bluuebunny/crossref_metadata_2025_split huggingface dataset:
TLR9 and Dendritic Cells Are Required for CD8+ T Cell Responses to the AAV Capsid
Authors: Rogers Geoffrey L. , Herzog Roland W 
Published: October 2019

Abstract
CD8+ T cell responses to the adeno-associated virus (AAV) capsid have posed a significant barrier to transduction in clinical trials of AAV-mediated gene therapy for hemophilia B, as reactivation of a memory CTL response to the capsid is capable of eliminating transduced hepatocytes in the absence of immunosuppression. Recently, it has been suggested that innate immune responses induced by the toll-like receptor (TLR) pathway can influence the development of adaptive immune responses to AAV-mediated gene transfer. In particular, reports have implicated TLR2 (AAV capsid), TLR9 (AAV genome), and MyD88 (downstream signaling adaptor of both these TLRs). Herein, we have used a modified AAV2 with an insertion of the immunodominant MHC class I epitope of ovalbumin into the capsid (AAV2-SIINFEKL) to study the mechanism of CD8+ T cell responses to the AAV capsid. Using an H2-Kb-SIINFEKL tetramer reagent, we determined that anti-capsid CD8+ T cell responses depended on the TLR9-MyD88 pathway. While the frequency of circulating capsid-specific CD8+ T cells peaked around 7-10 days post-injection and subsided after about 21 days in wild type (WT) mice, tetramer-positive cells were not detected in TLR9-/- or MyD88-/- mice. The kinetics and magnitude of the response was unaltered in TLR2-/- mice. Mice deficient in STING, a downstream adaptor of multiple cytoplasmic DNA sensing pathways, also developed comparable capsid-specific CD8+ T cell frequencies to WT mice, suggesting that this is not a general effect of pattern recognition of DNA. Interestingly, the frequency of capsid-specific CD8+ T cells was not reduced in AP3-/- mice, which are deficient in type I IFN signaling downstream of TLR9. Adoptively transferred OVA-specific OT-1 T cells proliferated in WT but not TLR9-/- mice that received AAV2-SIINFEKL, confirming the importance of TLR9. The effect was antigen-specific, as OT-1 cells in WT mice that received AAV2 lacking SIINFEKL showed minimal proliferation comparable to TLR9-/- mice. In addition to pattern-recognition receptors, we also assessed the role of antigen-presenting cells in the CD8+ T cell response to capsid. The formation of capsid-specific CD8+ T cells was unaltered in mice that received gadolinium chloride to inactivate macrophages, or in B cell-deficient μMT mice. Depletion of B cells in WT mice prior to vector administration also failed to affect the anti-capsid CD8+ T cell response. However, transient depletion of dendritic cells (DCs) in CD11c-DTR mice resulted in a delayed development of capsid-specific CD8+ T cells. Seven days post-injection, DC-depleted mice had a significantly reduced frequency of tetramer-positive CD8+ T cells which recovered to normal by 10 days, likely due to the repopulation of DCs before the input capsid was completely cleared. Overall, our results show that TLR9 signaling, most likely in DCs, is required for the formation of de novo anti-capsid CD8+ T cell responses.

Disclosures
Herzog: Genzyme: AAV-FIX technology Patents & Royalties.
Long Term Trends in Chronic Myeloid Leukemia (CML) Following the FDA Approval of Tyrosine Kinase Inhibitors (TKIs), on Bone Marrow Transplant Rates and Clinical Outcomes - a Study of the Nationwide Inpatient Sample Between 1998-2011
Authors: Patel Parshva , Varadarajan Indumathy , Varma Gaurav , Christina Trotta , Ward Kristine , Jain Maneesh , Styler Michael 
Published: October 2019

