bluuebunny/crossref_metadata_2025_split huggingface dataset:
Increasing CD28-FOXP3+CD8+ Treg and Senescent CD8+NK2GA+Eomes+ NK-like T Cells in Peripheral Blood of Patients with Multiple Myeloma
Authors: Kasakovski Dimitri , Zeng Xiangbo , Xu Ling , Li Yangqiu 
Published: October 2019

Abstract
Immune dysfunction in patients with multiple myeloma (MM) includes TGF-β induced dendritic cell dysfunction, regulatory T cell (Tregs)/Th17 cell imbalance, accumulation of Tregs and myeloid derived suppressor cells. These tumor-induced dysfunctions may contribute to immune escape and even suppress immune cells introduced in adoptive cellular immunotherapy. CD28 independent T cells of the effector memory (TEM) and CD45RA+ effector memory (TEMRA) population were shown to accumulate with age and contribute to the immunosuppressive tumor microenvironment in several solid tumors and hematological cancers. Especially in the CD8+ population, the loss of CD28 is associated with high cytotoxicity and regulatory function while showing high diversity and defective antigen-induced proliferation. In the CD4 subset, regulatory and senescent T cells were studied extensively, while in the CD8 positive subset their heterogeneity is still not clearly defined. Their potential immunosuppressive role and distribution in healthy individuals (HI) as well as patients with multiple myeloma (MM) remains to be observed. Furthermore, a recently characterized CD8+CD28- NK-like T cell subset showing expression of NK-related inhibitory receptors and TCR independent effector function is potentially of interest in the progression of MM. In the present study, we compared the changes of distribution of CD8+CD25+ and CD8+FOXP3+ regulatory T cells (Treg), CD28-CD57+ senescent T cells (Tsen), and CD8+KIR/NK2GA+EOMES+ NK-like T in peripheral blood (PB) between HI and patients with MM by multicolour flow cytometry (Gating strategy shown in Figure 1A). When comparing 35 HIs (Median age is 54) and 14 MM patients (Median age is 52), it was shown that there is no significant change in the proportion of senescent T cells in CD8 (P = 0.2452), TEM/CD8 (P = 0.1686) and TEMRA/CD8 (P = 0.4861) between HIs and the MM group, while both the CD25+FOXP3+ regulatory T cells of the CD4 population (P = 0.0031) and CD28-FOXP3+ regulatory T cells of the CD8 population (P = 0.0014) were shown to increase. There is no significant difference in the percentages of KIR/NK2GA+EOMES+ in the CD8 T cell and TEMRA/CD8 T cell population between HIs and the MM group. Remarkably, although there was no overall increase in senescent T cell in MM patients, senescent CD8+NK2GA+EOMES+ NK-like T cells increased in MM patients in comparison to HIs (P = 0.0068) (Figure 1B). In conclusion, the increase of regulatory T cells of both the CD4 and CD8 population as well as the increase of senescent NK-like T cells in the CD8 population potentially contributes to cancer progression through creation of suppressive microenvironments. Moreover, we found that regulatory CD8 T cells and CD8 NK-like T cells only contribute to a small part of the overall CD28- senescent T cell pool.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.
UBR5 Mutations in Mantle Cell Lymphoma Lead to Increased Proliferation through a Cyclin D1-Dependent Mechanism
Authors: Kutovaya Olga , Hung Stacy , Viganò Elena , Telenius Adele , Woolcock Bruce W , Meissner Barbara , Colborne Shane , Gascoyne Randy D. , Villa Diego , Morin Ryan D. , Scott David W. , Morin Gregg B , Steidl Christian 
Published: October 2019

