileri Stefano , Martinelli Giovanni , Baccarani Michele , Lemoli Roberto M. 
Published: October 2019

Abstract
Indoleamine 2,3-dioxygenase (IDO) enzyme, which catalyzes the conversion of tryptophan into kynurenine, has been identified as a novel immunosuppressive agent by inhibiting T-cell proliferation and is involved in tolerance induction to tumors. We have recently shown that IDO protein is constitutively expressed in a significant subset of newly diagnosed acute myeloid leukemia (AML) patients, resulting in tryptophan catabolism along the kynurenine pathway and in the inhibition of allogeneic T-cell proliferation. Moreover, we demonstrated that IDO-expressing AML cells are capable to promote the differentiation of new CD4+CD25+Foxp3+ T regulatory cells (Treg cells). AML cells may be differentiated into leukemic dendritic cells (AML-DCs) which have increased immunogenicity and may be used as vaccine against leukemia. We in vitro generated DCs from 7 AML samples according to standard procedures and tested IDO expression and activity. At baseline, 5/7 AML samples expressed IDO, whereas 2/7 did not. After differentiation into DCs, IDO+ AML samples showed an up-regulation of IDO mRNA and protein, and IDO− AML cells turned positive. IDO-expressing AML-DCs were capable to catabolize tryptophan into kynurenine metabolite and, functionally, they inhibited allogeneic T-cell proliferation through an IDO-dependent mechanism. Similarly to undifferentiated IDO+ AML blasts, IDO-expressing AML-DCs induced a population of CD4+CD25+Foxp3+ Treg cells, which were capable to suppress in vitro allogeneic naïve T-cell proliferation. These data identify IDO-mediated catabolism as a general tolerogenic mechanism in AML cells, including AML-DCs and raise several concerns for the use of AML-DCs as cellular vaccine against leukemia.
Malignant B Cells Skew the Balance between Treg Cell and TH17 Cell Differentiation in B-Cell Non-Hodgkin Lymphoma (NHL).
Authors: Yang Zhi-Zhang , Novak Anne J. , Witzig Thomas E. , Ansell Stephen M. 
Published: October 2019

Abstract
Numerous clinical therapies have attempted to modulate tumor cell immunity, but for the most part, have proven unsuccessful. The inability to produce or augment an effective immune response is due in part to regulatory T (Treg) cells, which inhibit CD4 and CD8 T cell function. Our group has recently shown that Treg cell numbers are elevated in NHL tumors and that NHL B cells induce the development of Treg cells thereby inhibiting anti-tumor responses. The ability of NHL B cells to direct the cellular composition of their microenvironment is critical to our understanding of tumor immunity and we therefore wanted to determine if NHL B cells also directed the expansion or reduction of other T cell populations. IL-17-secreting CD4+ T cells (TH17), a newly characterized CD4+ T helper cell lineage, promote inflammation and play an important role in autoimmune disease. IL-17 has been shown to inhibit tumor cell growth suggesting a potential role for TH17 cells in anti-tumor immunity. We therefore set out to determine if TH17 cells were present in NHL tumors and whether or not their numbers were regulated by NHL B cells. Using unsorted mononuclear cells from malignant lymph nodes, we were unable to detect IL-17 expression in resting CD4+ T cells or CD4+ T cells activated with PMA/Ionomycin stimulation (less than 1%). However, IL-17-secreting CD4+ T cells could be detected in significant numbers in inflammatory tonsil and normal PBMCs. Interestingly, depletion of CD19+ NHL B cells from mononuclear cells obtained from patient biopsies resulted in detection of a clear population of IL-17-secreting CD4+ T cells (5%). These results suggest that NHL B cells suppress TH17 cell differentiation. The frequency of IL-17-secreting CD4+ T cells could not be further enhanced by the addition of exogenous TGF-b and IL-6, a cytokine combination favoring for TH17 differentiation, suggesting a further impairment of TH17 cell differentiation in the tumor microenvironment. In contrast, Foxp3 expression could be detected in resting CD4+ T cells (30%) and could be induced in CD4+CD25−Foxp3− T cells activated with TCR stimulation (28%). Contrary to the inhibition of TGF-b-mediated TH17 differentiation, Foxp3 expression could be dramatically upregulated by TGF-b in intratumoral CD4+ T cells (35%). In addition, lymphoma B cells strongly enhanced Foxp3 expression in intratumoral CD4+CD25−Foxp3−. Furthermore, when added together, the frequency of Foxp3+ T cells and Foxp3-inducible cells reached up to 60% of CD4+ T cells in tumor microenvironment of B-cell NHL. These findings suggest that the balance of effector TH17 cells and inhibitory Treg cells is disrupted in B-cell NHL and significantly favors the development of inhibitory Treg cells. Our data indicate that lymphoma B cells are key factor in regulating differentiation of intratumoral CD4+ T cells toward inhibitory CD4+ T cells.
Surface Sialic Acid Prevents Loss of GPIbα during Platelet Storage and Rescues In Vivo Survival of Murine Platelets.
Authors: Jansen Gerard , Josefsson Emma C. , Hartwig John H. , Hoffmeister Karin M. 
Published: October 2019

