Invariant NKT Cells Recognize Leukemia Cells with T-Cell and NK Receptors in a CD1d-Independent Manner

Background: Invariant natural killer T (iNKT) cells are known as CD1d-restricted T cells that express the invariant T-cell receptors (TCR) Vα24 and Vβ11 in humans and specifically recognize glycolipid antigens such as α-galactosylceramide (αGalCer) presented by CD1d. iNKT cells show direct cytotoxicity toward CD1d-positive tumor cells presenting glycolipid antigens and indirect cytotoxicity by activating other cytotoxic immune cells or regulating CD1d-positive immunosuppressive cells in the tumor microenvironment. Although we previously reported that αGalCer-activated NKT cells exert a potent perforin-dependent cytotoxic activity against a wide variety of human tumor cell lines, the direct recognition of CD1d-negative tumors is controversial and the mechanism is unknown. Here we clarify whether iNKT cells recognize and exhibit cytotoxicity toward leukemia cells in a CD1d-independent manner and identify the molecule that recognizes CD1d-negative leukemia cells. Methods: Purified iNKT cells were generated from peripheral blood mononuclear cells (PBMCs) of healthy adult volunteer donors. PBMCs were cultured in complete RPMI 1640 medium for 9-14 days in the presence of 100 U/mL of recombinant human IL-2 and 200 ng/mL of αGalCer. The iNKT cells were then isolated with an autoMACS Pro separator using FITC-labeled anti-Vα24 antibody (clone, C15) and anti-FITC microbeads. We evaluated the cytotoxic activity of iNKT cells toward CD1d-negative leukemia cells within four days after isolation using a CD107a assay for degranulation, cytometric bead array for cytokine production, and cytotoxicity assay in vitro and in vivo. For in vivo cytotoxicity assays, NOG mice were inoculated with 1 × 106 K562-luc cells on day 0 and with 4 × 106 human iNKT cells on day 1. Gene knock-out (KO) was performed using a CRISPR/Cas9 system. T-cell or NK receptor-KO iNKT cells were used for experiments three or four days after electroporation of the Cas9 protein and guide RNA CRISPR ribonucleoprotein complex. Patient-derived leukemia cells were obtained from PBMCs or bone marrow mononuclear cells of pre-treatment pediatric patients. All studies were approved by the institutional review board and the Animal Care and Use Committee of Chiba University. Results: We observed that iNKT cells degranulated and released Th1 cytokines when co-cultured with CD1d-negative leukemia cells (K562, HL-60, REH, and CD1d-KO U937) as well as αGalCer-loaded CD1d-positive leukemia cells (Jurkat), and showed in vitro cytotoxicity toward these CD1d-negative leukemia cells. This CD1d-independent degranulation decreased over time after isolation and was not restored with re-stimulation by αGalCer. The cytotoxicity of iNKT cells toward K562 cells was confirmed in vivo by comparsion with survival curves of K562-inoculated NOG mice given iNKT cells or PBS alone (log-rank, p= 0.016). To identify the receptors contributing to the CD1d-independent recognition and cytotoxicity against CD1d-negative leukemia cells, we first focused on costimulatory receptors, which are also known as activating NK receptors and are expressed on iNKT cells such as NKG2D, DNAM-1, 2B4, LFA-1, and CD2, and analyzed cytotoxicity after blocking these receptors with antibodies. We found that all costimulatory receptors that we assessed contributed to cytotoxicity toward CD1d-negative leukemia cells. Next, we analyzed cytotoxicity of TCR-KO iNKT cells toward CD1d-negative leukemia cells to confirm the contribution of TCR to CD1d-independent recognition. Notably, TCR-KO iNKT cells showed decreased degranulation, Th1 cytokine release, and cytotoxicity toward K562 cells more so than iNKT cells with KO of NK receptors such as LFA-1(CD11a) or CD2. To assess the clinical application potential of adoptive iNKT cell immunotherapy for leukemia treatment, we analyzed degranulation of iNKT cells using patient-derived leukemia cells. We found iNKT cells degranulation using cells from four out of five myeloid leukemia cases, but only one out of eight BCP-ALL cases (p = 0.032). Conclusion: Primary iNKT cells activated by αGalCer can recognize and show anti-tumor effects toward leukemia cells in an unrestricted manner via CD1d. The TCR also has an important role in recognizing CD1d-negative leukemia cells and multiple NK receptors assist in cytotoxicity. Adoptive iNKT cell immunotherapy may be effective in treating myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

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PD-L1hi plasmablasts limit the T cell response during the acute phase of parasite infections

10.1101/2020.06.10.142067 ◽

2020 ◽

Author(s):

Melisa Gorosito Serrán ◽

Facundo Fiocca Vernengo ◽

Laura Almada ◽

Cristian G Beccaria ◽

Pablo F Canete ◽

...

