Cells Of Lymphoid Lineage Research Articles

L-CBM signaling in lymphocyte development and function

The nuclear factor-κB (NF-κB) plays a central role in the activation and survival of lymphocytes. NF-κB, therefore, is pivotal for acquired immunity, but the dysregulation of NF-κB signaling leads to inflammatory diseases and lymphomagenesis. Accumulating evidence has demonstrated that the mucosa-associated lymphoid tissue (MALT) lymphoma-related molecules, B-cell lymphoma 10 (BCL10) and MALT-lymphoma-translocation gene1 (MALT1), are essential signaling components for NF-κB and mitogen-activated protein kinase (MAPK) activation, mediated by the immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptors involved in both innate and adaptive immunity. CARMA1 (also referred to as CARD11 and Bimp3) is a crucial regulator for ITAM-mediated signaling as it forms a complex with BCL10-MALT1 in lymphoid lineage cells such as T, B, natural killer (NK), and natural killer T (NKT) cells, known as the lymphoid CARMA1-BCL10-MALT1 (L-CBM) complex. In this review, recent understanding of the molecular and biological functions and the signal regulation mechanisms of the L-CBM complex are described and its role in disease development and potential as a therapeutic target is further discussed.

Critical role for selectin receptor:ligand interactions in the development of IPS following allogeneic BMT (145.32)

Abstract Idiopathic pneumonia syndrome (IPS) is a frequently fatal complication of allogeneic BMT and limits successful outcomes. The pathophysiology of IPS involves the release of soluble inflammatory proteins and the recruitment of donor lymphoid and myeloid effectors into the lung. The entry of leukocytes into inflamed tissue is mediated by several molecules that are orchestrated in a sequential manner. We used well-established mouse BMT models to investigate the role of selectin (sel) receptor:ligand interactions in the development of IPS. Firstly, E and P sel ligands are up-regulated on donor T cells when activated by host APCs in vitro. Next, we found that sel receptor (mRNA) and ligand (protein) expression is significantly increased in the lungs of allo-BMT recipients during the development of IPS. BMT using donor cells deficient in E and P sel ligands resulted in a significant reduction in lung inflammation (cellularity, pathology, cytokines), and mixing studies uncovered a contribution to cells of both lymphoid and myeloid lineage. Experiments using isolated MHC class I or class II disparate strain combinations revealed a more significant effect on donor CD4+ vs. CD8+ T cells. Finally absence of E and P sel in BMT recipient mice conferred near complete protection from IPS especially during CD4+ mediated disease. These data demonstrate a critical role for selectin receptor:ligand interactions in the pathogenesis of IPS and uncover novel strategies to regulate disease severity.

Differential Geminin Requirement for Proliferation of Thymocytes and Mature T Cells

Stem/progenitor cells coordinate proliferation and differentiation, giving rise to appropriate cell numbers of functionally specialized cells during organogenesis. In different experimental systems, Geminin was shown to maintain progenitor cells and participate in fate determination decisions and organogenesis. Although the exact mechanisms are unclear, Geminin has been postulated to influence proliferation versus differentiation decisions. To gain insight into the in vivo role of Geminin in progenitor cell division and differentiation, we have generated mice that specifically lack Geminin in cells of lymphoid lineage through Cre-mediated recombination. T cells lacking Geminin expression upregulate early activation markers efficiently upon TCR stimulation in vitro and are able to enter the S phase of cell cycle, but show a marked defect in completing the cycle, leading to a large proportion of T cells accumulating in S/G2/M phases. Accordingly, T cells deficient in Geminin show a reduced ability to repopulate lymphopenic hosts in vivo. Contrary to expectations, Geminin deficiency does not alter development and differentiation of T cells in vivo. Our data suggest that Geminin is required for the proliferation events taking place either in vitro upon TCR receptor activation or during homeostatic expansion, but appears to be redundant for the proliferation and differentiation of the majority of progenitor T cell populations.