Abstract
Platelet processing and storage are associated with platelet lesion, e.g. shape change, activation, release reaction and apoptosis, which is partially due to loss of surface receptors. Surface sialic acid is considered to be a key determinant for the survival of circulating blood cells and glycoproteins. However, its role in platelet receptor loss and platelet survival is unclear. In this study, the relationship between surface sialic acid and platelet receptor loss was investigated in vitro and in vivo. Murine platelets stored at room temperature for 6 hours lost surface sialic acid, as evidenced by flow cytometry using FITC conjugated RCA I lectin, which recognizes exposed galactose residues. This loss correlated with a 30–60% loss of surface receptors GPIbα and GPV, but not GPIX and integrin αIIbβ3, as measured by flow cytometry. Treatment of murine platelets with the neuraminidase (NA) substrate fetuin partially decreases the loss of GPIbα and GPV to 10–20%. In vitro, sialic acid was cleaved from the platelet surface by adding NA (α2-3,6,8-NA (V. cholerae) or α2-3,6,-NA (C. perfringens)) to murine platelets. Removal of sialic acid correlated with the removal of 50–60% of surface GPIbα and GPV, but not GPIX and integrin αIIbβ3. Addition of fetuin, or the more specific NA inhibitor 2,3-dehydro-2-deoxy-, sodium salt (DANA), completely prevented this loss, as determined by both flow cytometry and Western blot analysis. Murine platelets treated with α2-3,6,8-NA (V. cholerae) ± the addition of DANA were labeled with the green dye CMFDA and transfused into age-, strain- and sex-matched C57BL/6 mice to measure platelet survival. NA-treated platelets were cleared within minutes after transfusion, whereas the addition of DANA rescued platelet survival to control-count increments. Our study shows that inhibiting the loss of surface sialic acid prevents platelet surface GPIbα and GPV loss during storage in vitro and rescues platelet survival in vivo.
Combined Inhibition of CDK and PI3K/Akt Pathways Induces Apoptosis of Mantle Cell Lymphoma.
Authors: Mohapatra Subhra , Chu Baoky , Zhao Xiuhua , Tao Jianguo , Sotomayor Eduardo , Pledger W. Jack 
Published: October 2019

Abstract
Mantle cell lymphoma (MCL) arises from the neoplastic transformation of naïve B cells in the mantle zone of the B cell follicle. It is an aggressive and incurable B cell neoplasm. It accounts for 5% to 8% of non-Hodgkin’s lymphomas. Patients with MCL respond initially to chemotherapy but ultimately relapse. Mean survival time is only three to four years. Thus, the need for new treatments that effectively combat MCL is obvious and imperative. Defects in cell cycle regulation and apoptosis are primary events in MCL. It is characterized by the presence of a chromosomal translocation t (11:14)(q13:q32), which results in deregulated cyclin D1 expression. Cyclin D1 overexpression in MCL is thought to play a major role in lymphomagenesis, although the precise mechanisms by which tumor formation and progression occur are not fully understood. Constitutive activation of the PI3K/Akt pathway contributes to the pathogenesis and survival of MCL. Activated AKT was found in cultured MCLs as well as in 100% of aggressive-blastoid variants of MCL tumors, compared to 30% of typical MCLs. Towards the search for novel therapies for MCLs, we examined the potential of roscovitine (rosc) or flavopiridol (flav), inhibitors of cyclin dependent kinase (CDK), as a single agent or in combination with LY294002 (LY), a PI3K/Akt inhibitor, in inducing apoptosis of various MCL lines as well as in MCL patient samples. CDK inhibitors modestly increased the percentage of apoptotic cells (∼30%), whereas LY had no effect. However, when added in combination, rosc/LY or flav/LY induced apoptosis in more than 70% of cells. In an effort to understand the mechanism of apoptosis, we identified three targets: cyclin D1, the anti-apoptotic proteins myeloid cell leukemia-1 (Mcl-1) and X-linked Inhibitor of Apoptosis (XIAP). CDK inhibitors eliminated Mcl-1 expression, slightly reduced XIAP abundance and had very little effect on abundance of cyclin D1. Conversely, LY reduced cyclin D1 expression and slightly reduced the abundance of Mcl-1 and XIAP. A larger decrease in XIAP abundance is seen in cultures treated with a combination of rosc/LY or flav/LY. On the basis of these findings, we suggest that agents that target Mcl-1, XIAP and cyclin D1 will be most effective in inducing apoptosis of human MCLs.
Efficacy of FDG-PET/CT Imaging To Guide Biopsies in the Detection of Richter’s Syndrome.
Authors: Le Gouill Steven , Bodet-Milin Caroline , Kraeber-Bodere Françoise , Moreau Anne , Campion Laurent , Dupas Benoit , Gastinne Thomas , Moreau Philippe , Harousseau Jean-Luc 
Published: October 2019

