bluuebunny/crossref_metadata_2025 huggingface dataset:
Relapse in Childhood “Very High Risk” Acute Lymphoblastic Leukemia Treated with BFM Chemotherapy Has a Dismal Outcome Even When Treated with Transplantation.
Authors: Balduzzi Adriana , Valsecchi Maria Grazia , Schrauder André , Silvestri Daniela , Buldini Barbara , von Stackelberg Arend , Testi Anna Maria , Klingebiel Thomas , Peters Christina , Rizzari Carmelo , Barisone Elena , Schrappe Martin
Published: October 2019

The definition “very-high risk” (VHR) in the setting of childhood acute lymphoblastic leukaemia (ALL) is usually adopted to identify children eligible for transplantation in first complete remission (CR1). The endpoint of this study is to assess whether, how many, and how VHR ALL children achieving CR1 and not transplanted in CR1 can be rescued in case of relapse. Eligiblity criteria to transplantation in AIEOP-BFM ALL 2000 study slightly changed overtime and differed between the BFM (Berlin Frankfurt Muenster) and AIEOP (Associazione Italiana di Ematologia ed Oncologia Pediatrica) Groups.. For this analysis VHR ALL was defined by one or more of the following criteria:

induction failure (IF), high levels (≥ 1x10−3) of minimal residual disease at day 78 (VHR-MRD), clonal abnormality t(4;11), prednisone poor response associated (PPR+) with one or more of the followings: hyperleukocytosis, T-immunophenotype, ≥ 25% marrow blasts at day 15, pro-B immunophenotype.

Patients carrying clonal abnormality t(9;22) were not included here since allocated in a specific study protocol since 2004. Out of 571 VHR patients, distributed among the four criteria (17%, 45%, 3%, 35%, as hierarchically listed), 249 (43%) were transplanted in CR1 (80%, 48%, 47%, 25% in each category) and 322 (57%) were not. Of the 322 patients treated with chemotherapy [5-year-EFS 53.5% (SE 3.1), 5-year-survival 66.6% (SE 2.9)], 19 died in CR1, 6 presented with a second malignancy, and 113 (35%) relapsed at a median of 15 months after diagnosis, mostly in the bone marrow (81%: 66% isolated, 15% combined) or in an extramedullary site (19%, 15% in the central nervous system) at a median time of 9 ms after diagnosis. The 3-year-survival after relapse was only 26.0% (SE 5.1) for the 113 patients overall, 38.7% (SE 7.8) for the 55 who underwent transplantation in CR2 and 16.2% (SE 5.3) for the 58 who were not transplanted in CR2. Interestingly the 3-year-survival after relapse by VHR criteria was only 7.1% (SE 9.3) in the IF, 28.6% (SE 7.1) in the VHR-MRD and 33.1% (SE 6.7) in the PPR+ subgroups. In conclusion very few VHR-ALL children could be rescued after relapse, particularly among those carrying most unfavourable criteria at the onset (IF and VHR MRD), confirming that transplantation should be performed in CR1. Transplantation in CR2 was feasible in only half of the patients and presumably their outcome was influenced by MRD response after relapse; this aspect is under investigation. New strategies are needed for this dismal subset of patients.
AML1-ETO Collaborates with the Homeobox Gene Meis1 in Inducing Acute Leukemia in the Mouse Bone Marrow Transplantation Model.
Authors: Naidu Vegi M. , Rawat Vijay P.S. , Schessl Christina , Petropoulus Konstantin , Cusan Monica , Deshpande Aniruddha J , Martinez Leticia Quintanilla , Hiddemann Wolfgang , Buske Michaela Feuring , Buske Christian
Published: October 2019

AML1-ETO is the most frequent fusion gene in human AML. Previously, we and others have demonstrated that the fusion is not able to cause leukaemia on its own in experimental murine models, but that it needs collaborative partners. However, although mutations such as the FLT3-length mutation and C-KIT mutations were defined as important collaborative genetic events in AML1-ETO positive AML, most human AML1-ETO cases do not carry these mutations, indicating the presence of unkown collaborative partners in these patients. On the other hand Meis1, a HOX gene co-factor, belonging to the TALE family of homeodomain proteins, has a well established function as a protooncogene with a strong collaborative potential in Hox gene associated AML in mice. First we confirmed expression of MEIS1 in some patients with AML1-ETO positive AML by real-time PCR. Based on this we sought to determine if AML1-ETO can collaborate with Meis1 in inducing acute leukemias: single constructs or both genes were co-transfected in 5-FU treated primary murine bone marrow cells by retroviral gene transfer, using MSCV retroviral constructs with an IRES–GFP or YFP cassette. Mice were transplanted with BM cells expressing Meis1 alone (n=10), with BM cells solely expressing the fusion gene (n=10) or EGFP (n=7, control) or with BM expressing both genetic alterations (n=14). None of the mice in the Meis1 and AML1-ETO as well as in the control group developed disease. In contrast, 14 mice transplanted with BM co-expressing AML1-ETO and Meis1 developed lethal disease after a median latency of 102 days. Three mice succumbed to a myeloproliferative syndrome and nine mice died by acute leukemia (6 mice developed AML, 3 mice ALL), which was serially transplantable into secondary recipients (median = 57 days). Immunohistochemistry of various organs of leukemic mice showed massive infiltration with blast cells. In MPS and AML 85 ± 9.3 % of the blast cells co-expressed Gr-1+ and Mac1+. In ALL cases 40 ± 19.9 % of the malignant cells co-expressed Mac1 and the lymphoid-associated B220 antigen. Analysis of retroviral integration did not reveal recurrent integration sites as an indication for insertional mutagenesis. In summary, our data demonstrate for the first time that AML1-ETO can collaborate with Meis1 and identify a novel collaborative partner in t(8;21) positive AML. Furthermore, our analyses demonstrate that Meis1 can collaborate with non-homeobox genes in inducing acute leukemia.
Results of a Randomized Phase 2 Study of Clofarabine Plus Cytarabine (CA) Vs Clofarabine Plus Idarubicin (CI) Vs Clofarabine, Idarubicin, Plus Cytarabine (CIA) as Salvage Therapy in Acute Myeloid Leukemia (AML).
Authors: Faderl Stefan , Borthakur Gautam , Ravandi Farhad , Huang Xuelin , Beran Miloslav , Ferrajoli Alessandra , Koller Charles A. , Thomas Deborah A. , Verstovsek Srdan , Kwari Monica , Kantarjian Hagop M.
Published: October 2019

