Novel Mouse Models of Chronic Lymphocytic Leukemia (CLL) Unravel the Molecular Mechanisms Controlling Bone Marrow Involvement by Leukemic B Cells.

Abstract Abstract 360 In CLL, the bone marrow (BM) represents a typical site of involvement and relapse, suggesting a preferential homing of leukemic cells to this anatomical site compared to other lymphoid organs, though the mechanisms controlling CLL cell migration and accumulation within the BM are unclear. In order to define the rules driving in vivo CLL cell re-circulation between the blood and tissutal compartments, we specifically generated two different mouse models and investigated the role played by HS1; this molecule, other than being a putative prognostic factor in CLL, is also involved in cytoskeleton reorganization of lymphocytes, and, potentially, in the control of cellular shape, migration and homing. First, we established a novel transplantable xenograft murine model of CLL by engrafting the cell line MEC1 into RAG2-/-γc-/- mice, at a variance with previous studies in nude mice where MEC cells failed to engraft. Likely due to the lack of B, T and NK cells (while nude mice retain NK cells), RAG2-/-γc-/- animals were successfully transplanted with the CLL cell line through either subcutaneous or intravenous routes, resulting in a systemic blood and tissutal involvement. When subcutaneous MEC1 cells silenced for HS1 expression were injected in these animals, we observed a preferential localization in the tumor draining axillary and inguinal lymph nodes and especially in the BM, when compared to controls. As we have previously demonstrated that CLL cases with hyper-phosphorylated HS1 show a worse clinical outcome, we took advantage of this mouse model to investigate the in vivo homing ability of primary CLL cells from patients showing different HS1 phosphorylation patterns. Purified leukemic cells from 4 patients with hyper-phosphorylated HS1 were labeled with high concentration of the dye CSFE, and each sample was paired and admixed with purified CLL cells obtained from patients with low levels of HS1 phosphorylation and separately labeled with low CSFE concentration. Each pair of samples was injected i.v. into RAG2-/-γc-/- mice recipients. When we analyzed the different organs of the animals by flow-cytometry, the differential expression of CFSE fluorescence (CFSE-high vs CFSE-low) allowed us to distinguish between the two leukemic cell populations with opposite HS1 phosphorylation status. In 3/4 experiments, CLL cells with hyper-phosphorylated HS1 revealed a preferential homing to the BM. Based on these results and on the in vitro evidence that B lymphocytes from HS1-/- mice have an impaired spontaneous migration, we have crossed HS1-/- (H-/-) mice with the Eμ-TCL1 transgenic (Ttg) mouse, an animal model that between 13 and 18 months of age develops a disease resembling human CLL. In the H-/-/Ttg mice, monoclonal CD19+CD5+ cells became evident earlier (at 7-13 months of age) and in significantly higher proportion as compared to Eμ-TCL1 transgenic mice. Cells preferentially localized in the BM where leukemic cells are usually observed at low frequencies in the Eμ-TCL1 mouse (mean value: 28%±16 vs 5%±2, respectively, p=0,008). These findings suggest that HS1 may have a relevant role in both normal and leukemic B-cells and in particular is crucial for cell migration, through its involvement in cytoskeleton organization. Accordingly, we also provide evidence that, in the absence of HS1, cells fail to form actin-myosin complexes, leading to an instability of the cell signalling complex. Our findings suggest a relationship between the expression of HS1 and the development and progression of CLL, most notably in terms of BM involvement, indicating that specific abnormalities in the cytoskeleton organization may be pivotal in regulating leukemic migration and infiltration in selected anatomical sites. This points at HS1 as a target for development of novel cancer treatments, aiming at interfering with the lymphoid tissue infiltration and invasion which is characteristic of the disease. In addition, these animal models could become very useful for evaluating the biological basis of CLL growth and dissemination as well as the efficacy of new therapeutic agents. Disclosures: No relevant conflicts of interest to declare.

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Expression of CD10 and CD7 Defines Distinct In Vivo Lymphoid Reconstituting Capacity within Human Cord Blood CD34+CD38-Cells.

Blood ◽

10.1182/blood.v108.11.3184.3184 ◽

2006 ◽

Vol 108 (11) ◽

pp. 3184-3184

Author(s):

Shuro Yoshida ◽

Fumihiko Ishikawa ◽

Leonard D. Shultz ◽

Noriyuki Saito ◽

Mitsuhiro Fukata ◽

...

