Journal articles on the topic 'Bone like-inducing graft'
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1
Krause, Daniela S., and Richard A. Van Etten. "Adoptive immunotherapy of BCR-ABL–induced chronic myeloid leukemia–like myeloproliferative disease in a murine model." Blood 104, no. 13 (December 15, 2004): 4236–44. http://dx.doi.org/10.1182/blood-2004-06-2229.
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Abstract Donor leukocyte infusion (DLI) can induce graft-versus-leukemia (GvL) reactions in patients with chronic myeloid leukemia (CML) relapsing after allogeneic bone marrow transplantation (BMT), but the mechanisms of the antileukemic effect of DLI are unknown, and the procedure is complicated by graft-versus-host disease (GvHD) and graft failure. Here, we adapted a murine retroviral BMT model of Philadelphia+ leukemia by combining allogeneic bone marrow (BM) from C57Bl/6 (H-2b) mice with BCR-ABL–transduced Balb/c (H-2d) BM, inducing mixed chimerism and myeloproliferative disease in recipients resembling relapse of CML following allogeneic BMT. Infusions of allogeneic splenocytes eliminated BCR-ABL–induced CML-like disease in the majority of mixed chimeras, with significant GvL effects mediated by both CD4+ and CD4- cells. BCR-ABL–induced acute B-lymphoblastic leukemia was also eradicated by DLI in major histocompatibility complex (MHC)–mismatched chimeras. Most DLI-treated mice converted to full allogeneic chimerism but succumbed frequently to GvHD or graft failure. When MHC-matched B10.D2 (H-2d) mice were the allogeneic donors, CML-like disease was more resistant to DLI. These results suggest that depletion of CD8+ cells from DLI could impair GvL against CML, while increased MHC disparity between donor and recipient may improve the responsiveness of Philadelphia+ B-lymphoblastic leukemia to DLI.
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Wang, Yanhua, Jun Ma, Lei Zhou, Jin Chen, Yonghui Liu, Zhiye Qiu, and Shengmin Zhang. "Dual functional selenium-substituted hydroxyapatite." Interface Focus 2, no. 3 (March 28, 2012): 378–86. http://dx.doi.org/10.1098/rsfs.2012.0002.
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Hydroxyapatite (HA) doped with trace elements has attracted much attention recently owing to its excellent biological functions. Herein, we use a facile co-precipitation method to incorporate selenium into HA by adding sodium selenite during synthesis. The obtained selenium-substituted HA products are needle-like nanoparticles which have size and crystallinity that are similar to those of the pure HA nanoparticles (HANs) when the selenium content is low. HANs are found to have the ability to induce the apoptosis of osteosarcoma cells, and the anti-tumour effects are enhanced after incorporation of selenium. Meanwhile, the nanoparticles can also support the growth of bone marrow stem cells. Furthermore, the flow cytometric results indicate that the apoptosis induction of osteosarcoma cells is caused by the increased reactive oxygen species and decreased mitochondrial membrane potential. These results show that the selenium-substituted HANs are potentially promising bone graft materials in osteosarcoma treatment due to their dual functions of supporting normal cell growth and inducing tumour cell apoptosis.
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Kadowaki, Masanori, Kazutoshi Aoyama, Motoko Koyama, Yukimi Sakoda, Kiyoshi Takeda, Chen Liu, Koichi Akashi, Mitsune Tanimoto, and Takanori Teshima. "T Cell Toll-Like Receptors Play a Critical Role in Graft-Versus-Host Disease and Graft-Versus-Leukemia." Blood 112, no. 11 (November 16, 2008): 63. http://dx.doi.org/10.1182/blood.v112.11.63.63.
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Abstract Graft-versus-host disease (GVHD), the major complication of allogeneic hematopoietic stem cell transplantation, is mediated by donor T cells recognizing host alloantigens expressed on antigen-presenting cells (APCs). Total body irradiation (TBI) activates and damages host tissue, produces inflammatory cytokines and damage-associated molecular patterns, and allows translocation of microbial products into the systemic circulation. It has been assumed that these products bind to toll-like receptors (TLRs) on APCs and monocytes/macrophages, activate inflammatory pathways, and further amplify GVHD. On the other hand, TLRs are also expressed on T cells but their roles in the adoptive T cell response remain to be investigated in vivo. TLR signals are mediated by both MyD88-dependent and independent TRIF protein pathways. We evaluated the role of T cell TLR signaling in T cell alloreactivity in a mouse model of allogeneic bone marrow transplantation using mice with defective TLR signaling induced by deletions of the TLR adaptor proteins TRIF and MyD88 (TRIF/MyD88 DKO mice); in these mice, the TLR-dependent signaling pathway was eliminated. Lethally irradiated B6D2F1 mice were injected with 2 × 106 T cells from wild-type (wt) or TRIF/MyD88 DKO mice together with 5 × 106 T cell-depleted bone marrow (TCD-BM) from wt or DKO mice. Morbidity and mortality of GVHD was significantly less in recipients of TRIF/MyD88 DKO T cells than in recipients of wt T cells, irrespective of donor bone marrow type (Table). Reduction of GVHD was not seen in recipients of MyD88−/− T cells or TRIF−/− T cells, suggesting that inhibition of signaling through TRIF or MyD88 alone in donor cells is not sufficient to reduce GVHD. Histopathologic examination of the small intestine, liver, and skin showed significantly reduced GVHD pathology in recipients of DKO T cells (Table). Thus, effects of TLR signaling in donor cells on GVHD primarily reside in the T cell compartment of the donor graft, not in donor accessory cells. In recipients of TRIF/MyD88 DKO T cells, reduction of GVHD was associated with impairment of donor T cell expansion and production of TNF-□ and IFN-□. In marked contrast though, TRIF/MyD88 DKO T cells were activated equally to wt T cells in unirradiated B6D2F1 mice after adoptive transfer, thus activation of DKO T cells was impaired only in recipient mice heavily pretreated with TBI. These results suggest that T cell TLR signaling provides critical co-stimulation for T cells in an inflamed environment. T cell TLR signaling was also required for optimal graft-versus-leukemia activity. These results challenge the current paradigm that TLR functions are primarily attributable to their role in APCs and monocytes/macrophages in GVHD, and reveal a previously unrecognized critical role for T cell TLRs in inducing GVHD and GVL after hematopoietic stem cell transplantation. Pathological scores T BM Survivals(%) Clinical scores Serum IFN □ (ng/ml) Liver Intestine Skin *P<0.05 vs wt, ND; not detected syn syn 100 0.2±0.2* ND 1.0±0.6 1.0±0.0 0.0±0.0 wt wt 27 6.2±0.9 4.3±1.2 18.3±2.3 16.7±3.0 1.7±0.8 wt DKO 25 5.5±0.8 DKO wt 100* 1.8±0.4* 0.4±0.1* 7.9±1.0* 10.9±1.7 0.1±0.1* DKO DKO 88* 2.2±0.4*
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Mutis, Tuna, Henk Rozemuller, Maarten E. Emmelot, Tineke Aarts-Riemens, Vivienne Verweij, Saskia Ebeling, Leo F. Verdonck, Henk Lockhorst, and Anton Martens. "The In Vivo Impact of Human Regulatory T Cells on the Graft Versus Tumor Effect." Blood 110, no. 11 (November 16, 2007): 349. http://dx.doi.org/10.1182/blood.v110.11.349.349.