Human Peripheral Lymphoid Tissues Contain Autoimmune Regulator-Expressing Dendritic Cells

Autoimmune regulator (AIRE) modulates the expression of tissue-restricted antigens (TSAs) and promotes central tolerance in the thymus. However, few autoreactive T cells escape negative selection and reach the periphery, where peripheral tolerance is required to avoid autoimmunity. Murine lymph nodes (LNs) have been shown to contain "stromal" cells expressing AIRE and TSAs. Here we report the occurrence of AIRE-expressing cells in human peripheral lymphoid tissues, including LNs, tonsils, and gut-associated lymphoid tissue, with the exception of the spleen. Notably, AIRE+ cells are absent in fetal LNs and, in postnatal life, they are more numerous in abdominal than in superficial LNs, thus suggesting that their development in periphery may depend on instructive signals from microenvironment and antigen challenge. Extrathymic AIRE+ cells show a dendritic morphology, consistently express human leukocyte antigen-DR (HLADR) and fascin, and are largely positive for CD11c and S100 and for the dendritic cell-activation markers CD40, CD83, DC-LAMP/CD208, and CCR7. Lymphoid, myelomonocytic, mesenchymal, and epithelial cell lineage markers are negative. The HLADRhigh/AIRE+ cell fraction isolated from mesenteric LNs expressed TSAs (insulin, CYP17A1, and CYP21A2), as well as molecules associated with tolerogenic functions, such as interleukin-10 and indoleamine 2,3-dioxygenase. Data indicate that AIRE+ cells in human peripheral lymphoid tissues correspond to a subset of activated interdigitating dendritic cells expressing TSAs and the tolerogenic molecules indoleamine 2,3-dioxygenase and interleukin-10, suggestive of a potential tolerogenic function.

Bone marrow lymphoid and myeloid progenitor cells are suppressed in 7,12-dimethylbenz(a)anthracene (DMBA) treated mice

In this study we used colony forming unit (CFU) assays to demonstrate rapid suppression (within 6 h) of lymphoid (CFU-preB) and myeloid (CFU-GM) progenitor cells in DMBA-treated mice. The duration of these changes were consistent with the blood levels of DMBA and its metabolites that were achieved by either IP or oral DMBA administration. CFU-GM and CFU-preB activities returned to control levels by 2 and 7 days after oral DMBA exposure, respectively, but remained suppressed through 7 days after IP DMBA administration. The continued presence of low levels of DMBA in the bloodstream following IP administration was associated with sustained suppression of CFU-preB, total bone marrow lymphoid cells and peripheral blood lymphocytes. The changes noted above were not observed in Cyp1b1 null mice, demonstrating the need for local DMBA metabolism in the bone marrow by Cyp1b1 to impair bone marrow CFU-preB and CFU-GM. Furthermore, these data provide evidence that myeloid-lineage cells are restored more quickly than lymphoid-lineage cells after DMBA exposure.

Origin and evolution of the adaptive immune system: genetic events and selective pressures

The adaptive immune system (AIS) in mammals, which is centred on lymphocytes bearing antigen receptors that are generated by somatic recombination, arose approximately 500 million years ago in jawed fish. This intricate defence system consists of many molecules, mechanisms and tissues that are not present in jawless vertebrates. Two macroevolutionary events are believed to have contributed to the genesis of the AIS: the emergence of the recombination-activating gene (RAG) transposon, and two rounds of whole-genome duplication. It has recently been discovered that a non-RAG-based AIS with similarities to the jawed vertebrate AIS - including two lymphoid cell lineages - arose in jawless fish by convergent evolution. We offer insights into the latest advances in this field and speculate on the selective pressures that led to the emergence and maintenance of the AIS.

Role of mTOR in Hematopoiesis and Hematopoietic Stem Cell Regulation.

