bluuebunny/crossref_metadata_2025 huggingface dataset:
Interleukin-4 Regulates Eomesodermin In CD8+ T Cell Development and Differentiation
Authors: Carty Shannon A. , Jordan Martha S
Published: October 2019

Cytokines regulate T cell development and function. Interleukin (IL)-4 is a cytokine classically associated with CD4+ T helper (TH) differentiation, specifically TH2 differentiation. However, IL-4 has recently been shown to drive the development of CD8+ innate-like lymphocytes (ILLs). CD8+ ILLs are non-conventional thymocytes that develop with characteristics typically associated with innate and/or memory immune cells, including surface expression of the activation/memory markers CD44 and CD122, expression of the T-box transcription factor Eomesodermin (Eomes) and the rapid production of interferon (IFN)-γ after ex vivo stimulation. Here we show that IL-4 is sufficient to promote Eomes expression in CD8 single-positive (SP) thymocytes in short-term in vitro culture and direct CD8+ ILL development in fetal thymic organ culture. Using genetic deficiency and pharmacologic inhibitors, we demonstrate that IL-4 up-regulation of Eomes in CD8SP thymocytes requires STAT6 and Akt signaling pathways. Next, we investigated the possibility that in addition to directing the fate of developing thymocytes, IL-4 might impact the function of mature CD8+ cells undergoing activation. We found that that in naïve peripheral CD8+ T the combination of IL-4 and low dose T cell receptor (TCR) stimulation is a potent inducer of Eomes. Futhermore, when combined, these stimuli promote the persistence of CD8+ T cells in an adoptive transfer model. Understanding how IL-4 directs CD8+ T cell differentiation may provide a novel way to enhance CD8+ T cell function in adoptive T cell therapies.

Disclosures:

No relevant conflicts of interest to declare.
Structural Studies Of The Factor VIII C2 Domain In a Ternary Complex With Two Inhibitory Antibodies Reveals Classical and Non-Classical Epitopes
Authors: Walter Justin D , Werther Rachel A , Brison Caileen M , Healey John F. , Meeks Shannon L. , Lollar John (Pete) S. , Clint Spiegel P.
Published: October 2019

The factor VIII C2 domain is a highly immunogenic domain, whereby inhibitory antibodies develop following factor VIII replacement therapy for congenital hemophilia A patients. Inhibitory antibodies also arise spontaneously in cases of acquired hemophilia A. The structural basis for molecular recognition by two classes of anti-C2 inhibitory antibodies that bind to factor VIII simultaneously has been investigated by small angle X-ray scattering and X-ray crystallography. The C2 domain/3E6 FAB/G99 FAB stable ternary complex, both in solution and in its crystalline state, illustrates that each antibody epitope resides on opposing faces of the factor VIII C2 domain. The 3E6 epitope is a classical antibody that forms direct contacts to the C2 domain at two loops consisting of Glu2181-Ala2188 and Thr2202-Arg2215, which inhibits the binding of the C2 domain to von Willebrand Factor and phospholipid surfaces. The G99 is a non-classical antibody that prevents proteolytic activation of factor VIII, and its epitope centers on Lys2227 and also makes direct contacts with loops Gln2222-Trp2229, Leu2261-Ser2263, His2269-Val2282 and Arg2307-Gln2311. Each binding interface is highly electrostatic, with positive charges present on both C2 epitopes and complementary negative charges on each antibody. A new model of phospholipid membrane association is also presented, where the 3E6 epitope faces the negatively charged membrane surface and Arg2320 is poised at the center of the binding interface. Furthermore, a 1.7 Å X-ray crystal structure of the porcine factor VIII C2 domain has also been determined, which supports the presented model for phospholipid binding. These results illustrate the complex nature of the polyclonal immune response against the factor VIII C2 domain, and further define the epitopes for both classical and non-classical inhibitory antibodies.

Disclosures:

No relevant conflicts of interest to declare.
Species-Specific Functions For Long Noncoding RNAs During Erythropoeisis
Authors: Paralkar Vikram R , Mishra Tejaswini , Luan Jing , Yao Yu , Kossenkov Andrew , Konuthula Neeraja , An Xiuli , Narla Mohandas , Bodine David M. , Hardison Ross C , Weiss Mitchell J.
Published: October 2019

Mammalian genomes encode thousands of long noncoding (lnc) RNAs, although the extent to which lncRNAs regulate biological functions is not known. Indeed, most lncRNAs show low levels of conservation among mammals, which could suggest limited or species-restricted functions. We used RNA-seq to identify 683 and 1189 lncRNAs expressed during mouse and human erythropoiesis respectively, of which ∼25% are erythroid-specific. Primary cell chromatin immunoprecipitation (ChIP) studies indicate that most mouse erythroid lncRNAs arise from classical promoters, and their genomic loci are bound by the hematopoietic transcription factors GATA1 and SCL/TAL1. Using the G1E-ER4 cell line, which has an inducible form of GATA1, we found that lncRNAs whose genomic loci are bound by GATA1 in primary cells are upregulated on GATA1 induction in the cell line, similar to coding genes.

We compared RNA-Seq transcriptomes of erythroblasts from 8 different mice strains and showed that lncRNA expression is highly conserved within strains of the same species. A comparison of the human and mouse lncRNA transcriptomes showed that only 15% of mouse erythroblast lncRNAs have detectable syntenic transcripts in human erythroblasts, and that most lncRNAs are not detectable in humans.