Keyword(s):

T Cell ◽

Cell Response ◽

Cell Activation ◽

Mononuclear Cells ◽

Plasma Cells ◽

Chronic Phase ◽

Surface Expression ◽

T Cell Response

ABSTRACTDuring infections with protozoan parasites or virus, T cell immunosuppression is generated simultaneously with a high B cell activation. Here, we show that in T. cruzi infection, all plasmablasts detected had higher surface expression of PD-L1, than other mononuclear cells. PD-L1hi plasmablasts were induced in vivo in an antigen-specific manner and required help from Bcl-6+CD4+T cells. PD-L1hi expression was not a characteristic of all antibody-secreting cells since plasma cells found during the chronic phase of infection express PD-L1 but at lower levels. PD-L1hi plasmablasts were also present in mice infected with Plasmodium or with lymphocytic choriomeningitis virus, but not in mice with autoimmune disorders or immunized with T cell-dependent antigens. PD-L1hi plasmablasts suppressed T cell response, via PD-L1, in vitro and in vivo. Thus, this study reveals that extrafollicular PD-L1hi plasmablasts, which precede the germinal center (CG) response, are a suppressive population in infections that may influence T cell response.Brief summaryPathogens develop different strategies to settle in the host. We identified a plasmablats population induced by pathogens in acute infections which suppress T cell response.

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Rapid Death of Adoptively Transferred T Cells in Acquired Immunodeficiency Syndrome

Blood ◽

10.1182/blood.v93.5.1506.405a38_1506_1510 ◽

1999 ◽

Vol 93 (5) ◽

pp. 1506-1510 ◽

Cited By ~ 2

Author(s):

Rusung Tan ◽

Xiaoning Xu ◽

Graham S. Ogg ◽

Pokrath Hansasuta ◽

Tao Dong ◽

...

Keyword(s):

T Cell ◽

Adoptive Transfer ◽

Acquired Immunodeficiency Syndrome ◽

Mononuclear Cells ◽

Virus Load ◽

Acquired Immunodeficiency ◽

Peptide Complexes ◽

Immunodeficiency Syndrome

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL) probably play the major role in controlling HIV replication. However, the value of adoptive transfer of HIV-specific CTL expanded in vitro to HIV+ patients has been limited: this contrasts with the success of CTL therapy in treating or preventing Epstein-Barr virus and cytomegalovirus disease after bone marrow transplantation (BMT). We investigated the fate of expanded HIV-specific CTL clones in vivo following adoptive transfer to a patient with acquired immunodeficiency syndrome (AIDS). Two autologous CTL clones specific for HIV Gag and Pol were expanded to large numbers (>109) in vitro and infused into an HIV-infected patient whose viral load was rising despite antiretroviral therapy. The fate of one clone was monitored by staining peripheral blood mononuclear cells (PBMCs) with T-cell receptor–specific tetrameric major histocompatibility complex (MHC)-peptide complexes. Although the CTL transfer was well tolerated, there were no significant changes in CD4 and CD8 lymphocyte counts and virus load. By tracking an infused clone using soluble MHC-peptide complexes, we show that cells bearing the Gag-specific T-cell receptors were rapidly eliminated within hours of infusion through apoptosis. Thus, the failure of adoptively transferred HIV-specific CTL to reduce virus load in AIDS may be due to rapid apoptosis of the infused cells, triggered by a number of potential mechanisms. Further trials of adoptive transfer of CTL should take into account the susceptibility of infused cells to in vivo apoptosis.

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Incorporating Hemoglobin Levels to Map Leukostasis Risk in Acute Leukemia Using Microvasculature-on-Chip Technologies

Blood ◽

10.1182/blood-2020-137804 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 9-10

Author(s):

Jamie Oakley ◽

Evelyn K. Williams ◽

Christina Caruso ◽

Yumiko Sakurai ◽

Reginald Tran ◽

...