Clofarabine is a second-generation deoxyadenosine analog with activity in adult AML. To improve single agent activity, various combinations of clofarabine are being studied. In two dose-finding phase I studies, we have previously established the MTD and DLT of the CI and CIA combinations in AML salvage (Blood2006; 108: 51a). Here we report the outcome of a subsequent adaptively (Bayesian) randomized phase 2 study comparing the two anthracycline-containing combinations CI and CIA with CA. Eligible were patients (pts) for first salvage therapy with first relapse or primary refractory AML. Drug doses and schedules were as follows: CI - clofarabine 22.5 mg/m2 iv d 1–5 and idarubicin 10 mg/m2 iv d 1–3; CIA – clofarabine 22.5 mg/m2 iv d 1–5, idarubicin 6 mg/m2 iv d 1–3, cytarabine 0.75 g/m2 d 1–5; CA - clofarabine 40 mg/m2 iv d 1–5 and cytarabine 1g/m2 iv d 1–5. Up to 2 induction and 4 consolidation cycles were allowed. Eighty-five patients were enrolled of whom 80 are evaluable. Pretreatment characteristics were similar with respect to median age (CI 57 yrs [range 25–73], CIA 56 [21–81], CA 55 [39–73]), proportion of pts with secondary AML (CI 33%, CIA 28%, CA 29%), proportion of pts with primary refractory disease (CI 31%, CIA 50%, CA 41%), prior median CR duration (CI 6 mos [1–108], CIA 6 [1–18], CA 10 [2–27]), prior exposure to at least intermediate doses of cytarabine (CI 78%, CIA 63%, CA 65%), and proportion of pts with FLT3/ITD abnormalities (CI 25%, CIA 17%, CA 18%). Thirty-three pts received CI, 31 CIA, and 16 CA. Overall, 31 (39%) pts responded (28% CR, 9% CRp). No difference in response was observed according to treatment arm (CI - CR 27%, CRp 12%; CIA - CR 29%, CRp 13%; CA - CR 25%, CRp 6%). Median CR duration was 6.4 mos (0.2–18) with CI, and not reached (1–12+) with CIA and CA (0.3–24) (p=0.07). Overall survival was 5.5 mos (0.3–25.5+) for CI, 5.1 mos (0.4– 13.4+) for CIA, and 5.1 mos (0.4–27+) for CA (p=0.94). Adverse events were comparable to the preceding phase 1 studies and as have been described with clofarabine in the past. Induction deaths occurred in 6% (CI), 16% (CIA), and 25% (CA), respectively (p>.05), and were due to myelosuppression-associated infectious complications. In summary, the response rate of the clofarabine combinations in AML salvage is 39% (CR+CRp). Response duration and overall survival are not significantly different between the treatment arms.
Phase 1 Clinical Trial of Prion-Filtered Red Cell Concentrates (pfRCC) in Patients Requiring Allogeneic Blood Transfusion.
Authors: Murphy Tracy , Cahill Mary R , Fagan Jim , Harry Croxon , Khan Mohammed , Gilligan Oonagh , McGrath Stephen , Murphy William G
Published: October 2019

Background; Transmission of vCJD by blood transfusion from pre-symptomatic blood donors has occurred in 4 reported cases to date. Screening blood donors for infectivity is unlikely to be feasible for several years. Removing infectivity from blood using selective filtration may provide a useful degree of protection from transfusion transmission of the disease. A filter that may remove infectivity from red cell concentrates has been developed and trialed in volunteers receiving autologous blood. No studies have been carried out to date of pfRCC in allogeneic transfusions in the clinical setting.

Aims; To establish safety and tolerability of transfusion of prion filtered red cell concentrates.

Methods; Twenty patients scheduled to receive transfusion were recruited following ethical approval and with informed consent. Prion filtered units were prepared by the Irish Blood Transfusion Service. A mean loss of 9 gm of haemoglobin per unit of RCC occurred during the filtration process. Each patient received one unit of pfRCC, and a median of 2 units overall (range 1 to 4 units) per transfusion episode. A cross-match sample, full blood count (FBC), renal and liver profile was taken from each patient prior to transfusion. Patients were observed for adverse reactions. After 24 hours, FBC, renal and liver profile were repeated. Six weeks after the transfusion a further sample was tested for red cell antibodies. Six of these patients have consented to undergo re-transfusion with pfRCC. Two re-infusions have taken place uneventfully six months after the first exposure to pfRCC and 4 more are planned. Results No serious adverse events were encountered during the study, or at 24 hour and 6 week follow up after the initial transfusion episode. Mean haemoglobin increment per unit transfused was 0.68g (SD 0.45g; range −0.5 to 1.35g ). Recruitment and follow-up is ongoing in patients exposed to repeat transfusion challenge. Summary The first clinical transfusions of pfRCC were well tolerated. Two patients were rechallenged with transfusions of pfRCC without adverse effect. Further studies with transfusions of prion filtered red cells are now warranted to extend the safety data and to determine whether efficacy is comparable to standard transfusions in adult and paediatric populations.
Human Adenylate Kinase 2 Deficiency Causes a Profound Haematopoietic Defect Associated with Sensorineural Deafness
Authors: Cavazzana-Calvo Marina , Lagresle-Peyrou Chantal , Six Emmanuelle M , Picard Capucine , Rieux-Laucat Frederic , Michel Vincent , Ditadi Andrea , Demerens-de-Chappedelaine Corinne , Morillon Estelle , Valensi Françoise , Simon-Stoos Karen L , Mullikin James C , Noroski Lenora M , Besse Céline , Wulffraat Nicolas , Ferster Alina , Abecasis Manuel M , Calvo Fabien , Petit Christine , Candotti Fabio , Abel Laurent , Fischer Alain
Published: October 2019