Abstract 1490Poster Board I-513The mammalian target of rapamycin (mTOR) integrates nutrients, growth factors, and cellular energy status to control protein synthesis that determines cell growth and metabolism. It is also known that mTOR plays an essential role in cell survival by regulating Akt/PKB signaling. By using the inhibitor rapamycin, mTOR has previously been suggested to regulate proliferation of megakaryocyte progenitors and late stage of megakaryocyte differentiation without a general impact on normal hematopoiesis or hematopoietic stem cell (HSC) function. Due to limitations of rapamycin and the early lethality of conventional mTOR gene targeted mice, the physiological role of mTOR in blood development remains undefined. In this study, we have utilized an inducible conditional mTOR knockout mouse model by crossbreeding mTORflox/flox mice with Mx-Cre mice that allow interferon-induced mTOR deletion in the bone marrow following a transplantation and polyI:C induction protocol, in an effort to determine the genetic role of mTOR in hematopoiesis. Depletion of mTOR drastically affected hematopoiesis in a blood cell autonomous manner in Mx-Cre;mTORflox/flox bone marrow transplant recipients: the mice showed marked reduction in BM cellularity and in the numbers of myeloid and lymphoid lineage cells, erythrocytes, and platelets in peripheral blood, bone marrow, and thymus, leading to bone marrow failure, blood cell exhaustion and lethality. In vitro colony-forming activities by bone marrow or spleen progenitors were completely abolished in the absence of mTOR. Interestingly, the number and frequency of HSCs in bone marrow (Lin−Sca-1+c-Kit+) increased transiently while the number of early progenitors (CMP, GMP, MEP, CLP) detected by cell surface markers remained unchanged or only mildly affected in the mutant mice within 14 days after polyI:C treatment. Concomitantly, mTOR deletion led to a massive egress of HSCs from bone marrow to distal organs including spleen (∼60-fold increase). Transplantation of mTOR−/− bone marrow cells into NOD-SCID mice or competitive transplantation of mTOR−/− bone marrow cells into BoyJ mice further demonstrated that mTOR deficiency caused a complete failure in HSC engraftment and repopulation. Surprisingly, at the cellular level these phenotypes are associated with increased proliferation of HSCs in vivo and in vitro by 60% and 2.5-fold, respectively, as assessed by 5-bromodeoxyuridine incorporation assays whereas the cell survival index appears to be unaffected. Moreover, mTOR−/− HSCs and progenitor cells displayed impaired adhesion to fibronectin CH296 fragment (∼30% decrease) and migration toward SDF-1α gradients (∼30% decrease). At the molecular level, gene chip microarray analysis of mTOR−/− HSCs revealed that the cell cycle regulators myb, wee1, FANCD2, and FANCE were significantly downregulated while Rb and E2F5 were upregulated, the survival/apoptosis regulators MCL1 and BCL2L1 were upregulated, and the actin cytoskeleton and cell extracellular matrix adhesion regulators Arp2/3 complex subunit 5, paxillin, laminin α5, integrin β3, and myosin light chain 6B were upregulated. Further, immunoblotting analysis of isolated Lin− cells showed that SCF-stimulated activation of translational regulators S6K and 4E-BP and survival regulator Akt were abolished upon mTOR deletion. Taken together, these data suggest that mTOR is a critical regulator of HSC quiescence, self-renewal, and engraftment through the regulation of cell cycle, survival and actin cytoskeleton signals, and is essential in multiple stages of hematopoiesis. DisclosuresCancelas: CERUS CO: Research Funding; CARIDIAN BCT: Research Funding; HEMERUS INC: Research Funding.

MT1-MMP Is Required for Hematopoietic Maturation in the BM Niche.

Abstract 3634Poster Board III-570Specialized niches, in which hematopoietic stem cells (HSCs) reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. Activation of the fibrinolytic pathway via matrix metalloproteinase-9 (MMP-9) resulted in the release of kit ligand (KitL) in the BM niche. Membrane type 1-MMP (MT1-MMP) can activate e.g. MMP-9. To investigate the role of MT1-MMP in hematopoiesis, we used MT1-MMP deficient mice. MT1-MMP−/− mice examined 12 days after birth showed pancytopenia and reduced numbers of bone marrow mononuclear cells (BMMCs) and splenocytes. BM cytospins from MT1-MMP−/− mice showed mild perturbations in erythropoiesis and a more severe impairment of myelopoiesis. Although all lineages were present, the ratio of erythroid to myeloid precursors increased from 0.36 in wildtype to 0.60 in MT1-MMP−/− mice. Myeloid and erythroid cell differentiation was impaired in MT1-MMP−/− BMMCs. The numbers of colony forming unit cells (CFU-C) was reduced in MT1-MMP−/− BMMCs. In contrary, the number of immature hematopoietic cells (CFU-S8, KSL cells) was augmented in MT1-MMP−/− BMMCs. FACS analysis of BM cells showed a decrease in the percentage of mature B cells with an increase number of Pro-B and immature B cells in MT1-MMP−/− BMMCs relative to controls. MT1-MMP−/− BM cells showed lower expression of CXCL12 and KitL, typical niche growth factors important for myelopoiesis and lymphopoisis. Thus, MT1-MMP is required for normal hematopoietic differentiation of lymphoid and myeloid lineage cells, most likely due to growth factor defective niche cells. Disclosures:No relevant conflicts of interest to declare.