Keyword(s):

T Cell ◽

Acute Leukemia ◽

Blood Viscosity ◽

Leukemia Cell ◽

Leukemia Cells ◽

Cell Counts ◽

On Chip ◽

Wbc Count

Hyperleukocytosis, most commonly defined as a white blood cell (WBC) count > 100,000/μL, is an oncologic emergency in acute leukemia that can lead to leukostasis, which occurs when leukemia cells obstruct the microvasculature resulting in significant morbidity and mortality from neurologic (CNS hemorrhage, thrombosis) or pulmonary (respiratory distress, hypoxia) symptoms. The underlying mechanisms are poorly understood but are thought to be related to increased blood viscosity, secondary to high WBC count, leukemia cell aggregation, and the abnormal mechanical properties, size, and cell-cell interactions of leukemia cells. Leukapheresis is a commonly used therapy for rapid cytoreduction in symptomatic patients, but the procedure is not without risks. No existing methods reliably predict leukostasis or guide treatment including the commonly used WBC count, which only loosely correlates with leukostasis and does not accurately describe the blood viscosity at the microvascular level. Importantly, while hematocrit/hemoglobin levels (Hgb) are known to be major contributors to blood viscosity, they have not been systematically assessed in leukostasis risk, and Hgb often decreases as leukemic cell counts rise, complicating the issue. Incorporating Hgb levels may better predict leukostasis and assist physicians balancing the risk of hyperleukocytosis compared to the interventions themselves. To that end, we investigated how the differing presentations of acute leukemia lead to microvessel occlusion, thereby affecting effective blood viscosity at the microvascular level using "microvasculature-on-a-chip" devices that mimic the microvascular geometry (Figure 1) developed by our laboratory. This physiologically relevant microvascular model allows for in vitro investigation as in vivo studies are nearly impossible due to difficulty in visualizing and manipulating the animal microvasculature and cell counts. The devices were microfabricated using polydimethylsiloxane (PDMS). Acute T-cell lymphoblastic (Jurkat) and acute monocytic (THP-1) cell lines were maintained via standard cell culture conditions. Red cells from healthy donors were isolated and mixed with leukemia cells to achieve target Hgb and WBC levels. Various physiologic leukemia "mixtures" were then perfused under physiologic microcirculatory flow conditions through the microvascular device and microchannels occlusion was tracked via videomicroscopy (Figure 2). With T-cell leukemia, Hgb levels affected the risk of "in vitro leukostasis." Specifically, with severe anemia and WBC count less than the hyperleukocytosis range, time to microchannel occlusion was longer, and was more dependent on Hgb rather than WBC count. However, in cases with severe anemia and WBC counts > 100k/μL, WBC count exhibited a stronger effect on occlusion with little dependence on Hgb (Figure 3). At Hgb > 8g/dL, microchannel occlusion was dependent on WBC count regardless of hyperleukocytosis or not. In contrast, our data to date shows that with myeloid leukemia, in vitro leukostasis is not associated with Hgb levels, and is consistent with how myeloid leukemias in vivo cause leukostasis symptoms at lower WBC counts than lymphoid leukemias, not only due to size but also adhesive interactions. These data suggest when determining risk for leukostasis, WBC count should not be the sole determinant. Here we show Hgb levels affect microvascular blood viscosity and propensity for microvascular occlusion, but it appears to have a greater impact with T-cell leukemias versus myeloid leukemias (Figure 4). These studies indicate Hgb is an important clinical parameter for leukostasis risk in acute leukemia and will help inform guidelines for leukapheresis and even phlebotomy, a much simpler and safer procedure, to mitigate hyperviscosity in acute leukemia. These results can also impact decisions regarding the need for red blood cell transfusions, which iatrogenically increase blood viscosity. Studies incorporating patient myeloid and lymphoid leukemia cells and microvasculature-on-chip devices integrating live endothelium to assess leukemia cell adhesion are ongoing. Figure Disclosures Lam: Sanguina, Inc: Current equity holder in private company.

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G-CSF Activated Granulocytes Inhibit Experimental Graft-versus-Host Disease.