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Abstract The curative Graft-vs-Tumor effect (GvT) of allogeneic Stem cell transplantation (SCT) is frequently complicated with life threatening Graft-vs-Host Disease (GvHD). In mice, prevention of GvHD, without abrogation of GvT is possible by co-transplantation of naturally occurring regulatory T cells (Tregs) with SC grafts. Consistent with these murine studies, we recently demonstrated that also human Tregs possess potent GvHD-downregulatory capacities in a xenogeneic(x) model, where x-GvHD is induced by infusion of autologous human T cells in RAG2−/−γc−/− mice (Mutis et al. Clin. Cancer Res.2006, 12: 5520–5525). Towards clinical application of Tregs, we now explored the impact of human Treg-administration on GvT in a bioluminescence imaging (BLI) based human-GvT model in the RAG2−/−γc−/− mice. In this model, mice inoculated with luciferase (LUC)-transduced human myeloma (MM) cell lines developed BLI-detectable, progressive, MM-like multifocal tumors exclusively in the bone marrow (BM). Full blown tumors were effectively eliminated by infusion of allogeneic human PBMC. This treatment also caused lethal x-GvHD as expected. In this setting, co-infusion of human PBMC with autologous, in vitro cultured Tregs at a 1:1 Treg: T effector cell ratio had no adverse effects on the development of GvT while significantly reducing the lethality of x-GvHD. In vitro analyses of sacrificed mice at day 21 revealed that administered Tregs homed to BM and spleen, significantly downregulated the total numbers of IFN-γ-producing CD4+ and CD8+ T cells responding to CD3 mediated signals, but had no downregulatory effect on the frequencies of IFN-γ-producing T cells responding to tumor cells. There was also no downregulation of cytotoxic activity against tumor cells in Treg-treated mice. Conclusively, these results showed that Tregs, at doses which are inhibitory for x-GvHD-inducing T cells, could maintain the GvT effect by allowing T cell reactivity against tumor cells. Human Tregs thus still hold promise as attractive cellular tools for separating GvT from GvHD.
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Dutt, Suparna, Diane Tseng, Tracy I. George, Joerg Ermann, Yinping Liu, C. Garrison Fathman, and Samuel Strober. "Allosensitized Memory CD4 T Cells Induce Chronic Graft Versus Host Disease." Blood 108, no. 11 (November 16, 2006): 449. http://dx.doi.org/10.1182/blood.v108.11.449.449.
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Abstract It has been reported that naive CD4+CD62LhiCD44lo T cells induce severe GVHD in a complete MHC mismatched allogeneic model of mouse bone marrow transplantation (BMT), but that effector memory CD4+CD62LloCD44hi T cells obtained from normal donors do not induce GVHD. The lack of GVHD-inducing capacity of effector memory cells from unprimed donors may reflect their lack of previous exposure to host alloantigens. We tested this hypothesis in a complete MHC mismatched allogeneic model of BMT by comparing the ability of effector memory T cells obtained from untreated C57BL/6 donors and donors immunized against host BALB/C alloantigens to induce GVHD. C57BL/6 donors were immunized by injecting 50 x106 host BALB/C spleen cells i.p. and after one week with 10x 106 cells. Both the unprimed and alloantigen primed CD4+ T cells expressed similar levels of lymph node homing chemokine receptor CCR7, and activation markers like CD69 and CD25. We sorted naive (CD62LhiCD44lo) and effector memory (CD62LloCD44hi) CD4+ T cell subsets from C57BL/6 donor mice four weeks after immunization, and compared their alloreactivity to BALB/C in an in vitro MLR. Interestingly effector memory CD4+ T cells from primed mice produced significantly higher levels of IFNγ compared to the effector memory from unprimed donors. We found that CD62LloCD44hi cells from unimmunized donors failed to induce GVHD in 85% of the hosts over 100 days while CD62LloCD44hi cells from immunized donors caused progressive weight loss and death in 100% of hosts (p <0.001). Whereas naive CD4+ T cells from unimmunized donors accumulated rapidly in the lymph nodes and spleen of irradiated hosts, effector memory CD4+ T cells had markedly reduced accumulation in these tissues. Furthermore, at day 6 after transplantation effector memory CD4+ T cells from primed mice, showed hundred fold higher accumulation in the host liver compared to unprimed effector memory donor CD4+ T cells. Long term surviving hosts transplanted with primed effector memory cells showed histopathological features of chronic GVHD in liver characterized by portal tract inflammation and lymphocyte infiltration with bile duct injury. In conclusion, memory CD4+ T cells from donors immunized to host alloantigens are able to induce chronic GVHD , but memory cells from unimmunized donors do not.
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Bassi, Giulio, Luciano Pacelli, Cedric Mènard, Francesco Bifari, Luc Sensebé, Frederic Deschaseaux, Fabien Guilloton, et al. "Microporous Biphasic Calcium Phosphate Granules (MBCP®) Retain Immunological Properties of Bone Marrow-Derived Mesenchymal Stromal Cells and Promote Osteoblastic Differentiation." Blood 118, no. 21 (November 18, 2011): 1924. http://dx.doi.org/10.1182/blood.v118.21.1924.1924.
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Abstract Abstract 1924 Bone is among the most frequently transplanted tissue with about 1 million procedures annually in Europe. Allografts and autografts account for more than 80% of total graft volume, despite their considerable disadvantages, including the risk of disease transfer and immunologic rejection, limited supply of bone, costs and complications. Significant growth opportunities exist for synthetic bone grafts in association with mesenchymal stromal cells (MSC) from autologous or allogeneic sources as alternatives to biological bone grafts in orthopaedic and maxillofacial surgery. The objective of REBORNE is to perform clinical trials using advanced biomaterials and cells triggering bone healing in patients. To reach this goal, five phase I clinical studies with 20 patients have been planned in 12 clinical Centers spread in 8 European countries. Aim of the Immunological Unit of Reborne is to assess the MSC immunomodulatory properties in presence of the biomaterial used as scaffold for MSC delivery. All the functional experiments were performed in parallel, by comparing the effects of standard culture conditions and three-dimensional culture setting using MBCP (Biomatlante). Material and methods: Bone marrow MSC were provided from REBORNE Consortium Centers. To perform proliferation assays, different immune effector cells (T, B and NK cells) were stained with CFSE according to manufacturer's protocol. Active caspase-3 cell staining was used for survival quantification of immune effector cells after co-culture experiments. Differentiation potential was evaluated by culturing MSC with two different media containing either bone morphogenetic protein 4 (BMP4) or dexamethasone. After three weeks, osteogenic differentiation was quantified by qRT-PCR, alkaline phosphatase activity and alizarin red staining. Results: We found that primed MSC, pre-treated with the inflammatory cytokines IFNg and TNFa, displayed upregulation of HLA-ABC, CD54, CD106 and de novo expression of HLA-DR, both in standard culture conditions and in association with MBCP. Immune effector cells could be cultured and collected even in presence of MBCP and no significant differences were found between standard- (MSC + effector cells) and 3D-coculture conditions (MSC + MBCP + effector cells), in terms of immune effector cell proliferation. In both experimental conditions MSC suppressed T and NK cell proliferation (% suppression: MSC + T = 68.4; MSC + MBCP + T = 62.4; MSC + NK = 17.5; MSC + MBCP + NK = 20.2) and increased B cell proliferation (MSC + B = +13%; MSC + MBCP + B = +12.3%). In addition, immune effector cells viability was not affected by MBCP and MSC co-culture increased their survival even in presence of MBCP; in fact, in each culture condition the percentage of inhibition of T, B and NK cell apoptosis was higher than 20% in comparison to immune effector cells cultured without MSC. Dexamethasone and BMP4 were capable of inducing MSC differentiation into osteoblast-like cells, as confirmed by qRT-PCR analysis. We demonstrated that BMP4-based medium led to fully differentiated osteoblasts (Osterix+, RUNX2+, DLX5+ and alkaline-phosphatase+). Moreover, MBCP was more efficient in increasing osteoblastic differention as compared to standard culture conditions, as shown by the higher expression of Osteocalcin and Osterix. These data show that the association of MBCP and MSC does not affect MSC properties and suggest that it could be a treatment of choice of bone defects instead of allograft and autograft transplantation. Disclosures: No relevant conflicts of interest to declare.
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Park, Jae H., Andrew J. Yee, Thomas R. Spitzer, Susan L. Saidman, David T. Ting, Andrea Pannone, Jessica Reid, Steven McAfee, and Bimalangshu R. Dey. "KIR Ligand Incompatibility in HLA-Identical Sibling Nonmyeloablative Hematopoietic Stem Cell Transplantation for Hematologic Malignancies." Blood 108, no. 11 (November 16, 2006): 5371. http://dx.doi.org/10.1182/blood.v108.11.5371.5371.