Generation of Functional Lymphoid (Natural Killer) Cells From Human ESC-Derived Hemangioblasts.

Abstract 1502Poster Board I-525Studies with human and mouse embryonic stem cells (ESCs) have shown that a common precursor to both vascular (endothelial and smooth muscle cells) and hematopoietic cell lineages called the hemangioblast can be produced from ESC-derived embryoid bodies in culture. We have developed a simple strategy to efficiently and reproducibly generate hemangioblasts from multiple hESC lines under serum- and stromal-free conditions, which will be important for their productive use in regenerative medicine. Previous work has shown that hESC-derived hemangioblasts can effectively differentiate into erythroid and myeloid lineages, but their ability to produce lymphoid lineage cells, including those with immunotherapeutic potential, is relatively unknown. Natural killer (NK) cells, which are part of the innate immune system, provide rapid, non-specific responses against viral infection and are involved in tumor cell detection and elimination. Interplay between various activating and inhibitory signals control the three main functions of NK cells, which are cytokine release, natural cytotoxicity, and antibody-dependent cellular cytotoxicity. Using hemangioblasts generated from both H7 and HuES-3 hESC lines, we have been able to produce mature CD56low/−CD16+ NK cells and found that their production does not require the use of stromal feeder layers. The differentiation procedure involves an initial 4 day culture to generate embryoid bodies, followed by a 12-14 day culture in methylcellulose supplemented with a set of cytokines and growth factors for the production and expansion of a hemangioblastic population. An additional 14-17 days in liquid culture plus human serum and a cocktail of cytokines allows for the differentiation of NK cells as assessed by flow cytometry. A non-radioactive cytotoxicity assay similar to the 51Cr release assay shows that these hemangioblast-derived NK cells harbor natural cytotoxicity function as they are able to effectively induce apoptosis in target K562 erythroblastic leukemia cells after a standard 4 hr co-culture. Using hemangioblasts as an intermediary cell source may enhance the capability and/or efficiency of hESCs to differentiate in vitro and importantly, allow for the development of feeder-free systems for the production of cells with immunotherapeutic potential. Disclosures:Kimbrel: Stem Cell & Regenerative Medicine International: Employment. Lu: Stem Cell & Regenerative Medicine International: Employment. Lanza: Stem Cell & Regenerative Medicine International/Advanced Cell Technology: Employment.

IL‐21: roles in immunopathology and cancer therapy

Cytokines are secreted signalling molecules with decisive effects on haematopoiesis, innate and adaptive immunity, and immunopathology. Interleukin (IL)-21 is a novel cytokine produced by activated CD4(+) T cells and natural killer T (NKT) cells. IL-21 is part of a family of cytokines which include IL-2, -4, -7, -9 and -15 that all share the common IL-2 receptor gamma chain (gamma(c)) in their individual receptor complexes. IL-21 receptor (IL-21R) is widely expressed on both myeloid and lymphoid cell lineages and IL-21 actions include co-stimulation of B cell differentiation and immunoglobulin (Ig) production, co-mitogen of T cells, and stimulation of NK and CD8(+) T cell cytotoxic function. Initially, IL-21 was recognized for its anti-tumour effects in several preclinical tumour models, warranting its currently ongoing clinical development as a cancer immunotherapeutic. More recently, IL-21 has been associated with the development of a panel of autoimmune and inflammatory diseases, where neutralization of IL-21 has been suggested as a potential new therapy. In this review, we will cover the latest discoveries of IL-21 as a cancer therapy and its implications in immunopathologies.