Blood ◽

10.1182/blood.v104.11.4989.4989 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4989-4989

Author(s):

Zilton F.M. Vasconcelos ◽

Julia Farache ◽

Bruna M. Santos Grad ◽

Tereza S. Palmeira Grad ◽

Luis Fernando Bouzas ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Mononuclear Cells ◽

Cell Activity ◽

Control Group ◽

Low Density ◽

Graft Versus Host ◽

The One

Abstract Acute Graft versus host diseas (aGVHD) is a major complication of stem cell transplantation. The disease is mediated by T cells and a higher incidence/severity would be expected when higher numbers of T cells are inoculated. However, the incidence of aGVHD in PBST, which carries about 10 times more T cells then BMT, is not higher than the one found in later. This finding indicates a modulatory role for G-CSF over T cell activity. We had previously shown that T cells from G-CSF treated PBSC donors do not produce g-IFN nor IL-4 and that this inhibition was mediated by low density, G-CSF activated, granulocytes. In order to test if in fact G-CSF activated granulocytes could inhibit disease, we first checked if G-CSF could generate low density granulocytes, in vivo and in vitro. Indeed, either in vivo(21mg /day - 5 days) or in vitro (150 ng -12hs) with G-CSF generates low density granulocytes which co-purify with the mononuclear cells in the ficoll® gradient. Moreover, as we had shown in humans, these low density cells, inhibit the production of g-IFN by anti-CD3 activated T cells on flow cytometry studies (17%-T cells alone versus 3% T cells with granulocytes 1:1). Radiation quimaeras were set with (B6 X BALB/c)F1 as hosts reconstituted with T cell depleted C57Bl6 bone marrow, in the presence or absence of nylon wool selected spleen cells (NWSC), as T cell source, from normal or G-CSF treated mice. As previously shown by others, NWSC from G-CSF treated mice diminishes the incidence of acute disease on day 20 post-transplant, from 75 to 25%. In order to investigate if this inhibition was dependent on the activated granulocytes present in the NWSC from G-CSF treated mice, granulocytes were depleted with anti-GR1 and complement. In this case, the incidence of disease is the same or even higher (75% experiment#1 and 100% in experiment #2) than the one observed on the control group (NWSC from control mice). These results strongly suggest that activated granulocytes could indeed inhibit aGVHD. We then generated activated granulocytes in vitro, by treating spleen derived high density granulocytes with 150ng of G-CSF for 12 hs. After the incubation period, a new ficoll® gradient was performed and the low density cells were obtained. T cell contamination on the second gradient was eliminated by anti-CD4 and CD8 complement lysis. These activated granulocytes were inoculated together with NWSC from control mice in the radiation quimaeras at a 1:1 ratio. In this case 100% disease inhibition was observed when compared to the positive control group, where 75% of the animals got sick. Our data indicate that activated granulocytes are the major mediators of the G-CSF immunossupressive effects and that these cells can be used as a novel immune modulator in clinical transplantation to prevent acute GVHD.

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Enhanced Growth Suppression of Philadephia1 Leukemia Cells in Mice by Targeting Both BCR-ABL and VEGF through the Antisense Strategy.

Blood ◽

10.1182/blood.v104.11.5257.5257 ◽

2004 ◽

Vol 104 (11) ◽

pp. 5257-5257

Author(s):

Zhong Chao Han ◽

Xiu Li Cong ◽

Bin Li ◽

Ren Chi Yang

Keyword(s):

