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Chimeric antigen receptors (CARs) can effectively redirect cytotoxic T cells toward highly expressed surface antigens on tumor cells. The low expression of several tumor-associated antigens (TAAs) on normal tissues, however, hinders their safe targeting by CAR T cells due to on-target/off-tumor effects. Using the multiple myeloma (MM)-associated CD38 antigen as a model system, here, we present a rational approach for effective and tumor-selective targeting of such TAAs. Using “light-chain exchange” technology, we combined the heavy chains of two high-affinity CD38 antibodies with 176 germline light chains and generated ∼124 new antibodies with 10- to >1,000-fold lower affinities to CD38. After categorizing them into three distinct affinity classes, we incorporated the single-chain variable fragments of eight antibodies from each class into new CARs. T cells carrying these CD38-CARs were extensively evaluated for their on-tumor/off-tumor cytotoxicity as well as CD38-dependent proliferation and cytokine production. We identified CD38-CAR T cells of ∼1,000- fold reduced affinity, which optimally proliferated, produced Th1-like cytokines, and effectively lysed CD382+ MM cells, but spared CD38+ healthy hematopoietic cells in vitro and in vivo. Thus, this systematic approach is highly suitable for the generation of optimal CARs for effective and selective targeting of TAAs.

Multiple myeloma is characterized by accumulation of malignant plasma cells in the bone marrow. Most patients suffer from an osteolytic bone disease, caused by increased bone degradation and reduced bone formation. Bone morphogenetic protein 4 (BMP4) is important for both pre‐ and postnatal bone formation and induces growth arrest and apoptosis of myeloma cells. BMP4‐treatment of myeloma patients could have the potential to reduce tumor growth and restore bone formation. We therefore explored BMP4 gene therapy in a human‐mouse model of multiple myeloma where humanized bone scaffolds were implanted subcutaneously in RAG2−/− γC−/−mice. Mice were treated with adeno‐associated virus serotype 8 BMP4 vectors (AAV8‐BMP4) to express BMP4 in the liver. When mature BMP4 was detectable in the circulation, myeloma cells were injected into the scaffolds and tumor growth was examined by weekly imaging. Strikingly, the tumor burden was reduced in AAV8‐BMP4 mice compared with the AAV8‐CTRL mice, suggesting that increased circulating BMP4 reduced tumor growth. BMP4‐treatment also prevented bone loss in the scaffolds, most likely due to reduced tumor load. To delineate the effects of BMP4 overexpression on bone per se, without direct influence from cancer cells, we examined the unaffected, non‐myeloma femurs by μCT. Surprisingly, the AAV8‐BMP4 mice had significantly reduced trabecular bone volume, trabecular numbers, as well as significantly increased trabecular separation compared with the AAV8‐CTRL mice. There was no difference in cortical bone parameters between the two groups. Taken together, BMP4 gene therapy inhibited myeloma tumor growth, but also reduced the amount of trabecular bone in mice. Our data suggest that care should be taken when considering using BMP4 as a therapeutic agent. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.

Acute leukemia is a clonal expansion of malignant hematopoietic cells leading to impaired production of normal blood cells in the bone marrow (BM). Acute leukemia can be classified, according to the lineage involved, into acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL).[1][1] AML

Dysregulation of polycomb repressive complex 2 (PRC2) promotes oncogenesis partly through its enzymatic function for inducing trimethylation of histone H3 lysine 27 (H3K27me3). However, it remains to be determined how PRC2 activity is regulated in normal and diseased settings. We here report a PRC2-associated cofactor, PHD finger protein 19 (PHF19; also known as polycomb-like 3), as a crucial mediator of tumorigenicity in multiple myeloma (MM). Overexpression and/or genomic amplification of PHF19 is found associated with malignant progression of MM and plasma cell leukemia, correlating to worse treatment outcomes. Using various MM models, we demonstrated a critical requirement of PHF19 for tumor growth in vitro and in vivo. Mechanistically, PHF19-mediated oncogenic effect relies on its PRC2-interacting and chromatin-binding functions. Chromatin immunoprecipitation followed by sequencing profiling showed a critical role for PHF19 in maintaining the H3K27me3 landscape. PHF19 depletion led to loss of broad H3K27me3 domains, possibly due to impaired H3K27me3 spreading from cytosine guanine dinucleotide islands, which is reminiscent to the reported effect of an “onco”-histone mutation, H3K27 to methionine (H3K27M). RNA-sequencing-based transcriptome profiling in MM lines also demonstrated a requirement of PHF19 for optimal silencing of PRC2 targets, which include cell cycle inhibitors and interferon-JAK-STAT signaling genes critically involved in tumor suppression. Correlation studies using patient sample data sets further support a clinical relevance of the PHF19-regulated pathways. Lastly, we show that MM cells are generally sensitive to PRC2 inhibitors. Collectively, this study demonstrates that PHF19 promotes MM tumorigenesis through enhancing H3K27me3 deposition and PRC2's gene-regulatory functions, lending support for PRC2 blockade as a means for MM therapeutics.

Polycomb proteins are classical regulators of stem cell self-renewal and cell lineage commitment and are frequently deregulated in cancer. Here, we find that the non-canonical PRC1.1 complex, as identified by mass-spectrometry-based proteomics, is critically important for human leukemic stem cells. Downmodulation of PRC1.1 complex members, like the DNA-binding subunit KDM2B, strongly reduces cell proliferation in vitro and delays or even abrogates leukemogenesis in vivo in humanized xenograft models. PRC1.1 components are significantly overexpressed in primary AML CD34(+) cells. Besides a set of genes that is targeted by PRC1 and PRC2, ChIP-seq studies show that PRC1.1 also binds a distinct set of genes that are devoid of H3K27me3, suggesting a gene-regulatory role independent of PRC2. This set encompasses genes involved in metabolism, which have transcriptionally active chromatin profiles. These data indicate that PRC1.1 controls specific genes involved in unique cell biological processes required for leukemic cell viability.

TH and MW contributed equally to this work. Background Multiple myeloma is caused by an accumulation of malignant plasma cells in the bone marrow. Myeloma is characterized by an osteolytic bone disease, caused by increased bone degradation and reduced bone formation. Bone morphogenetic proteins (BMPs) are members of the transforming growth factor (TGF)-β superfamily. BMP-signaling is important for both pre- and postnatal bone formation. Additionally, several BMPs induce growth arrest and apoptosis in myeloma cells. Thus, increasing BMP-signaling in myeloma patients may reduce tumor growth and restore bone formation. We therefore explored BMP4 gene therapy in a human-mouse model of multiple myeloma. Methods Calcium phosphate scaffolds with human mesenchymal stromal cells (MSCs) were implanted in RAG2-/-GC-/- mice and the MSCs were left to differentiate in vivo for 8 weeks to create a humanized bone microenvironment. Then, adeno-associated virus (AAV), AAV8-BMP4, which has tropism for liver cells and expresses murine Bmp4 under the control of the liver specific human α1-antitrypsin (hAAT1) promoter, were administered by tail-vein injection. Empty viral vectors, AAV8-CTRL, were used for the control group. After 2 weeks, when BMP4 was detectable in circulation, we injected fluorescently labelled KJON myeloma cells in 3 out of 4 scaffolds in each mouse. The KJON cells are hyperdiploid, have a relatively slow growth rate and rely on interleukin (IL)-6 supplementation in the absence of a supporting microenvironment, thus resembling primary human myeloma cells. Tumor growth was examined by weekly imaging until end-point, 6 weeks after tumor cell injection. Results At end-point, serum levels of BMP4 in AAV8-BMP4 mice were in the range of 50-200 ng/mL, but not detectable in AAV8-CTRL mice. Strikingly, tumor growth as quantified by imaging was significantly reduced in AAV8-BMP4 mice compared with the AAV8-CTRL mice (p We hypothesized that increased circulating BMP4 would also be beneficial for bone in this model. Human bone is generated on the scaffolds by osteoblasts that differentiate from human MSCs seeded on the scaffolds before implantation. In this model, myeloma cells inhibit osteoblast differentiation and bone formation. Indeed, in AAV8-CTRL mice we found less bone in tumor cell-containing scaffolds compared with scaffolds without tumor cells (p Conclusion Taken together, BMP4 gene therapy inhibited myeloma tumor growth, but also reduced trabecular bone formation in mice. Care should be taken when considering BMP4 as a therapeutic agent. If other BMPs that are also potent inhibitors of multiple myeloma cell survival and proliferation will have similar impact on bone remains to be investigated. Disclosures No relevant conflicts of interest to declare.