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Abstract Natural killer (NK) cell alloreactivity based on inhibitory killer immunoglobulin-like receptor (KIR)-ligand incompatibility (i.e., missing KIR ligand) in the graft-vs-host (GVH) direction has been described to favorably influence engraftment, GVHD, and graft-vs-tumor (GVT) effect following haploidentical or matched unrelated HSCT in patients with hematologic malignancies. The degree and effect of KIR-ligand incompatibility has recently been explored following HLA-identical sibling HSCT (Hsu et al., Blood2005;105:4878). Based on a murine model, we developed a clinical trial with a goal of deliberately inducing a GVHD-free mixed chimeric platform following nonmyeloablative conditioning (consisting of equine ATG, cyclophosphamide, thymic irradiation, and a brief course of cyclosporine) and HLA-matched sibling HSCT. Donor leukocyte infusions (DLI) were given as early as five weeks post-HSCT to patients without GVHD in an attempt to achieve a conversion to full donor chimerism (FDC) with the goal of fully capturing GVT effect with little or no GVHD. In this study we hypothesized that KIR-ligand incompatibility in the GVH direction and KIR-ligand compatibility in the host-vs-graft (HVG) direction would reduce the rate of graft failure, decrease the incidence of GVHD, and improve overall survival following HLA-matched sibling HSCT. Fourteen transplant recipients (bone marrow, n=9; peripheral blood stem cell, n=5) with refractory hematologic malignancies (NHL, n=7; HD, n=3; MM, n=2; CLL, n=1; AML, n=1) were analyzed. KIR typing was accomplished using PCR amplified DNA from both donor and recipient patient samples. Typing of the amplified DNA was performed using the Lifecodes KIR-SSO typing kits (Tepnel Lifecodes Corporation). By using the SSO (sequence-specific oligonucleotides) technology in conjunction with the Luminex Instrument, KIR loci were identified for each patient and donor sample. KIR-ligand (HLA) incompatibility in the GVH and HVG directions were assessed based on HLA and KIR genotyping (KIR2DL1, KIR2DL2, KIR2DL3 and KIR3DL1) of 14 donors and 12 of the 14 recipients. Six of the 14 patients eventually lost their grafts despite DLI. Seven patients spontaneously achieved FDC and one converted to FDC following DLI. The missing KIR ligand analysis showed 12 patients (86%, n=12/14) with KIR-ligand incompatibility in the GVH direction (1 missing ligand, n=9; 2 missing ligands, n=3) and 10 patients (83%, n=10/12) with KIR-ligand incompatibility in the HVG direction (1 missing ligand, n=9; 2 missing ligands n=1). The presence or the degree of KIR-ligand incompatibility in GVH or HVG direction was not found to be predictive of spontaneous FDC or graft rejection. There was no significant relationship between the number of missing KIR ligands in either direction and the development of acute or chronic GVHD. This study shows a higher rate of KIR-ligand incompatibility in the GVH (86%) and HVG (83%) directions in the setting of HLA-matched sibling HSCT than previously reported. Although the small number of patients does not allow for statistically meaningful conclusions regarding clinical outcome, the observation of a high incidence of KIR-ligand incompatibility in this population justifies the study of larger patient cohorts to determine the influence of NK cell alloreactivity on transplant outcomes.
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Montcuquet, Nicolas, Sylvain Perruche, Benjamin Shipman, Aliette Marandin-Decock, Francis Bonnefoy, Philippe Saas, Eric Robinet, and Pierre Tiberghien. "Infusion of Ex-Vivo Expanded Donor T Cells To Improve Graft-Derived T-Cell Reconstitution after Allogeneic Hematopoietic Stem Cell Transplantation." Blood 110, no. 11 (November 16, 2007): 3261. http://dx.doi.org/10.1182/blood.v110.11.3261.3261.
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Abstract Limitations resulting from the reduced availability of related donors have been solved by the development of haplo-identical transplantation or by the use of cord blood as an alternative source of hematopoietic stem cells (HSC) to the bone marrow or peripheral blood. However, these kinds of transplantation remain associated with an impaired immune reconstitution, leading to an increased risk of infection and require an efficient modulation of post-transplant alloreactivity. In this setting, we and others demonstrated the possibility to control the alloreactivity by suicide gene transfer into donor T cells after ex-vivo T-cell culture. Such ex vivo culture was associated with the acquisition of a memory-like phenotype and with a decreased alloreactivity of gene-modified T cells, leading to an impaired potential of GvHD induction in murine models of allogeneic bone marrow transplantation (BMT). Chen and al. (Blood 2004) showed in an allogeneic BMT murine models that memory T cells were less alloreactive than naive T cells, leading to a less severe GvHD, but improved the immune reconstitution as compared with mice transplanted with bone marrow cells (BMC) only. By analogy with these results, we investigated the potential of ex-vivo expanded T cells (consisting of Con-A-activated splenocytes cultured ex vivo for 12 days in the presence of 500 UI/ml IL-2) to improve immune reconstitution without inducing GvHD. As compared with recipients of T-cell-depleted (TCD) BMC only, the administration of 106ex-vivo-expanded splenocytes (T) from CD45.1 C57Bl/6 mice together with 106 TCD-BMC from CD45.2 C57Bl/6 donors into 8 Gy-irradiated Balb/c allogeneic recipients significantly increased survival of transplanted mice at day 45 (58.3% vs 23.4% for BMC + T vs BMC only; p=0.0012, log rank test). Improved survival was associated with accelerated lymphoid and myeloid reconstitution as evidenced by day 15 lymphocyte and granulocyte blood counts: 212 (median) [range: 15–991]) vs 135 [14–632] lymphocytes/μl (p=0.0220) and 802 [6–5648] vs 114 [5–2411] granulocytes/μl (p=0.0006) for BMC + T (n=61) vs BMC only (n= 55). Importantly, FACS analysis demonstrated that enhanced lymphoid and myeloid reconstitution induced by ex-vivo expanded donor T-cells was due to enhanced donor bone-marrow-derived cells (lymphocyte and granulocyte blood counts: 129 [0–932] vs 11 [0–603] lymphocytes/μl (p=0.0014) and 801 [2–5637] vs 114 [2–2409] granulocytes/μl (p=0.0007) for BMC + T vs BMC only) and not ex-vivo expanded donor cells or residual recipient cells. Within the lymphoid compartment, enhanced reconstitution was observed mainly for CD3+CD8+ cells. Co-infusion of ex-vivo expanded donor T-cells did not induce GvHD (no GvHD-induced mortality or weight loss) while co-infusion of fresh splenocytes from CD45.1 C57Bl/6 mice induced severe GvHD (p<0.001 vs BMC only). Our results establish that ex-vivo expanded donor T-cells have a graft-facilitating effect and that they could be considered as a new cell therapy product allowing improving immune reconstitution after hematopoietic stem cell transplantation. Mechanisms involved in this graft-facilitating effect of ex-vivo expanded donor T cells remain to be elucidated.
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Galaverna, Federica, Fabio Guolo, Daniela Pende, Alida Dominietto, Raffaella Meazza, Michela Falco, Carmen Di Grazia, et al. "Natural Killer (NK) Alloreactivity Seems Not to Play a Role in Preventing Leukemia Relapse in Unmanipulated Haploidentical Bone Marrow Transplantation with Post-Transplant Cyclophosphamide." Blood 126, no. 23 (December 3, 2015): 2033. http://dx.doi.org/10.1182/blood.v126.23.2033.2033.