To identify functional lncRNAs, we used RNA interference in primary erythroblasts to analyze the functions of 21 abundantly expressed murine erythroid lncRNAs, 15 of which are mouse specific and 6 of which have orthologous transcripts in humans. Suppression of seven different lncRNAs inhibited various aspects of erythroid maturation including loss of cell volume, down-regulation of the surface marker CD44, and enucleation. The suppression of enucleation ranged from 25% reduction to 75% reduction of enucleation demonstrated with at least 2 separate shRNAs for each of the 7 lncRNAs (p<0.0005) (Figure 1). Of the seven lncRNAs that have a demonstrable function in murine erythropoiesis, none are expressed in human erythroblasts, indicating that lack of conservation between different species does not necessarily predict a lack of function. These results reflect marked evolutionary differences between protein-coding genes and lncRNAs, and indicate that the latter exert tissue- and species-specific roles in mammalian development.Figure 1Figure 1.

Disclosures:

No relevant conflicts of interest to declare.
A Modified Dose Intensive R- CODOX-M/IVAC For HIV-Associated Burkitt and Atypical Burkitt Lymphoma(BL) Demonstrates High Cure Rates and Low Toxicity: Prospective Multicenter Phase II Trial Of The AIDS Malignancy Consortium (AMC 048)
Authors: Noy Ariela , Kaplan Lawrence , Lee Jeannette Y
Published: October 2019

Background

Concerns about the potential toxicity of the dose-intensive regimens typically used as treatment for BL in HIV seronegative patients prompted us to modify the CODOX-M/IVAC regimen with the goal of preserving efficacy while improving tolerability. The primary objective was to improve the 1 year overall survival (OS) from 65% based on historical reports to 85%. The early stopping rule for excessive treatment related mortality was not met.

Methods

Major modifications of the original US NCI regimen include the addition of rituximab, reduction in cyclophosphamide to 800 mg/m2 x 2 days, vincristine 2 mg cap, methotrexate 3000 mg/m2 on day 15, reduction of lumbar punctures using combination chemotherapy and continuous infusion of the IVAC portion for the high risk patients. Antibiotic prophylaxis and growth factor support were required. HAART therapy was continued at the discretion of the local investigator.

Results

Thirteen AMC centers entered 34 patients. Baseline characteristics included: Classical BL/unclassifiable between DLBCL and BL/other 71/21/9%; High Risk, 94% with stage III/IV 2 (6%)/25 (74%); Median age (range) 42 (19 – 55); CD4 count 195 (0 – 721), CD4 <100, 5 ( 27%); HIV viral load 1819 (Undetectable – 1,187,968). 12 patients had a viral load <100 and only 1 had undetectable plasma HIV RNA level. Only 14 patients were on HAART at study entry. Three/14 were on their second or greater antiretroviral regimen.

Treatment status

Patients with gr 3-5 treatment related toxicity: any 27; hematologic 20; infection 14 including febrile neutropenia 8; 6 metabolic including 1 tumor lysis syndrome; 4 neurologic; 2 thrombotic; and 1 each coagulation, GI or pain. Notably, only 2 patients had gr 1/2 stomatitis/mucositis and none had gr 3/4. Deaths: Seven patients died. There were three treatment-related deaths including encephalopathy with hepatic failure, hepatitis B and pneumonia (1); fungal infection (1) and complications of HIV (1). There were 2 deaths due to progressive lymphoma. Despite the 5 early treatment terminations, the 1 year overall survival was 83.1% (95% CI 63.9%, 92.6%) and the one year progression free survival was 74.1% (55.5%, 86.4%).

Conclusions

Modifications of the CODOX-M/IVAC regimen resulted in a gr 3/4 toxicity rate significantly lower than the 100% seen in the parent regimen, and no grade 3/4 mucositis was observed. Despite a protocol completion rate of only 68%, the 1-year survival rate was 83% which compares favorably with 2 reported studies that excluded HIV + pts. (Magrath et al, 1995; Mead et al, 2008) Correlative studies to improve our understanding of HIV-BL will be presented at the meeting.

Disclosures:

No relevant conflicts of interest to declare.
CD4+ Invariant Natural Killer T Cells Require NKG2D To Protect From Lethal Acute Graft-Versus-Host Disease
Authors: Schneidawind Dominik , Pierini Antonio , Alvarez Maite , Pan Yuqiong , Baker Jeanette , Meyer Everett , Negrin Robert
Published: October 2019

Invariant Natural Killer T (iNKT) cells are a rare cell population in humans (TCRα Vα24-Jα18) and mice (TCRα Vα14-Jα18) that are characterized by a rapid release of immunoregulatory cytokines upon stimulation. The tolerogenic impact of host iNKT cells on graft-versus-host disease (GVHD) after conditioning with total lymphoid irradiation and anti-thymocyte globulin (TLI/ATG) has been shown previously (Lan et al., BBMT 2003). Moreover, we recently described a subset of CD49b+ NKT cells overlapping with iNKT cells and providing protection from acute GVHD mainly via an IL-4-dependent mechanism (Leveson-Gower et al., Blood 2011). Here, we investigated the role of highly purified adoptively transferred donor-derived CD4+ iNKT cells. Balb/c recipient mice were transplanted with T cell-depleted bone marrow together with 1x106 CD4/CD8 T lymphocytes (Tcon) from C57Bl/6 donor mice after irradiation with 8 Gy. Mice co-injected with as low as 5x104 freshly isolated and highly purified (>99%) CD4+ iNKT cells showed a significant survival benefit compared to mice receiving Tcon alone (p=.0015). Consistently, weight and GVHD score improved in mice that received CD4+ iNKT cells. Signal intensity derived from expanding luciferase expressing alloreactive Tcon was significantly lower in animals treated with CD4+ iNKT cells demonstrating inhibition of proliferation of alloreactive Tcon through CD4+ iNKT cells (p<.0001). In vivo CFSE proliferation assay confirmed decreased Tcon proliferation in peripheral lymph nodes (p<.0001), mesenteric lymph nodes (p=.0277) and spleen (p=.0005). CD4+ iNKT cells showed a Th2-biased cytokine profile with high levels of IL-4 and IL-13 in the presence of alloreactive Tcon challenged with irradiated allogeneic stimulators. Co-injection of CD4+ iNKT cells that were expanded 5-fold in vitro with α-GalCer and IL-2 had the same protective effect from lethal acute GVHD compared to freshly isolated CD4+ iNKT cells (p=.7987). Interestingly, CD4+ iNKT cells derived from NKG2D-/- animals were significantly less effective in preventing acute GVHD lethality (WT vs. NKG2D-/-p=.0027). In conclusion, low numbers of highly purified freshly isolated and cultured CD4+ iNKT cells protect from lethal acute GVHD in mice and require NKG2D. Despite the fact that iNKT cells are a rare cell population, the feasibility of in vitro expansion with retained functionality of CD4+ iNKT cells provide the basis for clinical translation.

