Alloreactive NK-Cells Cure Tumor Stem Cell Containing Breast Cancer.

Abstract Abstract 4082 Poster Board III-1017 Background Patients with metastasized solid tumors can not be cured by current standard treatment options. One hypothesis is that slow cycling chemo-resistant tumor stem cells give rise to new tumors after cytoreductive treatment, ultimately leading to chemoresistant tumors. Acute myeloid leukemia tumor stem cells can be killed by alloreactive T- or NK-cells, as patients have a higher survival chance after allogeneic transplantation. In the past we published the curative effectiveness of allogeneic spleen and bone marrow transplantation in breast cancer bearing mice. Our current aim was to study if either alloreactive T- or NK-cells are able to cure these mice. In addition we wanted to know if the tumors contain slow cycling tumor stem cells. Methods The 4T1 breast cancer cell line was cultured under standard conditions. Fifty thousand 4T1 cells were injected s.c. in the flank of CB6F1 (H-2b/d) mice. At day 12 mice were treated with 2× 2Gy total body irradiation and 200 mg/kg cyclophosphamide (CY), followed by transplantation of spleen and bone marrow cells. B6CBAF1 (H-2b/k) mice were used as allogeneic donors. These donors harbour alloreactive NK-cells towards CB6F1 recipients as determined by an in vivo NK-cell alloreactivity assay. NK-cell or T-cell depleted grafts were obtained from donors injected with anti-AsialoGM1 or a combination of anti-CD4 and anti-CD8 respectively some days before spleen and bone marrow harvest. Tumor stem cells were defined as slow cycling (i.e. label retaining) Hoechst extruding cells. 4T1 tumor cells were labelled with Vybrant CM-Dil (Dil) and then injected s.c. in CB6F1 mice. Fourteen days later tumors were excised. Part of the tumor was prepared as single cell suspension. Subsequently the presence of Dil retaining tumor stem cells was determined by fluorescence microscopy. The other part of the tumor was snap frozen and studied by histochemistry and fluorescence microscopy. For in vitro cytotoxicity testing 4T1 cells were again labelled with Dil and then seeded in 96 well plates at 30 cells/well. Chemotherapeutics were added in the different plates: 4-OH-CY (the active metabolite), cisplatine and doxorubicin, in concentrations far above the lowest maximal lethal dose as determined in MTT assays. Wells were checked weekly for growth and presence of tumor stem cells. Hoechst staining was used in these cultures to underscore that Dil retaining chemorefractory cells are indeed stem cells. Results Ninety percent of CB6F1 mice injected with 4T1 breast cancer cells developed a tumor growing progressively to a size that made it necessary to sacrifice them. Growth in mice treated with radio- and chemotherapy only or additionally with CB6F1 spleen and bone marrow cells was delayed for 10 days compared with tumor growth in untreated mice, but the incidence was equally high (90% and 80% respectively). In contrast, but in concord with our previous published results, only 10% of 4T1 breast cancer bearing mice transplanted with haploidentical spleen and bone marrow cells did show progressive tumor growth (follow-up 99 days at the time of abstract submission, p<0.004 log-rank test)). Similarly, mice transplanted with spleen and BM cells from T-cell depleted donors did not develop tumors at all (p<0.004 log-rank test), in contrast to recipients of in vivo NK-cell depleted grafts (90% tumor incidence). Label retaining tumor stem cells are unmistakably present in tumors at the day of transplantation, evidenced by the presence of a minute number of Dil retaining cells in single cell suspension of the tumor and in tumor tissue sections. More direct evidence for chemo-resistance of 4T1 tumor stem cells is derived by results of in vitro co-culture experiments. When Dil labelled 4T1 cells are plated in 30 cells/well concentration, approx. 25% of the wells contain one or two label retaining cells while the other wells comprise non-label retaining cells only. After incubation with chemotherapeuticals growth is observed only in wells containing a label retaining cell. These label retaining cells are also Hoechst negative. Conclusions This report provides the first evidence that tumor stem cells can be eliminated in vivo by alloreactive NK cells resulting in cure of chemorefractory breast cancer. It is also shown in vitro that only tumor stem cell containing tumor colonies resist the effect of various chemotherapeuticals commonly used in oncology as they do in vivo. These results strongly encourage the exploration of clinical alloreactive NK therapy in patients with metastasized solid tumors. Disclosures: No relevant conflicts of interest to declare.