Abstract
Mantle cell lymphoma (MCL) is an aggressive type of non-Hodgkin lymphoma, with patient outcomes inferior to most other lymphoma subtypes. Recent progress in describing recurrent somatic gene mutations has led to a better understanding of MCL pathogenesis. However, the functional and clinical implications of many alterations remain to be elucidated. Here, to uncover the role of recurrent UBR5 gene mutations in lymphomagenesis, we studied a cohort of 248 MCL patients by targeted sequencing and performed genome editing of MCL-derived cell lines to investigate UBR5-mutation associated phenotypes in vitro. We identified deleterious UBR5 exon 58 hotspot mutations in 8% of MCL patients, all of which were mutually exclusive with CCND1 mutations. Proteomics analysis of Granta-519 and Jeko-1 cell lines with engineered UBR5 exon 58 indel mutations showed differential expression of genes involved in cell cycle and ubiquitination, and led to the discovery of decreased phosphorylation of CCND1 in the UBR5-mutated lines. Accordingly, in vitro studies of engineered genome-edited Granta-519, Jeko-1 and Mino cells revealed accumulation of cells in the S phase of the cell cycle, increased phosphorylation of retinoblastoma protein (Rb), and increased lymphoma cell proliferation. Our results demonstrate that UBR5 mutations, in addition to the hallmark t(11;14) translocation drive proliferation of MCL cells, potentially rendering mutation-carrying cells more sensitive to targeted therapies.

Disclosures
Gascoyne: NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies. Scott:NanoString: Patents & Royalties: Named Inventor on a patent licensed to NanoString Technologies, Research Funding; Roche: Research Funding; Celgene: Consultancy, Honoraria; Janssen: Research Funding. Steidl:Roche: Consultancy; Bristol-Myers Squibb: Research Funding; Nanostring: Patents & Royalties: patent holding; Juno Therapeutics: Consultancy; Seattle Genetics: Consultancy; Tioma: Research Funding.
Comparing Randomized Controlled Data with Grouped Data from the Literature to Inform Decision Making When Standard of Care Has Low Level of Evidence Available
Authors: Andreozzi Valeska , Vandewalle Björn , Parreira Jorge 
Published: October 2019

Abstract
Objectives: To apply indirect comparison methods to inform decision-making when standard of care has low level of evidence available.
Methods: Simulated treatment comparison is one of the methods available to generate indirect comparison of data in different formats and levels of evidence. It is particularly helpful when patient level data exists for one set of data and there is the need to compare it with single-arm studies. In theses cases evidence is deemed ''unanchored'' due to a lack of a common comparator. We use the case of second line treatment of chronic immune thrombocytopenic purpura (ITP). Splenectomy has been historically recommended in guidelines mainly due to the lack of evidence for other second-line therapies for ITP. We use individual patient data from eltrombopag clinical trials and one of the only studies published in the literature reporting long term outcomes for splenectomy to generate an indirect comparison of eltrombopag and splenectomy in second line treatment of ITP.
Results: A subset of patients (n=84) treated with eltrombopag but not subject to splenectomy in the randomised, phase 3 study RAISE (PMID:20739054) were indirectly compared with a large retrospective cohort of patients (n=233) who underwent splenectomy for ITP in the study by Vianelli et al (PMID:23144195). After controlling for gender, age, baseline platelet count and number of previous therapies, we estimate that the proportion of patients with ITP responding to second line therapy is higher with eltrombopag in comparison to splenectomy: complete response (platelet count >100x109/L) 68.6% vs 54.0%; partial response 18.6% vs 24.6%; no response (platelet count <30x109/L)) 12.8% vs 21.4%.
Conclusions: Indirect comparison with simulated treatment comparison is useful for decision making when no head to head data exists between new treatment options and standard of care and also when standard of care has low level of evidence available.