Immunotherapy with allogeneic natural killer (NK) cells offers therapeutic perspectives for multiple myeloma patients. Here, we aimed to refine NK cell therapy by evaluation of the relevance of HLA-class I and HLA-E for NK anti-myeloma reactivity. We show that HLA-class I was strongly expressed on the surface of patient-derived myeloma cells and on myeloma cell lines. HLA-E was highly expressed by primary myeloma cells but only marginally by cell lines. HLA-Elow expression on U266 cells observed in vitro was strongly upregulated after in vivo (bone marrow) growth in RAG-2−/−γc−/− mice, suggesting that in vitro HLA-E levels poorly predict the in vivo situation. Concurrent analysis of inhibitory receptors (KIR2DL1, KIR2DL2/3, KIR3DL1 and NKG2A) and NK cell degranulation upon co-culture with myeloma cells revealed that KIR–ligand-mismatched NK cells degranulate more than matched subsets and that HLA-E abrogates degranulation of NKG2A+ subsets. Inhibition by HLA-class I and HLA-E was also observed with IL-2-activated NK cells and at low oxygen levels (0.6 %) mimicking hypoxic bone marrow niches where myeloma cells preferentially reside. Our study demonstrates that NKG2A-negative, KIR–ligand-mismatched NK cells are the most potent subset for clinical application. We envision that infusion of high numbers of this subclass will enhance clinical efficacy. Electronic supplementary material The online version of this article (doi:10.1007/s00262-015-1694-4) contains supplementary material, which is available to authorized users.

Purpose: Novel therapeutic agents have significantly improved the survival of patients with multiple myeloma. Nonetheless, the prognosis of patients with multiple myeloma who become refractory to the novel agents lenalidomide and bortezomib is very poor, indicating the urgent need for new therapeutic options for these patients. The human CD38 monoclonal antibody daratumumab is being evaluated as a novel therapy for multiple myeloma. Prompted with the encouraging results of ongoing clinical phase I/II trials, we now addressed the potential value of daratumumab alone or in combination with lenalidomide or bortezomib for the treatment of lenalidomide- and bortezomib-refractory patients. Experimental Design: In ex vivo assays, mainly evaluating antibody-dependent cell-mediated cytotoxicity, and in an in vivo xenograft mouse model, we evaluated daratumumab alone or in combination with lenalidomide or bortezomib as a potential therapy for lenalidomide- and bortezomib-refractory multiple myeloma patients. Results: Daratumumab induced significant lysis of lenalidomide/bortezomib-resistant multiple myeloma cell lines and of primary multiple myeloma cells in the bone marrow mononuclear cells derived from lenalidomide- and/or bortezomib-refractory patients. In these assays, lenalidomide but not bortezomib, synergistically enhanced daratumumab-mediated multiple myeloma lysis through activation of natural killer cells. Finally, in an in vivo xenograft model, only the combination of daratumumab with lenalidomide effectively reduced the tumorigenic growth of primary multiple myeloma cells from a lenalidomide- and bortezomib-refractory patient. Conclusions: Our results provide the first preclinical evidence for the benefit of daratumumab plus lenalidomide combination for lenalidomide- and bortezomib-refractory patients. Clin Cancer Res; 21(12); 2802–10. ©2014 AACR. See related commentary by Laubach and Richardson, p. 2660

Objective Autologous stem cell transplantation (ASCT) induces long-term drug-free disease remission in patients with juvenile idiopathic arthritis. This study was undertaken to further unravel the immunologic mechanisms underlying ASCT by using a mouse model of proteoglycan-induced arthritis (PGIA). Methods For initiation of PGIA, BALB/c mice received 2 intraperitoneal injections of human PG in a synthetic adjuvant on days 0 and 21. Five weeks after the first immunization, the mice were exposed to total body irradiation (7.5 Gy) and received (un)manipulated bone marrow (BM) grafts from mice with PGIA. Clinical scores, T cell reconstitution, (antigen-specific) T cell cytokine production, and intracellular cytokine expression were determined following autologous BM transplantation (ABMT). Results ABMT resulted in amelioration and stabilization of arthritis scores. BM grafts containing T cells and T cell–depleted grafts provided the same clinical benefit, with similar reductions in PG-induced T cell proliferation and the number of PG-specific autoantibodies. In vivo reexposure to PG did not exacerbate disease. Following ABMT, basal levels of disease-associated proinflammatory cytokines (interferon-γ [IFNγ], interleukin-17 [IL-17], and tumor necrosis factor α [TNFα]) were reduced. In addition, restimulation of T cells with PG induced a strong reduction in disease-associated proinflammatory cytokine production. Finally, although the remaining host T cells displayed a proinflammatory phenotype following ABMT, IFNγ, IL-17, and TNFα production by the newly reconstituted donor-derived T cells was significantly lower. Conclusion Taken together, our data suggest that ABMT restores immune tolerance by renewal and modulation of the Teff cell compartment, leading to a strong reduction in proinflammatory (self antigen–specific) T cell cytokine production.

Item does not contain fulltext Bone marrow (BM) stromal cells (MSCs), also known as mesenchymal stem cells, display a high degree of heterogeneity. To shed light on the causes of this heterogeneity, MSCs were collected from either human BM (n=5) or adipose tissue (AT) (n=5), and expanded using 2 different culture methods: one based on fetal calf serum, and one based on human platelet lysate. After initial expansion, MSCs were frozen, and the vials were transported to 3 different laboratories and grown for 1 passage using the same brand of culture plastic, medium, and supplements. Subsequently, the cells were harvested and assayed for their gene expression profile using the Affymetrix exon microarray platform. Based on gene expression profiles, the most discriminative feature was the anatomical harvesting site, followed by culture methodology. Remarkably, genes in the WNT pathway were expressed at higher levels in BM-derived MSCs than in AT-derived MSCs. Although differences were found between laboratories, cell culture location only slightly affects heterogeneity. Furthermore, individual donors contributed marginally to the observed differences in transcriptomes. Finally, BM-derived MSCs displayed the highest level of similarity, irrespective their culture conditions, when compared to AT-derived cells.