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Abstract Background Natural Killer (NK) cells have been widely studied due to their non-major histocompatibility complex (MHC)-restricted cytotoxicity towards transformed or virally infected target cells. In the setting of hematopoietic stem cell transplantation (HSCT), donor NK cells may be "alloreactive" as their killer immunoglobuline-like receptors (KIRs) do not recognize their ligands on recipient human leukocyte antigen (HLA) class I molecules (i.e. KIR-ligands), leading to NK activation. NK alloreactivity can often occur in haploidentical HSCT (Haplo-HSCT), by means of KIR/KIR-L mismatch in graft versus host (GvH) direction, contributing to graft-versus leukemia (GvL) effect, clearing residual leukemic blasts. In the last decade, several studies have shown that NK cells alloreactivity plays a role in T-depleted Haplo-HSCT leading to higher disease free survival rates for patients transplanted from NK-alloreactive donors; recent studies have also shown that donors having KIR B haplotypes (characterized by the presence of more activating KIR) or expressing KIR2DS1 correlated with a better clinical outcome of transplantation. Thus, these NK cell features might be positively considered in the donor selection strategy. Materials and Methods: We analyzed NK-alloreactivity in the setting of unmanipulated Haplo-HSCT with post-transplant cyclophosphamide for patients affected by acute myeloid leukemia or myelodisplastic syndromes. 101 consecutive patients transplanted from September, 2010 to October, 2014 were enrolled, with the big majority of donors and patients studied for HLA-genotype and KIR. Results: Disease status at HSCT was the most relevant factor affecting outcome (p <0.0001), with 3 y 78% overall survival (OS) and 76% disease free survival (DFS) rates for "early" patients (CR1+CR2, n=61) versus 33% OS and 28% DFS rates for "advanced" patients (CR3 or active disease, n=40). Nor NK-alloreactivity nor the presence of donor KIR-B haplotype nor the presence of donor KIR2DS1 seemed to play a role in preventing leukemia relapse. NK alloreactive patients had DFS rate similar to non-NK alloreactive group (62% vs 59%, p 0.47) with a better, still non-significant trend in OS (72% vs 60%, p 0.14) for NK-alloreactive patients. Similarly, Haplo-HSCT from donors with KIR-B haplotype (with B-content score>=2) or who had KIR2DS1 was not associated with better outcome (p 0.67 and p 0.89, respectively). We observed an high expression of CD56 and inhibitory receptors such as NKG2A on surface of NK cells in post-HSCT samples, suggesting that NK-cell function could be inhibited in unmanipulated haploidentical setting. Conclusions NK alloreactivity seems not to play a role in preventing leukemia relapse in unmanipulated haploidentical transplantation with post-transplantation. The different immunosuppressive approach of this Haplo-HSCT setting compared to T-depleted Haplo-HSCT, with concomitant use of cyclosporine from early transplant days, which has been shown to interact and possibly inhibit NK cells in vivo, and post transplant cyclophosphamide effects, selectively killing activated T-cell and inducing long-term tolerance, could affect NK efficacy. Further studies are needed to better understand the complexity of this intriguing issue, leading to a more complete definition of NK cell functions in this Haplo-HSCT setting. Disclosures No relevant conflicts of interest to declare.
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Liu, Fang, Xiaofan Zhu, Wenyu Yang, Ye Guo, Xia Chen, and Xiaoyan Zhang. "An Encouraging Successful Result of Allogeneic Hematopoietic Stem Cell Transplantation in a Boy Both with Juvenile Myelomonocytic Leukemia and Osteogenesis Imperfecta : A Case Report." Blood 136, Supplement 1 (November 5, 2020): 28. http://dx.doi.org/10.1182/blood-2020-142710.
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Background: Juvenile myelomonocytic leukemia (JMML) is a rare hematologic malignancy in young children that is classified as a myelodysplastic/myeloproliferative neoplasm and not only characterized by young age, hepatosplenomegaly, thrombocytopenia and monocytosis, but also by molecular aberrations in the RAS-RAF-MEK-ERK signaling pathway and GM-CSF-hypersensitivity. Most children with JMML experience an aggressive clinical course and the only curative treatment option for these children is stem cell transplantation (SCT). Osteogenesis imperfect (OI) is also an orphan inherited monogenic bone fragility disorder that usually is caused by mutations in one of the two genes coding for collagen type I alpha chains, COL1A1 or COL1A2. A common issue associated with the molecular abnormality is a disturbance in bone matrix synthesis and homeostasis inducing bone fragility. In very early life, this can lead to multiple fractures and progressive bone deformities. Current multidisciplinary management could only improve quality of life for patients, including physical therapy, drug treatment and orthopaedic surgery. Innovative therapies, such as progenitor and mesenchymal stem cell or bone marrow transplantation, targeting the specific altered pathway rather than the symptoms, may develop new curative treatments. Here we report a 3-year-old boy who suffered from both JMML and OI, was successfully transplanted and kept presenting an encouraging outcome up to now. Aims:To investigate the possible efficacy and safety of Allogeneic Hematopoietic Stem Cell Transplantation in a boy both with Juvenile Myelomonocytic Leukemia and Osteogenesis Imperfecta. Methods:A 3-year-old boy presented with fatigue, fever, petechia and rash in Aug 2019, accompanying with loss of appetite, joint pain and severe hepatosplenomegaly. The boy had a special appearance of short stature and blue sclerae, meanwhile he suffered intermittent eczema and bone fracture twice since he was 2 years old. Similar characteristics were also positive in his grandmother, father and father's sister. The blood cell counts revealed anemia, thrombocytopenia and leukocytosis especially monocytosis. Bone marrow aspirate showed excessive proliferation of myelomonocytic cells and hypersensitivity to granulocyte-macrophage colony-stimulating factor in vitro. Somatic mutation of gene NF1, PTPN11 and COL1A1 were identified by Next Generation Sequencing.Therefore, the little boy was diagnosed with two rare diseases of Juvenile Myelomonocytic Leukemia and Osteogenesis Imperfecta at the same time. After 4 courses of hypomethylating agents therapy, the boy underwent haploidentical allogeneic bone marrow stem cell transplantation combined with allogeneic single umbilical cord blood transplant in May 2020. The myeloablative conditioning regimen was composed of Decitabine (20mg/m2/day, days -13 to -9), Cyclophosphamide (25mg/kg/day, days -8 and -7), Busulfan(100mg/m2/day, days -6 to -3), Fludarabine (40mg/m2/day, days -6 to -2) and Cytarabine (100mg/m2/day, days -6 to -2). Post-Cyclophosphamide (50mg/kg/day, days +3 and +4), tacrolimus and mycophenolate mofetil were used for prophylaxis of graft-versus-host disease (GVHD). Results:The number of infused TNCs from haplo-bone marrow and cord blood unit was 41.4×10^8/kg and 9.72×10^7/kg, respectively, while the number of infused CD34+ cells was 11.84×10^6/kg and 2.33×10^5/kg, respectively. The boy achieved sustained engraftment of both neutrophils and platelets at 16 days and 24 days, respectively, with complete haplo-donor chimerism of confirmed at 14 days. He developed grade III acute GVHD (skin, gut and liver) and recovered at 39 days after transplant. Clinical symptoms such as rash, joint pain and hepatosplenomegaly got complete remission, and the mutated genes like NF1, PTPN11 and COL1A1 all disappeared at 30 days. At the time of this report, the boy was alive with negative MRD and good quality of life with a follow-up of 3 months after HCT. Conclusion:To our knowledge, this is the first report that a child both with Juvenile Myelomonocytic Leukemia and Osteogenesis Imperfecta was cured by allogeneic hematopoietic stem cell transplantation.Our experience suggests that allogeneic bone marrow transplantation may be a novel safe and effective therapeutic strategy for OI patients. Disclosures No relevant conflicts of interest to declare.
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Wang, Ming, Ming Wang, Bixia Wang, Liping Guo, Yifei Cheng, Zhidong Wang, Yuqian Sun, Yu Wang, Yingjun Chang, and Xiaojun Huang. "PRDM1 Is Sufficient for Inducing Human Primary T Cell Hyporesponsiveness and Implicates Low Gvhd Occurrence after Allo-HSCT." Blood 138, Supplement 1 (November 5, 2021): 197. http://dx.doi.org/10.1182/blood-2021-149394.