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Effective Alloreactive NK Cell Therapy for Breast Cancer In Mice Requires Conditioning with Cyclophosphamide and Total Body Irradiation but Not Bone Marrow Engraftment

Blood ◽

10.1182/blood.v116.21.2085.2085 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2085-2085

Author(s):

Michel Van Gelder ◽

Peter Frings ◽

Catarina Matos ◽

Harry C. Schouten ◽

Gerard M.J. Bos

Keyword(s):

Breast Cancer ◽

Bone Marrow ◽

Nk Cells ◽

Nk Cell ◽

Spleen Cells ◽

Slow Cycling ◽

4T1 Breast Cancer ◽

Bone Marrow Engraftment

Abstract Abstract 2085 Background: Patients with metastasized breast cancer cannot be cured by current standard treatment options. One hypothesis is that slow cycling chemo-resistant tumor stem cells give rise to new tumors after cytoreductive treatment, ultimately leading to chemoresistant tumors. Last year we showed that the 4T1 mouse breast cancer model contains slow-cycling chemo-resistant cells that induce renewed growth of the tumor after chemo- and radiotherapy (abstract 4082). We also showed that haploidentical spleen and bone marrow transplantation (BMT) cures the mice and that donor NK cells are a prerequisite. Our current aim was to study the need of long term BM engraftment and to study the role of the conditioning in the curative process. Methods: The 4T1 breast cancer cell line, originating from a spontaneous Balb/c (H-2d) breast cancer, was cultured under standard conditions. Fifty thousand 4T1 cells were injected s.c. in the flank. For the experiments addressing the need for haploidentical BMT tumor bearing CB6F1 (H-2b/d) recipients were treated with 2x 2Gy total body irradiation and 200 mg/kg cyclophosphamide (CY+TBI) followed by in vitro NK cell enriched haploidentical B6CBAF1 (H-2b/k) spleen cell infusion with or without additional BM cells. Chimerism in tumor-free surviving recipients was measured by flowcytometry of spleens at least 100 days after the treatment. The role of the conditioning in the alloreactive NK cell effect was studied in fully H-2 mismatched B6CBAF1 mice. When indicated, in vivo NK cell depletion was by i.p. injection of anti-AsialoGM1. Results: Figure A shows overall survival of mice with breast cancer after various treatments (10 mice per group). Haploidentical BMT plus spleen cells cured 50% of tumor bearing mice after CY+TBI (♦, dashed line) and survival was at least as good when NK cell enriched spleen cells were co-transplanted (▴, solid line). Transplantation of spleen cells from NK cell depleted mice (•, dotted line) obliterated the beneficial effect of haploidentical transplantation and resulted is similar poor survival as syngeneic BMT plus spleen cells (▪, solid line). The majority of mice that received NK cell enriched spleen cells (10 out of 14 tested) had no bone marrow engraftment and in the other four only 1–5% donor cells were detectable at 150 days. Recipients of unmanipulated haploidentical spleen and BM cells had >90% donor chimerism in 10 out of 14 tested. The cure rate in both groups was nevertheless similarly high. In a subsequent experiment (Figure B, 10 mice per group) we infused haploidentical NK cells only after CY+TBI (▴, solid line); other groups received T cell depleted (x, solid line) or T cell replete (♦, solid line) haploidentical BMT, or syngeneic BMT (▪, solid line). This resulted in a similar superior tumor-free survival (80-90%) than in mice co-transplanted with haploidentical BM (90%), as compared with syngeneic BM and spleen cell transplantation (•, dotted line). We then planned to study the role of the conditioning in the curative process. For this purpose 4T1 breast cancer cells were injected in fully H-2 mismatched B6CBAF1 mice (H-2b/k). Surprisingly, 4T1 breast cancer is not rejected by B6CBAF1 mice despite the full MHC mismatch. Tumors are only rejected when the mice were treated with CY+TBI. Tumor rejection proved to be NK cell dependant and not a direct result of the conditioning as it was prevented by in vivo NK cell depletion. Conclusions: This report provides the first evidence that chemo resistant tumor cells can be eliminated in vivo by alloreactive NK cells resulting in cure without the need for long term donor bone marrow engraftment. Conditioning with CY+TBI seems essential for this effect. These results set the stage for the exploration of alloreactive NK therapy in patients with metastasized breast cancer. Disclosures: No relevant conflicts of interest to declare.

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Direct evidence that natural killer cells in nonimmune spleen cell populations prevent tumor growth in vivo

Journal of Experimental Medicine ◽

10.1084/jem.149.5.1260 ◽

1979 ◽

Vol 149 (5) ◽

pp. 1260-1264 ◽

Cited By ~ 105

Author(s):