Chronic Myeloid Leukemia ◽

Microvessel Density ◽

Myeloid Leukemia ◽

K562 Cells ◽

Chemotherapeutic Agents ◽

Leukemia Cells ◽

Vegf Expression ◽

Erythroleukemia Cell

Abstract The Philadephia chromosome (Ph1) translocation results in the formation of the BCR-ABL oncogene in over 95% patients with chronic myeloid leukemia (CML). VEGF levels are elevated both in the plasma of CML patients and in conditioned media taken from CML cells. Therefore, simultaneous targeting of BCR-ABL and VEGF might be a rational strategy for attempting treatment of Philadephia1 leukemia. To test this hypothesis, we used an antisense strategy to downregulate BCR-ABL and VEGF expression in K562 cells, a human erythroleukemia cell line. In vitro, combination of bcr/abl and VEGF antisense oligodeoxyribonucleotides (AS-ODNs) exerted a specific synergistic antiproliferative effect on K562 cells and prominently sensitized K562 cells to apoptosis-inducing stimuli. In vivo, nude mice injected with K562 cells were treated systemically with BCR-ABL or VEGF AS-ODNs or with both ODNs in combination. In comparison with the mice treated with individual agents, the mice treated with both ODNs showed a slower growth of leukemia tumors, a reduction of microvessel density and an increased apoptosis in the tumors. These results demonstrate that targeting both BCR-ABL and VEGF may represent an excellent strategy to overcome the resistance to chemotherapeutic agents and ultimately to augment the efficacy of chemotherapy in CML.

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Dasatinib-Induced Reduction of Tumor Growth Is Accompanied By the Changes in the Immune Profile in a Melanoma B16.OVA Mouse Model

Blood ◽

10.1182/blood.v124.21.1408.1408 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1408-1408

Author(s):

Mette Matilda Ilander ◽

Can Hekim ◽

Markus Vähä-Koskela ◽

Paula Savola ◽

Siri Tähtinen ◽

...

Keyword(s):

T Cells ◽

T Cell ◽

Tumor Growth ◽

Cd8 T Cells ◽

Research Funding ◽

K562 Cells ◽

Cell Cytotoxicity ◽

Cd8 Cells

Abstract Background: Dasatinib is a 2nd generation tyrosine kinase inhibitor (TKI) used in the treatment of chronic myeloid leukemia (CML). Its kinase inhibition profile is broad and includes several kinases important in the immune cell function such as SRC kinases. Furthermore, it is known that dasatinib has immunomodulatory effects in vivo. Recently, we observed that dasatinib induces a rapid and marked mobilization of lymphocytes, which closely follows the drug plasma concentration. The phenomenon is accompanied by an increase of NK-cell cytotoxicity. In addition, we have shown that dasatinib alters T-cell responses long-term favoring Th1 type of responses. Interestingly, the dasatinib induced immune effects have been associated with better treatment responses. We now aimed to characterize the dasatinib-induced antitumor immune responses in a syngeneic murine melanoma model to address whether dasatinib-induced immunoactivation affects tumor growth. Methods: Direct cytotoxic effect of dasatinib on B16.OVA melanoma cells in vitro was assessed with an MTS cell viability assay. T-cell cytotoxicity was assessed by preincubating splenocytes isolated from naïve and OT-I mouse spleen with 100 nM dasatinib and measured their cytotoxic capacity against B16.OVA cells. To further evaluate the dasatinib induced antitumor immune effects