Bone Marrow Lymphocytes Research Articles

CD84 is a survival receptor for CLL cells

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of CD5+ B lymphocytes in peripheral blood, lymphoid organs and bone marrow. The main feature of the disease is accumulation of the malignant cells due to decreased apoptosis. CD84 belongs to the signaling lymphocyte activating molecule family of immunoreceptors, and has an unknown function in CLL cells. Here, we show that the expression of CD84 is significantly elevated from the early stages of the disease, and is regulated by macrophage migration inhibitory factor and its receptor, CD74. Activation of cell surface CD84 initiates a signaling cascade that enhances CLL cell survival. Both downmodulation of CD84 expression and its immune-mediated blockade induce cell death in vitro and in vivo. In addition, analysis of samples derived from an on-going clinical trial, in which human subjects were treated with humanized anti-CD74 (milatuzumab), shows a decrease in CD84 messenger RNA and protein levels in milatuzumab-treated cells. This downregulation was correlated with reduction of Bcl-2 and Mcl-1 expression. Thus, our data show that overexpression of CD84 in CLL is an important survival mechanism that appears to be an early event in the pathogenesis of the disease. These findings suggest novel therapeutic strategies based on the blockade of this CD84-dependent survival pathway.

Circulating microenvironment of CLL: Are nurse-like cells related to tumor-associated macrophages?

B-cell chronic lymphocytic leukemia (B-CLL) is one of the most common hematologic malignancies in Western countries. Accumulation of leukemic lymphocytes in peripheral blood, bone marrow and secondary lymphatic organs of CLL patients is due to decreased apoptosis rather than to increased proliferation. The former is driven by signals from a specific microenvironment, created by stromal cells of mesenchymal origin, follicular dendritic cells, T lymphocytes and others. Nurse-like cells (NLCs) were first described to differentiate from peripheral blood mononuclear cells of CLL patients in vitro, then they have been also found in proliferation centers of their lymphatic tissues. Like tumor-associated macrophages (TAMs) in solid tumors, nurse-like cells promote survival of CLL lymphocytes. NLC gene expression patterns suggest their similarity to TAMs and differ between patients depending on ZAP70 protein expression status. NLC number in vitro corresponds with CD14 expressing cell count and beta-2-microglobulin serum level, and positively correlates with leukemic lymphocyte viability. As NLCs strongly express genes for adhesion molecules and secrete chemokines of antiapoptotic activity, they should be considered as a target for anti-microenvironment therapy of this incurable disease.

The Acute Immunological Response to Blood Transfusion Is Influenced by Polymicrobial Sepsis

Blood transfusion is a well-established risk factor for adverse outcomes during sepsis. The specific mechanisms responsible for this effect remain elusive, and few studies have investigated this phenomenon in a model that reflects not only the clinical circumstances in which blood is transfused, but also how packed red blood cells (PRBCs) are created and stored. Using a cecal ligation and puncture model of polymicrobial sepsis as well as creating murine allogeneic and stored PRBCs in a manner that replicates the clinical process, we have demonstrated that transfusion of PRBCs induces numerous effects on leukocyte subpopulations. In polymicrobial sepsis, these responses are profoundly dissimilar to the proinflammatory effects of PRBC transfusion observed in the healthy mouse. Transfused septic mice, as opposed to mice receiving crystalloid resuscitation, had a significant loss of blood, spleen, and bone marrow lymphocytes, especially those with an activated phenotype. Myeloid cells behaved similarly, although they were able to produce more reactive oxygen species. Overall, transfusion in the septic mouse may contribute to the persistent immune dysfunction known to be associated with this process, rather than simply promote proinflammatory or anti-inflammatory effects on the host. Thus, it is possible that blood transfusion contributes to the multiple known effects of sepsis on leukocyte populations that have been shown to result in increased morbidity and mortality.

Structural Basis for Galectin-1-dependent Pre-B Cell Receptor (Pre-BCR) Activation

During B cell differentiation in the bone marrow, the expression and activation of the pre-B cell receptor (pre-BCR) constitute crucial checkpoints for B cell development. Both constitutive and ligand-dependent pre-BCR activation modes have been described. The pre-BCR constitutes an immunoglobulin heavy chain (Igμ) and a surrogate light chain composed of the invariant λ5 and VpreB proteins. We previously showed that galectin-1 (GAL1), produced by bone marrow stromal cells, is a pre-BCR ligand that induces receptor clustering, leading to efficient pre-BII cell proliferation and differentiation. GAL1 interacts with the pre-BCR via the unique region of λ5 (λ5-UR). Here, we investigated the solution structure of a minimal λ5-UR motif that interacts with GAL1. This motif adopts a stable helical conformation that docks onto a GAL1 hydrophobic surface adjacent to its carbohydrate binding site. We identified key hydrophobic residues from the λ5-UR as crucial for the interaction with GAL1 and for pre-BCR clustering. These residues involved in GAL1-induced pre-BCR activation are different from those essential for autonomous receptor activation. Overall, our results indicate that constitutive and ligand-induced pre-BCR activation could occur in a complementary manner.