M Kasai ◽

JC Leclerc ◽

L McVay-Boudreau ◽

FW Shen ◽

H Cantor

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Tumor Growth ◽

Nk Cells ◽

Spleen Cell ◽

Cell Population ◽

The Body ◽

Nk Activity

Relatively large numbers of nonimmune spleen cells do not protect against the local growth of two lymphomas. However, this heterogeneous population of splenic lymphocytes contains a subset of cells that efficiently protects against in vivo tumor growth. This cell population (cell-surface phenotype Thyl.2(-)Ig(-)Ly5.1(+)) represents less than 5 percent of the spleen cell population and is responsible for in vitro NK-mediated lysis. Although these studies clearly and directly demonstrate that Ly5(+) NK cells selected from a heterogeneous lymphoid population from nonimmune mice can protect syngeneic mice against local in vivo growth of two different types of tumor cells (in contrast to other lymphocyte sets within the spleen), they do not directly bear upon the role of NK cells in immunosurveillance. They do indicate that highly enriched Ig(-)Thyl(-)Ly5(+) cells, which account for virtually all in vitro NK activity, can retard tumor growth in vivo. It is difficult to ascribe all anti-tumor surveillance activity to NK cells, because they probably do not recirculate freely throughout the various organ systems of the body. Perhaps NK ceils may play a role in prevention of neoplastic growth within discrete anatomic compartments where there is rapid differentiation of stem cells to mature progeny (e.g., bone marrow, spleen, and portions of the gastrointestinal tract)and may normally act to regulate the growth and differentiation of non-neoplastic stem cells. Long-term observation of chimeric mice repopulated with bone marrow from congenic or mutant donors expressing very low or very high NK activity may help to answer these questions.

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Bone marrow-derived mesenchymal stem cells inhibit T follicular helper cell in lupus-prone mice

Lupus ◽

10.1177/0961203317711013 ◽

2017 ◽

Vol 27 (1) ◽

pp. 49-59 ◽

Cited By ~ 10

Author(s):

X Yang ◽

J Yang ◽

X Li ◽

W Ma ◽

H Zou

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Flow Cytometry ◽

T Cells ◽

Lupus Nephritis ◽

Bone Marrow Cells ◽

Tfh Cells ◽

Follicular Helper

Background The objective of this paper is to analyze the role of bone marrow-derived mesenchymal stem cells (BM-MSCs) on the differentiation of T follicular helper (Tfh) cells in lupus-prone mice. Methods Bone marrow cells were isolated from C57BL/6 (B6) mice and cultured in vitro, and surface markers were identified by flow cytometry. Naïve CD4+ T cells, splenocytes and Tfh cells were isolated from B6 mice spleens and co-cultured with BM-MSCs. The proliferation and the differentiation of CD4+ T cells and Tfh cells were analyzed by flow cytometry. Lupus-prone MRL/Mp-lpr/lpr (MRL/lpr) mice were treated via intravenous injection with expanded BM-MSCs, the differentiation of Tfh cells was detected, and the relief of lupus nephritis was analyzed. Results MSCs could be successfully induced from bone marrow cells, and cultured BM-MSCs could inhibit T cell proliferation dose-dependently. BM-MSCs could prevent Tfh cell development from naïve CD4+ T cells and splenocytes. BM-MSCs could inhibit IL-21 gene expression and cytokine production and inhibit isolated Tfh cells and STAT3 phosphorylation. In vivo study proved that BM-MSCs intravenous injection could effectively inhibit Tfh cell expansion and IL-21 production, alleviate lupus nephritis, and prolong the survival rate of lupus-prone mice. Conclusions BM-MSCs could effectively inhibit the differentiation of Tfh cells both in vitro and in vivo. BM-MSC treatment could relieve lupus nephritis, which indicates that BM-MSCs might be a promising therapeutic method for the treatment of SLE.

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Short-Term Foamyviral Transduction with Virions Encoding Recombinant Corrects the Mitomycin C Hypersensitivity of Fancc −/− Progenitor Cells In Vitro and Restores Long Term Repopulating Activity of Fancc −/− Stem Cells In Vivo.

Blood ◽

10.1182/blood.v104.11.3171.3171 ◽

2004 ◽

Vol 104 (11) ◽

pp. 3171-3171

Author(s):

Yue Si ◽

Cordula Leurs ◽

Edward Srour ◽

Samantha Ciccone ◽

Helmut Hanenberg ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Progenitor Cells ◽