Keyword(s):

Bone Marrow ◽

T Cells ◽

B Cells ◽

Cord Blood ◽

Nk Cells ◽

Progenitor Cells ◽

Human Cord Blood ◽

Cd34 Cells

Abstract Human cord blood (CB) CD34+ cells are known to contain both long-term hematopoietic stem cells (LT-HSCs) and lineage-restricted progenitor cells. In the past, in vitro studies suggested that CD10, CD7 or CD127 (IL7Ra) could be candidate surface markers that could enrich lymphoid-restricted progenitor cells in human CB CD34+ cells (Galy A, 1995, Immunity; Hao QL, 2001, Blood; Haddad R, 2004, Blood). However, in vivo repopulating capacity of these lymphoid progenitors has not been identified due to the lack of optimal xenogeneic transplantation system supporting development of human T cells in mice. We aim to identify progenitor activity of human CB CD34+ cells expressing CD10/CD7 by using newborn NOD-scid/IL2rgKO transplant assay that can fully support the development of human B, T, and NK cells in vivo (Ishikawa F, 2005, Blood). Although LT-HSCs exist exclusively in Lin-CD34+CD38- cells, not in Lin-CD34+CD38+ cells, CD10 and CD7 expressing cells are present in Lin-CD34+CD38- cells as well as in Lin-CD34+CD38+ cells (CD10+CD7+ cells, CD10+CD7- cells, CD10-CD7+ cells, CD10-CD7- cells accounted for 4.7+/−2.7%, 10.5+/−1.9%, 7.6+/−4.4%, and 77.1+/−5.2% in Lin-CD34+CD38- CB cells, respectively). We transplanted 500–6000 purified cells from each fraction into newborn NOD-scid/IL2rgKO mice, and analyzed the differentiative capacity. CD34+CD38-CD10-CD7- cells engrafted long-term (4–6 months) in recipient mice efficiently (%hCD45+ cells in PB: 30–70%, n=5), and gave rise to all types of human lymphoid and myeloid progeny that included granulocytes, platelets, erythroid cells, B cells, T cells, and NK cells. Successful secondary reconstitution by human CD34+ cells recovered from primary recipient bone marrow suggested that self-renewing HSCs are highly enriched in CD34+CD38–CD10–CD7- cells. CD10–CD7+ cells were present more frequently in CD34+CD38+ cells rather than in CD34+CD38- cells. Transplantation of more than 5000 CD34+CD38+CD10–CD7+ cells, however, resulted in less than 0.5% human cell engraftment in the recipients. Within CD34+CD38–CD10+ cells, the expression of CD7 clearly distinguished the distinct progenitor capacity. At 8 weeks post-transplantation, more than 70% of total human CD45+ cells were T cells in the CD10+CD7+ recipients, whereas less than 30% of engrafted human CD45+ cells were T cells in the CD10+CD7– recipients. In the CD10+CD7- recipients, instead, more CD19+ B cells and HLA–DR+CD33+ cells were present in the peripheral blood, the bone marrow and the spleen. Both CD34+CD38–CD10+CD7+ and CD34+CD38–CD10+CD7- cells highly repopulate recipient thymus, suggesting that these progenitors are possible thymic immigrants. Taken together, human stem and progenitor activity can be distinguished by the expressions of CD7 and CD10 within Lin-CD34+CD38- human CB cells. Xenotransplant model using NOD-scid/IL2rgKO newborns enable us to clarify the heterogeneity of Lin-CD34+CD38- cells in CB by analyzing the in vivo lymphoid reconstitution capacity.

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Hijacking the Niche: The Role of Stromal Osteopontin in the Induction of Leukemic Dormancy within the Bone Marrow Microenvironment

Blood ◽

10.1182/blood.v118.21.754.754 ◽

2011 ◽

Vol 118 (21) ◽

pp. 754-754

Author(s):

Benjamin Boyerinas ◽

Ali Ekrem Yesilkanal ◽

Andrea Pontier ◽

Dorothy A. Sipkins

Keyword(s):