Prognostic Impact of the Levels of Expression of Cell Surface Proteins Commonly Expressed by Blasts and Hematopoietic Precursor Cells in De Novo AML Patients: A Report From the Spanish Cetlam Study Group

AbstractAbstract 1452▪▪This icon denotes a clinically relevant abstract Introduction:Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by the presence of acquired genetic alterations in hematopoietic progenitor and stem cells. The prognostic impact of immunophenotypic markers has long been controversial. Despite this, only a very limited number of studies have investigated the prognostic impact of the levels of cell surface membrane (Sm) proteins commonly expressed on AML blasts. In this study we analyzed the prognostic impact of the levels of expression of several markers associated with AML blasts and normal hematopoietic precursors, as asessed by Multiparameter Flow Cytometry (MFC) in de novo AML patients (pts). Methods:Overall, 122 adult de novo AML pts diagnosed between 12/2003 and 08/2011 were studied, after excluding acute promyelocytic leukemia. All cases were diagnosed and classified according to the WHO criteria and they were immunophenotyped using an extensive 4-color panel of monoclonal antibodies by MFC, as previously described. Patients were treated according to the multicenter CETLAM AML-03 trial. Blast cells were gated after excluding all other cell populations, as well-delineated clusters of SSC low/int/CD45dimevents (“blast gate”) using the merge function of the Infinicyt software (Cytognos SL, Salamanca, Spain). For each case, the reactivity for the following markers was evaluated in terms of mean fluorescence intensity (MFI; arbitrary units) in bone marrow samples: CD123, CD117, CD34 and CD7. Normal residual bone marrow lymphocytes were used as reference for internal quality control purposes. Cytogenetic and molecular risk stratifications were based on the European LeukemiaNet (ELN) recommendations and the molecular lesions were investigated using well established protocols. Overall survival (OS), disease-free survival (DFS) and relapse rate (RR) were measured by the Kaplan–Meier method and curves were compared with the log-rank test. Multivariate analysis was performed using the Cox regression model. P-values ≤0.05 were considered to be statistically significant. Results:Median AML patient age was of 54 years (range: 20–70 y) with a M/F ratio of 63/59. Twelve pts (10%) had good cytogenetic/molecular risk, 83 (68%) intermediate, 17 (14%) high risk and 10 (8%) pts were unknown. Fifteen cases (12%) had NPM1mut, 33 (27%) showed FLT3-ITDmut, 5 (4%) pts carried CEBPAmut, 41 (33.6%) pts with no mutations and 28 (23%) pts were unknown. The median follow-up of pts alive was 19 months (range 0–97 months) and the 5-year OS of all pts was 42±5%. Univariate analysis of prognostic factors showed an association between higher CD45 (MFI >232; p=.01), CD117 (MFI>259; p=.008), CD34 (MFI >242; p=.007) and CD7 (MFI> 19; p=.03) expression and both a shorter OS and DFS. In addition, high CD123 expression (MFI >248; p=.04) was associated with a shorter DFS. In multivariate analysis only a high CD45 (p=.03) and a high CD34 (p=.03) expression were identified as independent predictors for a shorter OS. In turn, higher levels of CD123 (p=.03) and of CD34 (p=.02) were independent predictors for DFS and relapse, even when age, gender and WBC were taken in account. Conclusion:In this study, we show that higher levels of expression of immunophenotypic markers associate with immature myeloid precursors (CD45, CD117, CD34 and CD7) as assessed by MFC, have a significantly adverse prognostic impact in AML, both as regards OS and DFS. Accordingly, higher levels of CD45 and CD34 were independent predictors for both a shorter OS whereas, greater CD123 and CD34 values were associated with an increased risk of relapse and a shorter DFS, supporting the adverse prognostic impact of a more immature blast cell phenotype in de novo AML. Disclosures:No relevant conflicts of interest to declare.

RIT with 90Y Ibritumomab Tiuxetan in Patients with Non-Hodgkin Lymphoma Over 65 Years.