Mitomycin C ◽

Bone Marrow Cells ◽

Genetic Disorder ◽

Foamy Virus ◽

Alkylating Agents

Abstract Fanconi anemia (FA) is a complex autosomal recessive genetic disorder characterized within the hematological system by progressive bone marrow aplasia, a high propensity to develop acute myeloid leukemia, and hypersensitivity to alkylating agents including mitomycin c. The identification of individual FA genes raises the potential of using gene transfer technology to express/introduce the functional cDNA in/into deficient autologous stem cells. We have previously shown that in the absence of genetic correction with a retroviral mediated Fancc transgene, ex vivo culture of Fancc−/− stem/progenitor cells (HSPC) predisposes uncorrected Fancc−/− HSPC cells to clonal hematopoiesis (Haneline, Blood 2003). Therefore we examined the potential of a helper-free human foamy virus (HFV) derived construct that encodes both the human FANCC and EGFP transgenes to transduce murine Fancc−/− HSC in the absence of prestimulation. In initial experiments, we determined that 40–80% of progenitors were transduced following a single overnight HFV infection using a 20:1 moiety of infection. Subsequent studies demonstrated that HFV efficiently transduced primitive hematopoietic progenitors in G0 and G1 phases of the cell cycle as evidenced both by using multicolor fluorescence activated cell sorting and subsequent culture of sorted cell populations in high proliferating potential (HPP-CFC) and low proliferating potential colony forming assays. Aliquots of HFV transduced cells that were transduced with the construct encoding both Fancc and EGFP, or the reporter transgene only were transplanted into irradiated recipient mice. Four months following transplantation, bone marrow cells were isolated from the reconstituted recipients and clonogenic assays were established in a range of mitomycin c (MMC) concentrations. In these experiments, the MMC hypersensitivity of Fancc−/− progenitors was corrected to wild-type levels. To assess quantitatively the potential of HFV expressed FANCC to correct stem cell repopulating ability, we next utilized the competitive repopulating assay. In two replicate experiments, we determined that the repopulating activity of HFV-transduced Fancc−/− stem cells was comparable to wildtype controls six months following transplantation in primary and secondary recipients. Collectively, these data provide in vivo evidence that the HFV vector is an efficient vehicle for introducing a functional hFANCC transgene into quiescent Fancc−/− HSC in the absence of prestimulation and for complementing the cellular FA defect in vitro and in vivo.

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The IL-15 Super-Agonist ALT-803 Promotes Superior Activation and Cytotoxicity of Ex Vivo Expanded NK Cells Against AML

Blood ◽

10.1182/blood.v126.23.3090.3090 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3090-3090 ◽

Cited By ~ 1

Author(s):

Folashade Otegbeye ◽

Nathan Mackowski ◽

Evelyn Ojo ◽

Marcos De Lima ◽

David N. Wald

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Nk Cells ◽

Myeloid Leukemia ◽

Nk Cell ◽

Ex Vivo ◽

Leukemia Cells ◽

Activating Receptor

Abstract Introduction: A crucial component of the innate immune response system, natural killer (NK) cells are uniquely competent to mediate anti-myeloid leukemia responses. NKG2D is an activating receptor on the surface of NK cells that engages stress ligands MICA and MICB, typically upregulated on myeloid leukemia cells. Adoptive transfer of NK cells is a promising treatment strategy for AML. Strategies to optimize the anti-leukemia effect of NK cell adoptive transfer are an area of active research. These include attempts to enhance NK cell activity and to maintain the activation status and proliferation of the NK cells in vivo. Traditionally, IL-2 has been used to maintain the in vivo proliferation of adoptively transferred NK cells, but it leads to unwanted proliferation of regulatory T cells and suboptimal NK cell proliferation. IL-15 may be superior to IL-2, without the effects on T regulatory cells. The IL-15 superagonist, ALT-803 exhibits >25 fold enhancement in biological activity as compared to IL-15. ALT-803 is a fusion protein of an IL-15 mutant and the IL-15Rα/Fc complex that has recently entered clinical trials as a direct immunomodulatory agent in cancer clinical trials We hypothesized ALT-803 would augment the activity and/or proliferation of adoptively transferred NK cells in vitro and in a mouse model system.. Methods: Human NK cells were isolated from healthy donor peripheral blood and were expanded over a 21-day period in co-culture with irradiated K562 cells genetically modified to express membrane-bound IL-21. (Somanchi et al. 2011 JoVE 48. doi: 10.3791/2540) The NK cells were expanded with IL-2 (50mU/mL) and/or ALT-803 (200ng/mL). On Day 21, NK cells were examined for cytotoxicity against AML cells as well as by flow cytometry for expression of known activating receptors. An NSG murine xenograft model of human AML was developed to test the in vivo function of NK cells expanded above. Briefly, NSG mice (n=5 per group) were non-lethally irradiated and each injected IV with 5 x106 OCI-AML3 leukemic cells. Two days later, each mouse received weekly NK cell infusions for 2 weeks. Mice that received NK cells expanded with IL2 got cytokine support with IL-2 (75kU IP three times a week). Mice infused with ALT-803 expanded cells (alone or in combination with IL2) received ALT-803 (0.2mg/kg IV weekly). One control group received OCI cells but were infused weekly only with 2% FBS vehicle, no NK cells. Leukemic burden in each mouse was assessed by flow cytometry of bone marrow aspirates on day 28 following start of NK cell infusions). This time point was chosen as the control mice appeared moribund. Results: ALT-803 did not have any differential effect on the proliferation of the NK cells ex vivo as compared to IL-2. However, the presence of ALT-803 either alone or in combination with IL-2 resulted in a significant increase (30% increase, p<0.0001) in the cytotoxic activity of the NK cells against leukemia cells as compared with IL-2 alone in vitro (figure 1). In addition, the percentages of NK cells that express the activating receptor NKG2D as well as CD16 were significantly higher (p<0.001 for both) after ALT-803 exposure (figure 1). Finally, in the murine xenograft AML model, ALT-803 expanded NK cells, which were also supported in vivo with ALT-803, resulted in an 8-fold reduction in disease burden in the bone marrow (p<0.0001). Importantly the efficacy of NK cells in the ALT-803 injected mice was significantly higher (3-fold, p= 0.0447) than IL-2 treated mice (figure 2). Discussion: Our results suggest that the presence of ALT-803 during ex-vivo expansion of NK cells results in increased activation and cytotoxicity against AML cells. In addition our results using a murine model of human AML show that the use of ALT-803 in combination with adoptively transferred NK cells provides a significant anti-leukemic benefit as compared to IL-2. Future studies to test larger panels of leukemia cells as well as other cancer cell lines are currently in progress. It is hoped that this work will lead to an improvement in the efficacy of adoptively transferred NK cells for AML patients due to an improvement in survival and activity of the NK cells. Disclosures Wald: Invenio Therapeutics: Equity Ownership.