Bone Marrow ◽

Cell Line ◽

In Vivo Imaging ◽

Tumor Burden ◽

Bone Marrow Microenvironment ◽

Leukemic Cells ◽

Hematopoietic Stem ◽

Bone Marrow Biopsies ◽

Stromal Microenvironment

Abstract Abstract 754 Introduction: Acute lymphoblastic leukemia (ALL) is a treatable malignancy where initial induction chemotherapy achieves clinical remission in the majority of patients. Relapsed disease, however, occurs in many patients and is significantly more difficult to treat. The majority of relapsed cases are a result of minimal residual disease (MRD) that persists within the bone marrow (BM) after initial chemotherapy. Our evolving knowledge of the importance of the host microenvironment in tumor progression suggests that the stromal microenvironment can protect leukemic cells from chemotherapeutic assault, and that inhibiting the supportive relationship between leukemic blasts and the bone marrow microenvironment (BMM) will provide novel therapeutic opportunities. We therefore aimed to identify and characterize novel stromal signaling mechanisms that retain and support blasts within the malignant BMM. Our preliminary data suggest that osteopontin (OPN), normally secreted by osteoblasts within the marrow, is one such signaling chemokine that is highly upregulated in the leukemic niche. OPN has well-defined roles in both solid tumor metastasis and normal hematopoietic stem cell function within the BMM. Specifically, OPN expression at the endosteal bone surface functions to recruit hematopoietic progenitors to bone where they are induced to become quiescent and maintain long term repopulating potential. We hypothesized that a similar relationship exists between leukemia and OPN resulting in a quiescent population of chemoresistant leukemic blasts at the BM endosteum. Here, we demonstrate that stromal OPN negatively regulates leukemia cell proliferation in the BMM. Methods: A GFP expressing clone of the pre-B ALL cell line Nalm-6 (10 × 106 cells) was engrafted into SCID hosts (6-8 weeks old) via tail vein injection. In vivo imaging was accomplished in live anesthetized mice by reflecting the scalp and imaging the calvarial marrow compartment using real-time multi-photon confocal microscopy. OPN expression in the malignant marrow was imaged by injecting engrafted mice with fluorescently conjugated anti-OPN antibodies 18hrs prior to imaging. For OPN neutralizing experiments, engrafted mice were injected with a cocktail of anti-mouse and anti-human OPN antibodies at a dose of 3 mg/kg. Results: Using PCR, Western blot and ELISA assays, we show that the ALL cell line Nalm-6 expresses OPN and secretes large quantities of OPN into conditioned media. Flow cytometric analysis demonstrates that Nalm-6 also express the cell surface OPN receptors VLA-4 and VLA-5. Furthermore, Nalm-6 cells specifically adhere to OPN in vitro via specific engagement of these integrin receptors. In vivo imaging demonstrates that OPN expression in the BM increases as leukemia progresses and that OPN is highly expressed adjacent to areas of high tumor burden. Specifically, a significant amount of OPN is detected in bony tunnels surrounding the vasculature at the invading tumor front. Using Q-PCR and western analysis, we demonstrate that both host-derived and leukemia-derived OPN are upregulated in malignant BM. In vivo inhibition of the OPN signaling axis in the Nalm-6 xenograft model using neutralizing antibodies directed at both human and murine OPN increased overall tumor burden two-fold as measured by flow cytometry and in vivo imaging (p=0.02, N=7) while simultaneously increasing the Ki-67 positive proliferative tumor population (p=0.029, N=4). Furthermore, IHC analysis of a panel of diagnostic bone marrow biopsies from a diverse cohort of ALL patients demonstrated high OPN expression in the marrow of these patients. Conclusion: Leukemic blasts that have hijacked normal stromal interactions to become quiescent may represent a major source of MRD and patient relapse in ALL. Our data demonstrate that the interaction of leukemic blasts with OPN in the stromal microenvironment reduces the number of cycling blasts and constrains tumor proliferation within the marrow. Current investigations are aimed at combining OPN neutralization with an in vivo model of MRD to determine whether OPN neutralization induces cycling of quiescent blasts, ultimately rendering them sensitive to chemotherapy. The ultimate goal of this work is the development of clinically relevant therapies designed to render leukemic cells more susceptible to chemotherapy by disengaging them from protective interactions with the BM microenvironment. Disclosures: No relevant conflicts of interest to declare.

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Establishment and Characterization of a Panel of Patient-Derived Acute Leukemia Mouse Models As Emerging Platform for Translational Research

Blood ◽

10.1182/blood.v126.23.2538.2538 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2538-2538

Author(s):

Eva Oswald ◽

Gabriele Greve ◽

Kerstin Klingner ◽

Benedikt Hammerich ◽

Dorothee Lenhard ◽

...