in vivo, B16.OVA cells were implanted subcutaneously in C57BL/6J mice. The mice (n=6/group) were treated daily i.g. either with 30 mg/kg dasatinib or vehicle only. Blood was collected before tumor transplantation, before treatment, and on treatment days 4, 7 and 11. Tumor volumes were measured manually and specific growth rate was calculated based on the first and the last day of the treatment. In addition to white blood cell differential counts, immunophenotyping of blood and tumor homogenate was performed by flow cytometry using antibodies against CD45.1, CD3, CD4, CD8b, NK1.1, CTLA4, PD-1 and CD107. Immunohistochemical staining of CD8+ T-cells was performed from the paraffin embedded tumor samples. Results: In vitro incubation of B16.OVA cells with dasatinib showed only a moderate unspecific cytotoxicity with the two highest concentrations of dasatinib (1- and 10 µM), whereas in K562 cells (a CML blast crisis cell line) almost complete killing was observed already with the 100nM concentration. The cell viability of B16.OVA cells was 90% with at 100 nM of dasatinib concentration (as compared to 21% of K562 cells) suggesting that there was no direct dasatinib sensitive target oncokinase in this cell line. In contrast, a significant enhancement in the cytotoxic capacity of splenocytes was observed when they were pretreated with 100nM dasatinib (60% of target cells were alive when incubated with dasatinib pretreated naïve splenocytes compared to 100% with control treated splenocytes, p=0.004). The in vivo tumor experiments demonstrated that the tumor volumes were smaller in dasatinib group, and there was a significant decrease in the specific tumor growth rate (0.06 vs. 0.18, p=0.01) on the 11th day of treatment. Interestingly, dasatinib treated mice had increased proportion of CD8+cells in the circulation (17.9% vs. 14.4%, p=0.005) and the CD4/CD8 ratio was significantly decreased (1.39 vs. 1.52, p= 0.04). During the tumor growth the mean CTLA-4 expression on CD8+ cells in PB increased from 1.2% to 9% in the control group, whereas, in dasatinib group the increase was more modest (1.2% to 5.7%). When the tumor content was analyzed, dasatinib treated mice had significantly more tumor infiltrated CD8+ T-cells (median 17 vs. 4/counted fields, p=0.03). In dasatinib group 80% of the tumor infiltrating CD8+ cells expressed PD-1 antigen compared to <5% of PD1 positive CD8+ cells in the peripheral blood suggesting either tumor induced CD8 T-cell exhaustion or the presence of tumor-reactive effector cells. Lastly, when CD4 and CD8 cells were depleted before tumor inoculation, dasatinib was no longer able to slow down the tumor growth. Conclusions: Dasatinib treatment slowed the tumor growth in a B16.OVA mouse model. The growth retardation was due to immunomodulatory properties of dasatinib as the drug was not directly cytotoxic and depletion of T-cells abolished the effect. Dasatinib may be a therapeutically useful immunomodulatory agent for targeting tumor-associated anergy, particularly in combination with novel checkpoint inhibitors and tumor-targeting drugs. Disclosures Hemminki: Oncos Therapeutics Ltd: Shareholder Other; TILT BioTherapeutics Ltd: Employment, Shareholder, Shareholder Other. Porkka:BMS and Novartis: Honoraria, Research Funding; Pfizer: Research Funding. Mustjoki:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding.