Disclosures
No relevant conflicts of interest to declare.
Long-Term Effect of Mesenchymal Stromal Cell Derived Extracellular Vesicles on the Restoration of Engraftment of Stem Cells in Radiation Exposed Mice
Authors: Wen Sicheng , Goldberg Laura R , Dooner Mark S , Pereira Mandy , Del Tatto Michael , Papa Elaine , Cheng Yan , Quesenberry Peter J. 
Published: October 2019

Abstract
Extracellular vesicles (EVs) including exosomes and microvesicles, have been found to deliver both mRNA and transcriptional modulators to target cells and affect their phenotype. Vesicles derived from mesenchymal stem cells (MSC) have been shown to affect the phenotype and induce healing of many different cell types. Our recent published work has shown that pretreated irradiated murine bone marrow stem cells with human or murine MSC-EV in vitro, could significantly improve the engraftment capacity of radiation-damaged stem cells up to 9 months post-transplantation. Interestingly, the restoration of engraftment was not significantly observed within the first month of post-transplant, the predominant reversal effect occurred on later period of post-transplant from 3 months up to 9 months. This is indicating a long-term effect of MSC-EVs on reversal of radiation damage of stem cell engraftment capacity. To confirm this hypothesis, in our current study, the effect of human MSC-EVs on reversal of engraftment capacity of bone marrow stem cells post-radiation was investigated by an in vivo study. C57BL/6 mice were exposed to 500 cGy total body irradiation. MSC-EVs or vehicle were then injected intravenously 24, 48 and 72 hours after irradiation. The whole bone marrow were harvested at 6, 12, 26 and 53 weeks post EV-injection and then transplanted into 950 cGy exposed B6.SJL mice and engraftment evaluated at 1 and 3 months post-transplantation. In those transplanted mice at 6 weeks post-EV injection, there was slight increase in the restoration of engraftment rate (the percent of irradiated mice with EV/Vehicle treatment engraftment rate compared to healthy non-irradiated mice engraftment rate) in EV treated mice (17.58±2.32% compared to untreated mice (13.80±1.41%) after 1 month post-transplantation. However, for those mice transplanted at 12, 26, and 53 weeks post-EV injection, there were the significant higher restorations of engraftment rate in EV treated mice (40.48±6.03%, 33.93±3.76%, and 56.62±3.635) compared to untreated mice (12.39±1.30%, 15.14±2.21%, 36.21±3.63%) after 1 month transplantation respectively. The similar restorations of engraftment were also seen in 3 months post-transplantation. Our study also showed that there was a significant inhibition of stem cell engraftment at 53 weeks post 500cGy whole body radiation mice which was 36.21±3.63% of engraftment rate from healthy mice. Thus our data suggest that there is a long-term effect of MSC-EVs on the restoration of engraftment of stem cells in radiation-exposed mice.

Disclosures
No relevant conflicts of interest to declare.
Favorable Outcome of Sequential HLA-Haploidentical Transplantation Using Ptcy As GvHD Prophylaxis in High Risk AML and MDS of the Elderly
Authors: Fraccaroli Alessia , Prevalsek Dusan , Häbe Sarah Elena , Bücklein Veit , Schulz Christoph , Drolle Heidrun , Fritsch Susanne , Ledderose Georg , von Bergwelt Michael , Hiddemann Wolfgang , Tischer Johanna 
Published: October 2019