Abstract 2742 Introduction:90Y Ibritumomab tiuxetan (90Y-IT) has become an efficient alternative to therapy in non-Hodgkin Lymphoma, mainly in elderly patients. The aim of this study is to analyse our updated information of patients treated with 90YIbritumomab/tiuxetan in a prospective study according clinical practice setting and to analyse treatment outcome. Subjects and Methods:39 non Hodgkin lymphoma patients were included in a clinical protocol conducted by a multidisciplinary team and treated in the same centre. According the inclusion criteria: patients over 65 years old diagnosed as CD20+ NHL with neutrophils ≥ 1,5 × 109/L, platelets ≥ 100 × 109/L, bone marrow lymphocytes CD20+ ≤ 25%. All patients received 0,3 or 0,4 mCi /kg IV (88%) of 90YIbritumomab/tiuxetan and response evaluation was performed 12 weeks after. Period of study: September 2005/July 2012. The 90Y-IT was administered as consolidation of first line therapy (Rituximab alone, R-COP, R-CHOP21) or in relapsed/refractory status. Endpoints:Objective response rate (ORR), time to relapse (PFS) overall survival (OS) and safety. Other clinical prognostic factors were observed to assess their possible influence upon treatment value. Results:Until May 2012, 39 patients had received treatment with 90YIbritumomab/tiuxetan and completed the evaluation protocol and were considered to analysis; M/F 18/21 mean age 72.8 years (65–87); ECOG 0–1 92.3%. According OMS classification: NHL-follicular 27 (69.2%), mantle cell Lymphoma 7 (17.9%), DLBCL 4 (10.3%) and 1MALT (2.6%). Score distribution: low risk 19 (48.7%), intermediate 12 (30.8.2%) and advanced 8 (20.5%). Previous therapy schedules ≤2 (66.7%), >2 (33.3%). The median follow-up time: 42.0 months (95% CI: 4.0; 62.0), mean PFS: 38.1 months (95% CI: 30.8; 45.4) median NR. 13 patients received 90Y-IT as consolidation of first line therapy (33.3%) and 26 relapsed/refractory (66.6%). ORR was 84.6 % CR: 29 (74.3%); PR 4 (10.2%) and 6 failures (15.4%) in relapsed/refractory disease. Mean estimated OS since 90Y-IT: 54.4 months (95% CI: 49.4; 59.3) and mean estimated OS since diagnosis 159 months. Median PFS was NR. The mean PFS for patients in consolidation therapy was 54.2 months (95% CI: 47.4; 61.1). Safety: thrombocytopenia being the most frequent, G3–4 (35.9%), median time to developed haematological toxicity: fourth week, and neutropenia G3–4 (41.0%), the median time to recover normal values was 4.2 and 2.6 weeks respectively. In 5 (12.9%) of patients red blood cell transfusion was required, and 10 platelet transfusions (25.6%). The most frequent non haematological toxicity was asthenia. One patient developed a severe mucositis. Four patients have concomitant associated tumours (colon, breast, lung and prostate) and two patients over 77 years developed a rectum carcinoma after 18 months of 90Y-IT and another prostate and renal tumour after 8 years. Comments:In our experience 90Y Ibritumomab tiuxetan is a safety and effective therapy in patients with NHL over 65 years. According to obtained PFS results, it seems like the use of this kind of therapy as used in early part of therapy offers good and maintained response rate with lower toxicity in this fragile population. The OS in this population was not inferior to observed in younger NHL patients. Disclosures:No relevant conflicts of interest to declare.

Restricted expression of new guanine nucleotide exchange factor Zizimin2 in aged acquired immune system.

The activity of various biological functions, such as nervous, endocrine and immune systems including acquired immunity, is known to decline along with aging. To elucidate the molecular mechanism of this phenomenon, we here compared the number of thymocytes, splenocytes, and bone marrow lymphocytes in young and aged mice and found the age-related functional fragility of the immune system. However, the molecular mechanisms or even the key molecules remain elusive. Therefore, we further focused on a candidate for immunosenesence-related molecules, Zizimin2, which we have recently isolated and identified as a novel guanine nucleotide exchange factor that is highly expressed in murine splenic germinal center B cells after immunization with a T cell-dependent antigen. Here, we showed that endogenous Zizimin2 protein as well as mRNA expression levels in immune organs are strictly suppressed in aged mice. We further observed that the serum antigen specific antibody response is hampered in aged mice compared to that in young animals. Moreover, the Zizimin2 mRNA expression level was not activated after immunization in aged mice. Taken together, these data suggested that Zizimin2 is associated with the reduction of immune response in acquired immunity along with aging.