The combination of scaffolds and mesenchymal stromal cells (MSCs) is a promising approach in bone tissue engineering (BTE). Knowledge on the survival, outgrowth and bone-forming capacity of MSCs in vivo is limited. Bioluminescence imaging (BLI), histomorphometry and immunohistochemistry were combined to study the fate of gene-marked goat and human MSCs (gMSCs, hMSCs) on scaffolds with different osteoinductive properties. Luciferase–GFP-labelled MSCs were seeded on hydroxyapatite (HA) or β-tricalcium phosphate (TCP), cultured for 7 days in vitro in osteogenic medium, implanted subcutaneously in immunodeficient mice and monitored with BLI for 6 weeks. The constructs were retrieved and processed for histomorphometry and detection of luciferase-positive cells (LPCs). For gMSCs, BLI revealed doubling of signal after 1 week, declining to 60% of input after 3 weeks and remaining constant until week 6. hMSCs showed a constant decrease of BLI signal to 25% of input, indicating no further expansion. Bone formation of gMSCs was two-fold higher on TCP than HA. hMSCs and gMSCs control samples produced equal amounts of bone on TCP. Upon transduction, there was a four-fold reduction in bone formation compared with untransduced hMSCs, and no bone was formed on HA. LPCs were detected at day 14, but were much less frequent at day 42. Striking differences were observed in spatial distribution. MSCs in TCP were found to be aligned and interconnected on the surface but were scattered in an unstructured fashion in HA. In conclusion, the spatial distribution of MSCs on the scaffold is critical for cell–scaffold-based BTE

Interactions within the hematopoietic niche in the BM microenvironment are essential for maintenance of the stem cell pool. In addition, this niche is thought to serve as a sanctuary site for malignant progenitors during chemotherapy. Therapy resistance induced by interactions with the BM microenvironment is a major drawback in the treatment of hematologic malignancies and bone-metastasizing solid tumors. To date, studying these interactions was hampered by the lack of adequate in vivo models that simulate the human situation. In the present study, we describe a unique human-mouse hybrid model that allows engraftment and outgrowth of normal and malignant hematopoietic progenitors by implementing a technology for generating a human bone environment. Using luciferase gene marking of patient-derived multiple myeloma cells and bioluminescent imaging, we were able to follow pMM cells outgrowth and to visualize the effect of treatment. Therapeutic interventions in this model resulted in equivalent drug responses as observed in the corresponding patients. This novel human-mouse hybrid model creates unprecedented opportunities to investigate species-specific microenvironmental influences on normal and malignant hematopoietic development, and to develop and personalize cancer treatment strategies.

Although mesenchymal stromal cells (MSCs) have been applied clinically to treat cardiac diseases, it is unclear how and to which extent transplanted MSCs exert their beneficial effects. To address these questions, pre-clinical MSC administrations are needed for which pigs appear to be the species of choice. This requires the use of porcine cells to prevent immune rejection. However, it is currently unknown to what extent porcine MSCs (pMSCs) resemble human MSCs (hMSCs). Aim of this study was to compare MSC from porcine bone marrow (BM) with human cells for phenotype, multi-lineage differentiation potential, immune-modulatory capacity and the effect on cardiac function after transplantation in a mouse model of myocardial infarction. Flow cytometric analysis revealed that pMSC expressed surface antigens also found on hMSC, including CD90, MSCA-1 (TNAP/W8B2 antigen), CD44, CD29 and SLA class I. Clonogenic outgrowth was significantly enriched following selection of CD271+ cells from BM of human and pig (129 ± 29 and 1961 ± 485 fold, respectively). hMSC and pMSC differentiated comparably into the adipogenic, osteogenic or chondrogenic lineages, although pMSC formed fat much faster than hMSC. Immuno-modulation, an important feature of hMSC, was clearly demonstrated for pMSC when co-cultured with porcine peripheral blood cells stimulated with PMA and pIL-2. Finally, pMSC transplantation after myocardial infarction attenuated adverse remodelling to a similar extent as hMSC when compared to control saline injection. These findings demonstrate that pMSCs have comparable characteristics and functionality with hMSCs, making reliable extrapolation of pre-clinical pMSC studies into a clinical setting very well possible.

Graft-versus-host disease (GVHD) remains a frequently occurring and difficult-to-treat complication in human allogeneic stem cell transplantation. Murine transplantation models are often used to study and understand the complex pathogenesis of GVHD and to explore new treatment strategies. Although GVHD kinetics may differ in murine and human models, adequate models are essential for identification of the crucial factors responsible for the major pathology in GVHD. We present a detailed description of the specific histological features of a graft-versus-host-induced fibrotic response in xenogeneic RAG2(-/-)γc(-/-) mice after total body irradiation and injection with human peripheral blood mononuclear cells. We describe the full morphological features of this reaction, including a detailed analysis of the specific tissue infiltration patterns of the human peripheral blood mononuclear cells. Our data show the development of fibrosis, predominantly near blood vessels, and reveal different cell populations and specific cell migration patterns in the affected organs. The combination of immunohistochemical cell characterization and mRNA expression analysis of both human (donor)- and murine (host)-derived cytokines reveals an interaction between host tissues and donor-derived cells in an entangled cytokine profile, in which both donor- and host-derived cytokines contribute to the formation of fibrosis.

Lgr5 stem cells reside at small intestinal crypt bottoms, generating both the enterocyte and secretory lineage. Entry into the latter epithelial lineage requires silencing of Notch signaling. The Notch ligand Dll1 is strongly up-regulated in a small subset of immediate stem cell daughters. Lineage tracing utilizing a novel Dll1GFP-ires-CreERT2 knock-in mouse reveals that single Dll1high cells generate small, short-lived clones of all four secretory cell types. In culture, sorted Dll1high cells can form long-lived organoids when briefly exposed to Wnt3A. When Dll1 cells are genetically marked prior to tissue damage, significant numbers of stem cell tracing events occur. Lineage specification therefore occurs already in the earliest stem cell daughters through Notch lateral inhibition. Yet, specified secretory progenitors display plasticity and can regain stemness upon tissue damage.

Previously, we have reported that our human bone marrow (BM)-like scaffold xenograft model allows the engraftment and outgrowth of normal and malignant hematopoiesis (e.g. multiple myeloma (MM), acute myelocytic/lymphocytic leukemia (AML/ALL) and MDS (Groen et al. Blood 2012; Gutierrez et al. JCI 2014 and data not shown). Whereas the presence of osteoblasts and bone of human origin mimics a human BM-like niche more closely than the murine BM in standard xenotransplant models (e.g. NOD-SCID/NSG mice), still some essential components of the human BM niche, i.e. human blood vessels, are missing. To this end, in addition to human mesenchymal stromal cells we now incorporated cord blood-derived endothelial progenitor cells (CB-EPCs) in the hybrid scaffold production process, to create a multi-tissue compartment that "maximally humanizes" the BM-like niche of our scaffolds. Towards successful implementation of a human vascular system we compared: i) scaffold material composition (biphasic calcium phosphate (BCP) vs. tricalcium phosphate (TCP)); ii) scaffold shape (particles vs. tubes); iii) different types of matrigel for CB-EPC embedding. Histological analysis of the humanized scaffolds, eight weeks after implantation in mice, showed a large number of functional human blood vessels, as indicated by hCD31+ staining and the presence of erythrocytes within. Comparison of the composition and the shapes of the scaffolds indicated superiority of TCP and tube-shaped scaffolds in supporting the formation of vessels. Further analysis of scaffolds for CD44, CD146, LEPR and nestin-positive cells, revealed the presence of other stromal niche cells besides human osteoblasts and endothelial cells. Irradiation of mice carrying these humanized implants did not have a significant deleterious effect on the established human vessels, allowing their further functional evaluation in xenotransplantation. Additionally, mice carrying tubes with and without human CB-EPC derived vessels (on either flank) were subsequently inoculated with adult BM-derived CD34-positive cells by intracardiac injection. Upon analysis 12 weeks later, all tubes showed multi-lineage hematopoietic outgrowth. Interestingly, CB-EPC embedment resulted in increased numbers of CD45+ (2-fold), CD13+ (4-fold) and CD7+ (2-fold), while CD19+ cell numbers were equal. In contrast, in mouse BM almost only CD19+ cells could be detected. Moreover, we observed that the use of CB-EPCs in our scaffolds provides faster kinetics of in vivo engraftment and growth of both patient-derived MM or AML cells. With the addition of both human CB-EPCs and human BM stromal cells, our scaffold systems now simulate both human endosteal and vascular niches of the BM, thereby more closely recapitulating the human hematopoietic niche. Disclosures Yuan: Xpand Biotechnology BV: Employment. de Bruijn:Xpand Biotechnology BV: Employment. Mitsiades:TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Novartis: Research Funding. Martens:Johnson & Johnson: Research Funding. Groen:Johnson & Johnson: Research Funding.

