Characterization of heterotypic adherence between transformed human lymphoblastic cells and marrow stromal cells: VCAM-1 is a ligand for one of the leukemia cell adhesion proteins.

Marrow stromal cells are important in normal myelopoiesis and support growth of leukemia/lymphoma (LL) cells in vitro. We have previously described the heterotypic adherence of a human B-lymphoblastic cell line (UTMB-460) to marrow stromal cells (MSC). We have extended these observations to a human T-lymphoblastic cell line (CEM) and characterized the heterotypic adherence of B- and T-lymphoblastic cell lines to human MSC. Electron microscopy demonstrated UTMB-460 cells were in very close apposition to the MSC, but no specific intercellular junctions were noted. Under the conditions employed, these MSC express extracellular fibronectin, collagen types I and IV, intracellular laminin, and vimentin, but no factor VIII-R antigen. In addition, the MSC had receptors for the lectin Ulex europaeus agglutinin I. UTMB-460 and CEM cells do not adhere to extracellular matrix (ECM) proteins secreted by the MSC, i.e., fibronectin, collagen types I, III, or IV, or laminin. Monoclonal antibodies (MoAbs) against CD11a, CD11b, CD18, and CD54 and a polyclonal anti-human fibronectin antibody do not inhibit attachment of either B- or T-lymphoblastic cells to MSC. Peptides GRGES and GRGDS did not inhibit adherence of UTMB-460 and CEM cells to MSC. In contrast, the anti-vascular cell adhesion molecule (VCAM)-1 MoAb (4b9) caused significant inhibition (p < 0.01) of the adherence of both UTMB-460 and CEM cells to normal human MSC monolayers. These data suggest: (1) that MSC to which lymphoblastic cells adhere are specialized mesenchymal cells; (2) that the membrane interactions between T- and B-lymphoblastic cells and MSC involve close apposition of cell membranes of MSC and the lymphoblastic cells; (3) that the heterotypic adherence between B- and T-lymphoblastic cell lines (UTMB-460 and CEM) and MSC does not involve the RGD recognition sequence of the integrin family, the B2 leukocyte integrins, CD44, LAM-1, or the ECM proteins examined; and (4) that VCAM-1 may at least be partially responsible for heterotypic adherence between human MSC and B- and T-lymphoblastic cells.     

E-selectin and very late activation antigen-4 mediate adhesion of hematopoietic progenitor cells to bone marrow endothelium

 Adhesion of CD34⁺ hematopoietic progenitor cells (HPCs) to sinusoidal endothelium probably plays a key role in homing of transplanted CD34⁺ HPCs to the bone marrow (BM). We have investigated the role of various adhesion molecules in the interaction of purified CD34⁺ HPCs derived from BM or peripheral blood (PB) and a human BM-derived endothelial cell line. Adhesion of CD34⁺ HPCs to endothelial cells was measured with the use of a double-color flow microfluorimetric adhesion assay. In this assay, adhesion is measured under stirring conditions, simulating blood flow in sinusoidal marrow vessels. Adhesion of PB CD34⁺ cells to human BM endothelial cells (HBMECs) was observed only after interleukin (IL)-1β prestimulation of the endothelial cells. This adhesion was strongly increased after addition of phorbol-myristate acetate (PMA). Adhesion of PB CD34⁺ cells to IL-1β-prestimulated HBMECs was inhibited by blocking monoclonal antibodies (mAbs) against E-selectin and by neuraminidase treatment of the PB CD34⁺ cells. mAbs against very late activation antigen (VLA)-4 inhibited adhesion only when the E-selectin-mediated interaction was prevented. No clear inhibiting effect was found with blocking mAbs against β₂-integrins. Stimulation with the β₁-integrin-activating mAb, 8A2, induced adhesion of CD34⁺ cells to endothelial cells. In conclusion, stimulation of both endothelial cells and CD34⁺ HPCs is necessary for adhesion of CD34⁺ HPCs to endothelial cells. We furthermore demonstrated that E-selectin and VLA-4 mediated this adhesion. Received: 26 April 1999 / Accepted: 8 February 2000     

Mobilization of CD34+ haematopoietic stem cells is associated with a functional inactivation of the integrin very late antigen 4