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Identification of a subset of murine natural killer cells that mediates rejection of Hh-1d but not Hh-1b bone marrow grafts.

Journal of Experimental Medicine ◽

10.1084/jem.170.1.191 ◽

1989 ◽

Vol 170 (1) ◽

pp. 191-202 ◽

Cited By ~ 86

Author(s):

C L Sentman ◽

J Hackett ◽

V Kumar ◽

M Bennett

Keyword(s):

Bone Marrow ◽

Natural Killer Cells ◽

Nk Cells ◽

Natural Killer ◽

Tumor Cells ◽

Bone Marrow Cells ◽

Immune Functions ◽

Infected Cells

NK cells demonstrate many immune functions both in vitro and in vivo, including the lysis of tumor or virus-infected cells and the rejection of bone marrow allografts. However it remains unclear whether or not all NK cells can mediate these various functions or if NK cells exist in functionally distinct subsets. We have developed a new NK-specific mAb, SW5E6, which binds to approximately 50% of murine NK cells. The 5E6 antigen identifies a distinct and stable subset of NK cells and is expressed on about one-half of fresh or rIL-2-activated murine NK cells. Both 5E6+ and 5E6- NK cells are capable of lysing YAC-1 tumor cells in vitro and in vivo. By treating animals with SW5E6, we demonstrate that the 5E6+ subset is necessary for the rejection of H-2d/Hh-1d but not H-2b/Hh-1b bone marrow cells. Thus NK cells exist as functionally separable subsets in vivo.

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Absence of NKG2D Ligands Defines Human Acute Myeloid Leukaemia Stem Cells and Mediates Their Immune Evasion

Blood ◽

10.1182/blood-2018-99-118047 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 769-769

Author(s):

Anna M Paczulla ◽

Kathrin Rothfelder ◽

Simon Raffel ◽

Martina Konantz ◽

Julia Steinbacher ◽

...

Keyword(s):