Major limitations of currently investigated αβT cells redirected against cancer by transfer of tumor-specific αβTCR arise from their low affinity, MHC restriction, and risk to mediate self-reactivity after pairing with endogenous α or βTCR chains. Therefore, the ability of a defined γ9δ2TCR to redirect αβT cells selectively against tumor cells was tested and its molecular interaction with a variety of targets investigated. Functional analysis revealed that a γ9δ2TCR efficiently reprograms both CD4+ and CD8+ αβT cells against a broad panel of cancer cells while ignoring normal cells, and substantially reduces but does not completely abrogate alloreactivity. γ9δ2TCR-transduced αβT cells reduced colony formation of progenitor cells of primary acute myeloid leukemia blasts and inhibited leukemia growth in a humanized mouse model. Thereby, metabolites of a dysregulated mevalonate pathway are targeted and the additional application of widely used biphosphonates is crucial for in vivo efficacy most likely because of its modulating effect on cytokine secretion of γ9δ2TCR-transduced αβT cells. Expression of NKG2D ligands and F1-ATPase contributed to the activity of γ9δ2TCR-transduced αβT cells but were not mandatory. In summary, γ9δ2 TCRs are an attractive alternative to broadly redirect αβT cells against cancer cells with both an improved efficacy and safety profile compared with currently used αβTCRs.

Purpose: The essential role of CD4+ T cells as helpers of anticancer immunity is indisputable. Little is known, however, about their capacity to serve as effector cells in cancer treatment. Therefore, we explored the efficacy of immunotherapy with sole CD4+ cytotoxic human T cells directed at a hematopoietic-restricted minor histocompatibility antigen (mHag). Experimental Design: In macrophage-depleted Rag2−/−γc−/− mice, which were also devoid of T, B, and natural killer cells, mHag-specific native T cells or tetanus toxoid (TT)-specific T cells transduced with the mHag-specific T-cell receptor (TCR) were injected to treat full-blown mHag+ human multiple myeloma tumors. Results: mHag-specific antitumor responses were achieved after injection of native or mHag-TCR-transduced T cells. Although the therapy completely eradicated the primary tumors in the bone marrow, it failed to control extramedullary relapses, even after repeated T-cell injections. Detailed analyses ruled out mHag or MHC downregulation as mechanisms of extramedullary tumor escape. Impaired T-cell survival in vivo or defective homing to the tumor site were also ruled out as mechanisms behind extramedullary relapses, because injections of TT-loaded antigen presenting cells could facilitate homing of long-term surviving T cells to s.c. tumor sites. Moreover, intratumoral treatment of extramedullary tumors with 3AB11 was also ineffective. Conclusions: Taken together, these results for the first time show the feasibility of immunotherapy of primary bone marrow tumors with sole CD4+ human T cells directed to a tumor-associated mHag. Extramedullary relapses, probably due to microenvironment-dependent inhibitory mechanisms, remain a challenging issue towards effective cellular immunotherapy of hematologic malignancies. Clin Cancer Res; 16(22); 5481–8. ©2010 AACR.

Tissue engineering of bone, by combining multipotent stromal cells (MSCs) with osteoconductive scaffolds, has not yet yielded any clinically useful applications so far. The fate and contribution of the seeded cells are not sufficiently clarified, especially at clinically relevant locations. Therefore, we investigated cell proliferation around the spine and at ectopic sites using noninvasive in vivo bioluminescence imaging (BLI) in relation to new bone formation. Goat MSCs were lentivirally transduced to express luciferase. After showing both correlation between MSC viability and BLI signal as well as survival and osteogenic capacity of these cells ectopically in mice, they were seeded on ceramic scaffolds and implanted in immunodeficient rats at two levels in the spine for spinal fusion as well as subcutaneously. Nontransduced MSCs were used as a control group. All rats were monitored at day 1 and after that weekly until termination at week 7. In mice a BLI signal was observed during the whole observation period, indicating survival of the seeded MSCs, which was accompanied by osteogenic differentiation in vivo. However, these same MSCs showed a different response in the rat model, where the BLI signal was present until day 14, both in the spine and ectopically, indicating that MSCs were able to survive at least 2 weeks of implantation. Only when the signal was still present after the total implantation period ectopically, which only occurred in one rat, new bone was formed extensively and the implanted MSCs were responsible for this bone formation. Ectopically, neither a reduced proliferative group (irradiated) nor a group in which the cells were devitalized by liquid nitrogen and the produced extracellular matrix remained (matrix group) resulted in bone formation. This suggests that the release of soluble factors or the presence of an extracellular matrix is not enough to induce bone formation. For the spinal location, the question remains whether the implanted MSCs contribute to the bone regeneration or that the principal mechanism of MSC activity is through the release of soluble mediators.

Background Incorporation of the chimeric CD20 monoclonal antibody rituximab in the treatment schedule of patients with non-Hodgkin’s lymphoma has significantly improved outcome. Despite this success, about half of the patients do not respond to treatment or suffer from a relapse and additional therapy is required. A low CD20-expression level may in part be responsible for resistance against rituximab. We therefore investigated whether the CD20-expression level related resistance to rituximab could be overcome by a new group of CD20 mAbs (HuMab-7D8 and ofatumumab) targeting a unique membrane-proximal epitope on the CD20 molecule.Design and Methods By retroviral transduction of the CD20 gene into CD20-negative cells and clonal selection of transduced cells a system was developed in which the CD20-expression level is the only variable. These CD20 transduced cells were used to study the impact of rituximab and HuMab-7D8 mediated complement-dependent cytotoxicity. To study the in vivo efficacy of these mAbs an in vivo imaging system was generated by retroviral expression of the luciferase gene in the CD20-positive cells.Results We show that HuMab-7D8 efficiently killed CD20low cells that are not susceptible to rituximab-induced killing in vitro. In a mouse xenograft model, we observed a comparable increase in survival time between HuMab-7D8 and rituximab-treated mice. Most significantly, however, HuMab-7D8 eradicated all CD20-expressing cells both in the periphery as well as in the bone marrow whereas after rituximab treatment CD20low cells survived.Conclusions Cells that are insensitive to in vitro and in vivo killing by rituximab as the result of their low CD20-expression profile may be efficiently killed by an antibody against the membrane-proximal epitope on CD20. Such antibodies should, therefore, be explored to overcome rituximab resistance in the clinic.

Antibody–Fc receptor (FcR) interactions play an important role in the mechanism of action of most therapeutic antibodies against cancer. Effector cell activation through FcR triggering may induce tumor cell killing via antibody-dependent cellular cytotoxicity (ADCC). Reciprocally, FcR cross-linking of antibody may lead to the induction of apoptotic signaling in tumor cells. The relative importance of these bisecting pathways to in vivo antibody activity is unknown. To unravel these roles, we developed a novel mouse model with normal FcR expression but in which FcR signaling was inactivated by mutation of the associated γ-chain. Transgenic mice showed similar immune complex binding compared with wild-type mice. In contrast, ADCC of cells expressing frequently used cancer targets, such as CD20, epidermal growth factor receptor, Her2, and gp75, was abrogated. Using the therapeutic CD20 antibodies ofatumumab and rituximab, we show that FcR cross-linking of antibody-antigen immune complexes in the absence of γ-chain signaling is insufficient for their therapeutic activity in vivo. ADCC therefore represents an essential mechanism of action for immunotherapy of lymphoid tumors. Cancer Res; 70(8); 3209–17. ©2010 AACR.

