scholarly journals Adeno-associated virus mediated gene delivery: Implications for scalable in vitro and in vivo cardiac optogenetic models

2017 ◽  
Author(s):  
Christina M. Ambrosi ◽  
Gouri Sadananda ◽  
Aleksandra Klimas ◽  
Emilia Entcheva
Keyword(s):  
Gene Expression ◽  
Cell Surface ◽  
Transgene Expression ◽  
Neonatal Rat ◽  
Cell Surface Receptor ◽  
Specific Gene ◽  
Neuraminidase Treatment ◽  
Rat Hearts

ABSTRACTAimsAdeno-associated viruses (AAVs) provide advantages in long-term, cardiac-specific gene expression. However, AAV serotype specificity data is lacking in cardiac models relevant to optogenetics. We aimed to identify the optimal AAV serotype (1, 6, or 9) in pursuit of scalable rodent and human models for cardiac optogenetics and elucidate the mechanism of virus uptake.MethodsIn vitro syncytia of primary neonatal rat ventricular cardiomyocytes (NRVMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) were infected with AAVs 1, 6, and 9 containing the transgene for eGFP or channelrhodopsin-2 (ChR2) fused to mCherry. In vivo adult rats were intravenously injected with AAV1 and 9 containing ChR2-mCherry.ResultsTransgene expression profiles of rat and human cells in vitro revealed that AAV1 and 6 significantly outperformed AAV9. In contrast, systemic delivery of AAV9 in adult rat hearts yielded significantly higher levels of ChR2-mCherry expression and optogenetic responsiveness. We tracked the mechanism of virus uptake to purported receptor-mediators for AAV 1/6 (cell surface sialic acid) and AAV9(37/67kDa laminin receptor, LamR). In vitro desialylation of NRVMs and hiPSC-CMs with neuraminidase significantly decreased AAV1,6-mediated gene expression, but interestingly, desialylation of hiPSC-CMs increased AAV9-mediated expression. In fact, only very high viral doses of AAV9-ChR2-mCherry, combined with neuraminidase treatment yielded consistent optogenetic responsiveness in hiPSC-CMs. Differences between the in vitro and in vivo performance of AAV9 could be correlated to robust LamR expression in the adult and neonatal rat hearts, but no expression in vitro in cultured cells. The dynamic nature of LamR expression and its dependence on environmental factors was further corroborated in intact adult human ventricular tissue slices.ConclusionThe combined transgene expression and cell surface receptor data may explain the preferential efficiency of AAV1/6 in vitro and AAV9 in vivo for cardiac delivery and mechanistic knowledge of their action can help guide cardiac optogenetic efforts.

Serial analysis of gene expression (SAGE) during porcine embryo development

2004 ◽  
Vol 16 (2) ◽  
pp. 87 ◽  
Author(s):  
Le Ann Blomberg ◽  
Kurt A. Zuelke
Keyword(s):  
Gene Expression ◽  
Functional Genomics ◽  
Embryo Development ◽  
Molecular Mechanisms ◽  
Physiological Role ◽  
Transgenic Animals ◽  
Specific Gene ◽  
Research Strategies

Functional genomics provides a powerful means for delving into the molecular mechanisms involved in pre-implantation development of porcine embryos. High rates of embryonic mortality (30%), following either natural mating or artificial insemination, emphasise the need to improve the efficiency of reproduction in the pig. The poor success rate of live offspring from in vitro-manipulated pig embryos also hampers efforts to generate transgenic animals for biotechnology applications. Previous analysis of differential gene expression has demonstrated stage-specific gene expression for in vivo-derived embryos and altered gene expression for in vitro-derived embryos. However, the methods used to date examine relatively few genes simultaneously and, thus, provide an incomplete glimpse of the physiological role of these genes during embryogenesis. The present review will focus on two aspects of applying functional genomics research strategies for analysing the expression of genes during elongation of pig embryos between gestational day (D) 11 and D12. First, we compare and contrast current methodologies that are being used for gene discovery and expression analysis during pig embryo development. Second, we establish a paradigm for applying serial analysis of gene expression as a functional genomics tool to obtain preliminary information essential for discovering the physiological mechanisms by which distinct embryonic phenotypes are derived.

scholarly journals A Dual Role for Oncostatin M Signaling in the Differentiation and Death of Mammary Epithelial Cells in Vivo