Formula Feeding Skews Immune Cell Composition toward Adaptive Immunity Compared to Breastfeeding

The ontogeny of the immune system and the effect thereon by type of infant feeding is incompletely understood. We analyzed frequencies and composition of immune cells in blood of breastfed (BF) and formula-fed (FF) infants at 1.5, 4, and 6 mo of age. Three formulas with the same protein concentration but with varying levels of alpha-lactalbumin and caseinoglycomacropeptide were compared. Twenty-nine exclusively BF infants served as reference, and 17 infants in each formula group completed the study. Whole blood and PBMCs were analyzed by flow cytometry and immunoflow cytometry, respectively. Leukocyte count of BF infants increased with time due to increased frequency of neutrophils. Lymphocyte count was high at 1.5 mo and was unchanged over time, as were the relative proportions of CD4+ alphabetaT cells, CD8+ alphabetaT cells, B cells, NK cells, and gammadeltaT cells. Most CD45R0+CD3+ cells were HLA-DR- and hence memory cells. Compared with breastfeeding, formula feeding resulted in a significant decrease in proportion of NK cells, but a significant increase in naive CD4+ alphabetaT cells and an elevated CD4-to-CD8 ratio, that is, 3.3 in the combined FF groups compared with 2.6 in the BF group. No significant differences were found between the three groups of FF infants. In conclusion, blood cells of lymphoid lineage did not change significantly in frequencies or composition from 1.5 to 6 mo of age in BF infants. In contrast, FF infants displayed an ongoing maturation of adaptive immunity cells and a delayed recruitment of innate immunity cells as compared with BF infants.

Molecular Genetics of Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is mainly a disease of childhood that arises from recurrent genetic insults that block precursor B and T cell differentiation and drive aberrant cell proliferation and survival. Recurrent defects including chromosomal translocations, aneuploidies, and gene-specific alterations generate molecular subgroups of B- and T-ALL with differing clinical courses and distinct responses to therapy. Recent discoveries arising from genome-wide surveys and adoptive transfer of leukemia-initiating cells have uncovered multiple gene copy number aberrations and have yielded new insight into at least one type of ALL-originating cell. Our understanding of the pathogenesis of ALL has benefited from genetically modified mouse models that recapitulate cellular transformation at specific developmental stages of lymphoid lineage cells. Here, we review the spectrum of genetic aberrations that promote acute B and T cell leukemias and the mechanisms of cell transformation and malignant progression that are reinforced by mouse models of human ALL.

Glucocorticoid-regulated microRNAs and mirtrons in acute lymphoblastic leukemia

Glucocorticoids (GCs) induce apoptosis in lymphoid lineage cells and are therefore used in the therapy of acute lymphoblastic leukemia (ALL) and related malignancies. MicroRNAs (miRNAs) and the related mirtrons are ~22 nucleotide RNAs derived from polymerase-II transcripts and implicated in the control of essential biological functions, including apoptosis. Whether GCs regulate miRNA-encoding transcription units is unknown. We investigated miRNA/mirtron expression and GC regulation in 8 leukemia/lymphoma in vitro models and 13 ALL children undergoing systemic GC monotherapy using a combination of expression profiling techniques, real time reverse transcription (RT)-PCR and northern blotting to detect mature miRNAs and/or their precursors. We found that mature miRNA regulations can be inferred from expression data of their host genes. Although a simple miRNA-initiated canonical pathway to GC-induced apoptosis or cell cycle arrest did not emerge, we identified several miRNAs/mirtrons that were regulated by GC in patients and cell lines, including the myeloid-specific miR-223 and the apoptosis and cell cycle arrest-inducing miR15 ~ 16 clusters. In an in vitro model, overexpression of miR15b ~ 16 mimics increased and silencing by miR15b ~ 16 inhibitors decreased GC sensitivity. Thus, the observed complex changes in miRNA/mirtron expression during GC treatment might contribute to the anti-leukemic GC effects in a cell context-dependent manner.

Humanized Sc(Fv)2 Minibody against C-Mpl Successfully Increased Platelet Number in Monkey and Increased Engraftment of Human Umbilical Cord Blood Cells in NOD/SCID Mouse.