Microenvironmental Interactions in Chronic Lymphocytic Leukemia: Hints for Pathogenesis and Identification of Targets for Rational Therapy

Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by the accumulation/expansion of a clonal population of small mature B lymphocytes in blood, bone marrow, and lymphoid organs. Although initial genetic events are considered primarily responsible for the first step(s) of neoplastic transformation, the development and progression of the CLL clone are thought to be affected by various micro-environmental signals that regulate proliferation and survival of malignant B cells. In the present review, we focus on specific interactions of CLL cells with the microenvironmental component, as they occur through the usage by CLL cells of specific molecular structures whose expression has been associated with prognosis, including: i) interactions of CLL cells via the surface BCR and dependent on specific molecular features of the BCR itself and/or the presence of the BCR-associated molecule ZAP- 70; ii) non-BCR-dependent proliferative and/or pro-survival interactions of CLL cells by CD49d and CD38. An overview of the putative drugs that could be employed to target specific molecules involved in CLL cells/tumor microenvironment interactions is also proposed in the closing chapter of the review.

Differential protein analysis of lymphocytes between children with acute lymphoblastic leukemia and healthy children

We identified differential proteins in lymphocytes between patients with childhood acute lymphoblastic leukemia (c-ALL) and healthy children. Samples of bone marrow lymphocytes from children with c-ALL and peripheral blood lymphocytes from healthy children were collected, and total proteins were extracted and separated from these samples followed by two-dimensional gel electrophoresis for comparative analysis. The differential protein spots in c-ALL cells were digested in situ, and then analyzed with matrix-assisted laser desorption ionization/time of flight mass spectrometry (MALDI-TOF-MS) followed by identification using the relevant database. Fifteen differential expression proteins were obtained by comparative proteomics analysis. Of the 15 differential proteins, eight were identified. Of the eight proteins, two had high expression and six low expression in c-ALL cells. The eight differential proteins are expected to become new diagnostic markers and drug targets for c-ALL.

A NUP98-HOXD13 leukemic fusion gene leads to impaired class switch recombination and antibody production

Myelodysplastic syndrome is a clonal process characterized by ineffective hematopoiesis and progression to acute leukemia. Although many myelodysplastic syndrome and leukemic patients have compromised immunity, the role of underlying mutations in regulating immune function is poorly understood. Recent studies show that NUP98-HOXD13 (NHD13) fusion gene results in myelodysplastic syndrome and impairs lymphocyte differentiation in transgenic mice. In our studies, we sought to elucidate the mechanism by which NHD13 affects B-lymphocyte development and function. Based on our preliminary findings that transgenic mice had increased levels of IgM and reduced IgG1 and IgE, we hypothesized that the fusion gene might impair class switch recombination (CSR). Mice were immunologically challenged with dinitrophenol. NHD13 mice showed a marked reduction in B-lymphocyte differentiation in their bone marrow and spleen following dinitrophenol stimulation and had reduced production of dinitrophenol-specific antibodies. Spleen follicles from these mice were small and hypocellular, indicating failure of clonal expansion. When isolated NHD13 B lymphocytes were stimulated in vitro using Escherichia coli lipopolysaccharide or lipopolysaccharide + interleukin-4, they failed to undergo sufficient CSR and proliferation. Taken together, our findings show that expression of NUP98-HOXD13 impairs CSR and reduces the antibody-mediated immune response, in addition to its role in leukemia. Further delineation of the NUP98-HOXD13 transgene may reveal novel pathways involved in CSR.

Signals controlling rest and reactivation of T helper memory lymphocytes in bone marrow.

Established views on the maintenance of immunological memory have been challenged recently by the description of memory plasma cells and memory T helper (Th) lymphocytes residing in the bone marrow (BM) in dedicated survival niches, resting in terms of proliferation and migration. While memory plasma cells are no longer reactive to antigen, memory Th lymphocytes are in a state of attentive rest, and can be reactivated fast and efficiently. Here, we discuss the signals controlling these resting states, which the memory lymphocytes receive from their microenvironment.

Three Cases of Aggressive Natural Killer Cell Leukemia with a Lethal Hemorrhagic Complication

Aggressive natural killer cell leukemia (ANKL) is a rare malignant disease of NK cells that has a median survival of less than 2 months and a strong association with the Epstein-Barr virus. Herein, we report three Japanese cases of the disease. A 21-year male patient, a 31-year female patient, and a 76-year female patient presented with high fever, lymphadenopathy, hepatosplenomegaly, and severe liver damage. All three cases had granular lymphocytes in both peripheral blood and bone marrow. The phenotype of these cells was CD2(+)CD3(-)CD56(+)HLA-DR(+). All cases had a high copy number of serum Epstein-Barr virus DNA in the peripheral blood and were diagnosed with ANKL. Case 1 and Case 2 were treated with chemotherapy, but suffered from gross intestinal bleeding or massive bleeding in the cerebellum, resulting in death. Although not treated with chemotherapy, Case 3 also suffered gross bleeding from an atypical duodenal ulcer and died from hemorrhagic shock 15 days after admission. There have been no previous reports of such acute lethal hemorrhagic complications with ANKL. The present cases suggest that patients with ANKL need a sufficient supply of coagulation factors, and that chemotherapy for this disease should be carefully designed with promising agents. [J Clin Exp Hematopathol 52(2) : 101-106, 2012].