2008 ◽  
Vol 22 (12) ◽  
pp. 2677-2688 ◽  
Author(s):  
Paul G. Tiffen ◽  
Nader Omidvar ◽  
Nuria Marquez-Almuina ◽  
Dawn Croston ◽  
Christine J. Watson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Epithelial Cell ◽  
Epithelial Cells ◽  
Mammary Epithelial Cells ◽  
Mammary Epithelial ◽  
Oncostatin M ◽  
Specific Gene ◽  
Mammary Involution

Abstract Recent studies in breast cancer cell lines have shown that oncostatin M (OSM) not only inhibits proliferation but also promotes cell detachment and enhances cell motility. In this study, we have looked at the role of OSM signaling in nontransformed mouse mammary epithelial cells in vitro using the KIM-2 mammary epithelial cell line and in vivo using OSM receptor (OSMR)-deficient mice. OSM and its receptor were up-regulated approximately 2 d after the onset of postlactational mammary regression, in response to leukemia inhibitory factor (LIF)-induced signal transducer and activator of transcription-3 (STAT3). This resulted in sustained STAT3 activity, increased epithelial apoptosis, and enhanced clearance of epithelial structures during the remodeling phase of mammary involution. Concurrently, OSM signaling precipitated the dephosphorylation of STAT5 and repressed expression of the milk protein genes β-casein and whey acidic protein (WAP). Similarly, during pregnancy, OSM signaling suppressed β-casein and WAP gene expression. In vitro, OSM but not LIF persistently down-regulated phosphorylated (p)-STAT5, even in the continued presence of prolactin. OSM also promoted the expression of metalloproteinases MMP3, MMP12, and MMP14, which, in vitro, were responsible for OSM-specific apoptosis. Thus, the sequential activation of IL-6-related cytokines during mammary involution culminates in an OSM-dependent repression of epithelial-specific gene expression and the potentiation of epithelial cell extinction mediated, at least in part, by the reciprocal regulation of p-STAT5 and p-STAT3.

scholarly journals Epigenetic regulation of neural cell differentiation plasticity in the adult mammalian brain

2008 ◽  
Vol 105 (46) ◽  
pp. 18012-18017 ◽  
Author(s):  
Jun Kohyama ◽  
Takuro Kojima ◽  
Eriko Takatsuka ◽  
Toru Yamashita ◽  
Jun Namiki ◽  
...  
Keyword(s):  
Gene Expression ◽  
Target Genes ◽  
Epigenetic Modification ◽  
Mammalian Brain ◽  
Cytokine Signaling ◽  
Specific Gene ◽  
Neural Cells ◽  
Specific Gene Expression

Neural stem/progenitor cells (NSCs/NPCs) give rise to neurons, astrocytes, and oligodendrocytes. It has become apparent that intracellular epigenetic modification including DNA methylation, in concert with extracellular cues such as cytokine signaling, is deeply involved in fate specification of NSCs/NPCs by defining cell-type specific gene expression. However, it is still unclear how differentiated neural cells retain their specific attributes by repressing cellular properties characteristic of other lineages. In previous work we have shown that methyl-CpG binding protein transcriptional repressors (MBDs), which are expressed predominantly in neurons in the central nervous system, inhibit astrocyte-specific gene expression by binding to highly methylated regions of their target genes. Here we report that oligodendrocytes, which do not express MBDs, can transdifferentiate into astrocytes both in vitro (cytokine stimulation) and in vivo (ischemic injury) through the activation of the JAK/STAT signaling pathway. These findings suggest that differentiation plasticity in neural cells is regulated by cell-intrinsic epigenetic mechanisms in collaboration with ambient cell-extrinsic cues.

scholarly journals OncogenicGata1causes stage-specific megakaryocyte differentiation delay

2019 ◽  
Author(s):  
Gaëtan Juban ◽  
Nathalie Sakakini ◽  
Hedia Chagraoui ◽  
Qian Cheng ◽  
Kelly Soady ◽  
...  
Keyword(s):  
Gene Expression ◽  
Es Cells ◽  
Erythroid Differentiation ◽  
Detailed Examination ◽  
S Phase ◽  
Myeloproliferative Disorder ◽  
Specific Gene ◽  
Specific Stage