Keyword(s):

Acute Leukemia ◽

Cell Line ◽

Mouse Models ◽

Molecular Diversity ◽

Human Cells ◽

Leukemic Cells ◽

Serial Transplantation ◽

Pdx Models

Abstract Many efforts are made to treat acute leukemia effectively, but still; new treatment options are urgently needed. In the past, mainly chemically induced and cell line-derived mouse models were used to evaluate the efficacy of new drugs. The high failure rate of 96% in early clinical development illustrates the urgent need for more predictive preclinical models as a major prerequisite for rapid bench-to-bedside translation of investigational anticancer therapies. Transplantable patient-derived xenograft (PDX) models of leukemia offer a strong preclinical tool for drug screening and biomarker development as they represent the complex clinical tumor heterogeneity and molecular diversity of human cancer. Between 2011 and 2015 we collected 91 samples of peripheral blood (PB) or bone marrow (BM) from patients diagnosed with acute myeloid or lymphoblastic leukemia (AML, ALL) and injected them intratibially (i.t.) into NOG (NOD/Shi-scid/IL-2Rγnull) mice (n=1-5/patient sample). Infiltration of human leukemic cells was determined by flow cytometry in murine PB, BM and spleen. If more than 5% of hCD45+, hCD33+, hCD34+, hCD38+, and/or HLA-ABC+ cells were detectable in one sample, it was classified as engrafted. Results were confirmed by histo-pathological examination. FISH analyses confirmed the cytogenetical concordance with the donor patient where available. 8 models were treated with cytarabine and results were compared to patient´s outcome and treatment experiments using cell line derived models of AML. Whole exome sequencing analyses of the transplantable models are underway for a deeper characterization of the respective leukemic clone which adapted to the murine microenvironment. Specimens of 44 female and 47 male patients (median age: 59 years, median BM infiltration: 39%) were collected. The donor patient cohort covered a broad range of different molecular subtypes: amongst others 5 MLL-rearrangements, 1 MLL-Deletion, 2 t(8;21) translocations, 20 cytogenetically normal and 7 complex karyotypes were included. Up to now 12 PDX models were in passage (P) 5 and higher, where P1 represents first implantation of human cells into the murine BM. 15 PDX models grew in P3 - P4, 21 in P2, 36 in P1 and 7 samples showed no engraftment. Engraftment capacity of the leukemic cells did not correlate significantly with any of the patient characteristics. BM engraftment ranged from 30% - 80%. Spleen and PB depicted 5 - 30% of leukemic cells. Infiltration rate in different organs and immuno-phenotypic characteristics of the human cells were specific for a defined model and preserved during serial transplantation. Take rates within one mouse cohort in serial transplantation were ≥98% for all transplantable PDX, similar to cell line derived models thereby qualifying the PDX approach as a suitable preclinical platform. Overall survival (OS) in P1 ranged from 52 to > 310 days (d). Models which could be serially transplanted showed model specific median OS ranging from 32 - 150 d. Comparable cell line derived models depicted median OS of 13 - 45 d. Of note, cell line derived models (KG-1, NOMO-1, MOLM-13, THP-1, MV4-11 or HL60, n ≥ 10 mice/cell line) induced hind limb paresis in all recipient mice. These symptoms could not be observed in PDX and most important are not part of the clinical picture. Cytarabine induced a significantly prolonged OS in 8/8 tested PDX models. Respective donor patients showed hematologic response under cytarabine based therapy highlighting the excellent predictivity of the in vivo platform. In contrast none of the investigated cell line derived models showed sensitivity towards cytarabine, although representing similar subtypes of AML as the investigated PDX models. Taken together a constantly expanding panel of well characterized AML/ALL PDX covering a broad range of different subtypes of the disease is available for drug development and tumorbiology studies. The comparison with cell line derived in vivo models revealed significant advantages of the PDX approach as the latter represents the molecular diversity more in detail, mimics the clinical signs of leukemia more realistic and most important mirrors sensitivity towards standard of care in a direct comparison with the donor patient´s clinical outcome. Therefore, we strongly believe that the AML/ALL PDX platform is a robust and predictive tool to address translational challenges in oncology research. Disclosures Oswald: Oncotest GmbH: Employment. Klingner:Oncotest GmbH: Employment. Lenhard:Oncotest GmbH: Employment. Schueler:Oncotest GmbH: Employment.