Stem Cells ◽

Nk Cells ◽

Immune Evasion ◽

Nk Cell ◽

Surface Expression ◽

Leukemic Cells ◽

Pdx Models ◽

Acute Myeloid

Abstract Patients with acute myeloid leukaemia (AML) often achieve remission but subsequently die of relapse driven by chemotherapy resistant leukemic stem cells (LSCs). To initiate and maintain cancer, LSCs must also escape immunosurveillance. However, in vivo studies on human LSCs largely disregard lymphocyte mediated anti-tumor immunity due to the use of immunocompromised mice. Here we investigate the immunosurveillance mediated by NKG2D, a danger detector expressed by cytotoxic lymphocytes such as natural killer (NK) cells that recognizes stress-induced ligands (NKG2DL) of the MIC and ULBP protein families on AML cells. Staining of n=175 de novo AML with antibodies against MICA, MICB and ULB2/5/6 or an NKG2D-Fc chimeric protein recognizing pan-NKG2DL expression revealed NKG2DL to heterogeneously express among leukemic cells of the same patient (Fig. 1a). As expected, NKG2DLpos AML cells were efficiently cleared by natural killer (NK) cells, while NKG2DLneg leukemic cells escaped NK cell lysis. Interestingly, these NKG2DLneg AML cells also showed immature morphology, enhanced in vitro clonogenicity (39±47 colonies vs. 1±4, p<0.001, n=32 AML cases) and selective abilities to initiate leukemia in NSG mice devoid of functional NK cells (NKG2DLneg, 33/35, 94%; NKG2DLpos, 0/35, 0%; p<0.001, n=13 AML cases, Fig. 1b) and to survive chemotherapy in vivo. In mice, NKG2DLneg AML cells generated both NKG2DLpos and NKG2DLneg progeny of which again only latter induced leukemia in re-transplant assays. Even though similar leukemia-specific mutations were retrieved in NKG2DLneg and NKG2DLpos AML cells derived from the same patient (n=12 analysed patients), published LSC, HSC and 17-genes stemness score signatures were specifically enriched in NKG2DLneg fractions. Mechanistically, expression of poly-ADP-ribose polymerase 1 (PARP1) was identified as enriched in NKG2DLneg compared to NKG2DLpos leukemic subpopulations, and PARP1 inhibition (PARPi) using either siRNAs or pharmacological inhibitors such as AG-14361, veliparib, talazoparib or olaparib, increased NKG2DL mRNA transcripts between 6 and >50 fold. PARP1 binding sites were identified by in silico analysis in NKG2DL promoters and binding was confirmed by chromatin immunoprecipitation in the promoters of MICA and MICB. Importantly, treatment with PARPi induced NKG2DL surface expression on LSCs in vitro and in vivo and co-treatment with PARPi and NK cells efficiently suppressed leukemogenesis in patient derived xenograft (PDX) models (Fig. 1c). These data suggest that PARP1 inhibition sensitizes LSCs to NK cell mediated elimination. Finally, NKG2DL surface expression was found to inversely correlate with favorable molecular AML characteristics (favorable ELN risk group vs. other: p=0.034; inv(16) versus other: p=0.023), complete remission rates after induction chemotherapy (all patients: p=0.002, patients <65 years: p=0.004) and patient overall survival (patients <65 years: p=0.028). Enhanced PARP1 expression in leukemic cells furthermore associated with poor clinical outcome (TCGA data set, p=0.0038). In summary, our data link the concept of LSCs to immune escape in human AML and propose the absence of immunostimulatory NKG2DL as a novel method to identify LSCs across genetic AML subtypes (including CD34 negative AMLs). This LSC specific mechanism of immune evasion could be overcome by treatment with PARP1 inhibitors, which in conjunction with functional NK cells holds promise to eradicate LSCs and promote immune-mediated cure of AML. Fig. 1: Human AML contain NKG2DLpos as well as NKG2DLneg subpopulations but only latter display leukemia initiation capacity (a: left, analysis of n=175 AML cases using NKG2D-Fc staining, right: exemplary flow cytometry plots; b: leukemic infiltration and survival in mice transplanted with NKG2DLneg or NKG2DLpos AML cells sorted from the same AML cases). PARP1 inhibition with AG-14361 up-regulates NKG2DL on CD34+ LSCs, and in vivo co-treatment with AG-14361 and polyclonal allogeneic NK cells suppresses leukemogenesis in PDX models (c). Figure. Figure. Disclosures Salih: Several patent applications: Patents & Royalties: e.g. EP3064507A1.

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High Level Expression of a Membrane-Anchored Inhibitor of HIV Infection in Murine Hematopoietic Stem and Progenitor Cells Does Not Pertubate Serial Multilineage Repopulation.

Blood ◽

10.1182/blood.v104.11.1758.1758 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1758-1758

Author(s):

Axel Schambach ◽

Bernhard Schiedlmeier ◽

Jens Bohne ◽

Dorothee von Laer ◽

Geoff Margison ◽

...

Keyword(s):

Stem Cells ◽

Bone Marrow ◽

Transgene Expression ◽

Bone Marrow Cells ◽

Hematopoietic Stem ◽

Murine Bone Marrow ◽

Hiv 1 ◽

Marrow Cells

Abstract T20 is a 36-amino-acid peptide that binds to HIV-1 gp41 and thereby acts as a fusion inhibitor, thus mediating potent and selective inhibition of HIV-1 entry in vitro and in vivo. An extended peptide expressed as an artificial, membrane-bound molecule (mbC46) efficiently inhibits HIV infection of primary human T-cells following retroviral vector mediated gene transfer (Egelhofer et al., J Virol, 2004). To develop an even more stringent approach to HIV gene therapy, we targeted hematopoietic stem cells. In 3 experimental groups of C57BL/6 mice (9 animals/group), we investigated the long-term toxicity of murine bone marrow cells transduced with M87o, a therapeutic vector designed to coexpress mbC46 and an HIV-derived RNA RRE-decoy to inhibit HIV replication. As controls we used the same vector containing an inactive C46 peptide and mock-transduced cells. Blood samples were collected monthly. Donor chimerism and transgene expression in multiple lineages were determined by FACS analysis and transgene integration was measured by real time PCR. Six months after transplantation, 4 mice per group were sacrificed and the remaining 5 mice per group were observed for another 6 months. In addition to the parameters mentioned above, we performed complete histopathology, blood counts and clinical biochemistry. Donor chimerism in all groups ranged from 82 – 94% (day 190 and day 349). In the M87o group, 60% of donor cells expressed mbC46. FACS data showed persisting transgene expression in T-cells (CD4, CD8, 65%), B-cells (B220, 46%), myeloid cells (CD11b, 68%), platelets (CD41, 19%), and RBC (60%) of the peripheral blood and bone marrow cells. Highly sustained gene marking (2–4 copies/genome) was noticed on day 190. To reveal latent malignant clones potentially originating from side effects of the genetic manipulation, 1x106 bone marrow cells from 4 primary recipients were transplanted into lethally irradiated secondary recipients (3 recipients/primary mouse) and these mice were observed for 8 months. All together, we could not observe any evidence for leukemogenic capacity. Analysis of peripheral blood and bone marrow showed a similar transgene expression pattern compared to the primary mice. To generate a complete chimerism of transgenic cells, we chose the human drug resistance gene methylguanine-methyltransferase (MGMT, P140K) to select for mbC46-transduced stem cells in vitro and in vivo. Different coexpression strategies were tested. Function of the MGMT protein was confirmed in a quantitative alkyltransferase assay and in a cytotoxicity assay using BCNU or temozolomide. In vitro selection of transduced 32D and PM1 cells with benzylguanine and BCNU showed >95% positive cells with evidence of polyclonal survival. Transduced PM1 cells underwent an HIV challenge assay. In vivo experiments in a murine bone marrow transplantation setting are ongoing to determine the potency and safety of combined retroviral expression of mbC46 and MGMT in relevant preclinical models. Successful conclusion of these studies will hopefully result in a phase I clinical trial testing the concept of generating an HIV-resistant autologous hematopoiesis.