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Abstract T cell hyporesponsiveness is crucial for functional immune system and prevent the damage induced by alloreactive T cells in autoimmune pathology and transplantation. As one of the most important regulators during B cell development, PRDM1 (also known as BLIMP-1) has been demonstrated its essential role for maintaining T cell hyporesponsiveness and homeostasis, evidenced by Prdm1 deficient mice accumulating activated T cells and developing multiorgan inflammatory disease. However, the mechanism of PRDM1 regulating T cell hyporesponsiveness is still ambiguous. In this study, we took advantage of multiomics technologies and systemic report the central role of PRDM1 in inducing human primary T cell hyporesponsiveness. Firstly, we overexpressed PRDM1 in human primary T cells and found increased ratio of CD4 +CD25 +FOXP3 + Treg cell subsets and increased IL-4 secretion. In parallel, inhibited PRDM1 expression level in human T cells decreased ratio of Treg cells and secretion of IL-4. Meanwhile, transcriptome analyses revealed that overexpressed PRDM1 enriched negative regulation of cell proliferation signaling pathway and resulted in a global reduction in IL-2 and inflammatory response signaling pathways. Furthermore, overexpressed PRDM1 in primary T cells upregulated several negative regulators of T cell function like EOMES, KLF2, LILRB1, KLRB1 and CD244, indicating a pioneer role of PRDM1 in inducing T cell hyporesponsive. To further investigate the regulation role of T cell hyporesponsiveness of PRDM1, we performed CUT&Tag and ATAC-seq in PRDM1 overexpressed primary T cells. CUT&Tag analysis showed PRDM1 could directly upregulated T cell hyporesponsiveness related gene such as KLF2, CD244 and KLRD1. Importantly, we observed consistent changes of IL-2, central regulator of T cell activation, in PRDM1 overexpressed T cell from ATAC-seq, CUT&Tag and RNA-seq data. We found PRDM1 could binding to IL-2 locus and decreased the chromatin accessibility of IL-2, consequently downregulated the expression level of IL-2 in human primary T cells. Moreover, altered open chromatin regions (OCRs) in PRDM1 overexpressed T cells enriched the similar transcription factors (TFs) with PRDM1 binding sites, indicating PRDM1 might be a pioneer TF in T cell hyporesponsiveness. These results demonstrated PRDM1 is sufficient for inducing T cell hyporesponsiveness in human primary T cells. To further validate the coexpression relationship between PRDM1 and Treg cell central TF FOXP3, we upregulated PRDM1 expression level on Jurkat T cells lines. The results also showed that elevated FOXP3 both in mRNA and protein level accompanied with upregulated PRDM1 expression level. To analyze the mechanism of PRDM1 regulating FOXP3 expression level, CUT&Tag data analyses showed that PRDM1 might upregulated FOXP3 by directly binding to the enhancer region of upstream of FOXP3 locus. Meanwhile, PRDM1 indirectly upregulated FOXP3 by upregulated KLF2, evidenced by inhibiting KLF2 in PRDM1 overexpressed primary T cells downregulated FOXP3 expression level. To further investigate the clinical implication of PRDM1 inducing T cell hyporesponsiveness, we detected the relationship of PRDM1 expression level and acute graft-versus-host disease(aGVHD) occurrence after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Our results showed that patients with aGVHD (n=7) exhibited lower PRDM1 expression level than those without aGVHD in the same period after HSCT (n=7). Furthermore, we detected the expression level of PRDM1 in CD4 + T cell and CD8 + T cells from bone marrow allografts (BM) or peripheral blood allografts (PB) and followed up the occurrence of GVHD after HSCT for 2 years (n=18). There are low expression levels of PRDM1 in CD4 + T cells both from BM or PB grafts corelated with aGVHD occurrence in patients after allo-HSCT compared with those without aGVHD occurrence. In conclusion, our study provides the global regulatory model of PRDM1 in human primary T cell. We introduced PRDM1 as a sufficient regulator in T cell hyporesponsiveness induction, which is altering the chromatin accessibility and directly upregulated T cell inhibitory signals and downregulated T cell activated signals. The negative relationship between PRDM1 expression level with GVHD occurrence indicated it might be a potential biomarker for indicating HSCT prognosis. Disclosures No relevant conflicts of interest to declare.
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Perez, Lia, Nancy Parquet, Ken Shain, Ramadevi Nimmanapalli, Melissa Alsina, Claudio Anasetti, and William S. Dalton. "Hematopoietic Stroma Confers Resistance to Immune Control by TRAIL in Multiple Myeloma through Induction of FLIP." Blood 108, no. 11 (November 16, 2006): 3424. http://dx.doi.org/10.1182/blood.v108.11.3424.3424.
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Abstract TNF-related apoptosis inducing ligand/Apo-2L (TRAIL), a member of the TNF superfamily of death ligands, is preferentially cytotoxic against neoplastic cells, while sparing normal tissue. TRAIL has been implicated in tumor immune-surveillance and mediates murine allogeneic graft-versus-tumor responses. The bone marrow microenvironment provides tumor protection from chemotherapy and Fas-death receptor mediated apoptosis through a process known as environmental mediated death resistance (EM-DR). We tested whether the tumor microenvironment inhibits TRAIL-mediated killing of myeloma cells, as this process may contribute to multiple myeloma escape from immune-surveillance. Three drug-sensitive myeloma cell lines (RPMI-8226, U266 and MM1s) exhibit apoptosis resistance to recombinant human (rh) TRAIL while adhered to HS5 stromal cells that expresses green-fluorescence-protein (HS5-GFP). Apoptosis resistance to TRAIL was time- and dose- dependent. Evaluation of TRAIL apoptosis in a transwell (TW) assay, with HS5-GFP cells on the bottom and RPMI-8226 cells in the upper well (TW+HS5) revealed that HS5-GFP stromal cells blocked TRAIL-induced apotosis through soluble factors. We then assessed the modifications in the TRAIL signaling pathway induced by TW+HS5 and responsible for the observed phenotype. RPMI-8226 treated in TW+HS5 exhibited attenuated pro-caspase-8, pro-caspase-3, PARP, and BID cleavage, with diminished mitochondrial membrane potential changes, but without alterations on TRAIL receptors and other Bcl-2 family members. We found increased levels of Fas-associated death domain like IL-1 converting enzyme-like inhibitory protein (FLIP), a regulatory factor that competes with caspase-8 inhibiting apoptosis. Subcellular fractionation of RPMI-8226 cells showed that FLIPL is maintained in or associated with organelle membranes and is released to the cytosol when exposed to soluble factor (TW+HS5) suggesting that that soluble factor signaling may influence FLIPL localization and availability. Furthermore, FLIP reduction by FLIP-RNA interference increases TRAIL sensitivity of RPMI-8226 treated in TW+HS5. Bortezomib is a proteosome inhibitor that inhibits NF-KB and reduces FLIP levels. To this end, pretreatment of RPMI-8226 cells with bortezomib for 20 hours followed by TRAIL for 4 hours overcame TRAIL EM-DR through inhibition of FLIP, thus providing rationale for testing this drug combination in a clinical trial. In addition, rh-IL-6 induced FLIP levels in RPMI-8226 cells conferring resistance to TRAIL mediated apoptosis. Our results suggest that IL-6 and other soluble factors produced by marrow stromal cells promote myeloma cell survival by upregulating FLIP, thereby mitigating the influence of the microenvironment on TRAIL-induced apoptosis. We conclude that the cytotoxic effect of TRAIL may be enhanced by FLIP inhibitors.
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Sun, Zejin, Rikki Enzor, Paula Rio, D. Wade Clapp, and Helmut Hanenberg. "Generation Of FANCA-/- Human CD34+ Hematopoietic Stem Cells By shRNA Knockdown." Blood 122, no. 21 (November 15, 2013): 2903. http://dx.doi.org/10.1182/blood.v122.21.2903.2903.
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Abstract Fanconi anemia (FA) is a recessive DNA repair disorder characterized by bone marrow (BM) failure, genomic instability, and a predisposition to malignancies. Natural gene therapy due to molecular self-correction of hematopoietic stem cells (HSCs) has been reported in a minority of FA patients, suggesting that due to the in vivo selection advantage of the corrected cells, FA is an excellent model disease for stem cell gene therapy. However, the scarcity of autologous HSCs from FA patients for research purposes is one of the major road blocks to preclinical studies with human cells. Here, we developed a lentiviral vector with EGFP as marker gene that co-expresses two distinct shRNA sequences against FANCA under two different human promoters (H1 and U6). In vitro analysis in primary human fibroblasts showed that stable integration of this construct was highly efficient to induce the typical FA cellular phenotypes as assessed by (1) FANCD2 ubiquitination deficiency and (2) a characteristic G2/M arrest upon DNA damage induced by DNA crosslinking reagent Mitomycin C (MMC). We then transduced human cord blood (CB) CD34+ cells with this lentiviral vector and demonstrated a reduced survival of clonogenic cells in progenitor assays at 20nM MMC: 70% (scrambled control shRNA) vs. 23% (FANCA shRNA). This vector pseudotyped with a foamyviral envelope was then used to transduce CD34+ CB cells on fibronectin CH296. The next day, genetically modified cells were transplanted into NOD.Cg---Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. When analyzing the percentage of EGFP+ cells in the human graft (hCD45+ cells), we noticed a progressive decline of EGFP+ cells from 29% on day 5 to 5% at 4 months after transplantation in the peripheral blood of the recipient mice, mimicking the progressive BM failure in FA patients. In contrast, engraftment over time was stable in CD34+ cells transduced with scrambled control shRNA vector (33% on day 5 vs. 34% at 4 months). The human progenitors isolated from the BM of NSG recipient mice at sacrifice 4 months after initial transduction and transplantation are still hypersensitive to MMC, with a much lower survival rate of 34% at 20nM MMC in the FANCA shRNA group as compared to 78% in the scrambled control shRNA group, thus confirming the knockdown by the lentiviral shRNA construct is stable. In summary, the novel double shRNA lentiviral vector is capable of inducing all major hallmarks of FA cells in normal human CB CD34+ cells, thus providing unlimited FA-like cellular materials including NSG mice-repopulating HSCs for preclinical gene therapy and basic stem cell biology research in FA. Disclosures: No relevant conflicts of interest to declare.