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Daratumumab, a Novel Human Anti-CD38 Monoclonal antibody shows Anti-Tumor Activity In Mouse Models Of MCL, FL and CLL

Blood ◽

10.1182/blood.v122.21.378.378 ◽

2013 ◽

Vol 122 (21) ◽

pp. 378-378 ◽

Cited By ~ 5

Author(s):

Alba Matas-Céspedes ◽

Anna Vidal Crespo ◽

Vanina Rodriguez ◽

Gael Roue ◽

Armando Lopez-Guillermo ◽

...

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Mouse Models ◽

Cell Lines ◽

Treatment Initiation ◽

Cell Homing ◽

Therapeutic Setting ◽

Cell Inoculation

Abstract Daratumumab (DARA) is a human CD38 antibody with broad-spectrum killing activity. DARA induces killing of tumor cells, mainly via complement-dependent cytotoxicity (CDC) and antibody-dependent cell-mediated cytotoxicity (ADCC) (de Weers M, J Immunol 2011). DARA is currently being evaluated in phase I/II clinical trials in patients with multiple myeloma. In these clinical studies the adverse events have been manageable and marked reductions in paraprotein and bone marrow plasma cells have been observed. We have previously reported (Blood (ASH annual meeting abstracts). Nov 2012, 120 (21): 3935) that DARA induces cytotoxic activity in vitro via ADCC in primary cells and cell lines from Mantle Cell Lymphoma (MCL), Follicular Lymphoma (FL) and Chronic Lymphoctic Leukemia (CLL). CDC induction was low, which is associated to high expression of the complement inhibitors and reduced number of CD38 molecules per cell in these indications. This suggests a threshold for CD38-targeted CDC lysis. In addition, based on known interactions between CD38-CXCR4, we also demonstrated that in the CLL subtype, with high CD38 and more migratory capacity, DARA (10-30 µg/mL) inhibited in vitro CXCL12/SDF1α-mediated migration up to 70%. Here, we present the first preclinical in vivo results of DARA in mouse models of MCL, transformed FL(tFL) and CLL. We generated heterotopic and systemic mouse models of these entities by subcutaneous or intravenous inoculation of tumor cell lines in SCID mice, that retain both NK cells and macrophages as potential effector cells. In MCL (REC cell line) and tFL (RL cell line) subcutaneous mouse models, we tested DARA activity in both prophylactic (treatment initiation simultaneous to lymphoma cell inoculation) and therapeutic settings(treatment initiation one week after lymphoma cell inoculation, when tumors were about 100 mm3 in size). In the prophylactic setting, mice received 3 doses of DARA or control IgG bi-weekly (20/10/10 mg/kg). In the therapeutic setting, treatment was intensified to 4 doses of DARA or control IgG weekly (20/10/10/10 mg/kg). In both schedules, mice were sacrificed one week after the last dose. DARA completely abrogated tumor growth of REC or RL cells in the prophylactic setting. Moreover, in the therapeutic setting, DARA induced total tumor regression of REC tumors in 4 out of 6 mice, and prevented the splenomegaly observed in control IgG treated mice. In the case of tFL and therapeutic setting, DARA reduced 60% of tumor growth compared to control IgG treated mice at day 32, when experiment was finished. In CLL, we analyzed the effect of DARA on cell homing to lymphoid organs together with its therapeutic properties in a systemic CLL mouse model. Using NOD/SCID/gamma null mice (lacking NK cells and effective macrophages), we analyzed the effect of DARA on primary CLL cell migration from Peripheral Blood (PB) to bone marrow (BM) and Spleen. In this system, NSG mice were pretreated (day 0) with DARA, control IgG or anti-CXCR4 as positive control for inhibition of cell homing, followed by fresh CLL cell inoculation (50×106 cells/per mice) on day 1. PB, BM and spleen cells were isolated on day 2 and CLL cells were identified by staining for CD45/CD19/CD5 and counted using a flow cytometer. Cell counting showed that CLL cells mainly migrate to the spleen, and that DARA significantly reduced this migration (55% inhibition on average, p<0.05). Migration of CLL cells to BM was limited and was not affected by pretreatment of mice with DARA. Finally, we tested DARA therapeutic activity in a systemic CLL mouse model (MEC2 cell line), following the schedule described before (4 doses of control IgG/ DARA weekly (20/10/10/10 mg/kg)), and assessed efficacy on mice overall survival. Mice treated with control IgG started to lose weight and showed signs of disease between days 30-40 and were sacrificed for ethical reasons. In the DARA treated group, in 7 out of 8 mice survival was extended up to day 90, when the experiment was stopped. In conclusion, DARA demonstrated strong in vivo activity in immunocompromised mouse models of MCL, tFL and CLL cell lines and interfered with homing of primary CLL cells to the spleen. These results warrant further investigation of DARA in clinical trials for these indications. Disclosures: Lopez-Guillermo: Roche: Membership on an entity’s Board of Directors or advisory committees. Lammerts van Bueren:Genmab: Employment, Stocks Other. Bakker:Genmab: Employment, Stocks Other. Parren:Genmab: Employment, Stocks Other. Perez-Galan:Genmab: Research Funding.