Multiple myeloma (MM) is an incurable B-cell neoplasia in which progressive skeletal lesions are a characteristic feature. Earlier we established an animal model for human MM in the immune-deficient RAG2-/-ãc-/- mouse, in which the growth of luciferase-transduced MM cells was visualized using noninvasive bioluminescence imaging (BLI). This model appeared well suited to study disease progression and response to therapy by identifying the location of various foci of MM tumor growth scattered throughout the skeleton and at subsequent time points the quantitative assessment of the tumor load by using BLI. We report here on the corresponding high-resolution X-ray micro-computed tomographic (micro-CT) analysis to study skeletal defects in the mice with full-blown MM. Several anatomical derangements were observed, including abnormalities in geometry and morphology, asymmetrical bone structures, decreased overall density in the remaining bone, loss of trabecular bone mass, destruction of the inner microarchitecture, as well as cortical perforations. Using the combination of BLI, micro-CT imaging, and immune-histopathological techniques, we found a high correlation between the micro-CT-identified lesions, exact tumor location, and infiltration leading to structural lesions and local bone deformation. This confirms that this animal model strongly resembles human MM and has the potential for studying the biology of MM growth and for preclinical testing of novel therapies for MM and for repair of MM-induced bone lesions.

Ligament tissue engineering based on cell-seeded biomechanically functional constructs is a commonly studied strategy toward native anterior cruciate ligament replacement. Little is known about the survival and differentiation of the seeded cells after the transplantation. We applied retroviral genetic marking to trace implanted cells and studied their differentiation by species-specific immunolabeling of the extracellular matrix produced. Goat bone marrow stromal cells were transduced with a MoMuLV-based vector encoding the DeltaLNGFR gene. Transduced cells were seeded onto poly(lactic-co-glycolic acid) (PLGA) fibers and implanted subcutaneously into nude mice and left for various periods up to 6 weeks. Immunohistochemistry for LNGFR expression showed survival of the seeded cells after transplantation for up to 6 weeks. Immunohistochemistry for collagen type I and III showed the production of fibrous tissue inside the scaffolds. Moreover, using a goat-specific anti-collagen type III, donor-derived matrix could be demonstrated. We conclude that bone marrow stromal cells survived in vivo and at least partially differentiated after implantation.

BACKGROUND: The development and preclinical testing of novel immunotherapy strategies for multiple myeloma can benefit substantially from a humanized animal model that enables quantitative real-time monitoring of tumor progression. Here we have explored the feasibility of establishing such a model in immunodeficient RAG2(-/-)gammac(-/-) mice, by utilizing non-invasive bioluminescent imaging for real-time monitoring of multiple myeloma cell growth.DESIGN AND METHODS: Seven multiple myeloma cell lines, marked with a green fluorescent protein firefly luciferase fusion gene, were intravenously injected into RAG2(-/-)gammac(-/-) mice. Tumor localization and outgrowth was monitored by bioluminescent imaging. The sensitivity of this imaging technique was compared to that of free immumoglobulin light chain -based myeloma monitoring. Established tumors were treated with radiotherapy or with allogeneic peripheral blood mononuclear cell infusions to evaluate the application areas of the model.RESULTS: Five out of seven tested multiple myeloma cell lines progressed as myeloma-like tumors predominantly in the bone marrow; the two other lines showed additional growth in soft tissues. In our model bioluminescent imaging appeared superior to free light chain-based monitoring and also allowed semi-quantitative monitoring of individual foci of multiple myeloma. Tumors treated with radiotherapy showed temporary regression. However, infusion of allogeneic peripheral blood mononuclear cells resulted in the development of xenogeneic graft-versus-host-disease and a powerful cell dose-dependent graft-versus-myeloma effect, resulting in complete eradication of tumors, depending on the in vitro immunogenicity of the inoculated multiple myeloma cells.CONCLUSIONS: Our results indicate that this new model allows convenient and sensitive real-time monitoring of cellular approaches for immunotherapy of multiple myeloma-like tumors with different immunogenicities. This model, therefore, allows comprehensive preclinical evaluation of novel combination therapies for multiple myeloma.

Mesenchymal stem cells (MSCs) represent a powerful tool for applications in regenerative medicine. In this study, we used in vivo bioluminescence imaging to noninvasively investigate the fate and the contribution to bone formation of adult MSCs in tissue engineered constructs. Goat MSCs expressing GFP-luciferase were seeded on ceramic scaffolds and implanted subcutaneously in immune-deficient mice. The constructs were monitored weekly with bioluminescence imaging and were retrieved after 7 weeks to quantify bone formation by histomorphometry. With increasing amounts of seeded MSCs (from 0 to 1 � 10 6 MSC/scaffold), a cell-dose related increase in bioluminescence was observed at all time points, correlating with increased bone formation at 7 weeks. To investigate the relevance of MSC proliferation to bone deposition, cell-seeded scaffolds were irradiated. The irradiated cells were functional with respect to oxygen consumption but no increase in bioluminescence was observed in vivo, and only minimal bone was produced. Proliferating MSCs are likely required for initiation of bone formation in tissue engineered constructs in vivo. Bioluminescence is a useful tool to monitor cellular responses and predict bone formation in vivo. 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res

Daratumumab (DARA) is a human CD38-specific IgG1 antibody that is in clinical development for the treatment of multiple myeloma (MM). The potential for IgG1 antibodies to induce macrophage-mediated phagocytosis, in combination with the known presence of macrophages in the tumor microenvironment in MM and other hematological tumors, led us to investigate the contribution of antibody-dependent, macrophage-mediated phagocytosis to DARA's mechanism of action. Live cell imaging revealed that DARA efficiently induced macrophage-mediated phagocytosis, in which individual macrophages rapidly and sequentially engulfed multiple tumor cells. DARA-dependent phagocytosis by mouse and human macrophages was also observed in an in vitro flow cytometry assay, using a range of MM and Burkitt's lymphoma cell lines. Phagocytosis contributed to DARA's anti-tumor activity in vivo, in both a subcutaneous and an intravenous leukemic xenograft mouse model. Finally, DARA was shown to induce macrophage-mediated phagocytosis of MM cells isolated from 11 of 12 MM patients that showed variable levels of CD38 expression. In summary, we demonstrate that phagocytosis is a fast, potent and clinically relevant mechanism of action that may contribute to the therapeutic activity of DARA in multiple myeloma and potentially other hematological tumors.

Daratumumab is an anti-CD38 monoclonal antibody with lytic activity against multiple myeloma (MM) cells, including ADCC (antibody-dependent cellular cytotoxicity) and CDC (complement-dependent cytotoxicity). Owing to a marked heterogeneity of response to daratumumab therapy in MM, we investigated determinants of the sensitivity of MM cells toward daratumumab-mediated ADCC and CDC. In bone marrow samples from 144 MM patients, we observed no difference in daratumumab-mediated lysis between newly diagnosed or relapsed/refractory patients. However, we discovered, next to an expected effect of effector (natural killer cells/monocytes) to target (MM cells) ratio on ADCC, a significant association between CD38 expression and daratumumab-mediated ADCC (127 patients), as well as CDC (56 patients). Similarly, experiments with isogenic MM cell lines expressing different levels of CD38 revealed that the level of CD38 expression is an important determinant of daratumumab-mediated ADCC and CDC. Importantly, all-trans retinoic acid (ATRA) increased CD38 expression levels but also reduced expression of the complement-inhibitory proteins CD55 and CD59 in both cell lines and primary MM samples. This resulted in a significant enhancement of the activity of daratumumab in vitro and in a humanized MM mouse model as well. Our results provide the preclinical rationale for further evaluation of daratumumab combined with ATRA in MM patients.