AbstractThe megakaryocyte/erythroid Transient Myeloproliferative Disorder (TMD) in newborns with Down Syndrome (DS) occurs when N-terminal truncating mutations of the hemopoietic transcription factor GATA1, that produce GATA1short protein (GATA1s), are acquired early in development. Prior work has shown that murine GATA1s, by itself, causes a transient yolk sac myeloproliferative disorder. However, it is unclear where in the hemopoietic cellular hierarchy GATA1s exerts its effects to produce this myeloproliferative state. Here, through a detailed examination of hemopoiesis from murine GATA1s ES cells and GATA1s embryos we define defects in erythroid and megakaryocytic differentiation that occur relatively in hemopoiesis. GATA1s causes an arrest late in erythroid differentiationin vivo, and even more profoundly in ES-cell derived cultures, with a marked reduction of Ter-119 cells and reduced erythroid gene expression. In megakaryopoiesis, GATA1s causes a differentiation delay at a specific stage, with accumulation of immature, kit-expressing CD41himegakaryocytic cells. In this specific megakaryocytic compartment, there are increased numbers of GATA1s cells in S-phase of cell cycle and reduced number of apoptotic cells compared to GATA1 cells in the same cell compartment. There is also a delay in maturation of these immature GATA1s megakaryocytic lineage cells compared to GATA1 cells at the same stage of differentiation. Finally, even when GATA1s megakaryocytic cells mature, they mature aberrantly with altered megakaryocyte-specific gene expression and activity of the mature megakaryocyte enzyme, acetylcholinesterase. These studies pinpoint the hemopoietic compartment where GATA1s megakaryocyte myeloproliferation occurs, defining where molecular studies should now be focussed to understand the oncogenic action of GATA1s.Scientific CategoryHaematopoiesis and Stem CellsKey PointsGATA1s-induced stage-specific differentiation delay increases immature megakaryocytesin vivoandin vitro, during development.Differentiation delay is associated with increased numbers of cells in S-phase and reduced apoptosis.

scholarly journals CD162 Is a Key E-Selectin Receptor Promoting Acute Myeloid Leukemia Chemo-Resistance in the Bone Marrow Niche

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 907-907
Author(s):  
Johanna D Erbani ◽  
Joshua Tay ◽  
Valerie Barbier ◽  
Jean-Pierre Levesque ◽  
John L. Magnani ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Surface ◽  
Disease Free Survival ◽  
Therapy Resistance ◽  
Confocal Imaging ◽  
Cell Surface Receptor ◽  
Bone Marrow Niche ◽  
Vascular Adhesion

We have recently found that the vascular adhesion molecule E(endothelial)-selectin is a critical bone marrow niche component mediating acute myeloid leukemia (AML) chemo-resistance. Clinical trials involving the use of E-selectin mimetics to improve efficacy of conventional AML therapy are in progress. In this study we investigate the identity of the AML cell surface receptors mediating vascular E-selectin-induced chemo-resistance. E-selectin has two well-characterised receptors, CD162 also known as P-Selectin Glycoprotein Ligand-1 (PSGL-1), and CD44 isoform HCELL (Hematopoietic Cell E-selectin/L-selectin Ligand) with other cell surface glycoproteins (such as ESL-1) and glycolipids also binding E-selectin after Sialyl Lewisx/a glycosylation. To investigate which of these AML cell surface receptors are responsible for mediating vascular E-selectin survival signalling, we first investigated if each co-localized with E-selectin on AML cell surface by confocal imaging. Human CD34+ AML KG1a cells were labelled ± adhesion to fluorescently E-selectin-IgM. Confocal imaging revealed that although both canonical CD44 and CD162 receptors co-localized with E-selectin at the site of cell contact, only CD162 became strongly polarized at E-selectin binding site, while CD44 remained widely distributed across the cell surface. To dissect a functional role for each of these canonical receptors in human AML, CRISPR-Cas9 gene editing was used to selectively delete CD44 and/or CD162 from human AML KG1a cells. We found that although deletion of both CD44 and CD162 receptors reduced KG1a E-selectin-IgM binding potential (3-fold), deletion of either receptor alone did not. Next, we investigated whether deletion of either receptor reversed E-selectin-mediated chemo-resistance in an in vitro chemosensitivity assay. KG1a cells were seeded in wells pre-coated with a range of vascular adhesion molecules commonly expressed in the bone marrow niche, then monitored for cell survival after 48hr treatment ± cytarabine. In this in vitro assay, we found that adhesion to E-selectin significantly increased parental KG1a survival to chemotherapy (p=0.0035). No similar increase in survival was observed following adhesion to P-selectin, or with integrin ligands ICAM-1 and PE-CAM-1. When we repeated the assay using Crispr CD44 deleted KG1a AML we found significant E-selectin-mediated chemo-resistance was still observed (p=0.027) even in the absence of CD44. These results suggest CD44 is not the receptor mediating AML chemo-resistance. In contrast E-selectin-mediated chemo-resistance was abrogated in the CD162 Crispr deleted human KG1a AMLs. Together these data suggest CD162/PSGL-1 expressed on the surface of human AML KG1a appears to be the receptor mediating vascular E-selectin chemo-resistance. This would be a completely novel role described for CD162 which is conventionally known as a homing molecule. To confirm this new role for CD162 in mediating AML chemo-resistance can be replicated in pre-clinical models in vivo, we next generated (11q23-rearranged) AML from CD44-/- and/or CD162-/- gene-deleted mice by retroviral transduction of murine hematopoietic stem cells with MLL-AF9 which then were transplanted into wildtype mice. Cohorts of leukemic mice (n=8/gp) were administered induction therapy (cytarabine/doxorubicin) to monitor impact on disease-free survival. In contrast to AMLs from wildtype, we found absence of CD162 in murine AMLs lead to a pronounced chemo-sensitisation in vivo resulting in a significant (6-fold, p=0.0004) extension in overall disease-free survival duration, compared to either no chemotherapy gene-deleted AML controls, or to treated wildtype AML controls. These in vivo murine data confirm the identification of an exciting new role for CD162 as an important cell surface receptor mediating therapy resistance in AML. In conclusion, we describe a novel form of niche-mediated chemo-resistance and identify CD162 as a key AML cell surface receptor involved in both human and mouse AML therapy resistance. CD162/PSGL-1 expression has not previously been implicated in direct therapy resistance. Together these findings help extend our knowledge on the potential mechanisms by which therapeutic blockade of vascular E-selectin can significantly improves therapy outcomes. Disclosures Levesque: GlycoMimetics: Equity Ownership. Magnani:GlycoMimetics Inc: Employment, Equity Ownership. Winkler:GlycoMimetics: Patents & Royalties.