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The Sooner the Better: Rapid Transduction Enhances the Engraftment/Selection of Gene Corrected Fanconi Anemia HSC.

Blood ◽

10.1182/blood.v110.11.194.194 ◽

2007 ◽

Vol 110 (11) ◽

pp. 194-194 ◽

Cited By ~ 1

Author(s):

Lars U.W. Muller ◽

Michael Milsom ◽

Chad E. Harris ◽

Jeff Bailey ◽

David A. Williams

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Bone Marrow ◽

Stem Cell ◽

Reporter Gene ◽

Fanconi Anemia ◽

Peripheral Blood ◽

Bone Marrow Cells

Abstract Fanconi anemia (FA) is amenable to genetic correction of hematopoietic stem cells (HSCs). However, as demonstrated in previous clinical gene therapy trials, successful extension of murine studies into human therapies is limited by low numbers of target HSC and poor engraftment of transduced FA HSC (Kelly et al., Mol Ther, 2007). To examine the potential biological consequences/benefits of shortened transduction we used a FA mouse model in which HSC are deficient and prone to excessive loss during in vitro manipulation. We applied a rapid transduction protocol (Mostoslavsky et al., Mol Ther, 2005) utilizing lentiviral vectors and demonstrate that this shortened transduction preserves engraftment of FA HSC to the level of C57BL/6 wt cells. Lin− Sca-1+ c-Kit+ bone marrow cells were isolated from Fanca−/− CD45.2 mice and underwent 4-hr rapid (RT) vs. 96-hr conventional (CT) transduction. An equivalent number of transduced cells were transplanted into lethally irradiated CD45.1 BoyJ mice. Analysis of engraftment chimerism three months post transplantation revealed a significantly higher level of engraftment in animals receiving RT vs. CT cells (90% +/− 14% vs. 26% +/− 31%, respectively, p=<0.01). Rapid transduction also resulted in a significant reduction of engraftment failure (0/36 animals RT vs. 20/36 animals CT). Importantly--emphasizing the FA disease-specific stem cell phenotype, RT vs. CT of C57BL/6 wt cells was associated with no significant difference in engraftment of these cells (93% +/− 1.2% RT vs. 84 +/− 19% CT, p=0.33). Analysis of peripheral blood cells expressing the proviral enhanced green fluorescent protein (eGFP) reporter gene revealed a normal distribution of B-lymphocytes (B220), T-lymphocytes (CD3 epsilon), and granulocytes (MAC-1), indicating multi-lineage engraftment of gene modified cells. In spite of this engraftment advantage, transduction efficiency was low (<30%) using RT. The 6-benzylguanine (6-BG) resistant P140K mutant of O6-methylguanine DNA methyltransferase (MGMTP140K) confers a selective advantage to tranduced HSC treated with alkylating drugs. Following RT with a MGMTP140K/ eGFP expressing lentivirus, 5/6 mice treated with 6-BG and the alkylating drug temozolomide showed a significant rise in the percentage of GFP reporter gene expression in peripheral blood. We extended this approach to the FA model by generating a tri-cistronic lentiviral vector expressing the FANCA cDNA, MGMTP140K, and eGFP. Despite modest in vivo gene marking with this vector, up to 37-fold selection (85% GFP-positive cells) was achieved following exposure of bone marrow of transplant recipients to 6-BG and the alkylating drug temozolomide in vitro. Concurrently, phenotypic correction of mitomycin C hypersensitivity of transduced Fanca−/− bone marrow cells was observed. These data suggest that RT improves stem cell engrafting capacity of FA stem cells in a relevant animal model of stem cell gene therapy. The combination of RT and in vivo selection may allow more successful reconstitution of the lympho-hematopoietic system in gene therapy applications.