CD3 and EBER double positive cells in bone marrow are a diagnostic aid for EBV-positive T-cell lymphoproliferative disorders of childhood

We describe a case of Epstein–Barr virus (EBV)-positive T-cell lymphoproliferative disorders (EBV+ T-cell LPD) of childhood. The patient presented with high fever, jaundice, hepatosplenomegaly, pancytopenia, and disseminated intravascular coagulation (DIC). On admission, liver biopsy could not be conducted due to DIC. The small but definitive number of abnormal lymphocytes in bone marrow (BM) was indicative of malignant lymphoma or related diseases. The diagnosis of EBV+ T-cell LPD of childhood was tentatively made on the basis of the presence of CD3 and EBER double positive cells in BM. We started chemotherapy, which promptly resolved the symptoms and DIC. Later the diagnosis was confirmed by the T-cell receptor rearrangement and a single band for the EBV terminal repeat region by Southern blot analysis of BM cells. The detection of EBER-positive CD3 cells in BM is a diagnostic aid for EBV+ T-cell LPD of childhood.

Effects of Diclofenac, Piroxicam and α-Tocopherol on Monoamine-lymphopoietic Interfacing in Mice

The non-steroidal anti-inflammatory drugs (NSAIDs) diclofenac (CAS 15307-79-6) and piroxicam (CAS 36322-90-4) were shown in previous works to induce bone marrow lymphopoiesis. The present work aimed at evaluating the extent to which lymphopoietic effects of the above mentioned drugs as well as their interactions with alpha-tocopherol (CAS 10191-41-0) may be reflected in changes in the tissue levels of norepinephrine (NE), dopamine (DA) and serotonin (5-HT). The study was performed in male adult mice. The doses were given once daily for 7 days. The evaluations were performed in 2 phases. First, dose-response relationships of each of diclofenac, piroxicam and alpha-tocopherol were evaluated using bone marrow lymphocytes counts and monoamines levels in plasma, brain and spleen. Then, interactions of alpha-tocopherol (10 mg/kg) with diclofenac (20 mg/kg) and piroxicam (3 mg/kg), respectively, were evaluated. The interaction evaluations included effects on bone marrow lymphocytes count, monoamines levels in plasma, brain, spleen and thymus, as well as spleen weight. The obtained findings revealed that diclofenac, piroxicam and alpha-tocopherol decreased NE and DA levels in plasma, brain and spleen. There was some correlation between the decrease in plasma NE and the bone marrow lymphopoiesis. Pre-administration of alpha-tocopherol failed to maintain monoamines levels at the range of normal values in plasma, brain and spleen of NSAIDs-treated mice except splenic DA levels. In addition, the decrease in NE level induced by administration of piroxicam or diclofenac, either alone or together with a-tocopherol, corresponded to the increase in bone marrow lymphopoiesis. The present work suggests that the diclofenac- and piroxicam-induced bone marrow lymphopolesis as well as their respective interactions with alpha-tocopherol are associated with parallel changes in monoamines levels, especially those of NE and 5-HT.

Analysis of the prognostic factors of very severe aplastic anemia treated with Chinese Kidney-invigorating drugs in combination with anti-lymphocyte globulin or anti-thymocyte globulin

To explore the prognostic factors for very severe aplastic anemia (VSAA) patients treated mainly with Chinese Kidney (Shen)-invigorating drugs (CKID) combined with anti-lymphocyte globulin (ALG) or anti-thymocyte globulin (ATG). Twenty-seven VSAA patients were treated with CSID+ALG/ATG therapy in conjunction with cyclosporine A, androgen, hemopoietic growth factor, etc. The relationship of the effectiveness and some factors (age of patients, course of illness, blood and bone marrow figures, etc.) were analyzed. In the 25 evaluated VSAA patients who had been followed up for over 1 year, 9 patients (36.0%) were basically cured, 5 (20.0%) remitted, 6 (24.0%) were markedly improved, and 5 (20.0%) were treated in vain, with the total effective rate of treatment being 80.0% (20/25). Better clinical therapeutic effects were shown in patients newly diagnosed with VSAA, of male sex (P=0.037), >20 years old (P=0.045), with an illness course [Symbol: see text] month (P=0.048), with peripheral neutrophil count >0.1 × 10(9)/L (P=0.023), and with reticulocyte count >10 × 10(9)/L (P=0.002). Platelet count (P=0.620) and bone marrow lymphocyte percentage (P=0.736) showed no correlation with the therapeutic effectiveness. Multi-factor analysis by the Kaplan-Meier procedure on the factors influencing survival showed that rather longer survival times occurred in patients > 20 years old, with peripheral neutrophil count [Symbol: see text] 0.1 × 10(9)/L, reticulocyte count [Symbol: see text]10 × 10(9)/L, and platelet count > 10 × 10(9)/L (all P=0.0001). Bone marrow lymphocyte percentage and the initiation time of ALG/ATG application (from onset of the illness) showed no significant influence on patients' survival time (P=0.085 and P=0.935, respectively). CSKD+ALG/ATG therapy for treatment of VSAA could enhance the current clinical therapeutic effects and elevate patients' survival rate. Conditions including male sex, age >20 years, illness course [Symbol: see text]1 month, neutrophil count >0.1 × 10(9)/L, and reticulocyte count >10 × 10(9)/L are the likely effective indices for predicting favorable therapeutic effectiveness in newly diagnosed VSAA patients.