Human adenylate kinase 2 deficiency causes the most profound human haematopoietic defect associated with sensorineural deafness. Reticular dysgenesis (RD) is an autosomal recessive form of human severe combined immunodeficiency (SCID) characterized by an early differentiation block in the myeloid lineage and a profound impairment in lymphoid maturation associated with bilateral sensorineural deafness. The lack of polymorphonuclear neutrophils in affected newborns is responsible for the occurrence of severe infections earlier than usually observed in the other forms of SCID. Furthermore, RD associated neutropenia is characterized by the lack of responsiveness to G-CSF. We have identified bi-allelic mutations in the adenylate kinase 2 (AK2) gene is seven patients affected with RD. These mutations resulted in the absence or a strong decrease in protein expression. Restoration of AK2 expression in the bone marrow cells of RD patients overcomes the neutrophil differentiation arrest, underlying its specific requirement in the development of a restricted set of haematopoietic lineages. Lastly, we established that AK2 is specifically expressed in the stria vascularis region of the inner ear. The function of this gene in the differentiation of a given set of cell lineages is rapidly in progress, showing that studies of primary immunodeficiencies continue to provide key information on human lympho-haematopoietic development. Moreover the AK2 enzyme seems a key molecule in different biological systems such as the lympho-haematopoiesis and the brain development.
Adoptive Immunotherapy with Memory T Cells.
Authors: Heslop Helen E.
Published: October 2019

Clinical adoptive cellular immunotherapy of malignancy and viral infection should transfer T cells that expand in vivo on exposure to antigen and can enter the memory compartment to persist long-term. A number of factors, including cellular phenotype, influence the behavior of the infused line. Primate studies have shown that antigen-specific CD8+ T cell clones only persisted long-term in vivo if they were derived from central memory T cells, but not from effector memory T cells, reacquiring phenotypic and functional properties of memory T cells.1 Other studies have suggested that adoptive transfer of ex vivo-expanded effector memory T cells will have poor survival and clinical efficacy, reporting instead that less differentiated T cells with longer telomeres exhibit longer persistence. These data imply that prolonged ex vivo expansion, required, for example, for T cell cloning, adversely affects subsequent in vivo expansion and survival. However, our trials administering ex vivo-expanded, polyclonal EBV-specific T cell lines demonstrated that expanded effector memory T cells, infused into a lymphodepleted host, can expand massively in vivo, enter the memory compartment, and persist for up to seven years after infusion. Furthermore, in a study infusing trivirus-specific CTLs with effector memory phenotype, we saw expansion of CTLs specific for the latent viruses CMV and EBV. By contrast, adenoviral-specific CTL persisted only in patients who were acutely infected with the agent2 We recently compared non-specifically activated T cells (ATC) with EBV-specific CTLs derived from the same initial peripheral blood collection and expressing distinguishable chimeric GD2-specific chimeric antigen receptors (CARATC and CAR-CTL). In this study, ATCs were cultured for 14 to 21 days. Between 0.9% and 6.1% retained a central memory (CCR7+, CD62L+) phenotype, up to 30% had an effector memory phenotype (CCR7−, CD62L+), and the remainder had a terminally/fully differentiated effector phenotype. By contrast, EBV-CTL were cultured for 30 to 44 days and expressed no CCR7, but up to 50% were CD62L+, and contained cells that were terminally/fully differentiated effectors and effector memory cells. These EBV-CTLs also all had a CD45RO memory phenotype, while about 13% to 60% of ATCs expressed CD45RA, a marker of naïve T cells. Despite these differences in memory subsets, it was the CAR-CTLs that had the clearly greater persistence and could be shown to retain functionality, while CAR-ATC rapidly disappeared from the circulation and could not be recovered. Hence, factors other than phenotype, such as antigenic stimulation and costimulation almost certainly influence cell fate after infusion, and determine whether or not effector memory cells can re-access the central memory pool. Ultimately, strategies that combine selection of optimal phenotype with the provision of antigen stimulation and co-stimulation and a cytokine milieu that favors homeostatic expansion will likely lead to the most effective outcomes following adoptive T cell transfer.
The Role of GM-CSF Signaling and CREB in Myeloid Proliferation and Survival.
Authors: Sakamoto Kathleen M.
Published: October 2019