Abstract
Sequential conditioning regimens, comprising cytoreductive chemotherapy shortly applied prior to reduced intensity conditioning are successfully used for high-risk (HR) AML/MDS in matched related and unrelated donor hematopoietic stem cell transplantation. However, few data are available for sequential conditioning in the context of HLA-haploidentical transplantation (haplo-HSCT), especially in the elderly. To investigate the relative merits of sequential haplo-HSCT in the elderly we retrospectively analyzed the outcome of thirty-five patients (pts) with advanced AML/MDS (>=50 years old).
Thirty-three pts suffering from HR AML, defined by refractory, relapsed or secondary leukemia, or complete remission with adverse-risk genetics according to ELN criteria and two pts with HR MDS according to IPSS-R, who underwent T-cell-replete haplo-HSCT at our institution between January 2009 and November 2016 were included. Disease was active in 29 pts while 6 had achieved CR. Pre-transplantation risk factors were scored using the hematopoietic cell transplantation-specific comorbidity index (HCT-CI) which was ≥3 in 13 pts (median HCT-CI:2, range 0-8). A sequential therapeutic concept using either FLAMSA (n=26) or clofarabine (n=9) for cytoreduction was used prior to RIC in all pts. Bone marrow (54%) and peripheral blood stem cells (46%) were both used as graft source. Post-grafting immunosuppression consisted of high-dose cyclophosphamide, tacrolimus and MMF in all pts.
Median age was 60 years (50-70). One graft rejection occurred. Three pts died early in aplasia. Neutrophil and platelet engraftment was achieved in 95% and 77% of evaluable pts, respectively at a median of 16,5 (13-89) and 31,5 (11-103) days.Acute GvHD grade I-III occurred in 25/32 of the pts (grade III n=2); no patient developed grade IV aGvHD. Chronic GvHD was observed in 13/29 pts and was most frequently assessed as mild (n=6) or moderate (n=5) while 2 pts developed severe cGvHD. No GvHD related death was observed. CI of NRM at day 100, 1-year and 3-years was 11%, 23% and 23%, respectively. CI of relapse at 1- and 3-years was 15% and 27%, with a median time to relapse of 152 days (20-413). At a median follow up of 27 months (4-74), estimated one- and three-year overall survival (OS) was 62% and 52% respectively. One- and three-year leukemia-free survival (LFS) was 59% and 52%.
Our results suggest that using a sequential therapeutic concept in PTCY-based haplo-HSCT is safe and properly tolerated while it provides a favorable disease control when treating elderly HR MDS/AML pts. Thus, sequential haplo-HSCT seems to be a valuable alternative in pts who lack a conventional donor or are in urgent need for prompt transplantation.

Disclosures
Tischer: Jazz Pharmaceuticals: Other: Jazz Advisory Board.
Association of CD47 with Integrin Mac-1 (αMβ2, CD11b/CD18) Regulates Macrophage Responses
Authors: Podolnikova Nataly , Balabiyev Arnat , Ugarova Tatiana P. 
Published: October 2019

Abstract
CD47 is a cell surface receptor, which is expressed by virtually all cells in the body, including immune cells. CD47 has originally been identified as an integrin-associated protein (IAP) and shown to associate with several integrins that belong to the β1 and β3 subfamilies. In addition, association of CD47 with a member of the β2 subfamily, integrin αLβ2, has also been reported. In neutrophils, CD47 mediates a number of integrin αvβ3-dependent functions, including adhesion, migration and phagocytosis. Surprisingly, the association of CD47 with integrin αMβ2 (Mac-1, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, has not been documented. Furthermore, while the major focus of recent studies was the mechanism by which CD47 on various host cells prevents phagocytosis by macrophages, the question as to how CD47 expressed on the surface of macrophages influences the responses of these cells has not been addressed. In the present study, we demonstrated that an association of CD47 with Mac-1 regulates Mac-1-dependent macrophage functions. In particular, adhesion of macrophages isolated from CD47-/- mice to fibrinogen and ICAM-1, the established physiological ligands of Mac-1 was significantly decreased compared to wild-type counterparts. In addition, spreading of CD47-deficient macrophages was decreased by four- and two-fold on fibrinogen and ICAM-1, respectively. Compared to wild-type macrophages, migration of CD47-deficient macrophages to the Mac-1 ligand, cathelicidin peptide LL-37 was significantly reduced. The lack of CD47 on the surface of macrophages impaired their ability to fuse in the presence of IL-4. Finally, the deficiency of CD47 also reduced phagocytosis of opsonized latex beads, a process fully dependent on Mac-1. The functional association of CD47 with Mac-1 implied that similar to other integrins, Mac-1 might form a complex with CD47. Indeed, co-immunoprecipitation experiments using peritoneal mouse macrophages, the IC21 murine macrophage cell line and Mac-1-expressing HEK293 cells revealed that Mac-1 forms a complex with CD47. The cis interaction between Mac-1 and CD47 was also detected using the proximity ligation assay. Together, these results indicate that Mac-1 forms a lateral complex with CD47, which regulates important macrophage functions. Studies to determine the structural requirements for the physical interaction between Mac-1 and CD47 are in progress.