scholarly journals Analysis of the Mouse CSF-1 Gene Promoter in a Transgenic Mouse Model1

2003 ◽  
Vol 51 (7) ◽  
pp. 941-949 ◽  
Author(s):  
Sherry L. Abboud ◽  
Maria Bunegin ◽  
Nandini Ghosh-Choudhury ◽  
Kathleen Woodruff
Keyword(s):  
Gene Expression ◽  
Transgenic Mice ◽  
Transgene Expression ◽  
Transcriptional Activity ◽  
Molecular Mechanisms ◽  
Tissue Expression ◽  
Cellular Level ◽  
Liver And Kidney

CSF-1 stimulates monocyte and osteoclast populations. However, the molecular mechanisms involved in regulating CSF-1 gene expression are unclear. To identify regulatory regions that control normal CSF-1 gene expression, a −774/+183-bp fragment of the murine CSF-1 promoter was analyzed in vitro and in vivo. Transcriptional activity was high in cultured osteoblasts that express CSF-1 mRNA compared to ARH-77 B cells that lack CSF-1 gene expression. Transient transfection of osteoblasts with promoter deletion constructs showed that the −774-bp fragment conferred the highest transcriptional activity and contained activator and repressor sequences. To assess the ability of the CSF-1 promoter to confer normal tissue expression of CSF-1, transgenic mice containing the −774/+183-bp region driving the E. coli β-galactosidase (lacZ) reporter gene were generated. β-Gal analysis of whole tissue extracts showed transgene expression in all tissues tested except liver and kidney. At the cellular level, the pattern of β-gal expression in the spleen, thymus, bone, lung, and testes of adult transgenic mice mimicked normal endogenous CSF-1 mRNA expression in non-transgenic littermates detected by in situ hybridization. This region also directed appropriate transgene expression to sites in other tissues known to synthesize CSF-1, with the exception of the liver and kidney. These findings indicate that the −774-bp fragment contains cis-acting elements sufficient to direct CSF-1 gene expression in many tissues. CSF-1 promoter/lacZ mice may be useful for studying the transcriptional mechanisms involved in regulating CSF-1 gene expression in tissues throughout development.