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Wilkinson, Kathryn N., Britt Anderson, Jennifer McNiff, Anthony Jake Demetris, Warren D. Shlomchik, and Mark Shlomchik. "A New TCR Transgenic Model of GVHD Reveals That, Independent of Repertoire, Effector Memory T Cells Are Severely Limited, and Central Memory T Cells Somewhat Limited, in Their Ability to Cause GVHD." Blood 114, no. 22 (November 20, 2009): 233. http://dx.doi.org/10.1182/blood.v114.22.233.233.
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Abstract Abstract 233 Graft versus host disease (GVHD) remains a major cause of morbidity and mortality in allogeneic stem cell transplantation (alloSCT). In murine models of alloSCT, effector memory (EM) T cells engraft, respond to antigen, and mediate graft versus leukemia, but do not cause GVHD. There are three potential explanations for EM T cells' inability to cause GVHD. First, unlike naïve T cells, EM T cells fail to traffic to lymph nodes and Peyer patches, areas which may be important for initiation of GVHD. Second, a more-restricted T cell receptor repertoire in the EM T cell pool may lead to a reduced ability to recognize alloantigens. Third, it is possible that EM T cells do not possess the necessary effector mechanisms or are incapable of the proliferation or survival required for induction of GVHD. We recently reported that the inability to migrate to lymph nodes and Peyer patches is not responsible for the inability of EM T cells to cause GVHD (Anderson et al, Blood. 2008). To date, the role of repertoire has been difficult to test because in existing models of GVHD the disease causing T cells are undefined. Furthermore, whether central memory (CM) CD4 T cells are also incapable of causing GVHD remains unclear, in part because it has been difficult to isolate pure populations of polyclonal CM CD4 T cells. In order to concurrently address the role of repertoire and determine if CM CD4 T cells can cause GVHD, we developed a novel T cell receptor transgenic GVHD model. In this model naïve CD4+ TS1 T cells on a RAG-deficient background, which recognize an epitope of influenza hemagglutinin (HA), are transferred, along with syngeneic bone marrow, into irradiated transgenic recipients that express HA in all tissues (HA104 mice). Within a week post transplant, HA104 recipients of naïve TS1 cells developed a GVHD-like condition characterized by weight loss, visible wasting, and pathology of the skin, colon, and liver. An advantage of this model is that the disease causing T cells are defined, enabling us to determine if naïve and memory T cells of identical specificity have inherent differences in their ability to cause GVHD. We generated memory TS1 cells using in vitro stimulation followed by transfer into RAG−/− mice, according to Farber and colleagues (Ahmadzadeh et al. PNAS 2002). After 2 to 3 months, pure populations of CD62L+ CM TS1 cells and CD62L- EM TS1 cells were isolated by FACS. Upon transfer into irradiated HA104 recipients, EM TS1 cells initially did not cause disease symptoms, however, 30 days post transplant, EM TS1 recipients developed mild weight loss. These results indicate that repertoire differences are not responsible for the inability of EM T cells to cause GVHD. Interestingly, CM TS1 cells caused more weight loss than EM T cells, though not as much as that caused by naïve TS1 cells. These findings indicate that, independent of repertoire, CM T cells are also inherently limited in their ability to cause GVHD, though they are not as disabled in this respect as EM T cells. A major issue in GVHD work has been the inability to track, quantify and characterize the actual alloreactive GVHD-inducing T cells. With a TCR transgenic model, this is now possible and we are currently exploiting this feature to determine the fate of naïve, EM, and CM T cells after transfer. Initial experiments demonstrated that, in comparison to naïve and CM TS1 cell recipients, the secondary lymph nodes of EM TS1 cell recipients contained fewer TS1 cells 60 days post transplant, suggesting that in the context of GVHD, EM cells are inherently limited in their ability to expand or survive. We are currently tracking naïve, EM and CM TS1 cells throughout the course of a GVHD experiment, and assessing how, when, and where the fates of these cell types diverge. Results from these ongoing experiments will be presented. Disclosures: No relevant conflicts of interest to declare.
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Al Malki, Monzr M., Sumithira Vasu, Dipenkumar Modi, Miguel-Angel Perales, Lucy Y. Ghoda, Donna Bui, Vineetha Edavana, et al. "Phase 1/2 Study of Nexi-001 Donor-Derived Multi-Antigen Specific CD8+ T Cells for the Treatment of Relapsed Acute Myeloid Leukemia (AML) after Allogeneic Hematopoietic Transplantation." Blood 138, Supplement 1 (November 5, 2021): 4819. http://dx.doi.org/10.1182/blood-2021-152419.
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Abstract Patients who relapse after allogeneic HCT have a poor prognosis and few effective treatment options. Responses to salvage therapy with donor lymphocyte infusions (DLI) are driven by a graft versus leukemia (GvL) effect. However, relapses and moderate to severe graft versus host disease (GVHD) are common. Therapies that increase the GvL effect without inducing GVHD are needed. The NEXI-001 study is a prospective, multicenter, open-label phase 1/2 trial designed to characterize the safety, immunogenic, and antitumor activity of the NEXI-001 antigen specific T-cell product. This product is a donor-derived non-genetically engineered therapy that consists of populations of CD8+ T cells that recognize HLA 02.01-restricted peptides from the WT1, PRAME, and Cyclin A1 antigens. These T cells consist of populations with key memory phenotypes, including stem-like memory, central memory, and effector memory cells, with a low proportion (<5%) of potentially allogeneic-reactive T-naïve cells. Patients enrolled into the first cohort of the dose escalation phase received a single infusion of 50 million (M) to 100M cells of the NEXI-001 product. Bridging anti-AML treatment was permitted during the manufacture of the cellular product with a wash-out period of at least 14 days prior to lymphodepletion (LD) chemotherapy (intravenous fludarabine 30 mg/m 2 and cyclophosphamide 300 mg/m 2) that was administered on Days -5, -4, and -3 prior to the infusion of the NEXI-001 product up to 72 hours later (Day1). Lymphocyte recovery to baseline levels occurred as early as three days after the NEXI-001 product infusion with robust CD4 and CD8 T cell reconstitution after LD chemotherapy. NEXI-001 antigen specific T cells were detectable in peripheral blood (PB) by multimer staining and were found to proliferate over time and to traffic to bone marrow. The phenotype composition of detectable antigen specific T cells at both sites was that of the infused product. T-cell receptor (TCR) sequencing assays revealed T cell clones in the NEXI-001 product that were not detected in PB of patients tested at baseline. These unique clones subsequently expanded in PB and bone marrow (BM) and persisted over time. Neutrophil recovery, decreased transfusion burden of platelets and red blood cells, and increased donor chimerism were observed. Decreases in myeloblasts and reduction in the size of an extramedullary myeloid sarcoma were suggestive of clinical activity. One patient, a 23-year- old with MRD+ disease at baseline, received two doses of 200M NEXI-001 cells separated by approximately 2 months. Following the first infusion, antigen specific CD8+ T cells increased gradually in PB to 9% of the total CD3+ T cell population just prior to the second infusion and were found to have trafficked to bone marrow. By Day 2 following the second infusion, which was not preceded by LD chemotherapy, the antigen specific CD8+ T cells again increased to 9% of the total CD3+ T cell population in PB and remained at ≥5% until the end of study visit a month later. The absolute lymphocyte count increased by 50% highlighting continued expansion of the NEXI-001 T cells. These cells also maintained significant Tscm populations. Treatment related adverse events, including infusion reactions, GVHD, CRS, and neurotoxicity (ICANS), have not developed in these patients who have received 50M to 200M T cells of the NEXI-001 product either as single or repeat infusions. In conclusion, these results show that infusion of the NEXI-001 product is safe and capable of generating a cell-mediated immune response with early signs of clinical activity. A second infusion is associated with increasing the level of antigen specific CD8+ T cells and their persistence in PB and BM. TCR sequencing and RNA Seq transcriptional profiling of the CD8+ T cells are planned, and these data will be available for presentation during the ASH conference. At least two cycles of 200M NEXI-001 cells weekly x 3 weeks of a 4-week cycle is planned for the next dose-escalation cohort. Early data suggest that the NEXI-001 product has the potential to enhance a GvL effect with minimal GVHD-associated toxicities. Disclosures Al Malki: Jazz Pharmaceuticals, Inc.: Consultancy; Neximmune: Consultancy; Hansa Biopharma: Consultancy; CareDx: Consultancy; Rigel Pharma: Consultancy. Vasu: Boehringer Ingelheim: Other: Travel support; Seattle Genetics: Other: travel support; Kiadis, Inc.: Research Funding; Omeros, Inc.: Membership on an entity's Board of Directors or advisory committees. Modi: MorphoSys: Membership on an entity's Board of Directors or advisory committees; Seagen: Membership on an entity's Board of Directors or advisory committees; Genentech: Research Funding. Perales: Sellas Life Sciences: Honoraria; Novartis: Honoraria, Other; Omeros: Honoraria; Merck: Honoraria; Takeda: Honoraria; Karyopharm: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; MorphoSys: Honoraria; Kite/Gilead: Honoraria, Other; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Medigene: Honoraria; NexImmune: Honoraria; Cidara: Honoraria; Nektar Therapeutics: Honoraria, Other; Servier: Honoraria; Miltenyi Biotec: Honoraria, Other. Edavana: Neximmune, Inc: Current Employment. Lu: Neximmune, Inc: Current Employment. Kim: Neximmune, Inc: Current Employment. Suarez: Neximmune, Inc: Current Employment. Oelke: Neximmune, Inc: Current Employment. Bednarik: Neximmune, Inc: Current Employment. Knight: Neximmune, Inc: Current Employment. Varela: Kite: Speakers Bureau; Nexlmmune: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees.