Normal myelopoiesis is regulated by the delicate interplay between hematopoietic stem cells (HSCs) and the bone marrow microenvironment. Through the action of hematopoietic growth factors and their receptors, alterations of transcriptional regulation result in myeloid progenitor cell proliferation and survival. Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and Interleukin-3 (IL-3) are cytokines that regulate myelopoiesis. GMCSF and IL-3 induce signaling pathways that activate serine threonine kinases, including MEK, ERK, and pp90RSK, leading to expression of immediate early genes such as early growth response gene-1 (egr-1). Analysis of promoter regions of egr-1 demonstrated that the cAMP response element, CRE, was necessary and sufficient for egr-1 expression in myeloid cells. We previously demonstrated that the transcription factor, CREB (cAMP Response Element Binding Protein), recognizes the CRE in the egr-1 promoter and is phosphorylated at serine 133 in response to GM-CSF and IL-3 through a MEK-dependent, protein kinase A-independent pathways. CREB is a leucine zipper transcription factor that regulates proliferation, differentiation, and survival of neuronal cells and lymphocytes. The majority of patients with acute lymphoid and myeloid leukemias express higher levels of CREB in their bone marrow. Therefore, we hypothesized that CREB plays a critical role in the regulation of normal hematopoiesis. To this end, we studied the biological and molecular effects of CREB in myeloid cells. CREB overexpression results in increased proliferation and survival through induction of cyclin A1 and other target genes. Transgenic mice that overexpress CREB in Gr-1/Mac-1+ cells develop myeloproliferative disease after one year. Knockdown of CREB using shRNAs leads to decreased proliferation and differentiation of myeloid progenitor cells and increased apoptosis of HSCs. Cell cycle analysis demonstrate that CREB downregulation in HSCs leads to greater numbers of cells in G1 and fewer cells in S phase. Both cyclin D1 and A1 levels are decreased in CREB shRNA-transduced HSCs. However, CREB knockdown of bone marrow progenitors did not affect long-term engraftment in transplantation assays. Recent findings on downstream pathways of CREB and potential target genes will be presented.
CD28 Costimulation of Effector T Cells During An Anti-Tumor Attack Sustains Its Repression by Regulatory T Cells Which Can Be Overcome by Preventing Lck Activation.
Authors: Kofler David M , Chmielewski Markus , Riet Tobias , Hombach Andreas , Hallek Michael , Wendtner Clemens M , Abken Hinrich
Published: October 2019

Abstract 3569

Poster Board III-506

Background

Massive infiltrations of the tumor tissue with CD4+CD25highFoxP3+ regulatory T (Treg) cells render a productive T cell anti-tumor response ineffective resulting in unrestricted tumor growth despite the presence of tumor-specific, cytolytic T cells.

Methods

Using a human/CD1-deficient mouse model we investigated the impact of human regulatory T cells on redirected effector T cells expressing a tumor-specific chimeric antigen receptor. The chimeric antigen receptor consists of an antibody derived binding domain for antigens in the extracellular part and of the TCR/CD3zeta or the combined CD3zeta-CD28 signaling domain in the intracellular part. Upon antigen binding the chimeric antigen receptor transmits an activation signal via the CD3zeta or CD3zeta-CD28 domain to drive T cell activation, resulting in cytokine secretion, T cell proliferation, and cytolytic activity.

Results

We revealed that effector T cells redirected by a tumor-specific chimeric antigen receptor are more effectively repressed by Treg cells when they are activated through a combined CD3zeta-CD28 signal compared to a CD3zeta signal without CD28 costimulation. Mutations in the CD28 signaling domain of the chimeric antigen receptors resulted in abolished IL-2 secretion by prevention of CD28 mediated lck activation. Abolished IL-2 induction in redirected effector T cells expressing the modified CD3zetaCD28delta antigen receptor increased their in vivo efficacy in an anti-tumor response by reduced sustaining of Treg cell suppression.

Conclusions

While data indicate the dichotomy of CD28 costimulation in inducing full effector T cell activation and sustaining Treg repression, our findings provide a strategy to improve the efficacy of the T cell anti-tumor attack in the presence of Treg cells for use in adoptive immunotherapy of cancer.

Disclosures:

No relevant conflicts of interest to declare.
Establishment and Characterization of A New Leukemia Mouse Model Induced by MLL/AF4 Fusion Protein.
Authors: Tamai Hayato , Miyake Koichi , Miyake Noriko , Yamaguchi Hiroki , Dan Kazuo , Shimada Takashi , Inokuchi Koiti
Published: October 2019

Abstract 3573

Poster Board III-510

Background

MLL/AF4 fusion is the most common abnormality in infant leukemia and is associated with a poor prognosis. Although further study and treatment should be groped for acute lymphoblastic leukemia (ALL) with MLL/AF4, the lack of a proper animal model has impeded understanding of the molecular mechanism of leukemia associated with the MLL/AF4 fusion gene. Previous mouse models suggest that the tumorigenesis capacity of MLL/AF4 alone is insufficient for development mixed-lineage leukemia. MLL/AF4 is capable of inducing lymphoid malignancy with long latency, which is very different from aggressive pro-B ALL in humans. Recently, we have reported that enhancement of S100A6 (calcyclin) expression played an important role in MLL/AF4-leukemogenesis using murine 32Dc cell line. In this study, we tried to make improved MLL/AF4 transgenic (Tg) mice which reflect human leukemogenicity and analyzed molecular mechanism of MLL/AF4 fusion gene.

Methods

To get high and sustained expression of MLL/AF4 in hematological lineage, we used MSCV promoter to express MLL/AF4 fusion gene. After MLL/AF4 expression plasmids were injected into fertilized eggs of mice (C57BL/6), the manipulated embryos were transferred into the oviducts of pseudopregnant females to make MLL/AF4 Tg mice. Finally we characterized MLL/AF4 Tg mice and analyzed molecular mechanism of MLL/AF4-leukemogenesis.

Results

Our new established MLL/AF4 Tg mice had lymphoma earlier than previous MLL/AF4 knock-in mice (median 12M vs 17M). Infiltration of lymphoid cells was located in liver, lung, kidney, and spleen and they consist of pro B-cell (CD45R+CD43+CD19-) lineage. Leukemic change developed two months (median 14M) after appearance of lymphoma. More than 30% of abnormal lymphocytes and severe anemia (RBC: 276×104 ml, Hb: 4.9 g/ml) were found in peripheral blood in MLL/AF4 Tg mice. Western blot analysis showed that up-regulation of HoxA9 and S100A6 was detected in MLL/AF4 Tg mouse compared to wild type C57BL/6.