EM and RN contributed equally.

Disclosures:

No relevant conflicts of interest to declare.
Chimerism and Tolerance Without Gvhd In Mismatched Recipients Of Combined Hematopoietic Stem Cell/Kidney Transplants: Donor-Specific Hyporeactivity Is Not a Reliable Biomarker For Tolerance
Authors: Leventhal Joseph , Abecassis Michael , Miller Joshua , Gallon Lorenzo , Tollerud David J. , Elliott Mary Jane , Ildstad Suzanne T.
Published: October 2019

We recently reported that low-intensity conditioning combined with facilitating cell (FC)-enriched hematopoietic stem cell transplantation (FCRx) can safely achieve high levels of durable chimerism in unrelated and related mismatched kidney transplant (KTx) recipients. This is associated with stable renal function, complete absence of GHVD, and successful withdrawal of immunosuppression (IS). We herein present interim follow-up of now 19 subjects and present data to demonstrate that donor specific hypoactivity does not correlate with tolerance to the renal allograft if chimerism is not present.

HLA-mismatched related (n = 12) and unrelated (n = 8) living donor KTx recipients were conditioned with fludarabine (days -5,-4,-3), cyclophosphamide (50 mg/kg day -3, +3), 200 cGy TBI, (day -1), KTx (day 0), followed by infusion of cryopreserved G-CSF mobilized FCRx (day +1). Multilineage chimerism was assessed prospectively by STR analysis. Mixed lymphocyte reaction (MLR) and cell mediated lympholysis (CML) assays were performed to assess donor-specific hyporeactivity.

Subjects ranged in age from 18-65 years. 18 of 19 patients demonstrated peripheral blood macrochimerism at 1 month post-transplant (6% - 100%). The one patient who failed to engraft was highly sensitized (Class I PRA > 40%). No subjects developed acute or chronic GVHD or “engraftment syndrome.” Chimerism was transient in 4 subjects, each of which received either a suboptimal cell dose or had PRA > 20%. Durable whole blood and T cell chimerism was achieved in 14 subjects. All patients demonstrated in vitro donor-specific hyporesponsiveness (DSH) by MLR +/- CML post-transplant regardless of chimerism status. However, DSH in patients who lost chimerism was not predictive of successful IS weaning, as 3 of 4 transiently chimeric patients with DSH had subclinical Banff 1A rejection on protocol biopsy and while on tacrolimus monotherapy. Recurrence of underlying autoimmune disease has only occurred in subjects who were transiently chimeric. Protocol biopsies in chimeric subjects have exhibited normal histology for up to 3 years. Nine chimeric subjects are completely off IS from 1-34 months. The remainder are in the process of weaning.

The presence of durable, high level whole blood and T cell chimerism in combined kidney + FCRx recipients is a robust biomarker of tolerance. DSH alone without donor chimerism in the first post-transplant year does not predict successful IS withdrawal or tolerance.

Disclosures:

Tollerud: Regenerex, LLC: Equity Ownership. Ildstad:Regenerex, LLC: Equity Ownership.
A Randomized Phase II Study of Tosedostat in Combination with Either Cytarabine or Decitabine in Newly Diagnosed Older Patients with Acute Myeloid Leukemia or High-Risk Myelodysplastic Syndrome
Authors: Mawad Raya , Becker Pamela S. , Hendrie Paul C. , Scott Bart L , Wood Brent L. , Dean Carol , Sandhu Vicky , Deeg H. Joachim , Walter Roland B. , Wang Lixia , Singer Jack , Estey Elihu H. , Pagel John
Published: October 2019

Background: Tosedostat is an oral aminopeptidase inhibitor that has anti-neoplastic activity in a variety of malignancies. Pre-clinical assays have demonstrated synergy between tosedostat and both cytarabine and hypomethylating agents. We performed a randomized, open-label Phase II trial to determine the rates of complete remission (CR) and 4 month overall survival using tosedostat in combination with intermediate-dose cytarabine or decitabine in patients with untreated AML or high-risk MDS.

Methods: Patients ≥60 years old were randomized in a 1:1 fashion to receive tosedostat 120 mg daily by mouth days 1-21 with either 5 days of intravenous cytarabine 1 g/m2/day or decitabine 20 mg/m2/day delivered every 35 days for up to three cycles. Eight patients received 180 mg tosedostat daily continuously. Patients who obtained a CR or CR with incomplete blood count recovery (CRi) after 3 cycles of therapy were eligible to receive up to 2 additional cycles (maximum of 5).

Results: Thirty-four patients with a median age of 70 (range, 60-83) years were treated, with 17 each receiving either tosedostat/cytarabine or tosedostat/decitabine. Twenty-nine patients (85%) had AML and 5 (15%) had MDS RAEB-2. Nineteen patients (56%) had cytogenetics with intermediate-risk, 14 (41%) adverse-risk, and 1 (3%) favorable-risk by European Leukemia Net criteria. Seven patients had FLT3/ITD mutations with normal cytogenetics. Fifteen patients (44%) had secondary AML/MDS or antecedent hematologic disorder. The median duration of treatment was 3 months. The overall CR/CRi rate was 53% (9 in each arm), with 14 patients (41%) achieving a CR and 4 patients (12%) a CRi. CR/CRi was attained in 6 patients with adverse cytogenetics and 4 additional patients with FLT3 mutations. Fourteen patients (41%) were taken off study after a median of 2 cycles due to lack of response or disease progression. With a median follow-up of 11.2 months (range, 0.5-22.3 months), the median overall survival was 11.5 months (95% CI, 5.2-16.7). Twenty-three patients (67.6%) were treated as outpatients and 10 of these patients (44%) required hospitalization during treatment for febrile neutropenia. There were no Grade 3-4 non-hematologic toxicities requiring withdrawal from the study.