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EphB4/ephrinB2 ephrinB1 Interaction Mediated Chronic Myelogenous Leukemia Mesenchymal Stromal Cells Osteogenic Differentiation in Vitro and In Vivo

Blood ◽

10.1182/blood.v128.22.1901.1901 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1901-1901

Author(s):

Li Lin ◽

Xu Na ◽

Jiang Zhiwu ◽

Lu Qisi ◽

Zhou Xuan ◽

...

Keyword(s):

Bone Marrow ◽

Treatment Group ◽

Osteogenic Differentiation ◽

K562 Cells ◽

Leukemia Cells ◽

Control Group ◽

Vegf Mrna ◽

Stat3 Phosphorylation

Abstract Background and Objective: Osteoblasts, important of stromal cells in bone marrow microenvironment, maintain HSCs in resting state and protect its' functions. Osteoblasts derived from mesenchymal stem cells (MSCs), which can be differentiated into osteoblast in bone marrow under the regulation of cytokines. Recent studies have indicated that EphB4/ephrinB2 protein participates in the regulation of osteogenesis differentiation of MSCs in bone marrow microenvironment. Our previous study found that EphB4 receptor was over expressed in CML patients and cell lines, which played an important role to change characterize of Imatinib(IM)-resistant in chronic myeloid leukemia cells. Furthermore, we performed experiments to prove that osteogenic differentiation in MSCs from CML-initial patient significantly higher in contrast to normal human MSCs and the change of EphB4 molecules on leukemia cells may transform MSCs functions in vitro. However, the mechanism of these transformations of MSCs in vitro and what is change in vivo were still unclear. Therefore, we hypothesis that the change of EphB4 molecules on leukemia cells might play an important role to osteogenic differentiate in MSCs in vitro and in vivo, which support to leukemia progression and disruption of normal hematopoiesis. Methods and Results: MSCs were prepared from bone marrow mononuclear cells isolated from normal human or CML- chronic phase (CCP) patients' BM and cultures in Cyagen Bone marrow culture medium at 37 °C, 5% CO2 incubator. In vitro, after stimulated with different concentrations of EphB4-Fc (0, 5, 8, 10 ug/ml) for 21 days, visualized by Alizarin Red staining, MSCs (CCP) produced maximum calcium nodules (P<0.05, n=3) in EphB4-Fc (8 ug/ml) group in contrast with other groups, accompanied by increased ephrinB1 and STAT3 phosphorylation. In vitro osteogenesis condition, after treatment with EphB4-Fc (0, 8 ug/ml) 14 days, MSCs (CCP) incubated with K562 cells. After 48 h, the IC50 (0.842±0.065, P<0.05, P<0.05 ANOVA, n=4) of K562 cells in MSCs+EphB4-Fc (8ug/ml) group increased, S phase cells percentage(56.6±4.01, P<0.05, P<0.01, ANOVA, n=4) increased and cells apoptosis rate(P<0.01, P<0.001, LSD, n=4) declined compared with K562 (control group) and K562+MSCs+EphB4-Fc (0 ug/ml). In vivo, K562-R, K562-R+MSCs (normal) (5:1), K562-R-EphB4-sh, K562-R-EphB4-sh+MSCs (normal) (5:1), MSCs (normal) cells were injected respectively into bone cavity of NOG rat (NOD/SCID/ɣ c-/-, n=12) rat and blank control group were also established. Examined peripheral blood in rats while hCD45+ cells > 1% is considered as leukemia model. K562-R+MSCs mice were earliest to establish leukemia model (31.75±1.26d) and had the shortest survival time(4.25±1.71d) than other groups. After treatment with IM, survival times of K562-R+MSCs mice were not significantly extended (4.7±3.055 d, pared-samples T test, P>0.05). In bone marrow of K562-R+MSCs mice, RUNX2 mRNA (0.654±0.0278; P < 0.001) over expressed in contrast to other groups. After treatment with IM, expression level of RUNX2 mRNA was significantly increased than non-treatment group. Among four leukemia groups of mice, expression levels VEGF mRNA in bone marrow were no significantly difference and there was no statistical difference existed in treatment group and non-treatment group. The same cells lines above were subcutaneously injected to establish subcutaneous transplantation tumor, respectively, in NOG rat (NOD/SCID/ɣ c-/-, n=8) rat. K562-R+MSCs tumors were earliest to appear (17.333±1.154 d) and had the biggest tumors volume (13116.27±165.502 mm3, P<0.001) compared to other groups. After mice treated by IM, compared with non-treatment group, K562-R+MSCs tumors had significantly increased in volume (14703.14±309.333mm3, pared-samples T test, P<0.01). VEGF mRNA (0.861±0.0648; P<0.01) in K562-R+MSCs tumor over express than other groups. After treatment, the expression level was no significantly declined (0.796±0.0688, P>0.05). The level expression of RUNX2 mRNA in four groups of subcutaneous transplantation tumors are low and had no statistical difference. Conclusion: Our experiments in vitro and in vivo illustrated that EphB4 molecule on leukemia cells may transform MSCs osteogenic differentiation to change characterize of Imatinib(IM)-resistant in CML through ephrinB1 and STAT3 phosphorylation. Disclosures Lin: Natural Science Foundation of China: Research Funding. Na:Natural Science Foundation of China: Research Funding.

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The Novel Sulfonamidebenzamide ML291 Activates Apoptotic UPR Signaling in Pediatric Leukemia

Blood ◽

10.1182/blood.v128.22.3523.3523 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3523-3523

Author(s):

Danielle Garshott ◽

Nicole Melong ◽

Tania T. Sarker ◽

Yue Xi ◽

Amy Brownell ◽

...

Keyword(s):