Conclusion

We succeeded in establishment of improved more aggressive MLL/AF4 Tg mice which reflect MLL/AF4 leukemogenicity of humans. Appearance of lymphoma in our MLL/AF4 Tg mice needed short er latency than those of the previous knock-in mice. Enhancement of HoxA9 and S100A6 expression was involved in MLL/AF4-associated leukemogenesis. We believe that this new MLL/AF4 Tg mouse is useful for study of the molecular mechanisms of MLL/AF4 leukemogenesis and development of new therapy for mixed-lineage leukemia.

Disclosures:

No relevant conflicts of interest to declare.
Chronic Granulomatous Disease and Mcleod Syndrome: Single Center Report of Four Cases.
Authors: Mahlaoui Nizar , Dumont Marie-Dominique , Peyrard Thierry , Blanche Stephane , Fischer Alain
Published: October 2019

Abstract 3595

Poster Board III-532

Background

Chronic Granulomatous Disease (CGD) is an inherited disease affecting innate immunity and leading to increased susceptibility to severe invasive fungal and bacterial infections. The X linked form of CGD is the most frequent and is caused by mutations in the gp91phox subunit of the NADPH oxidase complex encoding gene CYBB. McLeod syndrome patients present with late-onset neuromuscular troubles and a mild chronic haemolytic anaemia with acanthocytes. It is defined by the lack of expression of Kell antigens on erythrocytes and caused by mutations in the KX gene located close to the CYBB gene. The association of CGD and McLeod syndrome is a rare event. It can also be associated with Duchenne muscular dystrophy (DMD), retinitis pigmentosa or ornithine transcarbamylase deficiency. Patients : Four CGD patients patients were recently diagnosed with McLeod syndrome in Necker Hospital on a 2 year period of time. Their clinical and main biological characteristics are reviewed and a review of recent litterature Observations: In 1 patient, McLeod was evoked because of association of Duchenne myopathy and CGD revealed at 10 months with major hypotonia and recurrent infections. Initially, no acanthocytes were seen but Kell antigens testings led to confirmation of McLeod. Three other infants were diagnosed with CGD because of lung infections. One had lung biopsies revealing Nocardia sp. infection and received packed blood red cells. Four months after, an allo immunisation against red cells and a weakened Kell antigen expression led to McLeod diagnosis. For other patients, diagnosis was evoked because of anaemia and acanthocytes on blood smear analysis. All had confirmed by CYBB mutations with complete deletion. None had muscle weakness or retinal abnormality. For 2 infants, iron therapy and erythropoietin led to the correction of anemia.

Conclusion

Management and evaluation of X-linked CGD patients should require the search of McLeod syndrome. Biological diagnosis is sometimes uneasy, especially because acanthocytes are sometimes hard to identify on blood smears. Kell antigen reactivity screening could be easy to perform. When McLeod syndrome is diagnosed, patients are included in the French national registry for rare erythrocyte phenotype to receive appropriate transfusion management. Erythropoietin therapy could be useful in preventing chronic anemia.

Disclosures:

No relevant conflicts of interest to declare.
Erythropoietin as a Novel Pro-Inflammatory Mediator of Macrophages.
Authors: Lifshitz Lilach , Tabak Galit , Gassman Max , Mittelman Moshe , Neumann Drorit
Published: October 2019

Abstract 3596

Poster Board III-533

The immunomodulatory effects of erythropoietin (EPO) on the cellular and humoral compartments of the immune system were originally described by our group in multiple myeloma patients and have been further elucidated in murine experimental models (Mittelman, 2001; Katz 2005; 2007; Prutchi-Sagiv, 2006). However, the mechanisms of action by which EPO affects lymphocyte number and function are still unknown, particularly since lymphocytes do not carry EPO receptors (EPO-R). We thus set to unravel mechanisms underlying the anti-neoplastic immunomodulatory action of EPO. These studies led us to the novel discovery that dendritic cells (DCs) express EPO-R, and that EPO enhances their survival and function (Prutchi-Sagiv, 2008; Lifshitz, 2009). Here we focus on macrophages as an additional EPO target, since in analogy to DCs, macrophages are also antigen presenting cells, and serve as key effectors of the innate immune response.

Using murine models, we first explored the in-vivo effects of EPO using recombinant human EPO (rHuEPO, EPREXR, JC)-injected mice, as well as transgenic mice over-expressing human EPO (termed tg6). EPO treatment was associated with an increased splenic macrophage population, detected by F4/80 expression, and an increased number of macrophages expressing CD11b, CD80 and MHC class II. We further explored the effect of in-vivo EPO administration in an inflammatory model exploiting thioglygollate injection to induce recruitment of peritoneal inflammatory macrophages. The inflammatory macrophages obtained from both EPO injected and from tg6 mice displayed increased expression of F4/80, CD11b, CD80 and MHC class II and augmented phagocytic activity, as compared to the control counterparts.

These results are supported by in-vitro studies in bone marrow derived macrophages (BMDMs). We show that BMDMs express EPO-R mRNA, as detected by RT-PCR. In-vitro stimulation of the BMDMs with rHuEPO activated multiple signaling pathways including STAT1, STAT5, MAPK, AKT and NFkB indicating macrophage activation via surface EPO-R. EPO treatment of the BMDMs up-regulated their surface expression of CD11b, F4/80 and CD80, as well as enhanced their phagocytic activity. EPO treatment of LPS-stimulated BMDMs augmented IL-12 secretion, and decreased IL-10 secretion.

In conclusion our results show that macrophages are direct targets of EPO and that EPO treatment enhances their pro-inflammatory activity and function. These findings point to the multifunctional role of EPO and may advance its clinical applications as an anti-neoplastic immunomodulator.