Thrombopoietin (TPO, the ligand for c-mpl) is a key factor for megakaryopoiesis. Several clinical trials of TPO have been conducted for thrombocytopenia without much success due to, in part, the production of neutralized antibodies against endogenous TPO, which causes thrombocytopenia. To overcome this problem, we previously demonstrated that mouse type minibody against c-mpl, with an amino acid sequence totally different from TPO, showed megakaryopoiesis and increased platelet numbers in monkey. This time, using CDR grafting, we generated a humanized sc(Fv)2VB22B minibody (huVB22B) against c-mpl for therapeutic use. The new minibody showed almost the same activity in vitro as TPO and the mouse type minibody, confirmed by both a human megakaryocyte cell (CD41+) differentiation assay and a proliferation assay with TPO-dependent cell line, M-07e. Single sc or iv administration of huVB22B to cynomolgus monkeys showed a dose-dependent increase in platelet numbers. Pharmacokinetic analysis showed that the plasma half-life (T1/2) of huVB22B at iv and sc administration to cynomolgus monkeys was 7–8 h and 11–16 h, respectively. After administration of huVB22B, the platelets of these monkeys increased and showed functional aggregation in response to ADP in vitro. Repeated administration of huVB22B (0.2, 2 and 20mg/kg/week) revealed that the increase in platelet level in cynomolgus monkeys was maintained for a month. Very slight reticular fibers in bone marrow were detected in a high dose group (20mg/ kg). No overt changes were detected by toxicity evaluations including clinical pathology and histopathology in 0.2 and 2mg/kg groups. No neutralized activities in plasma were observed during these experiments. Next, we examined the activities of huVB22B on human bone marrow-derived CD34-positive cells (BM-CD34+) and umbilical cord blood-derived CD34-positive cells (UCB-CD34+) in vitro. BM-CD34+ and UCB-CD34+ cells were cultured with huVB22B in serum free medium. HuVB22B induced differentiation of CD41+ cells from BM-CD34+ or UCB-CD34+ cells in a similar dose-dependent manner. However, UCB-CD34+ cells showed greater proliferation in response to huVB22B compared to BM-CD34+ cells. We then examined the in vivo activities of huVB22B on UCB CD34+ cells by treating NOD/SCID mice transplanted with human UCB-CD34+ cells with huVB22B and examining the bone marrow cells of the mice. The results showed that, compared with the control, administration of huVB22B showed an increase in the number of human hematopoietic progenitor cells (CD34+), lymphoid lineage cells (CD19+), and myeloid lineage cells (CD33+) in addition to human CFU-Meg cells (CD41+). These results suggest that c-mpl stimulation in vivo after transplantation might increase engraftment of progenitor cells in the bone marrow microenvironment and subsequently induce differentiation to multilineage cells. Umbilical cord blood transplantation faces frequent complications including a low-level stem/progenitor cell engraftment and delayed platelet recovery. Our results suggest that c-mpl stimulation might be used to increase the engraftment of UCB stem/progenitor cells and shorten the time of platelet recovery following UCB transplantation.

The Src-Like Adaptor Proteins, SLAP and SLAP2, Regulate Flt3-Dependent Dendritic Cell Development.