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Ghosh, Arnab, Amanda M. Holland, Yildirim Dogan, Nury Yim, Uttam K. Rao, Lauren F. Young, Odette M. Smith, et al. "Genetic Engineering of Donor T Cells for BMT Immunotherapy: Expression of TRAIL and PLZF Selectively Enhances GVT and Abrogates Gvhd." Blood 116, no. 21 (November 19, 2010): 730. http://dx.doi.org/10.1182/blood.v116.21.730.730.
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Abstract Abstract 730 Efforts to improve graft-versus-tumor (GVT) activity of alloreactive donor T cells are limited by a concomitant rise in graft-versus-host disease (GVHD). We employed experimental allogeneic bone marrow transplantation (allo-BMT) models to assess two novel strategies, in which T lineage cells were genetically engineered to enhance selective effector functions. In addition to the transgenic expression of these molecules on mature donor T cells, we also used genetically engineered precursor T cells as an “off the shelf” adoptive cell therapy. One strategy relies on T cell cytolytic molecule TNF-Related Apoptosis Inducing Ligand (TRAIL), which can induce apoptotic signals through death receptor (DR) 4 and 5 molecules (only DR5 in mice) expressed on target cells. Certain tumor cells are known to express high levels of surface DR5 making TRAIL an attractive candidate for genetic engineering of donor T cells to enhance GVT activity. Extending previous studies indicating that TRAIL expression in donor T cells is dispensable for the development of GVHD, we evaluated the effect of TRAIL over-expression in donor T cells (mature and precursor) on GVHD and GVT. Mature T cells derived from donor B6 splenocytes were transduced with a lentiviral TRAIL expression vector. The transduced TRAIL+ T cells were adoptively transferred on day 0 into lethally irradiated CBF1 recipients of a T cell depleted allo-BMT, bearing LB27.4 tumors, (B6 ^ CBF1+LB27.4) to assess their GVHD and GVT activity. TRAIL+ T cells displayed significantly enhanced antitumor immunity compared to T cells transduced with a control vector against LB27.4 tumor cell lines in vitro and upon transfer into tumor bearing allo-BMT recipients (p<0.01)(Fig 1A). Interestingly, the recipients treated with TRAIL+ T cells had significantly less GVHD. We observed increased DR5 expression on host antigen presenting cells (APC) soon after the total body irradiation used in our preparative regimens, suggesting that TRAIL+ donor T cells could potently eliminate host APC, resulting in less GVHD. Precursor T cells have the added benefits of reconstituting the T cell compartment without GVHD and “off the shelf” use. We generated TRAIL+ precursor T cells from transduced B6 murine hematopoietic stem cells and expanded them using the OP9-DL1 co-culture system. Adoptive transfer of B6 TRAIL+ precursor T cells into syngeneic-transplanted BALB/c mice could reconstitute the T cell compartment with TRAIL expressing T cells. When challenged with localized RENCA tumors, TRAIL+ precursor T cells showed enhanced antitumor activity (p<0.05) compared to mock (GFP)-transduced controls (Fig 1B). The second strategy explores the use of promyelocytic leukemia zinc finger (PLZF). PLZF is a member of the BTB-ZF (Broad complex, Tram track, Bric-^-brac-zinc finger) family of transcription factors. In NKT and NK cells, it controls key steps in development, activation and effector functions. Its ectopic expression on conventional T cell confers NKT and NK-like properties. Recent studies using transgenic mice have shown that T cells ectopically expressing PLZF spontaneously acquire an effector/memory phenotype and CD8+ T cells spontaneously express IFN-gamma. Using PLZF+ transgenic donor (B6) T cells in A20-tumor challenged B6 ^ BALB/c allo-BMT model, we found that PLZF+ T cells have less GVHD activity while GVT activity remained intact (p<0.001)(Fig 1C). Using adoptive transfer of CFSE-labeled donor T cells we observed that fast proliferating allo-responsive PLZF+ T cells died after only a few cell cycles. We then transduced donor T cells with an expression vector and confirmed that transduced PLZF expressing donor T cells have less GVHD activity and intact GVT activity (Fig 1D). In conclusion, our data suggest that adoptive therapy with genetically engineered TRAIL or PLZF expressing donor T cells exert less GVHD activity while displaying intact or enhanced GVT activity. Furthermore, the “off the shelf” use of genetically enhanced precursor T cells indicates a promising cell therapy strategy to enhance anti-tumor activity in both autologous and allogeneic BMT patients. Disclosures: No relevant conflicts of interest to declare.
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Hsu, Fu-Yin, Jheng-Jie Chen, Wen-Chieh Sung, and Pai-An Hwang. "Preparation of a Fucoidan-Grafted Hyaluronan Composite Hydrogel for the Induction of Osteoblast Differentiation in Osteoblast-Like Cells." Materials 14, no. 5 (March 2, 2021): 1168. http://dx.doi.org/10.3390/ma14051168.
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A suitable bone substitute is necessary in bone regenerative medicine. Hyaluronan (HA) has excellent biocompatibility and biodegradability and is widely used in tissue engineering. Additionally, research on fucoidan (Fu), a fucose- and sulfate-rich polysaccharide from brown seaweed, for the promotion of bone osteogenic differentiation has increased exponentially. In this study, HA and Fu were functionalized by grafting methacrylic groups onto the backbone of the chain. Methacrylate-hyaluronan (MHA) and methacrylate-fucoidan (MFu) were characterized by FTIR and 1H NMR spectroscopy to confirm functionalization. The degrees of methacrylation (DMs) of MHA and MFu were 9.2% and 98.6%, respectively. Furthermore, we evaluated the mechanical properties of the hydrogels formed from mixtures of photo-crosslinkable MHA (1%) with varying concentrations of MFu (0%, 0.5%, and 1%). There were no changes in the hardness values of the hydrogels, but the elastic modulus decreased upon the addition of MFu, and these mechanical properties were not significantly different with or without preosteoblastic MG63 cell culture for up to 28 days. Furthermore, the cell morphologies and viabilities were not significantly different after culture with the MHA, MHA-MFu0.5, or MHA-MFu1.0 hydrogels, but the specific activity and mineralization of alkaline phosphatase (ALP) were significantly higher in the MHA-MFu1.0 hydrogel group compared to the other hydrogels. Hence, MHA-MFu composite hydrogels are potential bone graft materials that can provide a flexible structure and favorable niche for inducing bone osteogenic differentiation.