Conclusions: Tosedostat in combination with cytarabine or decitabine likely provides a CR/CRi rate of >50% with acceptable toxicity in older patients with untreated AML/ MDS. This approach delivered predominantly as outpatient therapy suggests that further study in a larger, controlled trial may be warranted.

Disclosures

Off Label Use: Pravastatin is not FDA approved for treatment of AML. Wang:CTI Biopharma: Employment. Singer:CTI BioPharma, Corp: Employment, Equity Ownership.
Novel Approach for NK Cell Therapy for Cancer
Authors: Parameswaran Reshmi , Wald David N. , De Lima Marcos , Lee Dean A. , Moreton Stephen
Published: October 2019

Novel therapeutic approaches are urgently needed for many malignancies such as Acute Myeloid Leukemia (AML). We have developed a new therapeutic strategy based upon NK cell immunotherapy that exhibits high clinical potential based upon cell and animal studies. While the harnessing of NK cells for cellular therapy against malignancies has been a topic of interest for several decades, our approach overcomes a major hurdle of insufficient NK cell cytotoxic activity. We have identified that targeting the kinase GSK3 through pharmacologic and genetic approaches leads to the hyperactivation of human blood derived NK cells and a significant improvement in efficacy as compared to traditionally used activated NK cells or chemotherapy in our mouse AML model systems. Importantly this GSK3 inhibition can be achieved through a short ex-vivo incubation of NK cells with a GSK3 inhibitor paving the way for a rapid implementation into a clinical trial. Utilizing both in vitro studies with AML cell lines (ex. OCI-AML3 and HL-60)) and primary human AML cells we observe approximately a 50% increase in efficacy with GSK3 inhibited NK cells as compared to untreated NK cells. Further, we demonstrate significant efficacy of GSK3 inhibited NK cells in a mouse model of circulating human AML. After 4 weekly injections of human NK cells, there is a 50% greater reduction in human AML cells present in the mouse bone marrow with GSK3 inhibited NK cells as compared to vehicle treated NK cells.

Besides efficacy studies, our work has led a model of how GSK3-inhibition enhances NK cell activity as depicted in figure 1. GSK3 inhibition leads to a dramatic increase in adhesion of NK cells to target cells as demonstrated by a flow cytometric adhesion assay (49% vs 83% after 20 min incubation) as well as live cell imaging. Consistent with the increased adhesion, GSK3 inhibited NK cells as well as target cells (after co-incubation) exhibit increased expression of essential NK cell-target adhesion molecules including L-selectin (on NK cells) and ICAM (on target cells). The induction of ICAM on target cells is due to a marked induction in TNFa production from the NK cells upon incubation with target cells (>7 fold increase in TNFa production). TNFa neutralization impairs the NK activity of the GSK3 inhibited NK cells (~30%) but not vehicle treated cells. Finally, GSK3 inhibition also leads to changes in the NK cells that enhance activity such as increased expression of granzyme and perforin and secretion of IFNg. Overall, our work has a revealed a novel strategy for NK cell therapy that holds high clinical potential.

Figure 1. Model of how GSK3 inhibition leads to hyperactive NK cells. GSK3I - GSK3 inhibitor Figure 1. Model of how GSK3 inhibition leads to hyperactive NK cells. GSK3I - GSK3 inhibitor

Disclosures

No relevant conflicts of interest to declare.
Outcome of Allogeneic Hematopoietic Stem Cell Transplantation in Aplastic Anemia: A Comparison of 3 Conditioning Regimens in 202 Consecutive Cases
Authors: Ahmed Parvez , Chaudhry Qamar Un Nisa , Mahmood Syed Karman , Satti Tariq Mahmood , Khan Mehreen Ali , Shahbaz Nighat , Satti Humayoon Shafique
Published: October 2019

Objective: To compare outcome, disease-free survival and rejection in aplastic anaemia patients receiving: (A) ALG or ATG plus cyclophosphamide (CY) 200 mg/Kg ; (B) Fludarabine 120 mg/m2, ATG plus CY 300 mg/m2 and (C) Fludarabine 120 mg/m2, ATG plus CY 120 mg/Kg conditioning regimens.

Patients & Methods: The study included 202 consecutive aplastic anaemia patients undergoing hematopoietic stem cell transplantation from HLA matched sibling donors at this centre from July 2001 to April 2014. Overall 122 had very severe aplastic anemia, 76 severe and 4 non-severe aplastic anemia. Group A, B and C were compared for outcome, disease free survival and rejection. The stem cell source was bone marrow (39.6%); PBSC (7.9%) or both bone marrow plus PBSC (51.5%). GVHD prophylaxis consisted of cyclosporine (51% cases) and cyclosporine plus methotrexate (45.5% case). Chi-square test was used to compare categorical variables. Two way ANOVA was used to compare group means. Kaplan Meier survival curves with log rank test was applied to compare the groups for survival analysis.

Results: Group A included 99, group B 72 and group C 31 patients. Male to female ratio was 76/23 in group A; 56/16 in group B and 22/9 in group C. Median age of patients in group A, B and C were 16, 22 and 18 years respectively (p=0.001). At a median follow up of 1267 days the overall and disease free survival were 76.7% and 68.7% in group A, 70.8% and 69.4% in group B, 67.7% and 54.8% in group C (p=0.350, and p=0.412 respectively). The rejection rate was 11.1%, 9.7% and 35.5% in group A, B and C respectively (p=0.001). Frequency of chronic GVHD was 10%, 8% and 35.5 in respective groups (p<0.001). There was no significant difference between the groups for disease severity, gender and ABO mismatch, stem cell source and GVHD prophylaxis.