Plxdc2 Marks Hematopoietic Stem Cells and Th2 Cytokine-Producing Innate Lymphocytes in Adult Bone Marrow

Abstract 1278Hematopoietic stem cells (HSCs) have been highly enriched using combinations of more than 10 surface markers. However the simple method using a few positive markers is preferable to identify HSCs location in tissue section. We performed a stringent comparative gene expression profiling analysis to find genes preferentially expressed in the HSC population, and identified a total of 63 genes that are highly expressed in HSC among various hematopoietic cell population.In order to find HSC-specific marker we focused on genes encoding cell surface protein, and found that plexin domain containing 2 (Plxdc2) is highly expressed in CD34—c-Kit+Sca-1+Lineage−(CD34−KSL) HSC population using Plxdc2::GFP knock-in mice. Only 0.2% of whole bone marrow cells were Plxdc2+, and competitive repopulation assay clearly showed that all HSCs are included in the Plxdc2+ fraction. These results identify Plxdc2 as a new marker of HSCs. Plxdc2+ population contain not only HSCs but uncharacterized c-Kitlow/−Sca-1+Lineage−cells. To further purify HSCs, we investigated the additional positive marker. Throughout the screening of various known HSC-related marker, CD150 was selected. CD150 is already recognized as a positive HSC marker (Kiel, et al. Cell 2005). The Plxdc2+CD150+ fraction represented only 0.1%±0.002% in whole bone marrow, and 6% in c-Kit+Sca-1+Lineage− cells, respectively. To test whether the combination of Plxdc2 and CD150 with or without other markers can highly enrich long-term HSCs, we competitively reconstituted irradiated mice with single Plxdc2+CD150+ cells or single Plxdc2+CD150+c-Kit+Sca-1+Lineage− cells. One out of every 4.6 Plxdc2+CD150+ cells (22%), and one out of 2.2 Plxdc2+CD150+c-Kit+Sca-1+Lineage− cells (44%) engrafted and gave long-term multi-lineage reconstitution. The simple combination of Plxdc2 and CD150 significantly increased HSC purity. In addition, we found robust levels of PLXDC2 transcripts in purified human cord blood CD34+ HSCs.Next, we attempted to characterize the another Plxdc2+ fraction which is c-Kitlow/−Sca-1+Lineage−. Multicolor flowcytometric analysis revealed that Plxdc2+c-Kitlow/−Sca-1+Lineage− cells uniformly express CD45, IL7Rα, Thy-1.2, CD27, T1/ST2 (IL1RL1, a subunit of IL33R) and CD25. These cell surface phenotype indicated that this population is probably of lymphoid lineage. However, culturing Plxdc2+ c-Kit low/−Sca-1+Lineage− cells on OP9-DL1, which supports the development of T-cell progenitors to mature T-cells, did not induce T-cell differentiation. Plxdc2+c-Kitlow/−Sca-1+Lineage−cells also did not differentiate into B cells when co-cultured with OP9 stroma cell line. Furthermore Plxdc2+c-Kitlow/−Sca-1+Lineage− cells produce IL-5 and IL-13 in response to IL-33 or a combination of IL-2 and IL-25. These characteristics resemble that of “natural helper (NH) cells”, a recently identified cell population capable of producing large amounts of Th2 cytokines in fat-associated lymphoid clusters (Moro, et al. Nature 2010). Immunohistochemical staining of bone section to detect HSCs, and functional analyses to clarify why Plxdc2 specifically express in HSCs and bone marrow “NH cells” using Plxdc2-deficient mice are our ongoing tasks. Disclosures:No relevant conflicts of interest to declare.