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Chronic Lymphocytic Leukemic B Cells But Not Normal B Cells Are Rescued From Apoptosis by Contact With Normal Bone Marrow Stromal Cells

Blood ◽

10.1182/blood.v91.7.2387.2387_2387_2396 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2387-2396 ◽

Cited By ~ 9

Author(s):

L. Lagneaux ◽

A. Delforge ◽

D. Bron ◽

C. De Bruyn ◽

P. Stryckmans

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Lymphocytes ◽

Stromal Cells ◽

Cell Apoptosis ◽

Bone Marrow Stromal Cells ◽

Ex Vivo ◽

Marrow Stromal Cells ◽

Leukemic Cells

The leukemic B lymphocytes from chronic lymphocytic leukemic (CLL) patients have a long survival in vivo, although ex vivo they rapidly die by apoptosis. To further investigate the mechanism of this, we have studied the influence of bone marrow stromal cells from normal subjects on apoptosis of B-CLL cells and normal umbilical cord blood (UCB) B lymphocytes. After 48 hours of incubation in medium alone, leukemic and normal B cells showed, respectively, 22 ± 3% and 31 ± 5% of apoptosis. Cocultures with stromal cells reduced the percentage of leukemic cells undergoing apoptosis (8 ± 2%, P< .0005) and prevented the loss of bcl-2 protein expression. In contrast, stromal cells slightly increased normal B-cell apoptosis (37 ± 6%). Direct contact between leukemic cells and stromal cells was found to be essential for inhibition of leukemic cell apoptosis; indeed, separation of leukemic cells from stromal cells by microporous membrane increased spontaneous apoptosis, and comparable results were obtained with stromal cell conditioned medium. The difference in behavior observed between normal and leukemic B cells plated on stromal cells can be explained by the fact that only a few normal B cells adhere to stromal cells in comparison with B-CLL cells. B-CLL cell adhesion to stromal cells is mediated by β1 and β2 integrins acting simultaneously. Contact between B-CLL cells and bone marrow stromal cells seems to play a major role in the accumulation and survival of B-CLL cells in the bone marrow.

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Chronic Lymphocytic Leukemic B Cells But Not Normal B Cells Are Rescued From Apoptosis by Contact With Normal Bone Marrow Stromal Cells

Blood ◽

10.1182/blood.v91.7.2387 ◽

1998 ◽

Vol 91 (7) ◽

pp. 2387-2396 ◽

Cited By ~ 274

Author(s):

L. Lagneaux ◽

A. Delforge ◽

D. Bron ◽

C. De Bruyn ◽

P. Stryckmans

Keyword(s):

Bone Marrow ◽

B Cells ◽

B Lymphocytes ◽

Stromal Cells ◽

Cell Apoptosis ◽

Bone Marrow Stromal Cells ◽

Ex Vivo ◽

Marrow Stromal Cells ◽

Leukemic Cells

Abstract The leukemic B lymphocytes from chronic lymphocytic leukemic (CLL) patients have a long survival in vivo, although ex vivo they rapidly die by apoptosis. To further investigate the mechanism of this, we have studied the influence of bone marrow stromal cells from normal subjects on apoptosis of B-CLL cells and normal umbilical cord blood (UCB) B lymphocytes. After 48 hours of incubation in medium alone, leukemic and normal B cells showed, respectively, 22 ± 3% and 31 ± 5% of apoptosis. Cocultures with stromal cells reduced the percentage of leukemic cells undergoing apoptosis (8 ± 2%, P< .0005) and prevented the loss of bcl-2 protein expression. In contrast, stromal cells slightly increased normal B-cell apoptosis (37 ± 6%). Direct contact between leukemic cells and stromal cells was found to be essential for inhibition of leukemic cell apoptosis; indeed, separation of leukemic cells from stromal cells by microporous membrane increased spontaneous apoptosis, and comparable results were obtained with stromal cell conditioned medium. The difference in behavior observed between normal and leukemic B cells plated on stromal cells can be explained by the fact that only a few normal B cells adhere to stromal cells in comparison with B-CLL cells. B-CLL cell adhesion to stromal cells is mediated by β1 and β2 integrins acting simultaneously. Contact between B-CLL cells and bone marrow stromal cells seems to play a major role in the accumulation and survival of B-CLL cells in the bone marrow.