Disclosures:

Mittelman: BioGAL- Start up (inactive): Equity Ownership, Patents & Royalties. Off Label Use: Non erythroid effects: immune, anti-cancer (all under investigation).
Glia Maturation Factor-Gamma Mediates Lipopolysaccharide-Induced Inflammation Via p38 MAPK and NF-Kappab.
Authors: Aerbajinai Wulin , Chin Kyung , Zhu Jianqiong , Hyun Lee , Kumkhaek Chutima , Rodgers Griffin P
Published: October 2019

Abstract 3598

Poster Board III-535

Neutrophils are the most abundant leukocytes in the circulation and provide a primary innate immune defense function against bacterial pathogens before the development of a specific immune response. Activation of TLR4 signaling pathways by lipopolysaccharide (LPS) leads to the production of a broad array of cytokines and mediators that coordinate the immune response. The TLR4-mediated inflammatory response is tightly regulated, since excessive activation of TLR4 may induce a variety of inflammatory diseases, so the negative regulation of TLR-triggered inflammatory response attracts much attention. Glia maturation factor gamma (GMFG), a novel factor in actin cytoskeleton reorganization, is preferentially expressed in inflammatory cells, but its function in leucocytes is undefined. In this study, we investigated whether GMFG participates in the molecular events underlying the inflammatory reaction and how GMFG mediated neutrophils inflammatory response to LPS. We show here that small interfering RNA-mediated knockdown of GMFG expression in leukocytes exhibited greater than 80% inhibition of the endogenous expression levels of proinflammatory cytokines and chemokines, including TNF-alpha, IL-1beta, IL-6, and IL-8 using quantitative RT-PCR. These decreased cytokine productions could be partially restored by stimulation with TLR4 agonist LPS, but exhibit significantly lower level in silencing of GMFG cells compared to non-targeting silencing control cells (inhibition by 71%+/−3.9% of TNF-alpha, 68%+/−6.9% of IL-1beta, 50%+/−4.3% of IL-6 and 40%+/−3.1% of IL-8). Knockdown of GMFG expression results in decreased phospho-p38 MAPK levels as well as suppressed NF-kappaB by Western blot analysis. These data suggest that endogenous expression of GMFG is required for basal cytokine and chemokine response and their induced response to LPS stimulation. Moreover, immunofluorescence analysis revealed that gene silencing of GMFG inhibited LPS-induced nuclear translocation of NF-kappaB in myeloid HL-60 and THP-1 cells. In contrast, transient overexpression of GMFG significantly enhanced endogenous level of proinflammatory cytokines by 1.8∼2.2 fold, phosphor-p38 MAPK and nuclear translocation of NF-kappaB in the absence of LPS stimulation. These results suggest that GMFG-mediated regulation of inflammatory cytokines occurs through activation of p38 MAPK and NF-kappaB signaling pathway. Taken together, our findings identify GMFG as a novel factor that participates in the regulation of TLR4-mediated control of cytokine and chemokine expression through the p38 MAPK and NF-kappaB pathway. These data, if confirmed in in vivo experiments, implicates GMFG as a potential target for therapeutic intervention in neutrophil-mediated autoimmune disease.

Disclosures:

No relevant conflicts of interest to declare.
TRAF2 Ring Domain Plays An Essential Role in Inhibition of TNFα-Induced Cell Death Independent of NF-kB Activation.
Authors: Blackwell Ken , Zhang Laiqun , Shi Zhaohui , Habelhah Hasem
Published: October 2019

Abstract 3621

Poster Board III-557

The proinflammatory cytokine TNFα regulates immune responses, inflammation and programmed cell death by activating the JNK/c-Jun and IKK/NF-κB signaling pathways. TRAF2 and RIP1 play critical roles in TNFα-induced activation of JNK and IKK, and inhibition of apoptosis. In the field, TRAF2 RING domain-mediated RIP1 ubiquitination is believed to play an essential role in TNFα-induced IKK activation, and the RING domain-deleted TRAF2 (TRAF2-ΔN) has been widely used as a dominant negative to block TNFα-induced JNK and IKK activation in transient overexpression systems. Here, we report that stable expression of TRAF2-Δ[N in TRAF2 and TRAF5 double knockout (T2/5 DKO) cells at a physiological level almost completely restores TNFα-induced IKK activation, but does not restore RIP1 ubiquitination. In addition, stable expression of TRAF2-ΔN in T2/5 DKO cells efficiently inhibited TNFα-induced later-phase prolonged JNK activation, but failed to inhibit TNFα-induced cell death. The basal and inducible expression of antiapoptotic proteins in TRAF2-ΔN-expressing cells is totally normal, yet these cells are still sensitive to TNFα-induced cell death due to the inefficient recruitment of cIAP1 to the TNFR1 complex. Moreover, stable expression of TRAF2-WT, but not TRAF2-ΔN, in T2/5 DKO cells suppressed constitutive p100 processing. On the other hand, TRAF2-ΔN-expressing cells exhibited significant resistance to oxidative stress-induced cell death compared to TRAF2-WT-expressing cells. These data suggest that: i) the TRAF2 RING domain plays a critical role in the inhibition of cell death induced by TNFα, and is essential for suppression of p100 processing in unstimulated cells; ii) RIP1 polyubiquitination is not required for TNFα-induced IKK activation, but may play a role in inhibition of TNFα-induced cell death; and iii) prolonged JNK activation has no obligate role in TNFα-induced cell death.