Conclusion: Conditioning regimens using ALG or ATG with Cy 200 mg/kg; and Fludarabine 120 mg/m2, ATG plus Cy 120 mg/kg give comparable results while the regimen using Fludarabine 120 mg/m2, ATG plus Cy 300 mg/m2 is associated with high rejection rate and inferior survival in aplastic anemia following allogeneic stem cell transplantation.

Disclosures

No relevant conflicts of interest to declare.
Development of an Alpha/Beta T-Cell Depleted Allogeneic Stem Cell Transplantation Protocol Form Matched Related and Unrelated Donor Grafts in Patients with Poor Risk Leukemia
Authors: DeWitte Moniek A , Boome Liane te , Wagen Lotte van der , Kuball Jurgen H
Published: October 2019

Introduction: The outcome of allo-SCT in patients with poor risk leukemia is still hampered by GVHD and relapse. The innate immune system has been reported to contribute to tumor control, with lower incidence of GVHD. Specific depletion of αβ T- cells – key players in the development of GVHD – will render NK cells and γδ T cells within the allograft. Recently reported results have shown the great promise of this approach in haploindentical transplantations. Within this study, we aim to extend αβT- cell depleted allo-SCT to patients with a MRD or MUD.

Methods: Patients with either ‘poor-risk’ or ‘very poor-risk’ leukemia were included in this phase I study. Either HLA matched siblings (MRD) or fully matched HLA matched (10/10) unrelated donors (MUD) were eligible. abT-cell reduction was performed by negative selection with anti-abTCR antibodies in combination with magnetic microbeads, using the automated CliniMACS device (Miltenyi Biotec, Bergisch Gladbach, Germany). The maximal contamination with αβT-cells for all dose levels was 5x105/kg. Three conditioning regimens have been investigated (I): fludarabine 120 mg/m2 + cyclophosfamide 4800 mg/m2, (II): fludarabine 120 mg/m2 + busilvex AUC=90 and (III): ATG (Genzyme®) 4 mg/m2 + fludarabine 120 mg/m2 + busilvex AUC=90 followed by αβT- cell depleted grafts from matched related or unrelated donors. Within cohort II and III, no additional immune suppression was given after allo-SCT.

Results: Products for 14 patients have been successfully processed and used for αβT-cell depleted allo-SCT between 2011 and 2013. A ~4 log depletion of αβT-cells has been observed in the product with a recovery of ~75% of CD34+ cells. In cohort I and cohort II, 60% and 25% primary graft failures were observed, whereas in cohort III primary engraftment (chimerism > 95%) was observed in all patients. The combination of ATG/fludarabine/busilvex was well tolerated with a hematological recovery of within 3 weeks. In all 14 patients immune reconstitution primarily consisted of innate cells (NK cells and γδ T cells) the first 6 months post transplantation. In addition, no increase in CMV or EBV reactivations has been observed so far under the profound “innate control”.

Conclusion: ATG Busulfan Fludarabine is a low toxicity platform for abTCR-depleted transplantations, resulting in a swift reconstitution of innate cells (NK cells and γδ T cells) the first 6 months post transplantation. This transplantation strategy can serve as a tool for future immunological interventions such as a low dose DLI or genetically modified T cells.

Disclosures

No relevant conflicts of interest to declare.
CD70/CD27 Signaling Mediates Resistance of Chronic Myeloid Leukemia Stem Cells to Tyrosine Kinase Inhibitors By Compensatory Activation of the Wnt Pathway
Authors: Riether Carsten , Schürch Christian M. , Radpour Ramin , Hinterbrandner Magdalena , Huguenin Anne-Laure , Ochsenbein Adrian F.
Published: October 2019

The introduction of BCR/ABL-specific tyrosine kinase inhibitors (TKIs) a decade ago revolutionized chronic myelogenous leukemia (CML) therapy. However, disease-initiating leukemia stem cells (LSCs) in CML are resistant to TKIs despite BCR/ABL inhibition. Therefore, CML will ultimately relapse upon drug discontinuation. We have previously shown that blocking CD70/CD27 signaling targets LSCs by inhibiting the activation of the Wnt pathway. Here, we investigated a combination therapy of TKIs and CD70/CD27 blocking monoclonal antibodies in human and murine CML. We demonstrate that TKI-mediated BCR/ABL inhibition down-regulates miR-29, leading to increased expression of specificity protein 1 (SP1), a transcription factor with binding site in the CD70 promoter. In addition, TKI treatment reduced the expression of DNA methyltransferases resulting in de-methylation of the CD70 promoter. These combined effects resulted in CD70 up-regulation on LSCs, enhanced CD70/CD27 signaling and compensatory Wnt pathway activation. Combined BCR/ABL and CD70/CD27 inhibition synergistically reduced Wnt signaling and eradicated leukemia cells in vitro. More importantly, combination therapy effectively eliminated CD34+ CML stem/progenitor cells in murine xenografts and LSCs in a murine CML model. Therefore, TKI-induced CD70 up-regulation triggers CD70/CD27 signaling leading to compensatory Wnt activation. These findings identify an important targetable TKI resistance mechanism of CML LSCs and may lead to new therapeutic strategies to directly target LSCs to overcome treatment resistance.