Id1 Regulates the Choice of Hematopoietic Stem Cells to Self-Renew or Commit to Differentiation.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 270-270 ◽  
Author(s):  
Vladimir Jankovic ◽  
Alessia Ciarrocchi ◽  
Piernicola Boccuni ◽  
Robert Benezra ◽  
Stephen D. Nimer
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Self Renewal ◽  
Myeloid Progenitor ◽  
E Protein

Abstract Id proteins belong to the basic helix-loop-helix family of transcription factors and act as dominant negative forms of E-protein transcriptional activators. Id mediated E protein silencing has an essential role in restricting differentiation and maintaining self-renewal in embryonic stem cells. However, the role of Id1 in adult stem cells including HSCs has not been described thus far. Having detected relatively high levels of Id1 mRNA in murine adult HSCs (compared to the committed myeloid progenitor cells) we examined the in vivo HSC function in Id1 deficient mice. We observed a >2 fold reduction in HSC frequency in the bone marrow in 8-10w old Id1−/− mice compared to Id1+/+ animals, detected by both lin-c-kit+Sca-1+ (LKS) cell surface marker profile and Hoechst 33342 dye efflux - “side population” phenotype, as well as a ~25% decrease in total bone marrow cellularity. Although Id1 deficient HSCs show robust long-term competitive repopulating capacity in primary transplant recipients, they have markedly impaired hematopoietic function upon secondary transplantation. Id1 null HSCs show a higher rate of S-phase entry in vivo as measured by 3 day BrdU incorporation (ko: 79.0±3.9% vs. wt: 49.7±7.4) and faster initial doubling times in response to cytokine stimulation in vitro during the first 2 days of culture. This failure to maintain normal HSC numbers and the diminished repopulating capacity, in the presence of enhanced cell cycling, suggests a defect in the regulation of self-renewal in Id1 deficient HSCs. Considering the general function of Id1 as an inhibitor of differentiation, the observed effect of Id1 loss could be explained by the excessive recruitment of LKS cells into the actively proliferating differentiated progenitor pool, at the expense of their self-renewal capacity. Consistent with this, sorted Id1−/− HSCs show accelerated expression of cell surface lineage markers in vitro and an increased ratio of CFU-S8 /CFU-S12 in the in vivo spleen colony forming assay. Global gene expression profiling of Id1+/+ vs. Id1−/− hematopoietic cells (using Affymetrix MOE430 Plus chips) revealed insignificant transcriptional deregulation in the committed myeloid progenitor subsets (CMP, GMP, MEP) in the absence of Id1. Meanwhile, Id1−/− HSCs showed a marked change in gene expression pattern (more than 1500 genes with a ≥2 fold difference in expression levels). Differentially regulated transcripts in Id1+/+ vs. Id1−/− HSCs significantly overlap (~30%) with the observed changes in gene expression that accompany the transition of HSCs to the common myeloid progenitor phenotype. Specifically, genes such as c/EBPα and GATA1 are significantly upregulated in Id1 null immunophenotypic HSCs, consistent with an earlier than normal commitment to myelo-erythroid differentiation. In contrast, several known transcriptional regulators of HSC self-renewal (Bmi1, Gfi1, HoxB4) show no significant change in expression pattern. These data clearly indicate the unique role of Id1 in regulating HSC self-renewal by restricting the rate of HSC commitment to the myeloid progenitor cell fate.

Maturation Stage-Specific Regulation of Megakaryocytic Genes by Pointed-Domain Ets Proteins.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1737-1737
Author(s):  
Liyan Pang ◽  
Xun Wang ◽  
Yuhuan Wang ◽  
Gerd Blobel ◽  
Mortimer Poncz
Keyword(s):  
Gene Expression ◽  
Fetal Liver ◽  
Critical Role ◽  
Regulatory Region ◽  
Maturation Stage ◽  
Specific Gene ◽  
Mrna Levels ◽  
Specific Regulation

Abstract The pointed-domain Ets transcription factor Fli-1 has a critical role during megakaryocyte-specific gene expression. Previously, we demonstrated that Fli-1 occupies the early megakaryocyte-specific gene αIIb in vivo. Moreover, our work suggested a mechanism for Fli-1 function by showing that Fli-1 facilitates GATA-1/FOG-1 dependent expression of the αIIb gene. However, studies by others with a targeted disruption of the Fli-1 gene in mice showed that while Fli-1 is essential for normal megakaryocyte maturation, αIIb mRNA levels were not significantly reduced in the resulting megakaryocytes, suggesting that a related Ets factor(s) might compensate for the loss of Fli-1. Here we show that the widely expressed pointed domain Ets protein GABPα specifically binds in vitro to Ets elements from two early megakaryocyte-specific genes, αIIb and c-mpl. Chromatin immunoprecipitation (ChIP) experiments using primary murine fetal liver-derived megakaryocytes reveal that GABPα associates with αIIb and c-mpl in vivo. Moreover, GABPα is capable of mediating GATA-1/FOG-1 synergy in the context of αIIb promoter constructs. These results suggest that GABPα contributes to megakaryocyte-restricted gene expression and is capable of at least partially compensating for the loss of Fli-1. However, loss of Fli-1 leads to a pronounced decrease in the expression of the late megakaryocyte-specific gene GPIX, indicating that compensation by GABPα is incomplete. Consistent with this observation, ChIP experiments fail to detect significant levels of GABPα at the regulatory region of GPIX while Fli-1 is readily detected there. Together, these results point to a model in which Fli-1 and GABPα serve overlapping, but distinct roles, during the development of megakaryocytes. GABPα may be important during early megakaryopoiesis, but Fli-1 exerting an essential role during late stages of maturation.