Abstract
Introduction: Most lymphomas show a good FDG uptake, with significant difference in intensity according to different histopathological subtypes. Richter’s syndrome </DEL> (RS) is defined as the development of aggressive lymphoma in patients with low grade histopathological subtypes. Diagnosis requires histological findings. The aim of our study was to investigate prospectively FDG-PET/CT to guide biopsy in the diagnosis of aggressive transformation of low grade lymphomas.
Material and methods: Thirty-eight patients, presenting clinical and/or biological signs of aggressive transformation were included. The 38 underwent FDG-PET/CT. FDG-PET/CT scans were analysed visually and semi-quantitatively with maximal standardised uptake value (SUVmax) normalised for body mass. Guided-biopsy was performed 4 weeks after FDG-PET/CT, in the abnormal focus showing the highest SUVmax and histopathological analysis was performed.
Results: Histopathological analysis concluded to aggressive transformations in 17 cases (15 NHL and 2 Hodgkin diseases included one composite lymphoma) and low grade lymphomas in 21 cases. Size of lesions on CT was not predictive of RS (p=0.094). FDG-PET/CT images showed abnormal FDG uptake in the 38 included patients. SUVmax ranged from 11.7 to 41.2 in patients with Richter’s syndrome and from 1.7 to 17.0 in patients with no sign of transformation(p<0.0001). Gradient of SUVmax was significantly higher in patients with Richter’s syndrome (p<0.0001). A SUVmax threshold of 14.0 gave the better balance with sensitivity, specificity, predictive positive and negative values around 95.0%. This threshold of 14.0 correctly classified as transformed or not transformed 36/38 patients (94.7%).
Conclusion: FDG-PET/CT imaging is a very relevant tools to detect aggressive transformation of low grade lymphomas and to guide biopsy.
Human Hematopoietic and Mesenchymal Stem Cells Are Interconnected by Cadherin-Catenin Based Junctions.
Authors: Wuchter Patrick , Saffrich Rainer , Schubert Mario , Wagner Wolfgang , Lannert Heinrich , Eckstein Volker , Ho Anthony D. 
Published: October 2019

Abstract
The direct cell-cell contact between human hematopoietic stem cells (HSC) and their niche has been shown to control stem cell fate by regulating self-renewal versus differentiation. In previous studies we have demonstrated gap and adherens junctions arranged in complex villiform, vermiform, intercalating protrusions and invaginations among mesenchymal stem cells (MSC). Specific cadherin-catenin-molecules, among other junction proteins, were found in these contact systems. Using MSC-feeder-layer as an in vitro surrogate model for the hematopoietic stem cell niche, we have analyzed the intercellular junctional complexes between HSC and MSC in this study. Additionally, we characterized cell-cell-connections between leukemia stem cells (LSC) and MSC. MSC were derived from bone marrow aspirates from healthy voluntary donors. HSC were isolated from umbilical cord blood. Leukemia stem cells were obtained from bone marrow aspirates from patients suffering from acute myeloid leukemia either at the time of initial diagnosis or at relapse after chemotherapy. After 24–48 hours of co-cultivation, we have stained the cell-cell contacts with a panel of antibodies specific for various components of tight, gap and adherens junctions including alpha-/beta-catenin, N-cadherin, Cadherin 11, E-Cadherin, p120, Vinculin and Connexin 43. Using advanced confocal laser scanning microscopy in combination with deconvolution and volume rendering software, we were able to produce 3D-images of intercellular junctions between HSC/MSC as well as between LSC/MSC. We have confirmed previous observations that HSC demonstrated directed locomotion towards a MSC gradient. HSC then migrated around the MSC and some of them are then interconnected by podia formation to the MSC. These podia are directly linked to the MSC feeder layer and at the intimate contact zone we have identified N-cadherin as well as alpha-/beta-catenin as the main junction proteins. The specific function of this cadherin/catenin-complex is probably associated with redistribution of beta-catenin and hence to self-renewing division, similar to the findings in stem cells of drosophila model systems. Functional analyses are currently underway to confirm this hypothesis. We have also compared these findings with a similar setting consisting of human LSC co-cultured with MSC-feeder-layer.
Human Placental Derived Mesenchymal Stromal Cells (MSC) Grown in 3D-Culture (PLX-I), Promotes Engraftment of Human Umbilical Cord Blood (hUCB) Derived CD34+ Cells in NOD/SCID Mice.
Authors: Burger Ora , Ashtamker Galit F. , Bercovich Noam , Rusanovsky Marina , Pinzur Lena , Jacov Osnat M. , Carmi Nirit D. , Meiron Moran , Lior Limor , Meretzki Shai , Prather William R. , Kadouri Avinoam , Treves Avi J. , Rowe Jacob M. , Horwitz Edwin M. , Nagler Arnon 
Published: October 2019