The beta1 integrin very late antigen 4 (VLA-4) plays a central role in mobilization and homing of CD34+ cells. In this study, we examined the activation state of VLA-4 on CD34+ cells from bone marrow (BM) and peripheral blood (PB) by flow cytometry using a vascular cell adhesion molecule I-immunoglobulin (VCAM-I/IgG) fusion protein as soluble ligand. In an intraindividual analysis, we found a significantly reduced affinity and avidity of the VLA-4 receptor on CD34+ cells from PB during granulocyte colony-stimulating factor (G-CSF)-enhanced marrow recovery in comparison with steady-state BM. Moreover, the amount of circulating CD34+ cells during marrow recovery was inversely related to the activation state but not to the expression level of VLA-4, suggesting that a modulation of the functional state of VLA-4 is involved in the mobilization of CD34+ cells. Moreover, VLA-4 function on CD34+ cells from BM was associated with the maturation state of CD34+ cells as high-affinity VLA-4 receptors were observed on the vast majority of more primitive CD34+ cells. In addition, we found that Mg2+ ions as well as co-incubation of CD34+ cells with endothelial cells resulted in an activation of the VLA-4 receptor. In conclusion, modulation of the functional state of VLA-4 appears to be of relevance for the mobilization and homing of CD34+ haematopoietic stem cells.     

Fusion of human hematopoietic progenitor cells and murine cardiomyocytes is mediated by alpha 4 beta 1 integrin/vascular cell adhesion molecule-1 interaction

Fusion of transplanted stem cells and host cells has been proposed as a major mechanism for the generation of hepatocytes, Purkinje neurons, and cardiomyocytes. However, the mechanism of cell fusion has not been precisely defined. Furthermore, the consequence of cell fusion remains unclear. We have previously shown that adult peripheral blood CD34-positive cells injected into severe combined immune deficiency (SCID) mice can transform into cardiomyocytes, endothelial cells, and smooth muscle cells following experimentally induced myocardial infarction and that most of the newly formed cardiomyocytes result from cell fusion. We therefore undertook this study to define the mechanism and consequences of cell fusion. Here we show that hypoxia and cytokines increase fusion of human peripheral blood CD34-positive cells and murine cardiomyocytes in vitro by up to 7-fold, and this is blocked by anti-alpha4beta1 or anti-vascular cell adhesion molecule (VCAM)-1. In vivo, fusion of progenitor cells and cardiomyocytes can also be blocked by anti-alpha4beta1 or anti-VCAM-1, but not by anti-vascular endothelial growth factor. On the other hand, generation of human-derived endothelial cells is blocked by anti-vascular endothelial growth factor but not by anti-alpha4beta1 antibodies. Two months following transplant, a high percentage of fused cells expressed cyclin B1 and incorporated bromodeoxyuridine. Thus, hematopoietic progenitor cell and cardiomyocyte fusion is mediated by alpha4beta1/VCAM-1 interaction, leading to cell cycle reentry and cellular proliferation.     

Homing of human cells in the fetal sheep model: modulation by antibodies activating or inhibiting very late activation antigen-4-dependent function.

The mechanisms by which intravenously (IV)-administered hematopoietic cells home to the bone marrow (BM) are poorly defined. Although insightful information has been obtained in mice, our knowledge about homing of human cells is very limited. In the present study, we investigated the importance of very late activation antigen (VLA)-4 in the early phases of lodgment of human CD34(+) progenitors into the sheep hematopoietic compartment after in utero transplantation. We have found that preincubation of donor cells with anti-VLA-4 blocking antibodies resulted in a profound reduction of human cell lodgment in the fetal BM at 24 and 48 hours after transplantation, with a corresponding increase of human cells in the peripheral circulation. Furthermore, IV infusion of the anti-VLA-4 antibody at later times (posttransplantation days 21 to 24) resulted in redistribution or mobilization of human progenitors from the BM to the peripheral blood. In an attempt to positively modulate homing, we also pretreated human donor cells with an activating antibody to beta1 integrins. This treatment resulted in increased lodgment of donor cells in the fetal liver, presumably for hemodynamic reasons, at the expense of the BM. Given previous involvement of the VLA-4/vascular cell adhesion molecule (VCAM)-1 adhesion pathway in homing and mobilization in the murine system, our present data suggest that cross-reacting ligands (likely VCAM-1) for human VLA-4 exist in sheep BM, thereby implicating conservation of molecular mechanisms of homing and mobilization across disparate species barriers. Thus, information from xenogeneic models of human hematopoiesis and specifically, the human/sheep model of in utero transplantation, may provide valuable insights into human hematopoietic transplantation biology.     