The Src-like Adaptor Proteins, SLAP and SLAP2, are hematopoietic adaptor proteins that have been previously shown to act as negative regulators of T- and B-cell signaling. SLAP and SLAP2 work in conjunction with the E3 ubiquitin ligase, c-Cbl, to down regulate the T-cell receptor and other components of the T- and B-cell receptor signaling pathways including ZAP-70 and Syk. SLAP and SLAP2 are expressed in many hematopoietic cell types, including progenitor cells that give rise to cells of both myeloid and lymphoid lineages. Recent evidence indicates a role for SLAP and SLAP2 in regulating hematopoietic receptor tyrosine kinase (RTK) signaling. We have shown that SLAP and SLAP2 interact with CSF-1R/Fms and demonstrated that SLAP2 negatively regulates CSF-1 dependent differentiation of FD-Fms cells. Our recent work demonstrates that both SLAP and SLAP2 interact with the Flt3 receptor in an SH2 domain dependent manner and the interaction was mapped to pY589 and pY591 within the juxtamembrane region of the receptor. To examine the role of SLAP and SLAP2 in Flt3 regulation and signaling in vivo, we utilized Flt3 ligand (Flt3L) to generate dendritic cells from mice lacking both SLAP and SLAP2. Slap1−/− Slap2−/− mice have impaired development of Flt3L-dependent CD11c+ bone marrow-derived dendritic cells (BMDC). In contrast to wild-type mice, Slap1−/− Slap2−/− mice produce 47% ± 9.7% less CD11c+ BMDC after 10 days in culture with Flt3L. Whether the reduction in BMDC numbers is indicative of a defect in the proliferation of Slap1−/−Slap2−/− progenitor cells in response to Flt3L is currently being explored. Although the absolute number of Flt3L-generated BMDC from Slap1−/−Slap2−/− mice is reduced, there are no major differences in the subtype, myeloid (70–90% CD11b+) and lymphoid (4–8% B220+), of DC produced between wild-type and double knock-out mice. To determine whether the reduction in dendritic cell numbers is specific to those generated with Flt3L, we generated BMDC using GM-CSF and IL-4. Both wild-type and double knock-out mice produce similar numbers of CD11c+ BMDC when this combination of cytokines is used. The maturation and activation of Flt3L-generated BMDC from Slap1−/−Slap2−/− mice is being investigated. These data indicate a novel role for SLAP and SLAP2 in the differentiation of Flt3L-dependent dendritic cells.

Granulocyte Colony Stimulating Factor Increases Drug Resistance of Leukaemic Blast Cells to Daunorubicin

Acute leukaemia is known as the most common cancer in childhood. Febrile neutropenia is a common serious side effect of the cytostatic treatment of malignancies. The clinical use of Granulocyte Colony Stimulating Factor (G-CSF) has become widespread to minimize chemotherapy-induced myelosuppression and febrile neutropenia in childhood solid tumors, acute lymphoid leukaemia (ALL) and in several trials with AML. In case of ALL this seems to be reasonable because, due to the absence of G-CSF receptor (G-CSFR) on the surface of normal lymphoid cells, G-CSF does not have any influence on the pathways of proliferation and differentiation of lymphoid lineage cells. It has been suggested, however, that ALL blasts with B or T cell surface antigens as well as biphenotypic leukaemia cells express G-CSFR, and they are able to respond to exogenously added G-CSF with proliferation. In this study we investigated how G-CSF might influence the sensitivity of leukemic cells to daunorubicin induced cell death using MTT assay, flow cytometry and Western blot analysis. After pretreatment of KG-1 leukaemic cells with G-CSF a moderate increase in the resistance of these cells to daunorubicin could be observed. These results draw attention to the risk of G-CSF application as an adjuvant therapy of childhood ALL. In addition, adjuvant treatment of AML patients with G-CSF in order to prevent neutropenia, or its use in priming regimens might result resistance to daunorubicin.

Expression profiling of circulating non-red blood cells in embryonic blood

BackgroundIn addition to erythrocytes, embryonic blood contains other differentiated cell lineages and potential progenitor or stem cells homed to changing niches as the embryo develops. Using chicken as a model system, we have isolated an enriched pool of circulating non red blood cells (nRBCs) from E4 and E6 embryos; a transition period when definitive hematopoietic lineages are being specified in the peri-aortic region.ResultsTranscriptome analysis of both nRBC and RBC enriched populations was performed using chicken Affymetrix gene expression arrays. Comparison of transcript profiles of these two populations, with verification by RT-PCR, reveals in nRBCs an expression signature indicative of hematopoietic stem cells (HSCs) and progenitor cells of myeloid and lymphoid lineages, as well as a number of previously undescribed genes possibly involved in progenitor and stem cell maintenance.ConclusionThis data indicates that early circulating embryonic blood contains a full array of hematopoietic progenitors and stem cells. Future studies on their heterogeneity and differentiation potentials may provide a useful alternative to ES cells and perinatal blood.