Disclosures

No relevant conflicts of interest to declare.
Sphingosine 1-Phosphate (S1P)/S1P Receptor 1 Pathway Has an Essential Role for Sickle Cell Disease
Authors: Zhang Yujin , Zhao Shushan , Wu Hongyu , Hu Xia , Luo Renna , Parchim Nicholas , Kellems Rodney E , Xia Yang
Published: October 2019

Sickle cell disease (SCD) is a devastating hemolytic genetic disorder associated with high morbidity and mortality. In order to understand the pathogenesis of this disease, we have conducted non-biased metabolomic screening and found that circulating sphingosine-1-phosphate (S1P) was significantly elevated in mice and patients with SCD. S1P is an important bioactive lipid signaling molecule known to regulate inflammation. Our previous study demonstrated that reduced S1P level in plasma and erythrocytes by treatment with sphingosine kinase 1 (SPHK1) inhibitor, PF-543, significantly decreased sickling cells, hemolysis and inflammation in SCD mouse model, which indicated that S1P may play an important role in an SCD complication, especially in inflammation.

S1P engages five G-protein coupled receptors known as S1PR1-5. Targeting S1P signaling has been successfully applied in the treatment of the autoimmune disease-multiple sclerosis with the compound named FTY720. In order to understand the roles of S1P/S1PRs signal pathway in pathophysiology of SCD, we treated SCD mice with S1P receptors antagonist FTY720. The results showed that FTY720 successfully inhibited S1P receptors, especially S1P1 expression on immune cells from thymus and lymph node (P<0.05). Circulating white blood cells and inflammatory cytokines, such as CRP, IL-1β, TNF-α and IL-6, also decreased significantly measured by ELSIA kit. Additionally, FTY720 treatment significantly ameliorated organ damage. To investigate the roles of S1P1 receptor in SCD, we treated SCD mice with S1P1 specific antagonist, SEW2871. The results demonstrated that circulating white blood cells and inflammatory cytokines reduced significantly. Histologic studies revealed that the necrosis and congestion of multiple organs including kidney, lung and spleen were substantially reduced by SEW2841.Our studies demonstrate the elevated circulating S1P signaling via its receptor (likely S1PR1) directly contributes to inflammation and multiple tissue damage. Thus, it provides strong evidence that S1P/S1P1 pathway is likely a therapeutic target for SCD.

Disclosures

No relevant conflicts of interest to declare.
Bone Marrow Derived Mesenchymal Cells Secrete Granulopoietic Cytokines upon Danger Signaling
Authors: Wirths Stefan , Bugl Stefanie , Radsak Markus P. , Märklin Melanie , Müller Martin R. , Kanz Lothar , Kopp Hans-Georg
Published: October 2019

Granulopoietic homeostasis is regulated at steady-state to supply sufficient numbers of pooled and circulating neutrophils to maintain barrier function against commensal flora. In addition, upon pathogenic microbial challenge, an increased formation of neutrophils is induced, termed ‘emergency granulopoiesis’.

Antibody-mediated reduction of neutrophil numbers in steady-state induces a feedback loop leading to an increase of bone marrow granulopoiesis with expansion of hematopoetic stem and progenitor cells. This feedback loop was demonstrated to depend on TLR4 and TRIF, but not MyD88 signaling (Bugl et al. Blood 2013). In contrast, emergency granulopoiesis was shown to be dependent on MyD88 signaling in endothelial cells (Boettcher et al. Blood 2014).

Bone marrow mesenchymal stromal cells (MSC) are niche-forming cells, harboring and regulating hematopoiesis. Upon steady-state neutropenia an increase of niche size was observed. Here we investigated, whether niche-forming MSC act as sensors of pathogen-associated molecular patterns (PAMPs) and induce granulopoietic cytokines to stimulate expansion of adjacent hematopoietic stem and progenitor cells.

MSC of C57BL/6 and TLR4-KO mice were cultured in vitro and treated with LPS for 24 hours. Cells were harvested and qRT-PCR for G-CSF, TLR4, MyD88, TRIF, GM-CSF, IL-1β, IL-18 and Casp-1 was performed

After treatment with LPS, RNA of granulopoietic cytokines G-CSF and GM-CSF were massively up regulated in MSC of WT mice. Upstream regulating, inflammasome components IL-1ß and caspase-1 RNA levels increased as well, with little changes in IL-18, TLR4, MyD88 and TRIF.

Unexpectedly, TLR4-KO MSC up regulated transcription of IL-1β and G-CSF upon LPS stimulation as well, and caspase-1 was found to be strongly up-regulated in unstimulated TLR4-KO compared to WT MSC.

In summary, bone marrow stromal cells are found to be PAMP-sensing and secrete cytokines that regulate granulopoiesis. TLR4-independent sensing of LPS by MSC might correspond to the alternative noncanonical inflammasome pathway recently described (Kayagaki et al. Science 2013).

Disclosures

No relevant conflicts of interest to declare.
Further Research on the Activity of Ribonucleases in the Blood and Urine of Patients Suffering from Proliferative Hemocytopathia
Authors: ALEKSANDROWICZ J. , URBANCZYK J. , OSTROWSKA ALEKSANDRA , SIERKO J.
Published: October 2019

1. A simple quantitative method (based on McCarty’s method) of measuring the RN-ase activity in urine and blood is presented.

2. The RN-ase activity was examined in the blood and urine of patients suffering from chronic granulocytic leukemia, myeloblastic leukemia, lymphatic leukemia, granulocytopenia, lymphogranulomatosis, and in cases of infectious disease with an increased leukocytosis.

3. The RN-ase activity in the urine is increased in chronic granulocytic leukemia, while the RN-ase activity in the blood serum remains unchanged. In other diseases, in spite of an increased or decreased leukocytosis, no essential changes in the RN-ase activity in the urine and blood serum have been observed, which indicates that there is no close relationship between the number of leukocytes in the peripheral blood and the RN-ase activity in the 24-hour sample of urine.
Effect of Chemical Protection and Bone Marrow Treatment on Radiation Injury in Mice
Authors: URSO PAUL , CONGDON C. C. , DOHERTY D. G. , SHAPIRA RAYMOND
Published: October 2019

MEG (prepared from 9.0 mg. of AET) significantly modified the response of the bone marrow, peripheral blood leukocytes, spleen, thymus, body weight, hematocrit, and histology of the hematopoietic organs to lethal (900 r) and sublethal (450 r) x-irradiation in CAF1 mice.