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Target Transplantation of Mesenchymal Stem Cells into the Murine Femur to Support Hematopoiesis By Magic-TT: Both In Vivo and in Vitro Studies

Blood ◽

10.1182/blood.v126.23.5409.5409 ◽

2015 ◽

Vol 126 (23) ◽

pp. 5409-5409

Author(s):

Qianli Jiang ◽

Hao Huang ◽

Yongjun Zhou ◽

Qiuxia Zhang ◽

Xiaowei Sun ◽

...

Keyword(s):

Magnetic Field ◽

Stem Cells ◽

Bone Marrow ◽

Bone Marrow Cells ◽

In Vitro Study ◽

Nano Particle ◽

Cell Target ◽

Differential Ability

Abstract BACKGROUND: Mesenchymal stem cells (MSCs) are considered to be the most promising non-hematopoietic multipotent stem cells. They are widely applied in the treatment of aplastic anemia, graft failure and GVHD post transplantation etc. However, the mechanism of MSCs homing, proliferation and the interaction with microenvironment in bone remain unclear. Based on our previous work (2014ASH poster, No.2416), a novel cell target transplantation system is developed, which is named MagIC-TT (Magnetism-induced cell target transplantation). OBJECTIVE: To explore whether MagIC-TT can help the MSCs target homing into the bone marrow and support hematopoiesis. Methods : 1) Magnetized cells: The C57BL/6 RFP-MSCs were magnetized by Au@Fe nano-particle, sorted by MACS column. 2) In vitro study: Both magnetized and wild type cells were compared with morphology, cell proliferation curve, cell cycle and cell differential ability. Under the effect of magnetic field, magnetized MSCs viability, migration, proliferation and co-culture with scal-1+ GFP bone marrow cells in vitro, or within 3D artificial bone scafold culture system were studied. To understand the underlining mechanism , FACS, confocal microscopy and Lightsheet Z.1 Microimage (Carl Zeiss), immunohistological staining, real-time PCR for GFP and RFP cells, deep sequencing, as well as own derived semi-solid decalcification (SSD) technique were used. 3) In vivo study: Twenty wide type C57BL/6 and another 20 female C57BL/6-Tg(CAG-EGFP) mice were used, 10 mice in each group. Magnetized MSCs were transplanted into the femur cavity of the mice by MagIC-TT, intra-femur or i.v.. Following transplantation, the above mentioned research methods as well as bioluminescence by Xenogen IVIS Image System (Lumina) were performed. Results: 1) In-vitro study: There were no differences in cell morphology, growth curve and differentiation, etc. Prussian blue staining of MSCs showed intracytoplasmic Au@Fe nano-particle inclusions as dense blue-stained vesicles, the particles exist within or on the surface of magnetized cells (Fig.1). Magnetized cells can migrate through matrigel and transwell membranes much more efficiently under the magnetic field, 174¡À22 vs. 2¡À1 per 200X microscopic vision (P<0.0001). The cells advance toward the inner roof of 24-plate and undergo proliferation (Fig. 1). Scal-1+ GFP bone marrow cells can differentiate into megakaryocyte cells with the support of magnetized MSCs. 2) In vivo study: Bioluminescence assay by Xenogen IVIS Imaging System showed that MSCs were blocked in the lung in non-MagIC-TT group mice 15 minutes after cell injection, while no signal was found in the lung of MagIC-TT group mice (Fig. 2). More RFP+ cells were found in the MagIC-TT treated femur compared with the other femur in the same mouse by FACS (Table 1), pathological examination showed that lots of RFP-MSCs reside within the bone marrow MagIC-TT treated femur than the controls (Fig. 2), thereby demonstrating the success of MSCs target transplantation. The transplanted MSCs support hematopoiesis. Moreover, the relationship of RFP donor cells with surrounding cells was clearly illustrated within the bone (Fig. 2). Those MSCs were found to survive more than 3 months in the bone, lung, liver, spleen, etc. Conclusion: MSCs target transplanted into the murine bone marrow by MagIC-TT, provide ideal support towards hematopoietic proliferation and differential ability. This technique maybe helpful to treat aplastic anemia, graft failure and GVHD, as well as other cell therapies in the future. Table 1. Comparison of the number of RFP-MSCs in two groups within 1h and 24h by flow cytometry. group 1h£¨%£© p 24h£¨%£© p *LC **RT *LC **RT BMM 0.015¡À0.006 0.255¡À0.145 0.010 0.078¡À0.022 0.965¡À0.732 0.094 BMW 0.013¡À0.006 0.247¡À0.201 0.115 0.073¡À0.045 0.368¡À0.301 0.144 P 0.685 0.944 0.851 0.182 *LC: Control femur without magnetic field (W group); **RT: Treated femur with magnetic field (M group). Disclosures No relevant conflicts of interest to declare.

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