The MLL-AF9 fusion oncogene is found in pediatric leukemia of both the myeloid and lymphoid lineage, while in adults the same oncogene is predominantly associated with myeloid leukemia. We have been able to establish in vitro models that faithfully recapitulate these patient phenotypes by lentiviral transduction of MLL-AF9 into CD34+ cells from fetal Cord Blood and adult Bone Marrow. Upon transplantation of transduced MLL-AF9 expressing cells into NOD-SCID/NSG mice, serially transplantable lymphoid leukemia could be generated within 15-24 weeks, but the myeloid engraftment was more difficult to achieve and CD33+/CD19- myeloid clones were only observed in about 10% of the cases. Importantly, these CD33+/CD19- clones displayed impaired self-renewal as indicated by the lack of secondary transplantation capacity while lymphoid CD33-/CD19+ clones readily engrafted in secondary recipients to give rise to 2nd ALL. These results are in contrast with what we detect in vitro or in patients where both myeloid and lymphoid phenotypes are observed. We hypothesized that extrinsic and species-specific factors might dictate the lineage fate of the leukemic clone, and therefore we have started to use a new model in which human mesenchymal stem cells (MSCs) are coated on scaffolds, which are then implanted subcutaneously into NSG mice to allow vascularization and formation of a “human niche” (huScaffold). Within these human niches we observe both AML and ALL upon transplantation of MLL-AF9-transduced CB CD34+ cells. Moreover, the human niche also allowed engraftment and tumor formation of primary AML MLL-AF9 patient samples. These data indicate that extrinsic factors present in the human niche can dictate lineage fate of the leukemia. Future directions include further improvements of the human niche model like using MSCs that we have genetically engineered to express factors like IL3 and TPO. In addition, we took advantage of the CB MLL-AF9 in vitro and in vivo models to test the efficacy of a small molecule inhibitor of the BET family named I-BET151. I-BET151 targets BRD3/4 association to acetylated histones and thereby prevents MLL-AF9 binding to chromatin. We demonstrate that I-BET151 inhibits the growth of CB MLL-AF9 cells in an in vitro MS5 co-culture setting, under both myeloid and lymphoid conditions. Furthermore, we transplanted CD19+ MLL-AF9 clones into secondary recipients and we performed intra-scaffold injections of the inhibitor as soon as the tumors were palpable. Tumor volume was measured during the treatment and we observed a significant 2.5-fold reduction of tumor volume in a dose-dependent manner. In conclusion, our data indicate that extrinsic factors can dictate lineage fate of MLL-AF9-driven leukemia. The humanized in vivo xenograft model allows the faithful recapitulation of human disease and thus will aid in the development of novel therapeutic approaches to treat leukemias driven by oncogenes such as MLL-AF9. Ongoing studies include those in which we target the FLT3 pathway since our transcriptome studies indicated that this tyrosine kinase receptor is highly upregulated in MLL-AF9-positive leukemias. In vitro targeting of this receptor abrogated both initiation as well as maintenance of transformation by MLL-AF9. We analyzed FLT3L-initiated genome-wide transcriptional changes in MLL-AF9 cells and observed that inhibition of the FLT3 pathway resulted in reduced expression of genes such Bcl2 and CyclinD2, possibly involving loss of activity of the PI3K pathway, thereby inducing apoptosis in MLL-AF9 cells. Disclosures No relevant conflicts of interest to declare.

Background In mouse models for metastatic growth of colorectal carcinoma (CRC) cells in the liver, tumor growth is routinely measured by determining the area of liver tissue that has been replaced by tumor tissue (hepatic replacement area [HRA]). This technique has several major disadvantages. Modern visualization techniques make it possible to image tumor growth noninvasively. In the present report, we validated bioluminescence imaging of liver metastases by comparing it to standard HRA measurements and liver weight. Materials and methods BALB/c mice received an intrasplenic injection of luciferase-expressing C26 CRC cells and the spleen was subsequently removed. On days 5, 7, 9, and 11 after injection, luciferase activity was measured. After imaging, the mice were sacrificed and the livers was removed, weighed, and fixed. HRA was determined by analyzing liver tissue sections. Comparative trend analyses between luciferase activity, wet liver weight, and HRA were then performed. Results Luciferase activity, wet liver weight, and HRA all increased over time. Statistical analyses showed that all three types of measurements display a highly significant degree of correlation. Conclusions The measurement of tumor growth in the liver by imaging luciferase activity correlates well with the standard method of determining the HRA and with the increase in liver weight that results from tumor growth. Given the great advantages of measuring luciferase activity over measuring HRA, we conclude that bioluminescent imaging is a reliable and superior method for measuring experimental CRC growth in the liver.

OBJECTIVE: The ability of human cells to repopulate the bone marrow of nonobese diabetic immunodeficient mice (NOD/SCID) is commonly used as a standard assay to quantify the primitive human hematopoietic stem cell population. We studied the applicability of the immunodeficient RAG2(-/-)gammac(-/-) double-knockout mouse for this purpose. METHODS: RAG2(-/-)gammac(-/-) mice and NOD/SCID mice were injected intravenously (i.v.) with umbilical cord blood-derived CD34(+) cells and engraftment was quantified by determining the human CD45+ cell chimerism in bone marrow at several time points. RAG2(-/-)gammac(-/-) were pretreated with total-body irradiation and depleted of macrophages in liver, spleen, and bone marrow by i.v. injection of clodronate diphosphonate containing liposomes. RESULTS: We demonstrated that the frequency of chimerism and the level of engraftment in macrophage-depleted RAG2(-/-)gammac(-/-) largely resemble that in NOD/SCID mice. Also similar is the multilineage differentiation pattern in the two mouse strains at 7 weeks after transplantation, with a prominent outgrowth in RAG2(-/-)gammac(-/-) of CD19+ cells (88% +/- 10%). Cells of other lineages were clearly less frequent: 9% +/- 2% myeloid cells and 0.1% +/- 0.1% erythroid cells. As for immature progenitors, 6% +/- 1% of the human cells express the CD34 antigen and 0.4% +/- 0.1% have the CD34+,CD33,38,71(-) phenotype. The presence of human committed progenitors (i.e., CFU-GM/BFU-E) was evident. The persistence of human cells at 4 months after transplantation shows that the RAG2(-/-)gammac(-/-) support long-term maintenance of human hematopoiesis. CONCLUSION: Our findings indicate that macrophage-depleted RAG2(-/-)gammac(-/-) are a suitable model for studying human hematopoiesis including multipotential stem cells, and long-term repopulation

Introduction of the HSV-Tk suicide gene into allogeneic T cells offers the possibility to control developing host-reactive cells within the context of allogeneic bone marrow transplantation (BMT). Sensitive quantitative detection methods are a prerequisite to monitor genetically modified T cells in peripheral blood and tissues to study their involvement in graft-versus-host disease (GVHD)-induced lesions as well as their disappearance or persistence after ganciclovir (GCV)-induced suicide. We monitored the alloreactivity of HSV-Tk-transduced T cells after BMT by studying their in vivo distribution and quantity in peripheral blood and in tissues in a WAG/Rij into Brown Norway fully mismatched rat allogeneic BMT model. Genetically modified T cells were quantified in blood and tissues by fluorescence-activated cell sorting, immunohistochemical analysis, and real-time quantitative polymerase chain reaction (PCR) analysis. A significant increase in the number of allogeneic HSV-Tk(+) T cells was found in particular in spleen and lymph nodes and large numbers were found in tongue, skin, and intestines. In blood, an increase in HSV-Tk(+) T cells closely preceded clinical symptoms of GVHD. Real-time quantitative PCR proved to be a fast and accurate tool by which to quantify transduced T cells both in blood and tissues. This enables the study of the in vivo alloreactivity of retrovirus-transduced cells and the response of HSV-Tk-expressing T cells to GCV-induced suicide therapy. Furthermore, we showed the potential use to study specific cause-effect relationships in a broad range of animal and clinical studies involving genetically engineered cells.

International audience; Tumoral plasma cells has retained stemness features and in particular, a polycomb-silenced gene expression signature.Therefore, epigenetic therapy could be a mean to fight for multiple myeloma (MM), still an incurable pathology.Deazaneplanocin A (DZNep), a S-adenosyl-L-homocysteine hydrolase inhibitor, targets enhancer of zest homolog 2 (EZH2), acomponent of polycomb repressive complex 2 (PRC2) and is capable to induce the death of cancer cells. We show here that,in some MM cell lines, DZNep induced both caspase-dependent and -independent apoptosis. However, the induction of celldeath was not mediated through its effect on EZH2 and the trimethylation on lysine 27 of histone H3 (H3K27me3). DZNeplikely acted through non-epigenetic mechanisms in myeloma cells.In vivo, in xenograft models, andin vitroDZNep showedpotent antimyeloma activity alone or in combination with bortezomib. These preclinical data let us to envisage newtherapeutic strategies for myeloma.