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Mechanism of rejection of virus persistently infected tumor cells by athymic nude mice.

Journal of Experimental Medicine ◽

10.1084/jem.149.5.1117 ◽

1979 ◽

Vol 149 (5) ◽

pp. 1117-1133 ◽

Cited By ~ 96

Author(s):

N Minato ◽

B R Bloom ◽

C Jones ◽

J Holland ◽

L M Reid

Keyword(s):

Cytotoxic Activity ◽

Nk Cells ◽

Cell Line ◽

Tumor Cells ◽

Cell Lines ◽

Nude Mice ◽

Nk Cell ◽

Persistently Infected

Cell lines known to be tumorigenic in the nude mouse were modified by rendering them persistently infected (P.I.) with a variety of RNA viruses, including measles, mumps, vesicular stomatitis virus, and influenza. Although as few as 100 HeLa or BHK cells produced tumors in 100% of nude mice, as many as 2 x 10(7) of the same cells P.I. with viruses failed to produce tumors. An active host response responsible for restricting the growth of the P.I. cells was suggested by the findings of marked mononuclear cell infiltrates at the inoculation sites and the inability of irradiated nude mice to reject them. An analysis of the in vitro cytotoxic activity of spleen cells from normal nude mice indicated that: (a) P.I. cell lines, but not uninfected cell lines, were susceptible to spontaneous cytotoxicity; (b) in vivo inoculation of P.I. lines induced an enhanced cytotoxic activity for P.I. targets in vitro, and this induction was not specific either for inducing virus or cell line; and (c) the effector cell had the characteristics for natural killer (NK) cells. Although the specificity of recognition of the various P.I. cell lines remains unclear, cold competition experiments indicated that blocking the killing of one P.I. cell line, e.g. HeLa-measles, could be achieved only by unlabeled homologous cells, i.e. HeLa-measles, and not by uninfected cells or other P.I. lines. A variant subline of BHK cells P.I. with VSV was selected for its ability to withstand the rejection process in nude mice. These cells formed metastatic and invasive tumors in nude mice. Although they were the most potent inducers in vivo of NK cell activity against various P.I. targets, they were the most resistant of the P.I. lines to NK cell cytotoxicity in vitro. In this system there was a good correlation between tumor rejection in vivo and susceptibility to NK cells in vitro. The present results suggest that NK cells may play a significant role in both rejection of tumor cells, and in resistance to viruses, particularly persistent infections.

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Transformation-associated alterations in interactions between pre-B cells and fibronectin

Blood ◽

10.1182/blood.v76.11.2311.2311 ◽

1990 ◽

Vol 76 (11) ◽

pp. 2311-2320 ◽

Cited By ~ 22

Author(s):

FM Lemoine ◽

S Dedhar ◽

GM Lima ◽

CJ Eaves

Keyword(s):