Disclosures
No relevant conflicts of interest to declare.
Adoptive Therapy with Cord Blood Regulatory T Cells Does Not Impair Anti-Leukemia Activity of Infused Donor Cells
Authors: Ma Hongbing , Nishimoto Mitsutaka , Zeng Ke , Nguyen Tuongvan D , Schuler William , Kellner Joshua , Peedikayil Jaimol , Lyu Mi-Ae , Thangavelu-Devaraj Santhana , Parmar Simrit 
Published: October 2019

Abstract
Introduction
Cord blood derived ex vivo expanded regulatory T cells(CB-Tregs) have been verified that they can effectively prevent xenogenic graft-versus-host disease(GVHD) in NSG mice, which provide evidence to further investigate the effect of CB-Tregs in clinical setting. However, due to the potent suppression function of CB-Tregs, it needs to elucidate the concerning about whether CB-Tregs worsen leukemia or dampen anti-leukemia activity of donor cells. Beatrice et al. report donor derived in vitro expanded Treg can also prevent GVHD successfully while allowing a powerful graft-versus-leukemia(GVL) in murine models. We hypothesize cord blood derived ex vivo Treg are effective in preventing GVHD without compromising GVL.
Methods
Tregs are isolated from cord blood and expanded with CD3CD28 beads and IL-2 in SCGM medium. Then the cells are harvested on day14 after culturing. We use CD4+CD25+CD127low or - Foxp3+ to confirm Tregs, viabilty and purification on flow cytometry. The suppresive assay of CB-Tregs are based on CFSE. 3 or 10 million HL-60 cells are used to build AML model in NSG mouse. AML VS AML+Tregs or AML+PBMC/Tcons VS AML+PBMC/Tcons+Tregs are set up in each experiments. The primary end point is survival, secondary point is AML percentage and weight loss as well as the cytokines in the serum and tissue pathology.
Results
62-135 folds of CB-Tregs compared original number are harvested on day14, >90% cells are CD4+CD25+CD127low or - Foxp3+, There is no any difference in weight loss, AML percentage of peripheral blood and survival between AML+PBMC and AML+PBMC+Treg (Figure1 a, b,c and d,e,f). There is no significant difference in AML percentage between AML+PBMC/Tcons +Treg and AML+PBMC/Tcons, but mice in AML+Tcons+Treg survived longer than mice in AML+Tcons (Figure 2a,b,c,d).
Conclusion
CB-Tregs do not deteriorate the development of AML, they also do not impair anti-leukemia activity of PBMC or Tcons while they provide protection from GVHD. Our findings lay down the basis for testing impact CB-Tregs on CAR T cell activity in Vivo.

Disclosures
No relevant conflicts of interest to declare.
Aged Neutrophils in Sickle Cell Disease Impede Ischemic Repair
Authors: Okwan-Duodu Derick , Archer David R. , Taylor W Robert 
Published: October 2019