Licencia Creative Commons Reconocimiento-No comercial.-- et al., Hematopoietic stem cell transplantation (HSCT) using umbilical cord blood (UCB) progenitors is increasingly being used. One of the problems that may arise after UCB transplantation is an impaired engraftment. Either intrabone (IB) injection of hematopoietic progenitors or mesenchymal stem cell (MSC) coadministration has been proposed among the strategies to improve engraftment. In the current study, we have assessed the effects of both approaches. Thus, NOD/SCID recipients were transplanted with human UCB CD34+ cells administered either intravenously (IV) or IB, receiving or not bone marrow (BM)-derived MSCs also IV or IB (in the right femur). Human HSC engraftment was measured 3 and 6 weeks after transplantation. Injected MSCs were tracked weekly by bioluminescence. Also, lodgment within the BM niche was assessed at the latter time point by immunofluorescence. Our study shows regarding HSC engraftment that the number of BM human CD45+ cells detected 3 weeks after transplantation was significantly higher in mice cotransplanted with human MSCs. Moreover, these mice had a higher myeloid (CD13+) engraftment and a faster B-cell (CD19+) chimerism. At the late time point evaluated (6 weeks), human engraftment was higher in the group in which both strategies were employed (IB injection of HSC and MSC coadministration). When assessing human MSC administration route, we were able to track MSCs only in the injected femurs, whereas they lost their signal in the contralateral bones. These human MSCs were mainly located around blood vessels in the subendosteal region. In summary, our study shows that MSC coadministration can enhance HSC engraftment in our xenogenic transplantation model, as well as IB administration of the CD34+ cells does. The combination of both strategies seems to be synergistic. Interestingly, MSCs were detected only where they were IB injected contributing to the vascular niche., This study was supported in part by a grant from Gerencia Regional de Salud de Castilla y León (ref. GRS/222/A/08) and by a grant from Consejería de Educación de la Junta de Castilla y León (ref. HUS003A10-2). S.C. was supported by Junta de Castilla y Leon (FPI grant EDU/1878/2006).

Mesenchymal stromal cells (MSC) are multipotent cells with an immune suppressive capacity. In the last decade the therapeutic application of these cells has been tested in inflammatory diseases like graft versus host disease and rheumatoid arthritis. Injection of MSC can be a potential therapy for juvenile idiopathic arthritis patients refractory to conventional therapies.

The in vivo elimination of suicide gene-expressing tumor cells with prodrug treatment can induce protective immunity against wild-type tumors. In this study, we determined the efficacy and safety of the in vivo elimination of HSVtk expressing cells with ganciclovir treatment of a bystander cell killing-insensitive leukemic cell line. The retroviral construct pLTk+NeoDeltaMo, containing the HSVtk gene and the NeoR gene in a bicistronic unit, was introduced into rat leukemic LT12 cells. LT12/Tk+N cells showed a 1000- to 10 000-fold increased sensitivity to ganciclovir in vitro. In vitro mixing experiments demonstrated that LT12 cells were not susceptible to bystander cell lysis by LT12/Tk+N-2 cells exposed to ganciclovir. Rats injected s.c. with cloned LT12/Tk+N-2 cells developed tumors reaching a diameter of 3-4 cm after 40 days. Rats treated with gan- ciclovir twice daily for 5 consecutive days starting at day 7 did not develop s.c. tumors. Large established s.c. LT12/Tk+N-2 tumors completely regressed after ganciclovir treatment. However, recurrences of s.c. tumors were observed that were no longer sensitive to ganciclovir treatment. In vitro analysis of aspirates from the recurrent tumors demonstrated loss of HSVtk expression. In vitro culture of LT12/Tk+N-2 cells in soft agar in the presence of ganciclovir indicated that the frequency with which HSVtk-loss variants occurred is approximately one per 104 cells. The in vivo occurrence of HSVtk-loss variants escaping ganciclovir-induced elimination may have important implications for vaccination protocols using HSVtk gene expressing tumor cells that are not susceptible to bystander cell killing.

Background: Chromosome banding techniques and in situ hybridization reveal the majority of chromosomal aberrations. However, difficulties remain in cases of highly contracted chromosomes, poor quality of the metaphases or the presence of markers with the involvement of several chromosomes. Here, it is demonstrated that reverse painting can be applied successfully starting with bone marrow cells from primary acute myelocytic leukemias (AML). Methods: This was accomplished by culturing the leukemic cells with a cocktail of various growth factors, which yielded sufficient numbers of cells in cycle to harvest chromosomes for sorting. Aberrant chromosomes were flow-sorted and amplified by degenerate oligonucleotideprimed PCR. The resulting products were labeled by nick-translation and hybridized on normal metaphase spreads.

We studied the cell kill induced by granulocyte-macrophage colony-stimulating factor (GM-CSF ) fused to Diphtheria Toxin (DT-GM-CSF ) in acute myeloid leukemia (AML) samples and in populations of normal primitive hemopoietic progenitor cells. AML samples from three patients were incubated in vitro with 100 ng/mL DT-GM-CSF for 48 hours, and AML cell kill was determined in a proliferation assay, a clonogenic assay colony-forming unit-AML (CFU-AML) and a quantitative long-term bone marrow (BM) culture ie, the leukemic-cobblestone area forming cell assay (L-CAFC). To measure an effect on cells with in vivo leukemia initiating potential DT-GM-CSF exposed AML cells were transplanted into immunodeficient mice. In two out of three samples it was shown that all AML subsets, including those with long-term abilities in vivo (severe combined immunodeficient mice) and in vitro (L-CAFC assay) were reduced in number by DT-GM-CSF. Cell kill induced by DT-GM-CSF could be prevented by coincubation with an excess of GM-CSF, demonstrating that sensitivity to DT-GM-CSF is specifically mediated by the GM-CSF receptor. Therefore, binding and internalization of GM-CSF probably occur in immature AML precursors of these two cases of AML. The third AML sample was not responsive to either GM-CSF or DT-GM-CSF. The number of committed progenitors of normal bone marrow (burst-forming unit-erythroid, colony-forming unit granulocyte- macrophage, and cobble stone area forming cell [CAFC] week 2) and also the number of cells with long-term repopulating ability, assayed as week 6 CAFC, were unchanged after exposure to DT-GM-CSF (100 ng/mL, 48 hours). These studies show that DT-GM-CSF may be used to eliminate myeloid leukemic cells with long-term potential in vitro and in immunodeficient mice, whereas normal hemopoietic stem cells are spared.

Fetal cells are present in the circulation of pregnant women and can be isolated using density gradient centrifugation and magnetic cell sorting. In the present study, maternal cell preparations were depleted for CD45- and CD14-positive cells and enriched for CD71-positive cells. The number of fetal nucleated cells was determined using fluorescence in situ hybridization for X and Y chromosomes. Analysis of maternal blood samples taken before and after transabdominal chorionic villus sampling (TA-CVS) showed an increase in the number of fetal cells in 10 out of 19 male pregnancies after the invasive procedure. This cellular transfusion was found to correlate with elevated maternal serum alpha-fetoprotein levels. TA-CVS-induced cellular transfusion may form a good in vivo system to optimize fetal cell isolation procedures and to study fetal cell dynamics and characteristics. © 1997 John Wiley & Sons, Ltd.