Abstract
Background: Single-institution and international, retrospective studies have demonstrated a decrease in the number of allogeneic stem cell transplants (alloSCTs) in patients with CML in the first few years after the introduction of TKIs. However alloSCT remains the only curative therapy in patients who develop resistance to multiple TKIs, and in those who present with blast crises. The long-term impact of TKIs on the trend of SCTs in the United States in CML remains unexplored. Therefore, we sought to examine the trend in the number of SCTs, as well as the mortality and clinical complications for CML over the decade following the FDA approval of Imatinib.
Methods: We queried the Healthcare Cost and Utilization Project's Nationwide Inpatient Sample (NIS) between 1998 and 2011 using the ICD-9 code 205.1 for CML in the primary and secondary diagnosis fields. Only patients 18 years or older were included in the analysis. We monitored the trends in allo-SCT, mortality and clinical complications related to CML. A p value of <0.05 was considered significant. We have also attempted to analyze the trends in cost and length of hospitalization during this decade. Cost of hospitalization was adjusted for inflation in reference to the year 2011 and cost to charge ratio.
Results: A total of 53254 (weighted n=262964) hospitalizations for CML (Male 54.6% and average age 65.9+/-0.8 yrs) occurred from 1998-2011. There has been a 408% decrease in the rate of transplants and 79.9% decrease in in-hospital mortality since the introduction of TKIs (p<0.0001). There has also been a 48.3% reduction in the average length of hospital stay, despite which there has been a 17% increase in cost of hospitalizations for CML (p<0.0001). There have also been marked reductions in the complications related to CML with the advent of TKI therapy. There has been almost 200% reduction in blast crises (p<0.0001), a 120.8% decrease in splenic infarct/splenic vein thrombosis (p<0.0001), 56.1% reduction in epistaxis (p <0.0004).
Conclusion: This analysis supports the widely held belief that the introduction of TKIs has not only reduced the reliance on allogeneic transplants, but has also led to remarkable improvement in mortality and CML related complications. Increase in cost of hospitalization could be related to the overall increase in health care cost, 4.5% yearly from 1998-2000, 3.9% from 2000-2007 and 1.8% 2007-2010.

Disclosures
No relevant conflicts of interest to declare.
Characterisation of Novel Hypomorphic and Null Mutations in Klf1 Derived from a Genetic Screen for Modifiers of a-Globin Transgene Variegation
Authors: Huang Stephen , Gillinder Kevin R. , Ilsley Melissa , Magor Graham , Tallack Michael , Sorolla Anabel , Oey Harald , Harten Sarah , Clemens-Daxinger Lucia , Whitelaw Emma , Perkins Andrew C 
Published: October 2019

Abstract
Position-effect variegation of transgene expression is sensitive to the chromatin state. We previously reported a forward genetic screen in mice carrying a variegated a-globin GFP transgene to find novel genes encoding epigenetic regulators. We named the phenovariant strains "Mommes" for Modifiers of murine metastable epi-alleles. Here we report positional cloning of mutations in two Momme strains which result in suppression of variegation; i.e. an increased percentage of GFP+ circulating red blood cells. Both strains harbour point mutations in the erythroid specific transcription factor, Klf1. One (D11) generates a stop codon in the zinc finger domain. D11 homozygous mice die in utero of anaemia at 14.5DPC. The other (D45) generates an amino acid transversion (H350R) within a conserved linker between zinc fingers two and three. Homozygous MommeD45 mice have mild compensated microcytic anaemia which models the phenotype in a recently described human family. Mice Carrying the H350R mutation were interbred with Klf1+/- mice. Klf1H350R/- mice have severe perinatal haemolytic anaemia and marked splenomegaly. Furthermore blood haemoglobin content, haematocrit and red blood cell size (MCV) were significantly reduced in Klf1H350R/- mice compared to wildtype and D45 homozygous offspring of the same age. Analysis of Klf1H350R/- by flow cytometry showed an increase in circulating immature red blood cells. In the bone marrow, a lack of mature red blood cells was observed. Flow cytometric analysis of the spleen from Klf1H350R/- animals revealed an expansion of erythroid cells and a relative reduction in B and T Cells. Gel shifts assays of a recombinant Klf1 zinc finger protein with the H350R mutation showed normal binding to the b -globin promoter sequence but weak binding to the Alas2 intronic enhancer site. Furthermore b -globin gene expression was near normal whereas expression of other known Klf1 target genes was decreased. We will discuss how H350R disrupts function from ChIP-seq and RNA-seq in primary fetal liver tissue. Previous studies of the linkers in C2H2 zinc finger transcription factors have revealed their necessity as structural and regulatory components for the C2H2 class of transcription factors. Our results thus far show that the second linker of Klf1 has a role in maintaining the integrity of Klf1 function (at a subset of Klf1-occupied sites,) and does not act just as a spacer for the zinc fingers.