MEG reduced the effect of 900 r on the bone marrow, granulocytes of the blood, hematocrit, spleen, thymus, and body weight by a factor of approximately two.

Combined treatment (MEG and isologous bone marrow) of mice exposed to 900 r of x-rays demonstrated that MEG is primarily responsible for preventing the early destruction of the bone marrow, but bone marrow injection was primarily responsible for causing a more rapid recovery of the bone marrow.

In mice receiving combined treatment, recovery of the leukocytes and spleen was primarily influenced by the bone marrow injection; whereas recovery of the thymus and body weight was primarily influenced by MEG. The hematocrit values were normal after combined treatment.
A Rapid Method for Estimation of the Total Leukocyte Count
Authors: BENJAMIN NAOMI R.
Published: October 2019

A technic has been devised for the estimation of the white blood cell count by examination of a stained drop of blood of standard size. The drop is measured by immersing a pinhead in the blood and then touching onto a microscope slide the blood which adheres to the surface of the pinhead. The drop is dried and stained and the count is estimated by comparing its microscopic appearance with photographs or "standard" preparations. It was developed to be used as a screening procedure in case of atomic war, but it may prove useful in epidemics or as a routine screening method in the hematology laboratories of large hospitals.
A New Hereditary Hemoglobinopathy (the Lepore Trait) and Its Interaction with Thalassemia Trait
Authors: GERALD PARK S. , DIAMOND LOUIS K.
Published: October 2019

A new hemoglobinopathy, termed the "Lepore trait," is described. The Lepore trait is characterized by an altered erythrocyte morphology, resembling classical thalassemia trait,[unknown] and by the presence of a hitherto unreported abnormal hemoglobin occurring in low concentration (10-12% of the total hemoglobin). The trait is transmitted as if due to a single gene defect. Simultaneous inheritance of the Lepore trait and classical thalassemia trait occurred in one member of the pedigree studied and resulted in a severe hemolytic anemia clinically indistinguishable from thalassemia major.

On the basis of the erythrocyte morphology, the clinical findings, and the electrophoretic studies, it is conjectured that the Lepore trait may be more closely related to thalassemia than to the other abnormal hemoglobin syndromes.
The Anemia of Human Riboflavin Deficiency
Authors: LANE MONTAGUE , ALFREY CLARENCE P.
Published: October 2019

1. Riboflavin deficiency was induced in 8 adult males with a riboflavin deficient diet and a riboflavin antagonist, galactoflavin.

2. Each patient developed a normochromic normocytic anemia and reticulocytopenia.

3. The leukocyte counts and platelet counts remained within the normal range throughout the period of deficiency.

4. The bone marrow changes and ferrokinetic characteristics of this anemia have been described.

5. The anemia was reversed by riboflavin administration.
Intranuclear inclusions in Bence Jones lambda plasma cell myeloma
Authors: Cohen HJ , Lefer LG
Published: October 2019

A patient with plasma cell myeloma producing only Bence Jones lambda protein was found to have pale intranuclear inclusions in the majority of the bone marrow plasma cells. These inclusions, previously undescribed in myeloma patients producing only Bence Jones protein, contained Bence Jones lambda protein, were non-electron dense, bound by a single membrane,and contained no cytoplasmic structures. Intracytoplasmic inclusions were not present, and the perinuclear cistern was not dilated. Thus, the inclusions may represent intranuclear protein synthesis with anomalous release in the abnormal cells.
Effect of blood in the gut on measurements of endogenous carbon monoxide production
Authors: Brouillard RP , Conrad ME , Bensinger TA
Published: October 2019

Changes induced in measurements of endogenous carbon monoxide (CO) production by blood in the lumen of the gut were studied in five normal volunteers. The study was undertaken because exogenous heme is absorbed by intestinal mucosal cells where the porphyrin ring is split with the release of CO that could contribute to blood CO levels and lead to a fallacious diagnosis of hemolytic disease. Volunteers who consumed 200 ml of their own blood doubled their endogenous production of CO (0.69 versus 0.34 mumoles/kg/hr). This suggested that at least 3% of the ingested heme was degraded and recovered as CO within 2 1/2 hr. Measurements of serum bilirubin also showed a significant increase after ingestion of blood. These data indicate that blood in the gastrointestinal tract can interfere with quantification of heme and bilirubin turnover from measurements of either endogenous CO production or bilirubin and suggest that this might occur with the ingestion of meat.
Dibromomannitol in the treatment of chronic granulocytic leukemia: a prospective randomized comparison with busulfan
Authors: Canellos GP , Young RC , Neiman PE , DeVita VT Jr
Published: October 2019

Dibromomannitol (DBM) is a new agent for the treatment of chronic granulocytic leukemia. A propsective evaluation of the drug was undertaken in a randomized comparison with busulfan. Forty previously untreated, Philadelphia chromosome-positive cases were treated, with 20 patients in each treatment group. The protocol provided for continuous maintenance therapy after remission induction, with a crossover to the opposite drug in patients who became refractory to the primary agent but are without evidence of blastic tranformation. There were 14 remissions in the DBM group and 15 in those treated with busulfan. The rate of decrease of the elevated leukocyte count was more rapid with DBM, but prolonged disease control off treatment occurred in only three of 14 cases as opposed to nine of fifteen busulfan-treated patients who required a median delay of 12 mo before maintenance could be initiated. Hypoplasia occurred in one DBM patient and two busulfan cases. Following recovery, crossover to the opposite drug in two cases again resulted in hypopllasia. Increased skin pigmentation, amenorrhea, pulmonary fibrosis, and cytologic dysplasia, commonly associated with busulfan adminstration, were also noted with DBM. The median duration of disease control with busulfan was 34 mo and 26 mo with DBM. There was no signigicant difference in the incidence of blastic transformation, and median survival for both groups was 44 mo. DBM appears to be as effective as busulfan in the treatment of the chronic phase of CGL but with a more predictable myelosuppressive action. The principal advantage of busulfan over DBM is the fact that more than half the busulfan-treated patients experienced prolonged disease control off treatment.
Syndrome of recurrent increased secretion of antidiuretic hormone following multiple doses of vincristine
Authors: Stuart MJ , Cuaso C , Miller M , Oski FA
Published: October 2019