Disclosures:

No relevant conflicts of interest to declare.
Functional Analysis of Pegasus: The ‘Atypical’ Member of the Ikaros Gene Family.
Authors: John Liza B , Ward Alister C
Published: October 2019

Abstract 3645

Poster Board III-581

The Ikaros family of zinc finger transcription factors are an important class of hematopoietic regulators involved in the development of the immune system. However, the in vivo role of its most divergent ‘atypical’ member, Pegasus, remains unknown. Therefore, we have utilised zebrafish to explore the function of Pegasus. Bioinformatics analysis identified a single pegasus orthologue in zebrafish. Wholemount in situ hybridisation revealed an initial broad distribution of maternally-derived pegasus transcripts during early zebrafish embryogenesis, which was replaced by specific expression in the brain, eye, thymus, gut and pancreas during late embryogenesis. Morpholino-mediated gene knockdown of zebrafish pegasus resulted in a reduction in cells expressing markers for hematopoietic stem cells (lmo2), granulocytes (mmp9), macrophages (fms), as well as early (ikaros) and late (rag1) lymphoid cells while, conversely, those expressing early (spi1) and late (l-plastin) myeloid markers were increased. No difference was noted in the early erythroid (gata1) compartment, although mature red blood cells appeared reduced, as judged by o-dianisidine staining. During emergency hematopoiesis (elicited by LPS injection), numbers of granulocytes (mpo+) and macrophages (lyz+) were both significantly further reduced. In addition, morphants also showed anterior defects, including reduced forebrain, smaller eyes and otoliths, which corresponded with a reduction of expression of the brain markers pax2.1, krox20 and dlx1. Gut expression of ifabp also appeared affected. Collectively, these experiments suggest a clear role for pegasus in hematopoiesis, as well as novel functions in brain and gut development.

Disclosures:

No relevant conflicts of interest to declare.
Loss of the Gata1 Gene IE Exon Leads to Variant Transcript Expression and the Production of GATA1 Protein Lacking the N-Terminal Domain.
Authors: Kobayashi Eri , Shimizu Ritsuko , Kikuchi Yuko , Takahashi Satoru , Yamamoto Masayuki
Published: October 2019

Abstract 3648

Poster Board III-584

GATA1 is a transcription factor essential for the differentiation of erythroid cells and megakaryocytes. Since GATA1 regulates genes related to the survival, proliferation and differentiation of hematopoietic cells, regulation of the Gata1 gene expression is critically important for the understanding of hematopoiesis. The Gata1 locus contains multiple untranslated first exons plus five common coding exons. Of these first exons, erythroid first exon (IE exon) is important for the Gata1 gene expression in the hematopoietic lineages. However, due to the embryonic lethality of this IE exon knockdown mice, less is understood about the contribution of the IE exon to adult hematopoiesis. Here, we achieved specific deletion of the IE exon in adulthood by crossing the IE-floxed mice with the interferon-inducible Mx1-Cre transgenic mice. This conditional IE-deletion mouse (ΔIE mouse) showed severe thrombocytopenia with increased premature megakaryocytes similarly to the phenotypes reported in the conditional Gata1 knockout mice in which the entire Gata1 gene was deleted in adulthood. In addition, the ΔIE mice showed severe anemia with skewed erythroid maturation, and importantly this erythroid phenotypes substantially differed from those observed in the conditional Gata1 knockout mice. Further analyses revealed that the Gata1 mRNA level in the megakaryocytic lineage was significantly downregulated. By contrast, in the erythroid lineage, Gata1 mRNA was retained at a comparable level to that in control mice utilizing two alternative first exons; one was the IEb/c, which was previously reported as a first exon rarely used in hematopoietic cells, and the other was newly identified IEd exon located within the second intron. Surprisingly, in the ΔIE mice these transcripts failed to produce full-length GATA1 protein, but instead inefficiently yielded GATA1 lacking the N-terminal 83 amino acids. This form of GATA1 is often observed in Down syndrome-associated transient myeloproliferative disorder and acute megakaryoblastic leukemia. Of note, the transcript derived from exon IEb/c preserved the first translation initiation codon in exon 2 but lost the potential to select the first translation initiation codon or failed to produce full-length GATA1. The present study demonstrates that the IE exon is instrumental to adult erythropoiesis by regulating the proper level of transcription and by selecting the correct translation start site for production of adequate full-length GATA1 protein.

Disclosures:

No relevant conflicts of interest to declare.
Myeloid-Derived Suppressor Cells in Patients with Hodgkin Lymphoma.
Authors: Parrinello Nunziatina , La Cava Piera , Tibullo Daniele , Giallongo Cesarina , Di Bartolo Orazio , Spina Paolo , Fiumara Paolo , Schinocca Luciana , Conticello Concetta , Chiarenza Annalisa , Palumbo Giuseppe A , Di Raimondo Francesco
Published: October 2019

Abstract 3662

Poster Board III-598

Background

Immune suppression and angiogenesis are mechanisms key to tumour growth and progression. Myeloid-derived suppressor cells (MDSC) are a heterogeneous population of cells of myeloid origin and include immature macrophages, dendritic cells (DC) and other myeloid cells. In mice are phenotypically characterized as CD11b+Gr-1+ cells, while in human they have an immature phenotype, including lineage negative (Lin-), CD14-, HLA-DR-, CD15+, CD34+, CD11b+, CD33+, and CD13+ cells. MDSC reduce activated T-cell number and inhibit their function through different mechanisms including: L-arginine metabolism, nitric oxide (NO), up-regulation of reactive oxygen species (ROS), and secretion of immunosuppressive cytokines. MDSC also promote tumor-dependent angiogenesis as well as tumor metastasis.

Their accumulation has been described in patients affected by some solid tumors but information on haematological neoplasms are lacking. Our study investigated by flow cytometry the presence of MSDC in the peripheral blood of patients affected by Hodgkin Lymphoma (HL).