Disclosures
Perkins: Novartis Oncology: Honoraria.
Hematopoeitic Stem Cells and Their Progenitors Critically Require Autophagy to Promote Early Engraftment Following Allogeneic Stem Cell Transplantation
Authors: Lineburg Katie E , Leveque-El Mouttie Lucie , Le Texier Laetitia , Teal Bianca , Kuns Rachel D , Hill Geoffrey R , MacDonald Kelli P A 
Published: October 2019

Abstract
Stem cell transplantation (SCT) is the only curative therapy for the majority of hematological malignancies and relies on successful engraftment of donor hematopoietic stem cells (HSC) to reconstitute the patient's hematopoiesis and immunity. Graft-versus-host disease (GVHD) and graft failure are complications of SCT that contribute to significant morbidity and mortality. Recent studies have established a role for autophagy in the long-term survival and function of HSCs and as a regulator of hematopoiesis. We investigated the contribution of autophagy to HSC in the setting of allogeneic transplantation. Using LC3-GFP bone marrow as a graft source in the well-established B6 into F1 model of GVHD, together with imaging flow cytometry, we demonstrated increased autophagy (LC3-GFP punctae) within donor HSC and progenitor cells in GVHD compared to non-GVHD recipients. To assess the contribution of autophagy to HSC development and function we used mice deficient in Atg5, an essential protein for the autophagy pathway. In primary transplants, ATG5 KO foetal liver (FL) had reduced ability to engraft lethally irradiated congenic recipient mice compared to WT FL. Furthermore, competitive transplantation of 50% ATG5 KO FL cells together with 50% WT FL cells demonstrated that ATG5 KO cells had a reduced capacity to reconstitute compared to congenic WT FL cells. Finally, to address the role of autophagy in HSC in the setting of GVHD, we transplanted WT or ATG5 KO FL ± T cells into lethally irradiated B6D2F1 recipients. At D10 post-transplant ATG5 KO recipients failed to establish tri-lineage engraftment in peripheral blood and lacked expansion of myeloid precursor cells in the BM, leading to engraftment failure and significantly reduced survival compared to WT FL recipients (50% WT vs 10% Atg5 KO p<0.0001). This early engraftment failure was confirmed to be autophagy specific using VAVcre x Atg7fl/fl donor mice, in which Atg7 deficiency is restricted to hematopoietic cells. Furthermore, the essential requirement for autophagy in early progenitors and HSC was confirmed using LysMcre Atg7fl/fl mice, which ablate Atg7 from the granulocyte-macrophage progenitor population onwards. Survival, or engraftment, in mice receiving the LysMcre Atg7fl/fl donor graft did not differ significantly from those receiving a WT graft. We demonstrate that autophagy is increased in the GVHD setting and that without autophagy early myeloid precursors fail to provide short term reconstitution leading to primary graft failure and mortality. Thus, intervention to increase autophagy in these cells post-transplant may improve engraftment in the clinic.

Disclosures
No relevant conflicts of interest to declare.
Force-Induced Cooperative Unfolding of Two Distinctive Domains in a Single Gpibalpha Molecule
Authors: Chen Yunfeng , Ju Lining , Lou Jizhong , Zhu Cheng 
Published: October 2019

Abstract
GPIbα, a major member of the GPIb-IX-V complex, initiates a mechano-signaling pathway that leads to platelet intracellular calcium flux when binding to VWF at the A1 domain1. Exactly how this signal is transduced across the membrane is unknown. A recent work identifying the unfolding of a jextamembrane mechanosensitive domain (MSD) (Fig.1 A,C) suggested that this unfolding might play a role in the signal transduction of GPIbα2. Using molecular dynamics (MD) simulations to pull the GPIbα leucine-rich repeat domain (LRRD) from the VWF A1 domain, we observed unfolding of the LRRD (Fig.1 A) (manuscript under review).
Here we used a biomembrane force probe (BFP) to study single GPIbα unfolding (Fig. 1E,F). Platelet GPIbα was pulled by A1 coated on the probe through a ramping phase (pink, Fig. 1G) to a clamping phase to wait for bond dissociation under a 25-pN force (red, Fig. 1G). Two unfolding signatures were identified: i) ramped unfolding, featured by a sudden force kink in the ramping phase (dashed circle, Fig. 1G); ii) clamped unfolding, featured by an abrupt force drop in the clamping phase (dashed ellipse, Fig. 1G). Unfolding lengths from both signatures were measured from the force vs. time data (Fig. 1G), based on which the unfolding events of MSD (7-25 nm), LRRD (25-56 nm) or both together (56-80 nm) were differentiated. Intriguingly, LRRD unfolding was only observed in the ramping phase, while MSD could unfold in both ramping and clamping phases.
The frequency of observing both LRRD and MSD unfolding in the same cycle was much higher than the product of the frequencies for LRRD and MSD to unfold (the joint probability for both to unfold if they were independent), suggesting that the two GPIbα domains unfolded cooperatively (Fig 2A). Chi-square tests also rejected the hypothesis that MSD and LRRD unfolded independently (p =5.24×10-5). Separating the ramped and clamped unfolding of MSD and evaluating their respective cooperativity with LRRD unfolding demonstrated similar results (Fig 2A). Agreeing with these, both the ramped and clamped unfolding of MSD occurred much more frequently in the presence of LRRD unfolding (Fig 2B, left), and vice versa for the unfolding of LRRD (Fig 2B, right). Cooperative unfolding of LRRD and MSD may play a key role in transducing signals across platelet membrane.
References
1 Nesbitt, W. S. et al. The Journal of biological chemistry277, 2965-2972, doi:10.1074/jbc.M110070200 (2002).
2 Zhang, W. et al. Blood125, 562-569, doi:10.1182/blood-2014-07-589507 (2015).
Figure 1. Figure 1. Figure 2. Figure 2.