A “canonical” Npm1 mutation Knock-in Mouse Model Revealed Subtle but Definitive Myeloid Expansion with Poor HSC Niche Interaction

Abstract 762Nucleophosmin (NPM1) is a ubiquitous multifunctional phosphoprotein, which has nucleocytoplasmic shuttling activity. Somatic mutations in NPM1 gene result in cytoplasmic dislocation of NPM1 (NPM1c) and are frequently associated with acute myeloid leukemia (AML). The pathogenetic effects of mutated NPM1 protein have been explored by animal models including transgenic or “humanized” knock-in mouse models. Here, we demonstrate the first “canonical” mouse Npm1 mutant knock-in model. Different from the previously report of humanized NPM1 mutant knock-in model, we inserted TCTG after nucleotide c.857 of murine Npm1 coding sequence (c.854–857dupTCTG), a pattern identical to human NPM1 mutation, without any “humanized” sequence. This mutation caused a shift of peptide sequence from WQWRKSL* (amino acid 286–292) to LCLAVEEISLRKGFKQFEIFCLHFCNS* (amino acid 285 to 311), a pattern mildly different from the change in human NPM1 mutation, but is still predicted to generate a nuclear export signal. NPM1c+ genotype and protein accumulation in cytoplasm were confirmed with PCR and immunocytochemistry, respectively. We found that the homozygous NPM mutant (NPMc+/c+) mice were embryonic lethal before E10.5 day, while hetrozygote (NPMwt/c+) mice survived and were fertile, and born with Mendelian ratio. Most NPMwt/c+ mice had normal hematologic parameters and remained disease-free, however, these mice developed a delayed-onset aberration on the distribution of granulocyte-monocye progenitors (GMP), monocytes, and B lymphocytes in blood, spleen, and bone marrow. Colony forming unit assay showed normal hematopoietic development of marrow hematopoietic stem cells (HSC), but poor cobblestone formation while HSCs contacted with stroma microenvironment in NPMwt/c+ mice, suggesting the NPMc mutant may affect the ability of HSCs on contact signal expression. Three (12.5%) of 24 NPMwt/c+ mice developed leukocytosis and splenomegaly mimicking myeloproliferative neoplasm of human. Microscopic examination showed panmyelosis of the bone marrow and existence of hematopoiesis in the spleen. In addition, the immune activities of NPMwt/c+ mice’s splenocytes and thymocytes with mitogen stimulation were decreased. In summary, our “canonical” NPMwt/c+ mouse model demonstrated subtle but definitive phenotypes in hematopoietic cells and provided insight into the pathogenesis of NPM1 mutation in human acute myeloid leukemia. A second hit may be necessary for the development of AML in NPMwt/c+ mice since no AML was detected in these mice till 20 months of age. Disclosures:No relevant conflicts of interest to declare.

DISTINCT CLINICOLABORATORY CHARACTERISTICS of the t(14;19)(q32;q13)-POSITIVE SPLENIC MARGINAL ZONE LYMPHOMA

Abstract 5200 Background:Splenic marginal zone lymphoma (SMZL) is a well recognized B-cell neoplasm which is characterized by splenomegaly, bone marrow involvement, immunologically by typical phenotype of marginal zone cells. The most frequent cytogenetic findings are involvement of chromosomes 1, 3, 7(usually deleted in 7q) and 8. The t(14;19)(q32;q13) is a rare cytogenetic abnormality with bcl-3 rearrangement that has been reported in other B-cell lymphomas. Aims:To describe the clinical, morphological, immunophenotypic findings in SMZL associated with t(14;19)(q32;q13). Methods and results:In Hematological Research Centre, Moscow between January 2001 and May 2011 three cases SMZL with t(14;19)(q32;q13) were identified. All patients were males with age 51, 58, 67 y.o. Lymphoma presented with B-symptoms, high level of lactate dehydrogenase (LDH), hepatosplenomegaly and regional lymphadenopathy (enlarge splenic hilar lymphnodes). The hemoglobin was 92 g/l, 110 g/l, 122 g/l. All patients had normal count of leukocytes with an absolute lymphocytosis (lymphocytes count 72 × 109 g/l, 79 × 109 g/l, 83 × 109/l) and thrombocytopenia. Morphological examination of peripheral blood and bone marrow lymphocytes showed that all lymphocytes are atypical with wide cytoplasm and nuclear indentation. In all cases there was nodular type of bone marrow involvement, composed of majority medium sized cells. Immunophenotypic analysis has shown the expression of mature B-cells antigens (CD19, CD20, CD22, FMC7, sIg) and absence of ÑD10, CD23, CD5, CD43, CyclinD1.Two patients were treated with CHOP-regimen without any response. They progressed with spleen enlargement and decreased of thrombocytes counts. All 3 patients undergo splenectomy. Weight of spleen was 1800 g, 2083 g, 2850 g. Splenic section generally show massive nodular pattern (involvement of the white and red pulp) associated with diffuse invasion of the sinuses. In all cases discovered high Ki-67. All patients demonstrated progression after splenectomy during 3–6 months that was characterized by increase of leukocytes count (range 45,4 – 101,8 × 109 /l), high level of LDH, appearance of peripheral and visceral lymph nodes. Considering the increase leucocytes, presence of lymphadenopathy in all cases CHOP, FMC regimen were used. All patients died of disease progression and infectious complications. Time of observation was 21, 30, 34 months. Conclusions:The t(14;19)(q32;q13)-positive SMZL is distinct variant which is characterized by rapid progression after splenectomy, poor responses to chemotherapy and short survival. So t(14;19)(q32;q13) may be regarded as a poor prognostic factor. Disclosures:No relevant conflicts of interest to declare.