Abstract
INTRODUCTION: Repeated bout of tissue ischemia is a hallmark of sickle cell disease (SCD). Neutrophils are critical first responder cells that initiate host response to sterile injury, including ischemia. Of the numerous neutrophils in circulation, a small proportion undergo physiologic changes (termed aging) which directs their homing to the bone marrow for clearance. How neutrophil homeostasis is regulated in sickle cell disease (SCD) to influence ischemic repair is unknown.
HYPOTHESIS: We hypothesized that SCD may alter the homeostasis and function of aged neutrophil.
METHODS: Aged neutrophils from the Townes humanized sickle cell (SS) mice were compared to aged neutrophils from MerTK deficient mice (MerKO), which age in the circulation owing to defective efferocytosis. Hind limb ischemia (HLI) was used as model of vascular injury. LASER Doppler perfusion imaging (LDPI) measured perfusion recovery after HLI.
RESULTS: Compared to wild-type mice (7 ± 1.2%), the proportion of CXCR4HiCD62Llo aged neutrophils in circulation was markedly elevated in the SS (34 ± 4%) and MerKO mice (38 ± 4%). However, in contrast with aged neutrophils from MerKO mice, those from SS mice showed impaired phagocytosis, increased capacity to produce reactive oxygen species, and pronounced release of extracellular traps (NETs). In response to HLI, perfusion recovery in the MerKO and wild type mice were similar, but this was dramatically impaired in SS mice. Importantly, the phenotype of SS mice was reversed by treatment with the anti-oxidant N-acetyl cysteine (NAC), but NAC therapy had no impact on MerKO mice.
CONCLUSIONS: Although SS and MerKO mice demonstrated elevated numbers of aged neutrophils, our data suggest that oxidant stress causes aged CXCR4HiCD62Llo neutrophils in SS to impede vascular repair after ischemia. This specialized subset of neutrophils may be targeted to improve vascular health in SCD.

Disclosures
No relevant conflicts of interest to declare.
Adamts-13 Inhibitor Testing Is Often Negative on Initial Testing
Authors: Chaudhry Hina , Sholzberg Michelle , Pavenski Katerina 
Published: October 2019

Abstract
Background:
Thrombotic thrombocytopenic purpura (TTP) presents with microangiopathic hemolytic anemia and thrombocytopenia and is caused by severe ADAMTS13 deficiency. TTP can be the result of autoantibodies to ADAMTS13 or genetic defect in the ADAMTS13 gene. ADAMTS-13 is an enzyme that specifically cleaves unusually large von Willebrand Factor (VWF) multimers which mediate platelet thrombus formation under high shear. When ADAMTS13 is deficient, unusually large VWF multimers accumulate causing excessive platelet aggregation and thrombosis in the microvasculature.
Methods:
St. Michael's Hospital, Toronto is home to a large reference laboratory for special coagulation. We use a commercial ELISA Technoclone Technozym ADAMTS-13 activity assay for the determination of ADAMTS-13 activity and Technoclone Technozym ADAMTS-13 inhibitor assay to identify anti-ADAMTS-13 antibodies. We send samples to another Canadian laboratory for validation of our results as they use an in-house ELISA assay for ADAMTS13 activity and anti-ADAMTS13 antibody.
Results:
We performed a retrospective review of all ADAMTS13 activity tests performed by our laboratory between January 1, 2013 and June 30, 2018. The total number of tests was 466 from 203 unique patients. 24% had an ADAMTS-13 activity under 10% (N = 144) which is consistent with the diagnosis of TTP.
When specimens with severe ADAMTS-13 deficiency were tested for presence of anti-ADAMTS13 antibody, 46% were negative. Four of these specimens were sent to the other laboratory and all had detectable, albeit very low titre, inhibitors. Furthermore, on repeated testing over the study period, the vast majority of patients who presented with low ADAMTS13 activity and no detectable antibody subsequently became antibody positive. Fifty-two patients remained antibody negative by our internal and send-out testing. Five of them were known to have or were subsequently diagnosed with hereditary TTP (hTTP). Only one patient continues to have negative antibody but whose clinical course is not consistent with hTTP.
Conclusions:
We found that a commercial ADAMTS13 (Technoclone Technozym) antibody assay is falsely negative in a substantial proportion of patients with autoimmune TTP, the majority of which likely had a low titer inhibitor, below the threshold of test detection. More sensitive assays and/or repeated testing, presumably as inhibitor titre increases during the course of the disease, may detect antibody presence in the majority of samples of patients with autoimmune TTP. This is an important finding as this could impact the types of therapies offered to patients with negative antibody screens and may also avoid unnecessary, expensive genetic testing .

Disclosures
No relevant conflicts of interest to declare.