Disclosures
No relevant conflicts of interest to declare.
Liver Tropism and Factor VIII Transgene Expression after Intrauterine Chorionic Villus Stem Cell Transplantation
Authors: Miguelino Maricel G. , Kumar Priyadarsini , Pivetti Christopher , Lanford Lee , Powell Jerry S. , Wang Aijun 
Published: October 2019

Abstract
INTRODUCTION: Gestation is characterized by rapid mitosis and histogenesis in an immune naïve environment. Growth and development of vital organs are occurring continuously at different time points throughout pregnancy under the regulation of paracrine signals, transcription and growth factors. Appropriate timing to target the developing organ of interest during in utero stem cell transplantation (IUT) may result in high level engraftment in that organ as well as recognition of foreign cells as self. Hemophilia A, is an X-linked recessive bleeding disorder caused by a mutation of the gene that encodes Factor VIII (FVIII) which is primarily produced by the liver. We hypothesize that introduction of stem cells in utero during hepatogenesis in mice (e9.5-15 days) will result in localization and persistence of stem cells in the liver.
METHODOLOGY: We reported isolation and characterization of early preterm chorionic villus derived mesenchymal stem cells (PMSCs) at GA 9-13 weeks. PMSCs (n =5) were transduced with a lentiviral vector encoding B-domain deleted FVIII, GFP and luciferase reporter genes (pCCLc-MNDU3-eGFP/LUC-PGK-F8-WPRE) at an MOI (Multiplicity of Infection) of 3. Chromogenic assay was performed on cell supernatant of transduced PMSCs 72 hours after transduction. Prior to transplantation, PMSCs were plated at different densities ranging from 250,000 cells to 2 x10^6 cells and subjected to bioluminescence imaging. Intraperitoneal transplantation of PBS (control group) or with 1 x10^6 PMSCs (experimental group) was conducted in fetal mice at e12-14 days using a 33 gauge non-coring needle. Bioluminescence analysis was performed 24 hours after IUT on dams, and 12-15 days after transplantation. Mice were euthanized and liver tissue was harvested from all animals. Expression of FVIII was analyzed by RT-PCR from both groups.
RESULTS: Bioluminescence imaging revealed focal density and transgene expression for transduced cells in vitro, in vivo at 24 hours and in postnatal pups 12-14 days after transplantation. Postmortem bioluminescence analysis of IUT transplanted mice showed positive bioluminescence in the livers of pups injected with transduced PMSCs. RT-PCR analysis of the liver samples using human specific FVIII primers revealed FVIII expression in liver tissues in PMSC treated mice while negative for PBS treated mice.
CONCLUSION: These results demonstrate the functional survival and localization of genetically modified PMSCs in the liver after intrauterine transplantation for up to two weeks. This study provide better understanding of the cell fate of stem cells after IUT and suggest that appropriate timing of stem cell transplantation in utero may lead to immunological tolerance.

Disclosures
Powell: CSL Behring: Employment.
Binding of rhFVIIa (LR769) to the Endothelial Protein C Receptor
Authors: Grandoni Jerry A 
Published: October 2019