Methods

We studied 14 patients with HL at diagnosis and 10 age-matched healthy controls (HC). Peripheral blood mononuclear cells were stained with the following monoclonal antibodies:CD11b, CD13, CD14, CD34, CD45, for 20 minutes at room temperature. After lysing red cells, cells were analyzed by flow cytometry.

Results

we observed a increased number of MDSC (CD11b+,CD13+,CD34+,CD14-, CD45+) in the peripheral blood of patients with HL compared to HC (13,37 ± 17,77 ×109/l vs 1,45± 0,98 ×109/l, p=0,0007). We also found that patients with advanced-stage Hodgkin disease (III and IV) have higher number of MDSC, compared to patients stage I and II (p= 0,04).

Conclusion

These data suggest a role for myeloid-derived suppressor cells in promoting tumor cell proliferation in hodgkin lymphoma.

Disclosures:

No relevant conflicts of interest to declare.
Irradiation-Mediated Thymic Plasticity Is An Age-Related Phenomenon in Immunodeficient Mice.
Authors: Singh Kamaldeep , Myers Aaron , Prockop Susan E
Published: October 2019

Abstract 3677

Poster Board III-613

Successful T lymphopoiesis is essential for maintaining resistance to infection, mediating graft versus tumor activity, and as a platform for some immune based therapies. In mouse models of T cell immunodeficiency syndromes successful T cell reconstitution requires robust cellular reconstitution as well as remodeling of the thymic stroma. We have previously demonstrated that robust thymic lymphopoiesis in young IL-7 receptor deficient (IL7R−/−) mice can occur without prior irradiation and have linked this to the presence of intact stromal niches for DN3 precursors. In contrast to IL7R−/− mice, RAG−/− mice generate wild type T lymphocytes via thymic T lymphopoiesis after transfer of wild type precursors, but the thymus of transplanted RAG−/− mice remains hypocellular. As has been shown before, thymic cellularity of young RAG−/− mice can be rescued by irradiation followed by transfer of wild type precursors. We now demonstrate a profound difference between young and old RAG−/− mice in the thymic remodeling that occurs after irradiation and transfer of wild type hematopoietic stem cells. While the thymi of young RAG−/− mice undergo profound remodeling and develop normal cortico-medullary areas with segregation of single positive cells to the medulla, older RAG−/− mice do not undergo this remodeling and the cortical and medullary stroma remains less organized and more interdigitated. In addition, while the thymi of these older RAG−/− mice do develop small nests of medullary thymic epithelial cells (identified by flow cytometry and immunohistochemistry) not visualized in the untreated RAG−/− mice, these cells do not appropriately express MHC and these thymi do not appropriately segregate single positive developing T lymphocytes to these medullary areas. In vitro testing of sorted single positive thymocytes from these mice demonstrates failure of appropriate negative selection. Together these studies identify a specific defect in the thymic stroma of aging immunodeficient mice preventing appropriate de novo T lymphopoiesis.

Disclosures:

No relevant conflicts of interest to declare.
RIAM and RapL Regulate Distinct Signaling Events and Functional Outcomes Upon TCR-Mediated Activation.
Authors: Patsoukis Nikolaos , Lafuente Esther M , Boussiotis Vassiliki A
Published: October 2019

Abstract 3683

Poster Board III-619

Rap1, a small GTPase of the Ras superfamily, originally identified by its ability to reverse Ras-mediated transformation, is now known to regulate cytoskeletal reorganization, cell morphology, adherens junction positioning and adhesion. The best-studied function of Rap1 is inside-out activation of integrins and cell adhesion. Two newly identified Rap1 effectors, RapL and RIAM, have been implicated in Rap1 mediated in inside-out activation of integrins and cell adhesion. However, significant differences in the structure and interactions of these molecules indicate that they may mediate distinct signaling events. RIAM has a N-terminal coiled-coil region, central RA and pleckstrin homology (PH) domains, and proline-rich N-terminal and C-terminal regions, with multiple FPPPP motifs capable of interacting with the EVH1 domains of the actin regulatory proteins Ena/VASP, multiple XPPPP motifs interacting with Profilin and multiple PXXP motifs capable of interacting with SH3 domain containing proteins. Because of these properties RIAM is a regulator of actin polymerization but also interacts with components of the T cell signaling machinery. In contrast, RapL has an RBD (Ras binding domain-structurally similar to RA domain) and a C-terminal coiled-coil region and interacts with Rap1 via its RBD domain and its N-terminal region, and with the aL subunit of LFA-1 via its C-terminal domain. In the present study we investigated the role of RIAM and RapL in regulating signaling and functional events activated via the TCR. For this purpose we used RIAM-knockdown (KD) and RapL-KD Jurkat T cells in which endogenous RIAM and RapL, respectively, had been depleted by siRNA. Whereas activation of the extracellular signal regulated kinases MEK1/2 and Erk1/2 was impaired by depletion of RIAM, activation of these kinases was unaffected by depletion of RapL. In contrast, activation of p38 was unaltered in RIAM-KD cells but was abrogated in RapL-KD cells. Moreover, RIAM knockdown resulted in impaired activation of Ras and Rap1 due to defective activation of the calcium and diacylglycerole-dependent GEFs, RasGRP1 and CalDAG-GEFI. In contrast, RapL knockdown had no effect on these events compared to control Jurkat T cells. Strikingly, RIAM-KD cells displayed impaired IL-2 production in response to stimulation with SEE-loaded APC or to TCR/CD3-plus-CD28 crosslinking, whereas RapL-KD cells displayed a dramatic increase in IL-2 production upon stimulation under the same conditions. These results indicate that although both RIAM and RapL regulate Rap1-dependent LFA-1 activation, these molecules have distinct roles in regulating signaling and functional outcomes of T cell responses after T cell receptor triggering.

Disclosures:

No relevant conflicts of interest to declare.