CD38 Induces Homing of Chronic Lymphocytic Leukemia Cells to the Lymphoid Organs through a Functional Interplay with CXCR4.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2328-2328
Author(s):  
Silvia Deaglio ◽  
Semra Aydin ◽  
Tiziana Vaisitti ◽  
Luciana Bergui ◽  
Davide Rossi ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Cell Surface ◽  
Lymphoid Organs ◽  
Lymphocytic Leukemia ◽  
Cell Surface Receptor ◽  
Functional Responses ◽  
Short Period ◽  
Static Conditions

Abstract Abstract 2328 Poster Board II-305 Chronic lymphocytic leukemia (CLL) is characterized by the expansion of a monoclonal population of mature B lymphocytes expressing surface CD5, CD23 and low levels of immunoglobulin (Ig). Originating in the bone marrow (BM), neoplastic cells accumulate in blood (the circulating pool) and in lymphoid organs (the residential pool). Data obtained ex vivo and in patients suggest that the proliferative core of the disease dwells within the residential pool, while little or no proliferation is seen among circulating CLL cells. Threrefore, homing of chronic lymphocytic leukemia (CLL) cells to sites favoring growth is a critical step in disease progression. Human CD38 is a pleiotropic glycoprotein belonging to a complex family of genes coding for enzymes of the cell surface and involved in the catabolism of extracellular nucleotides. Along with the absence of mutations in the immunoglobulin variable region heavy chain gene (IgVH) and expression of the cytoplasmic kinase ZAP-70, CD38 expression above a critical threshold represents a reliable negative prognostic marker for CLL patients. The working hypothesis of our group is that CD38 is not merely a marker in CLL, but a cell surface receptor and adhesion molecule directly involved in the delivery of growth signals. Prolonged interactions between CD38 and its non-substrate ligand CD31 under static conditions increase survival of the CLL clone and sustain its proliferation in vitro. Here we show that CD38, is directly involved in chemokine-mediated homing of CLL cells. CD38 signals facilitate short- (ERK1/2 phosphorylation) and long-term (chemotaxis) effects induced by the CXCL12 chemokine. This was first shown by testing functional responses to CXCL12 in a large cohort of clinically and molecularly characterized patients. The superior chemotactic performance of CD38+ CLL cells was confirmed by comparing the CD38+ and CD38- leukemic components within the same patient presenting with a bimodal clone. Assays using i) a specific inhibitor of CD38 enzymatic activities, ii) a panel of agonistic and blocking monoclonal antibodies (mAbs) and iii) microarray technology indicate that increased homing depends on the receptor functions of CD38. Indeed, interactions between CD38 and its non-substrate ligand CD31 define a genetic signature characterized by modulation of pathways involved in proliferation and migration of CLL cells. Conversely, blocking CD38 with domain-specific mAbs significantly weakens CXCL12 responses. This is attributed to physical proximity on the cell membrane between CD38 and CXCR4 (the CXCL12 receptor). The existence of a CD38/CXCR4 complex was demonstrated by i) co-immunoprecipitation, ii) confocal microscopy and iii) direct measurement of CXCL12 binding to its receptor. The relevance of CD38 in homing is substantiated by results from an in vivo mouse model, where CLL cells are allowed to home to lymphoid organs for a short period of time. The compromised homing ability of CLL cells to spleen and BM in the presence of blocking anti-CD38 mAbs confirms in vivo the in vitro evidence. Furthermore, the lack of effects induced by an isotype-matched mAb with the same anti-CD38 specificity convincingly rules out the possibility that the effects observed are secondary to sequestration of mAb-coated cells. Future studies will address the issue of whether CD38 may become a therapeutic target for selected CLL patients. Disclosures: No relevant conflicts of interest to declare.

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