B Cells ◽

B Cell ◽

Cell Line ◽

Cell Lines ◽

Stromal Cells ◽

Stromal Cell ◽

Cell Attachment ◽

Attachment Site ◽

Receptor Antibodies

Abstract Marrow stromal elements produce as yet uncharacterized soluble growth factors that can stimulate the proliferation of murine pre-B cells, although close contact between these two cell types appears to ensure a better pre-B cell response. We have now shown that freshly isolated normal pre-B cells (ie, the B220+, surface mu- fraction of adult mouse bone marrow) adhere to fibronectin (FN) via an RGD cell-attachment site, as shown in a serum-free adherence assay, and they lose this functional ability on differentiation in vivo into B cells (ie, the B220+, surface mu+ fraction). Similarly, cells from an immortalized but stromal cell-dependent and nontumorigenic murine pre-B cell line originally derived from a Whitlock-Witte culture were also found to adhere to fibronectin (FN) via an RGD cell-attachment site. Moreover, in the presence of anti-FN receptor antibodies, the ability of this immortalized pre-B cell line to proliferate when co-cultured with a supportive stromal cell line (M2–10B4 cells) was markedly reduced (down to 30% of control). This suggests that pre-B cell attachment to FN on stromal cells may be an important component of the mechanism by which stromal cells stimulate normal pre-B cell proliferation and one that is no longer operative to control their more differentiated progeny. Two differently transformed pre-B cell lines, both of which are autocrine, stromal-independent, tumorigenic in vivo, and partially or completely differentiation-arrested at a very early stage of pre-B cell development, did not bind to FN. In addition, anti-FN receptor antibodies were much less effective in diminishing the ability of these tumorigenic pre-B cells to respond to M2–10B4 cell stimulation, which could still be demonstrated when the tumorigenic pre-B cells were co- cultured with M2–10B4 cells at a sufficiently low cell density. Analysis of cell surface molecules immunoprecipitated from both the nontumorigenic and tumorigenic pre-B cell lines by an anti-FN receptor antibody showed an increase in very late antigen (VLA) alpha chain(s) in both tumorigenic pre-B cell lines and a decrease in the beta 1 chain in one. Interestingly, all of the pre-B cell lines expressed similar amounts of messenger RNA for the beta 1 chain of the FN receptor. These results suggest that alteration of FN receptor expression on pre-B cells may represent a mechanism contributing to the outgrowth of leukemic pre-B cells with an autocrine phenotype and capable of stromal cell-independent, autonomous growth.

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A cloned cell line mediating natural killer cell function inhibits immunoglobulin secretion.

Journal of Experimental Medicine ◽

10.1084/jem.156.2.658 ◽

1982 ◽

Vol 156 (2) ◽

pp. 658-663 ◽

Cited By ~ 53

Author(s):

G Nabel ◽

W J Allard ◽

H Cantor

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Line ◽

B Lymphocytes ◽

Cell Function ◽

Killer Cell ◽

Major Histocompatibility Complex Gene ◽

Cell Surface Antigens

We previously described a cloned cell line that combines information for a unique display of cell surface antigens and specialized function similar to activated natural killer (NK) cells. In addition to conventional cellular targets such as the YAC-1 and MBL-2 lymphomas, this cloned line also lysed lipopolysaccharide-activated B lymphocytes. To determine whether some NK cells can inhibit B cell function, we tested the ability of NK-like clones to suppress Ig secretion in vitro and in vivo. These cloned cells suppressed Ig secretion when they constituted as few as 0.2% of the total cell population and inhibition did not require identity at the H-2 locus. We suggest that some NK cells might recognize non-major histocompatibility complex gene products on activated B lymphocytes and lyse these cells, and this might represent a fundamental cell-cell interaction that regulates antibody secretion by activated B cells.

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CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adult mice

Blood ◽

10.1182/blood-2010-04-277897 ◽

2011 ◽

Vol 117 (2) ◽

pp. 451-458 ◽

Cited By ~ 58

Author(s):

Mamiko Noda ◽

Yoshiki Omatsu ◽

Tatsuki Sugiyama ◽

Shinya Oishi ◽

Nobutaka Fujii ◽

...

Keyword(s):

Bone Marrow ◽

Nk Cells ◽

Nk Cell ◽

Cell Development ◽

Hematopoietic Stem ◽

Adult Mice ◽

Multipotent Progenitors ◽

Chemokine Signaling ◽

Chemokine Ligand

Abstract Natural killer (NK) cells are granular lymphocytes that are generated from hematopoietic stem cells and play vital roles in the innate immune response against tumors and viral infection. Generation of NK cells is known to require several cytokines, including interleukin-15 (IL-15) and Fms-like tyrosine kinase 3 ligand, but not IL-2 or IL-7. Here we investigated the in vivo role of CXC chemokine ligand-12 (CXCL12) and its primary receptor CXCR4 in NK-cell development. The numbers of NK cells appeared normal in embryos lacking CXCL12 or CXCR4; however, the numbers of functional NK cells were severely reduced in the bone marrow, spleen, and peripheral blood from adult CXCR4 conditionally deficient mice compared with control animals, probably resulting from cell-intrinsic CXCR4 deficiency. In culture, CXCL12 enhanced the generation of NK cells from lymphoid-primed multipotent progenitors and immature NK cells. In the bone marrow, expression of IL-15 mRNA was considerably higher in CXCL12-abundant reticular (CAR) cells than in other marrow cells, and most NK cells were in contact with the processes of CAR cells. Thus, CXCL12-CXCR4 chemokine signaling is essential for NK-cell development in adults, and CAR cells might function as a niche for NK cells in bone marrow.

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