Abstract
Congenital Hemophilia A and B treatment may be complicated by the development of inhibitors to the coagulation factors used in replacement therapies. In such cases, factor replacement becomes ineffective and use of a bypassing agent is needed to treat or prevent bleedings. A recombinant form of activated Factor VII has been available for this purpose. In this study, a new second generation recombinant human Factor VIIa (rhFVIIa, LR769) produced by LFB USA was studied. The goal is to provide patients with Hemophilia A and B who develop inhibitors an alternative treatment option. In work presented previously, we demonstrated that LR769 was similar to NovoSevenRT with respect to activation of FX and FIX, tissue factor binding, and inactivation by antithrombin. LR769 bound better to platelets than NovoSevenRT at saturation but had a similar binding constant(1). It is known that FVIIa binds to Endothelial Protein C Receptor (EPCR) with the same affinity as Protein C (2-4). Several studies suggest EPCR is involved in therapeutic mechanism rhFVIIa (5,6,7,8). In addition to its effect on thrombin generation, binding to EPCR may protect against permeability induced by VEGF and LPS (6). In the work presented here, the binding of LR769 to EPCR on the surface of cultured HUVEC cells was determined and compared to NovoSevenRT. A dose-dependent binding was observed at rhFVIIa concentrations expected in patients undergoing treatment with this product. The specificity for EPCR binding was confirmed by competition with Protein C, soluble EPCR, or anti-EPCR which reduced binding to these cells. Addition of soluble tissue factor increased the binding by approximately 20-30%. Overall, the results show that LR769 binds to primary human endothelial cells via EPCR and that tissue factor increases the binding affinity. Binding was found to be higher with LR769 than NovoSevenRT. The significance of the tissue factor effect on rhFVIIa binding with respect to Hemophilic joint arthropathy is discussed. The methods used will be used to further characterize the interactions of rhFVIIa with important components of the vascular system.
1) Grandoni, J.A. et al. (2013) ASH 2013 poster
2) Gosh et al. (2007) JBC 282 11849-11857
3) Lopez-Sagaseta et al. (2007) JTH 5 1817-1824
4) Preston et al. (2006) JBC 281 28850-28857
5) Pavani, G et al. (2014) Blood 7 1157-65
6) Sundarham et al. J. Thromb and Hemost (2014)12:690-700
7) Monroe DM, et al. (1997) Br J Haematol;99:542-7

Disclosures
Grandoni: LFB USA: Employment.
Biological Effects of Different Extracellular Vesicles Population on Reversal of Marrow Cells Radiation Damage
Authors: Wen Sicheng , Cheng Yan , Dooner Mark , Pereira Mandy , Del Tatto Michael , Papa Elaine , Goldberg Laura R. , Aliotta Jason M. , Quesenberry Peter J. 
Published: October 2019

Abstract
We have shown that Human or mouse mesenchymal stem cell (MSC) derived vesicles separated by differential centrifugation (300g, 10kg &100kg) can reverse radiation whole bone marrow (WBM) damage in vitro on the hematopoietic cell line FDC-P1 and in vivo on recovery of stem cells and differentiated blood cells after whole body irradiation. The vesicle separation is a classic method for preparations of exosomes. However, this separation isolates the smaller vesicles, discarding larger vesicles. Biologic healing effects of larger vesicles with or without smaller vesicles have not been investigated. Here, we investigated the healing effects of 3 different preparations of vesicles: 10k pellet (microvesicles), 100-10k pellet (exosomes) & 100k pellet (no 10k spin).
Three different fractions (10k pellet, 100-10k pellet &100k pellet) of murine/human MSC or murine WBM derived vesicles were isolated by differential centrifugation. FDC-P1 cells were exposed at 500cGy irradiation and cultured with or without the addition of 3 different preparations of murine WBM or human MSC-derived vesicles for 7 days. A significant increase in cell proliferation was observed after 3 different vesicle fractions treatments. However, exosomes were clearly inferior, microvesicles and the combined exosomes and microvesicles population were superior. The capacity of human MSC-derived vesicles of different fractions on reversal of murine bone marrow damage was also evaluated in vivo. Again, the combined fractions shown a significant increase donor chimerism in bone marrow at 6 month post-transplant with 5x's the level of engraftment compared to irradiation control. The 10k and 100-10k fractions showed intermediate healing. Exposure to vesicles in irradiated FDC-P1 cells downregulated phosphorylated H2AX, the DNA damage marker and decreased apoptosis marker, PARP cleavage part. We further investigated the effect of murine marrow MSC-derived vesicles on the gene expression in peripheral blood cells from radiation damaged mice. 28 genes were altered by irradiation, 22 showed partial or complete reversal of these alterations. The characterization of three different fractions of vesicles derived from murine bone marrow cells and human MSC were evaluated by miRNA realtime PCR array (total 750 miRNA). Several MSC-EV associated miRNA responsible for the reversal of radiation damage were evaluated. Our preliminary data shown that miR-221, miR654-3p and miR210-5p could partially reverse radiation damage in FDC-P1 cells
There are different biological effects in different extracellular vesicles populations. For reversal of radiation toxicity the most effective vesicle population would include both smaller (exosomes) and larger vesicles (microvesicles).
Acknowedgements: This work was support by the NIH grant 5UH2TR00080

Disclosures
No relevant conflicts of interest to declare.