Abstract
The use of UCB for transplantation in adults is limited due to the low cell number in each unit. Engraftment rates are lower and the time for neutrophil and platelet recovery is longer following CBT compared to bone marrow (BM) transplants. Co-transplantation of human hematopoietic stem/progenitor cells (HSC) with mesenchymal stromal cells in an irradiated NOD/SCID mice model has been demonstrated to promote HSC engraftment. Using PluriX technology, we expanded human placental derived MSC on 3D-carriers in a bioreactor (PLX-I) and evaluated its potential to promote UCB engraftment in the NOD/SCID mice model. Flow cytometer analysis detected high rates of expression of CD105, CD73, CD90 and CD29 and lacked expression of CD45, CD34 and CD19 surface markers in the PLX-I cells. PLX-I cells were capable of differentiation into bone, adipose tissue and cartilage under specific inductive conditions. Additionally, they possess both immune privileged and immune suppressive characteristics in a mixed lymphocyte reaction (MLR) assay. In this study 60 – 100x103 hUCB derived CD34+ cells were injected into the tail vein of 7–8 week old NOD/SCID mice along with 0.5x106 PLX-I cells. Human cell engraftment was tested in both sublethally irradiated (350 rad) and chemotherapy (50 mg/kg busulfan) treated NOD/SCID mice. Following 5–6 weeks, mice BM FACS analysis showed a significant increase in % hCD45+ rate in mice transplanted with PLX-I cells compared to mice transplanted with CD34+ cells alone: 13.6 vs. 31.7, p=0.01, (n=6) in the irradiation setting and 28.8 vs 6.3, p<0.05 (n=7) in the chemotherapy setting. These preclinical results demonstrate the potential of human placental-derived MSCs, grown as a 3D culture (PLX-I), to promote hUCB CD34+ cell engraftment in BM. Co-transplantation of PLX-I may be considered for improving the delayed engraftment using CB as the source of HSC.
Modulation of Fox-Regulated Alternative Splicing Events during Erythropoiesis.
Authors: Gallagher Thomas L. , Gee Sherry L. , Schluepen Christina L. , Conboy John G. 
Published: October 2019

Abstract
A switch in the alternative splicing pattern in protein 4.1R pre-mRNA, involving activation of exon 16 splicing, occurs in late erythropoiesis. Because the inclusion of exon 16 leads to synthesis of protein isoforms with high affinity for spectrin and actin, and mechanical strengthening of the membrane, this switch is critical for normal erythyroid differentiation. Previous studies have shown that Fox-2 protein is a splicing regulator that binds to intronic enhancer sequences downstream of exon 16, and that this binding stimulates the inclusion of exon 16. Fox binding sites are also located downstream of other tissue-specific alternative exons, some of which exhibit variable splicing efficiencies, leading us to investigate whether splicing switch strength is influenced by variations in the sequence of the Fox binding-site motif (UGCAUG) and by the number of these protein binding sites. A series of minigenes was created in which protein 4.1R E16 splicing efficiency was measured in transfection studies. Mutation of the first U residue of the Fox binding site resulted in a significantly weaker, but still Fox dependent activation of splicing, whereas mutation of the terminal G residue dramatically reduced enhancer activity. Insertion of two wild-type UGCAUG elements enhanced splicing substantially in a Fox protein concentration-dependent manner, and four elements gave even stronger inclusion. To test whether the reduced splicing efficiency observed for minigenes containing mutated enhancer elements was due to a reduction in Fox binding, surface plasmon resonance (SPR) was employed. Our binding studies indicate that full-length mouse Fox-2a bound to the UGCAUG sequence with high affinity (KD ∼100nM). Furthermore, in agreement with our functional splicing assays, Fox binding affinity for the hexameric sequence was reduced with mutations in position one, and almost eliminated with mutations in position six. There was an excellent correlation between binding affinity of Fox protein for the enhancer motif, and strength of enhancer activity measured in functional splicing assays. Additional studies demonstrated that Fox enhancer activity can be reduced by the presence of a closely linked binding site for hnRNP A1, a known splicing inhibitor. Taken together, these studies demonstrate that the composition and number of binding sites for Fox can significantly affect the efficiency of splicing for E16. It is likely that through these combinatorial mechanisms, Fox splicing factors are able to modulate the efficiency with which tissue specific exons in the erythroid alternative splicing program can be spliced into mature mRNA.