The syndrome of inappropriate antidiuretic hormone secretion (SIADH) has been recognized to occur following treatment with vincristine. None of the reports have provided information regarding its potential for recurrence on further challenge with vincristine (VCR), an agent generally required for repeated use in patients with malignancies. Symptomatic hyponatremia and SIADH that occurred 8 days following administration of VCR in a child with acute lymphatic leukemia was documented with specific radioimmunoassay of urinary ADH levels. The further occurrence of recurrent elevations in ADH excretion 8–10 days following repeated treatment with VCR was also observed. However, SIADH was prevented by prophylactic rigorous fluid restriction. The occurrence of SIADH following VCR therefore does not preclude the further safe usage of this drug.
Use of postpheresis plasma to improve granulocyte yields for transfusion
Authors: Roy AJ , Brivkalns A , Yankee RA
Published: October 2019

Humoral factors which stimulate release of mature granulocytes from body reserves are presumed to be the mechanism through which high yields of granulocytes are obtained from donors by filtration leukopheresis. Postpheresis plasma (PPP) obtained 2 hr after leukopheresis, when infused into normal rats, induced a peak granulocytosis at 3 hr of 22,000/cu mm above controls. A substance in the nylon filters, which caused a peak granulocytosis at 4 hr of 7600/cu mm above controls, was eliminated by washing the filter with 30 volumes of saline. Injection of PPP obtained following leukopheresis with washed filters resulted in an 8000/cu mm increase in granulocytes. One milliliter of PPP given 1 hr before pheresis increased the granulocyte yield from 4.3 to 8.7 times 10–7 granulocytes in a 2-hr run. We conclude that (1) a humoral substance elaborated by the host during filtration leukopheresis induces a granulocytosis in the donor, (2) a substance in commercial leukopaks, which can be eliminated by vigorous washing of the filters, may be responsible in part for granulocytosis observed during leukopheresis, (3) PPP may be used to increase granulocyte yields in donors undergoing leukopheresis.
Culture of autologous bone marrow in diffusion chambers: effect of host irradiation
Authors: Laissue JA , Chanana AD , Cronkite EP , Joel DD , Pavelec M
Published: October 2019

Autologous bone marrow (BM) cells were cultured in diffusion chambers (DC) implanted into whole-body irradiated, non-irradiated, or sham- irradiated goats. Proliferation was apparent in DC implanted in both irradiated and nonirradiated goats. However, cells in DC cultured in irradiated hosts increased in number beginning earlier, proceeded at a faster rate, and reached higher numbers than in DC in nonirradiated hosts. Growth enhancement could not have occurred as a result of radiation-induced immunosuppression in autologous hosts. The nonirradiated “target cells” in the DC therefore constituted an indicator system for stimulatory or inhibitory substances in the host. The simultaneous increase in the number of granulocytes in peripheral blood and in DC of irradiated hosts was paralleled by an initial rise in serum colony-stimulating factor (CSF). A second, prolonged period of severe granulocytopenia following irradiation of the host correlated with high levels of serum CSF. Increased numbers of megakaryocytes were seen in DC as thrombocytopenia developed in the irradiated host. DC erythropoiesis disappeared rapidly in nonirradiated goats; however, in DC of irradiated goats, the number of erythrocytic precursors increased exponentially during ablation of host erythroid marrow. Anemia did not develop in the host during the culture period. Proliferation of mononuclear cells in DC was markedly stimulated by irradiation of the host. Proliferation of macrophages appeared independent of host treatment. These observations provide strong evidence for diffusion of specific and/or nonspecific humoral hematopoietic stimulators from the host into the DC. This stimulation appears to be elicited and/or intensified by irradiation of the host.
Recovery of normal hematopoietic tissue and tumor following chemotherapeutic injury from cyclophosphamide (CTX): comparative analysis of biochemical and clinical techniques
Authors: Rosenoff SH , Bostick F , Young RC
Published: October 2019

Simultaneous alterations in the incorporation of 3H-thymidine (3H-TdR) into DNA are induced by CTX in normal host target tissues and L1210 ascites tumor. The timing of suppression and recovery of these nucleoside incorporation alterations was similar at the three CTX doses studied, but some evidence for a dose-response effect was seen as the magnitude of suppression of DNA synthesis increased with increasing dosage. A differential pattern of suppression and recovery of 3H-TdR incorporation in malignant and normal host tissues was observed. The pattern of suppression and recovery of the peripheral white blood count and bone marrow (BM) cellularity, two frequently studied clinical parameters of hematopoietic recovery, were out of phase with the recovery of BM-DNA synthesis and failed to accurately reflect the sensitivity of the BM to subsequent chemotherapeutic injury. In contrast, drug schedules based on the differential recovery patterns of the host tissues and tumor, reflected by their 3H-TdR incorporation into DNA, both reduced toxicity to normal mice and increased the survival of tumor-bearing animals.
A new method of measuring the deformability of the red cell membrane
Authors: Bull BS , Brailsford JD
Published: October 2019

Red cells moving in a stream of fluid can be arrested by means of a network of fibrin threads. Those cells which fold over a fibrin thread present to view a flat strap-like portion of membrane in which the strain can be easily observed and accurately measured both optically and by scanning electron microscopy. Preliminary results obtained by this method show elastic strains of nearly 300% and indicate that the stress required to produce these large strains is an order of magnitude greater than that reported by other methods.