Induction of Natural Killer Cell-dependent Antitumor Immunity by the Autographa californica Multiple Nuclear Polyhedrosis Virus

Wild-type Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects a variety of mammalian cell types in vitro, but does not replicate in these cells. We investigated the effects of AcMNPV in the induction of the immune response and tumor metastasis in mice. After intravenous injection, AcMNPV was taken up by the liver and spleen, and preferentially infected dendritic cells (DCs) and B cells in the spleen; costimulatory molecules CD40, CD80, and CD86 were upregulated in the DCs. The hepatic mononuclear cells (MNCs) in these animals were highly cytotoxic to natural killer (NK)-sensitive YAC-1 and B16 melanoma cells, but not to NK-resistant EL4 cells. Intravenous injection of AcMNPV-induced NK cell proliferation in the liver and spleen, and enhanced antitumor immunity in mice with B16 liver metastases. Furthermore, such treatment increased the survival of C57BL/6, J alpha 281 (-/-), and interferon (IFN)-gamma (-/-) mice that were previously injected with B16 tumor cells. AcMNPV injection did not enhance the survival of NK cell-depleted mice. Moreover, one AcMNPV treatment effectively prolonged survival in a B16 liver metastasis model, and was equivalent to five treatments with recombinant interleukin-12 (IL-12) protein. These findings suggest that AcMNPV efficiently stimulates NK cell-mediated antitumor immunity.

Maternal microchimerism in human fetal tissues

The aim of the present study was to analyze the presence of maternal cells in human fetal tissues in the second trimester. Tissues from 11 second-trimester fetuses terminated because of social reasons or because of malformations and/or trisomy were investigated. By cell sorting and polymerase chain reaction amplification, we studied the presence of maternal CD3+, CD19+, CD34+, and CD45+ in different fetal tissues and in placenta. In the group of fetuses with normal karyotype and normal autopsy findings, 4 of 5 fetuses were positive for maternal microchimerism. In the group in which the fetuses were diagnosed with trisomy 21 and/or malformations, we found cells of maternal origin in 3 of 6 fetuses. The results from this study indicate that maternal microchimerism is a common phenomenon in the second-trimester fetuses. Maternal cells of lymphoid and myeloid lineages and hematopoietic progenitors are widely distributed in the second-trimester fetuses.

The blood stem cell's pool modulation in remote period improved health status of Chernobyl clean-up workers

Correction of the psychosomatic disorders in numerous CUW population in remote period after the Chernobyl catastropheremains an issue in medical support of the mentioned population cluster. Earlier, we reported on the possibility of improving of somatic status of the CUWs achieved through vibroacoustic mobilization of autologous haemopoetic stem cells into the bloodstream from the bone marrow haemopoetic zones. Material: A random sampling of CUW persons under long-term dispensary observation (n=40, male, age 49,8±0,7) have been examined under dispensary conditions. The following group consisted of 36 males of the same age, also under dispensary observation but without impact of small radiation dozes in past history. Methods: The quality of life has been examined by Medical Outcome Study 36-Item Short-Form Health Survey (SF-36) method. The quality of life parameters in both CUW and following groups have been compared. A course of vibroacoustic mobilization of autologous haemopoetic stem cells into the bloodstream from the bone marrow haemopoetic zones has been conducted with the purpose of correction of psychosomatic status of CUWs. The following group has not undergone such vibroacoustic course. Various mononuclear cell subpopulations in peripheral blood were estimated by laser cross flow-cytometry method (FACScan, Beckton-Dickinson) with monoclones (DAKO, BD). The SF-36 test was estimated either after the end of the modulation course and 6 months later. Results: Initial self-certification of the quality of life in the monitored CUW group was certainly lower than both the one of the following group and of the population levels. Vibroacoustic modulation has in the CUW group lead to a certain improvement of the quality of life indices directly after the course and has brought the majority of the indices nearer to their population levels. The following group has not demonstrated any similar effects. In 6 months, the indices of the quality of life in the CUW group remained certainly higher than those in the following group. The extent of explication of the correcting index of the quality of life throughout the CUW group has expressed direct correlation with the extent of induced increase of the circulation pool of CD34+ and TdT+ -cells after the modulation course. Conclusions: Deviations of psychosomatic condition of CUWs in remote period after the impact of the complex of factors of a nuclear catastrophe can be corrected by noninvasive mobilization of autologous haemopoetic stem cells into the blood flow and by induction of their subsequent differentiation, including the differentiation towards lymphoid lineage cells.