Cell Lines ◽

Leukemia Cell ◽

Leukemic Cell ◽

K562 Cells ◽

Leukemia Cells ◽

Wild Type ◽

Leukemia Cell Lines ◽

Proliferation Assays

Abstract Background: Acute leukemias are the most common cancers in childhood. Despite multi-agent chemotherapy protocols and the introduction of novel molecularly targeted therapies which have resulted in improved survival over the last few decades, relapsed acute lymphoblastic leukemia remains the second most common pediatric cancer diagnosis. In addition, morbidities from current chemotherapy regimens are unacceptably high. Abundant evidence point to a major role for mediators of the unfolded protein response (UPR) in normal and leukemic white blood cell biology. We have demonstrated that activation of the UPR is a productive approach to inhibit the proliferation of solid tumor cell lines in vitro and to reducing xenograft burden in vivo. The UPR consists of genetically distinct mechanisms that serve to clear misfolded proteins from the endoplasmic reticulum (ER) and enhance protein folding, or induce apoptosis if the initiating stress is prolonged or robust. ML291 is a novel UPR-inducing sulfonamidebenzamide, identified through cell-based high throughput screening and iterative SAR-guided chemical synthesis, that overwhelms the adaptive capacity of the UPR and induces apoptosis in a variety of solid cancer models. Objective: To determine the ability of ML291 to activate the UPR and induce apoptosis in a panel of leukemia cell lines, and to use CHOP-null K562 cells to elucidate the relative contribution of the UPR. We hypothesized that ML291 might activate the PERK/eIF2a/CHOP (apoptotic) arm of the UPR and reduce leukemic cell burden in vitro and in vivo. Methods: MTT and luciferase-based proliferation assays, flow cytometry and RT-qPCR were used to evaluate cell growth, UPR activation and apoptosis in a panel of leukemia cell lines that included AML, ALL and CML in cells exposed to ML291. CRISPR-Cas9 genome editing was used to delete CHOP in K562 (human myeloid leukemia) cells. Deletion was validated by immunoblot analysis and these cells were subjected to the same proliferation and gene analyses described above. The in vivo response to ML291 therapy was evaluated in an established zebrafish xenograft assay (Corkery et al. BJH 2011) in which embryos were xenotransplanted with wild type or CHOP knockdown K562 cells and embryos bathed in ML291. Results: Immunoblot and RT-qPCR analysis revealed an accumulation of proteins and increased gene expression for downstream UPR genes, including CHOP, GRP78/BiP, GADD34 and XBP1 in leukemia cells following ML291 treatment, indicating the activation of the UPR. Increased expression of the apoptotic genes, NOXA, PUMA and DR5 was also observed post-treatment with ML291; and dose response proliferation assays performed after 24 hours revealed IC50 concentrations of 1 - 30µM across cell lines. CHOP deleted K562 cells were protected from cell death when cultured with increasing concentrations of ML291, and were significantly less able to translocate phosphatidylserine across the cell membrane and activate the caspase cascade. When zebrafish embryos xenotransplanted with K562-wild type or -CHOP-null cells were bathed in water containing 5mM ML291 for three days, there was a significant reduction in leukemia cell burden exclusively in theK562 wild type xenografts. Conclusion: Collectively these data indicate that intact PERK/eIF2a/CHOP signaling is required for efficient leukemic cell apoptosis in response to ML291 in vitro and in vivo, and support the hypothesis that small molecule enforcement of the UPR might be a productive therapeutic approach in leukemia. Disclosures No relevant conflicts of interest to declare.

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Mitogenicity of the Recombinant Mycobacterial 27-Kilodalton Lipoprotein Is Not Connected to Its Antiprotective Effect

Infection and Immunity ◽

10.1128/iai.72.6.3383-3390.2004 ◽

2004 ◽

Vol 72 (6) ◽

pp. 3383-3390 ◽

Cited By ~ 9

Author(s):

Avi-Hai Hovav ◽

Liuba Davidovitch ◽

Gabriel Nussbaum ◽

Jacob Mullerad ◽

Yolanta Fishman ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

T Cell ◽

B Cell ◽

Interleukin 2 ◽

Independent Manner ◽

Lipid Moiety ◽

High Gamma ◽

Type Response

ABSTRACT We reported previously that even though immunization with the recombinant mycobacterial 27-kDa lipoprotein (r27) induced a Th1-type response in mice, the vaccinated mice became more susceptible to challenge with Mycobacterium tuberculosis. In this study we show that r27 stimulates naive splenocytes to proliferate. Acylation of r27 was crucial for this effect, since a nonacylated mutant of r27, termed r27ΔSP, failed to stimulate splenocytes either in vitro or in vivo. Depletion experiments indicated that only B cells were proliferating in a T-cell-independent manner. We also found that r27 is recognized by TLR2, which is involved in mitogenic stimulation. Interestingly, r27 but not r27ΔSP induced high gamma interferon levels in splenocyte supernatants, whereas no significant interleukin-2 levels were detected. Since B-cell polyclonal activation might aggravate pathogen infection, we asked whether the antiprotective effect of the r27 lipoprotein is associated with its mitogenicity. We showed that, as in the case of r27, immunization of mice with the nonmitogenic r27ΔSP lipoprotein resulted in increased M. tuberculosis multiplication. We conclude that the antiprotective effect of the r27 lipoprotein must be linked to properties of the polypeptide portion of the lipoprotein rather than to its lipid moiety and its mitogenicity.

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