Activation of mitogen-activated protein kinase through alpha5/beta1 integrin is required for cell cycle progression of B progenitor cell line, Reh, on human marrow stromal cells

OBJECTIVE: Attachment to bone marrow (BM) stromal cells is crucial for the normal growth and development of B-cell progenitors (pro-B). However, the molecular mechanisms by which contact facilitates the proliferation of pro-B cells are not completely understood. This study was performed to investigate this interaction. MATERIALS AND METHODS: A model pro-B cell line (Reh) and a human BM stromal cell line (KM102) were used. Flow cytomery was used for cell cycle analysis. Western Blotting and immunoprecipitation were utilized to examine the levels of cyclin-dependent kinase (cdk) and p27(Kip1). RESULTS: Attachment to both KM102 and normal BM stromal cells significantly promoted the growth of Reh cells. Pretreatment of Reh cells with anti-integrin beta1 or alpha5 monoclonal antibody (mAb), but not alpha4 or ICAM-1 mAb, abrogated this enhancement of proliferation. Furthermore, stroma attachment resulted in shortening of the G(1) phase of cell cycle, significant increases cdk2 activity, degradation of cdk inhibitor p27-GST protein, and decrease in levels of p27(Kip1) protein. In addition, solid-phase cross-linking of alpha5 via immobilized antibody also resulted in extracellular signal-regulated (ERK)-2 kinase phosphorylation, increase in cdk2 activity, decrease in levels of p27(Kip1) protein, and enhanced proliferation that was inhibited by treatment with PD98059, a specific ERK inhibitor. CONCLUSION: Integrin alpha5beta1-mediated stroma contact promotes the proliferation of B-cell progenitors through the activation of ERK-2, which in turn modulates cell cycle regulation machinery including induction of cdk2 activity and degradation of p27(Kip1) and contributing to acceleration of the G(1) phase of cell cycle progression.     

Outcomes after late bone marrow and very early central nervous system relapse of childhood B-acute lymphoblastic leukemia: a report from the Children’s Oncology Group phase III study AALL0433

Outcomes after relapse of childhood B-acute lymphoblastic leukemia (B-ALL) are poor, and optimal therapy is unclear. The children’s Oncology Group study AALL0433 evaluated a new platform for relapsed ALL. Between March 2007 and October 2013 AALL0433 enrolled 275 participants with late bone marrow or very early isolated central nervous system (iCNS) relapse of childhood B-ALL. Patients were randomized to receive standard versus intensive vincristine dosing; this randomization was closed due to excess peripheral neuropathy in 2010. Patients with matched sibling donors received allogeneic hematopoietic cell transplantation (HCT) after the first three blocks of therapy. The prognostic value of minimal residual disease (MRD) was also evaluated in this study. The 3-year event free and overall survival (EFS/OS) for the 271 eligible patients were 63.6±3.0% and 72.3±2.8% respectively. MRD at the end of Induction-1 was highly predictive of outcome, with 3-year EFS/OS of 84.9±4.0% and 93.8±2.7% for patients with MRD <0.1%, versus 53.7±7.8% and 60.6± 7.8% for patients with MRD ≥0.1% (P<0.0001). Patients who received HCT versus chemotherapy alone had an improved 3-year disease-free survival (77.5±6.2% vs. 66.9 + 4.5%, P=0.03) but not OS (81.5±5.8% for HCT vs. 85.8±3.4% for chemotherapy, P=0.46). Patients with early iCNS relapse fared poorly, with a 3-year EFS/OS of 41.4±9.2% and 51.7±9.3%, respectively. Infectious toxicities of the chemotherapy platform were significant. The AALL0433 chemotherapy platform is efficacious for late bone marrow relapse of B-ALL, but with significant toxicities. The MRD threshold of 0.1% at the end of Induction-1 was highly predictive of the outcome. The optimal role for HCT for this patient population remains uncertain. This trial is registered at clinicaltrials.gov (NCT# 00381680).