Significance of Increased Blastic-Appearing Cells in Bone Marrow Following Myeloablative Unrelated Cord Blood Transplantation in Adult Patients

An abnormal increase of nonleukemic blastic-appearing lymphocytes in bone marrow (BM) specimens has been reported after unrelated cord blood transplantation (UCBT). This study analyzed the incidence, chronology, biological features, and clinical significance of elevated numbers of these cells in a series of 165 consecutive adult patients demonstrating myeloid engraftment after myeloablative UCBT in a single institution. The patients' BM samples were routinely evaluated by cytomorphology at different time points after UCBT. When ≥5% of blastic-appearing cells were detected by cytomorphology in the BM, samples were also evaluated by multiparametric flow cytometry to characterize these cells. Systematic chimerism analyses of BM samples using PCR amplification of short tandem repeat markers were performed. Forty-three patients (cumulative incidence, 26.1%) demonstrated ≥5% of nonmalignant blastic-appearing cells in BM after a median of 101 days after UCBT (range, 28-377 days). All of these patients had full-donor chimerism and a clinical course without leukemic relapse. Multiparametric flow cytometry analyses performed in 36 of the 43 patients showed a polyclonal expansion of B lymphocytes with a broad spectrum of maturation stages. An increased number of nonmalignant blastic-appearing cells was significantly associated with a high number of lymphocytes infused at the time of UCBT and with low rates of acute and chronic extensive graft-versus-host disease, suggesting a potential immunoregulatory role of these cells. The observation of ≥5% nonmalignant blastic-appearing cells in BM samples after myeloablative UCBT is frequent, and these should be distinguished from malignant blasts.

N-Acetylcysteine Increases the Frequency of Bone Marrow Pro-B/Pre-B Cells, but Does Not Reverse Cigarette Smoking-Induced Loss of This Subset

BackgroundWe previously showed that mice exposed to cigarette smoke for three weeks exhibit loss of bone marrow B cells at the Pro-B-to-pre-B cell transition, but the reason for this is unclear. The antioxidant N-acetylcysteine (NAC), a glutathione precursor, has been used as a chemopreventive agent to reduce adverse effects of cigarette smoke exposure on lung function. Here we determined whether smoke exposure impairs B cell development by inducing cell cycle arrest or apoptosis, and whether NAC treatment prevents smoking-induced loss of developing B cells.Methodology/Principal FindingsGroups of normal mice were either exposed to filtered room air or cigarette smoke with or without concomitant NAC treatment for 5 days/week for three weeks. Bone marrow B cell developmental subsets were enumerated, and sorted pro-B (B220+CD43+) and pre-B (B220+CD43−) cell fractions were analyzed for cell cycle status and the percentage of apoptotic cells. We find that, compared to sham controls, smoke-exposed mice have ∼60% fewer pro-B/pre-B cells, regardless of NAC treatment. Interestingly, NAC-treated mice show a 21–38% increase in total bone marrow cellularity and lymphocyte frequency and about a 2-fold increase in the pro-B/pre-B cell subset, compared to sham-treated controls. No significant smoking- or NAC-dependent differences were detected in frequency of apoptotic cells or the percentage cells in the G1, S, or G2 phases of the cycle.Conclusions/SignificanceThe failure of NAC treatment to prevent smoking-induced loss of bone marrow pre-B cells suggests that oxidative stress is not directly responsible for this loss. The unexpected expansion of the pro-B/pre-B cell subset in response to NAC treatment suggests oxidative stress normally contributes to cell loss at this developmental stage, and also reveals a potential side effect of therapeutic administration of NAC to prevent smoking-induced loss of lung function.