The potential to selectively eliminate acute myeloid leukaemia (AML) cells with the GM-CSF-diphtheria toxin fusion protein (DT-GM-CSF) was studied under conditions of autonomous proliferation in vitro with no growth factors (GFs) added and after growth stimulation with a mixture of human (hu)G-CSF, huIL-3 and huSCE DNA synthesis was maximally inhibited after 48 h exposure to DT-GM-CSF, Cell viability and AML colony forming ability in vitro were reduced. 18/22 samples were found to be sensitive to DT-GM-CSF, with 50%, inhibition of DNA synthesis (ID 50 ) at concentrations ranging from 0.1 to 16 ng/ml, and four samples were minimally or not sensitive to DT-GM-CSF (ID 50 ≥ 99 ng/ml). From the 15 samples which showed autonomous proliferation. 13 were sensitive to inhibition of proliferation by DT-GM-CSF. The level of GM-CSF receptor (GM-CSFR) expression was determined by flow cytometry after labelling with specific antibodies for the alpha and beta subunits. Although the toxicity to DT-GM-CSF was specifically mediated by the GM-CSFR, no correlation was found between the level of expression of the GM-CSFR alpha or beta subunit and the sensitivity for DT-GM-CSF. These in vitro studies show that the DT-GM-CSF fusion protein can be used for specifically targeting and eliminating leukaemic cells in the majority of AML cases.

The NSG [NOD/Lt-scid/IL2Rγnull] xenotransplantation mouse model is currently the model of choice to evaluate human hematopoietic engraftment and to study development of human leukemia. Indeed, we have previously shown that co-expression of BCR-ABL together with the polycomb repression complex 1 (PRC1) member BMI1 in human cord blood (CB) derived CD34+ cells was sufficient to induce a serially transplantable lymphoid leukemia (Rizo et al., Blood 2010). This leukemia was characterized by high levels of CD34+/CD19+/CD20-/IgM-/CD33-/CD15- lymphoid blasts in the bone marrow and a high degree of infiltration of blasts in spleen and liver. Clonal analysis revealed that similar clones gave rise to leukemia in primary and secondary recipients. Although in vivo no myeloid leukemias were observed, in vitro both lymphoid as well as myeloid immortalized long-term cultures could readily be established, in line with phenotypes observed in chronic myeloid leukemia patients whereby a chronic myeloid phase can egress into a myeloid or lymphoid blast crisis. It is very plausible that differences between murine and human hematopoietic stem cell niches underlie these observed differences. Human engraftment in NSG mice is typically lymphoid biased, and since many growth factors and cytokines are species-specific it is clear that the murine niche is not ideal to evaluate human hematopoietic engraftment and leukemic transformation potential. In our current study we have evaluated the in vivo leukemic transformation potential of human CB derived CD34+ cells expressing BCR-ABL and/or BMI1 in NSG mice in which scaffolds coated with culture-expanded human mesenchymal stromal cells (MSCs) were implanted subcutaneously 8 weeks prior to injection of transduced cells, to allow the development of a humanized niche containing mineralized bone-matrix, osteoblasts, stromal cells, as well as appropriate vascularization (Groen et al., Blood 2012). BCR-ABL/BMI1 transduced human CB derived CD34+ cells or primary blast crisis CML patient cells were injected either intravenously or directly into the humanized scaffolds, and leukemia development was evaluated. Our data indicate that in a humanized niche, in contrast to a murine niche, BCR-ABL was sufficient to induce leukemia as a single hit without overexpression of exogenous BMI1. Furthermore, both ALL as well as erythro/myeloid leukemias could be induced. The ALL could be transplanted to secondary recipients and besides the lymphoid marker CD19, the cells also expressed CD33 and CD15, but not CD11b or GPA. These data are in sharp contrast to results obtained in xenograft mouse models without human niches, where BCR-ABL expression alone in human cells was not sufficient to induce leukemia, and secondary hits such as BMI1 were essential. Efficient engraftment of a blast-crisis CML patient sample was also observed in the human niche model, whereby the immature blast-like phenotype was maintained in the human scaffold niche, while more differentiated cells were observed in the mouse bone marrow niche. In vitro, long-term self-renewing cultures could readily be established with cells retrieved from the human scaffold niche of these leukemic mice, while no long-term cultures could be initiated with cells retrieved from the murine bone marrow niche, from the same mouse. These data indicate that a human niche is required to maintain appropriate in vivo self-renewal of human BC CML cells. Interestingly, the endogenous BMI1 levels were significantly higher in cells retrieved from the human scaffold niche as compared to the mouse BM niche. In conclusion, our data indicate that BCR-ABL transformed cells needs secondary event such as over expression of oncogene like BMI1 for its full transformation potential, most likely to overcome or repress oncogene-induced senescence. The mouse environment is not able to provide these secondary events in human cells, whereas the human niche is able to provide signals that together with BCR-ABL are sufficient to fully transform human cells in xenograft models. Disclosures: No relevant conflicts of interest to declare.

Purpose: Cellular immunotherapy frequently fails to induce sustained remissions in patients with multiple myeloma, indicating the ability of multiple myeloma cells to evade cellular immunity. Toward a better understanding and effective therapeutic modulation of multiple myeloma immune evasion mechanisms, we here investigated the role of the tumor microenvironment in rendering multiple myeloma cells resistant to the cytotoxic machinery of T cells. Experimental Design: Using a compartment-specific, bioluminescence imaging-based assay system, we measured the lysis of luciferase-transduced multiple myeloma cells by CD4+ or CD8+ CTLs in the presence versus absence of adherent accessory cells of the bone marrow microenvironment. We simultaneously determined the level of CTL activation by measuring the granzyme B release in culture supernatants. Results: Bone marrow stromal cells from patients with multiple myeloma and healthy individuals, as well as vascular endothelial cells, significantly inhibited the lysis of multiple myeloma cells in a cell–cell contact-dependent manner and without substantial T-cell suppression, thus showing the induction of a cell adhesion-mediated immune resistance (CAM-IR) against CTL lysis. Further analyses revealed that adhesion to accessory cells downregulated Fas and upregulated the caspase-3 inhibitor survivin in multiple myeloma cells. Reconstitution of Fas expression with bortezomib enhanced the CTL-mediated lysis of multiple myeloma cells. Repressing survivin with the small-molecule YM155 synergized with CTLs and abrogated CAM-IR in vitro and in vivo. Conclusion: These results reveal the cell adhesion-mediated induction of apoptosis resistance as a novel immune escape mechanism and provide a rationale to improve the efficacy of cellular therapies by pharmacologic modulation of CAM-IR. Clin Cancer Res; 19(20); 5591–601. ©2013 AACR.

Aim: One of the main limitations for an effective cell therapy for the heart is the poor cell engraftment after implantation, which is partly due to a large percentage of cell death in the hostile myocardium. In the present study, we investigated the utilization of Necrostatin-1 (Nec-1) as a possible attenuator of cell death in Sca-1+ cardiac progenitor cells (CPCs). Methods and Results: Under oxidative stress conditions in vitro, Nec-1 pretreatment reduced necrotic cell death in Sca-1+ CPCs. In a mouse model of myocardial infarction, survival of CPCs 3 days after intra-myocardial injection was 39% higher (BLI signal, 71,665 ± 11,165 vs 117,138 ± 18,567 ph/s/cm2/sr) in cells pretreated with the Nec-1 compound. However, the increase in cell number did not translate into improved cardiac function at one month follow-up (EF %, 20.6±2.1 vs 21.4±2.5 for vehicle and Nec-1 treated CPC, respectively). We extensively investigated, but did not find, alternative effects to the pro-survival properties of the compound on the CPCs. Furthermore, CPCs rescued by Nec-1 remained functionally competent. Conclusion: A pharmacological pretreatment approach to solely enhance cell survival on the short-term does not seem to be effective strategy to improve cardiac cell therapy with Sca-1+ CPCs. Since the initial cell retention directly after injection is low (~10%), survival strategies could conceivably be more useful in a tissue engineering setting, in which they can exert their effects on larger number of cells.