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Martin-Antonio, Beatriz, Maria Suarez-LLedo, Montserrat Arroyes, Francesc Fernandez-Avilés, Carmen Martinez, Montserrat Rovira, Ildefonso Espigado, et al. "A Gene Variant in IRF3 Impacts On the Clinical Outcome of Acute Myeloid Leukemia (AML) Patients Submitted to Allogeneic Stem Cell Transplantation (allo-SCT)." Blood 120, no. 21 (November 16, 2012): 468. http://dx.doi.org/10.1182/blood.v120.21.468.468.
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Abstract Abstract 468 AML is worldwide the most frequent indication for allo-SCT. This is most likely due to the curative potential of the graft versus leukemia (GVL) effect associated with the procedure. Unfortunately, GVL and GVHD are intimately linked. Thus, it is important to identify markers predictive of severe GVHD, to balance the risk of this complication and the risk of relapse in a particular AML patient. The innate immune system, as the initial regulator of the inflammatory response, mediates an important role in these reactions. After conditioning regimen, toll-like receptors initiate the innate inflammatory response by activating intracellular signaling cascades that converge on the activation of NF-κB and interferon regulatory factor-3 (IRF3). IRF3 activation in bone marrow derived dendritic cells (DCs) results in natural killer (NK) cell activation inducing an anti-tumor NK response. We hypothesized that genetic variability in IRF3 in either the donor or the recipient could impact on the degree of the inflammatory response and on GVHD and GVL effect allo-SCT. We analysed the effect of two frequent single nucleotide polymorphisms (SNPs) in IRF3 (rs7251 and rs2304205), which are inherited in a haplotype, on the GVHD and GVL in 249 patients diagnosed with AML and submitted to HLA-identical sibling allo-SCT. Those patients with a donor carrying dominant GG gene variant in rs7251 (45% of the donors) had, as compared to GC (44%) and CC (11%) variants, lower aGVHD III-IV incidence (4% vs 11% vs 27%; p=0.0078) (Figure 1A), higher relapse incidence (49% vs 35% vs 26%; p=0.018) (Figure 1B), and lower TRM (7% vs 24% vs 18%; p=0.0065). This clinical impact on severe aGVHD, relapse, and TRM was retained at multivariate analysis. Further, GG gene variant in rs7251 when present in the patient was also associated with lower aGVHD III-IV incidence (4% vs 13% vs 24%; p=0.009), higher relapse incidence (50% vs 34% vs 31%; p=0.014), and with a trend for a lower TRM (9% vs 19% vs 23%; p=0.064). Patients carrying AA dominant gene variant in rs2304205 in IRF3 presented a higher relapse incidence than the rest of genotypes (50% vs 39% vs 18%, p=0.0068). However, this impact was not retained at multivariate analysis. Functional studies in 180 healthy individuals showed that after stimulation of peripheral blood with cytomegalovirus (CMV) peptides, GG gene variant in rs7251 presented lower IFN-γ serum production than the rest of individuals, and GG gene variant was associated with lower number of IFN-γ producing mature NK cells, lower number of cytotoxic NK cells against K562 cell line and lower proliferation of T cells after antigen presentation by DCs. In conclusion, we show that a particular gene variant in IRF3 in the donors is associated with a low incidence of severe aGVHD and high incidence of relapse in AML patients submitted to allo-SCT. This finding could be explained by its effect in the inflammatory and adaptive immune response, with lower IFN-g production, lower lymphocyte proliferation after antigen presentation by DCs and lower mature NK cell response. Thus, these results suggest that, if possible, when transplanting an AML patient with a low risk of relapse it might be preferable to select a donor harbouring GG in rs7251 in IRF3, and when transplanting an AML patient with a high risk of relapse after the transplant it might be preferable to consider select a donor GC or CC in rs7251 in IRF3. Disclosures: No relevant conflicts of interest to declare.
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Chu, J. L., P. Ramos, A. Rosendorff, J. Nikolić-Zugić, E. Lacy, A. Matsuzawa, and K. B. Elkon. "Massive upregulation of the Fas ligand in lpr and gld mice: implications for Fas regulation and the graft-versus-host disease-like wasting syndrome." Journal of Experimental Medicine 181, no. 1 (January 1, 1995): 393–98. http://dx.doi.org/10.1084/jem.181.1.393.
Full textAbstract:
Fas-deficient lpr and gld mice develop lymphadenopathy due to the accumulation of T cells with an unusual double negative (DN) (CD4-CD8-) phenotype. Previous studies have shown that these abnormal cells are capable of inducing redirected lysis of certain Fc receptor-positive target cells. Since the Fas ligand (FasL) has recently been shown to be partly responsible for T cell-mediated cytotoxicity, lymph node cells from lpr and gld mice were examined for the expression of FasL mRNA. Northern blot analysis revealed that lymph node cells obtained from lpr and gld mice had a striking increase in the level of expression of FasL mRNA predominantly due to expression in the DN T cells. Furthermore, lpr, but not gld lymph node cells killed the B cell line, A20, in a Fas-dependent manner. These findings indicate that Fas mutations result in a massive up-regulation of FasL which, most likely, results from repetitive exposure to (self) antigen. This phenomenon could explain the lpr-induced wasting syndrome observed when lpr bone marrow-derived cells are adoptively transferred to wild-type recipients.
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Li, Hongmei, Catherine Matte-Martone, Anthony Jake Demetris, Jennifer McNiff, Dhanpat Jain, and Warren D. Shlomchik. "Redundant Mechanisms for Dendritic Cell Activation in GVHD Induction: Signalings Via TLRs, TNF-α, IL-1 and CD40 Are Not Required." Blood 112, no. 11 (November 16, 2008): 3509. http://dx.doi.org/10.1182/blood.v112.11.3509.3509.
Full textAbstract:
Abstract Graft-versus-host disease (GVHD) is initiated by antigen-presenting cells (APCs) that prime alloreactive donor T cells. Amongst APCs, dendritic cells (DCs) are the most potent at priming naïve T cells. A paradigm of DC function holds that instructive maturation signals transform “immature” DCs into “mature” DCs which are optimized for T cell activation. Signaling via Toll-like receptors (TLRs) has emerged as a central mechanism for inducing this maturation in anti-pathogen responses. However, in allogeneic stem cell transplantation, there is no specific pathogen and the signals that induce DC maturation in GVH responses have not been defined. To study potential maturation stimuli we performed GVHD experiments in model systems wherein host APCs are essential for GVHD induction (C3H.SW (H-2b)→B6 and B6bm12→B6). We then compared GVHD in wild type (wt) recipients to that in hosts genetically unable to respond to various DC maturation signals. We first used as hosts mice deficient in TLR4, CD40, IL1-R or TNFR1/R2 and found that in C3H.SW→B6 model, GVHD in each of these was similar. Likewise, GVHD was similar in wt and mice deficient in MyD88, essential for signaling via all TLRs except TLR3 and TLR4. To focus our studies on APCs, we used as hosts radiation bone marrow (BM) chimeras in which wt B6 (CD45.1) mice were reconstituted with BM from CD45.2 wt, MyD88−/−, TRIF−/− (required for signaling via TLR3 and, in-part, TLR4), and MyD88/TRIF−/− mice, in which signaling by all TLRs, IL-1 and IL-18 is completely blocked. When retransplanted with C3H.SW BM and CD8 cells, all chimeras developed similar clinical and histologic GVHD. To exclude that T cells were being primed by residual wt host APCs in BM chimeric recipients, we used as hosts B6bm12 mice that were reconstituted with BM from B6 wt, MyD88−/−, TRIF−/− or MyD88/TRIF−/− mice. These chimeras were then retransplanted with B6bm12 BM and CD4 cells. Because residual host APCs in these BM chimeric hosts were B6bm12 and therefore syngeneic with the donor cells, T cell priming could only occur on B6 background APCs derived from the first transplant. As in the prior experiments, all chimeras developed similar clinical GVHD; pathology scoring is underway. Therefore in contrast to nearly all adaptive T cell responses, GVHD induction in both MHC-matched and MHC-mismatched systems did not require APC maturation signals mediated by TLRs. Absent any phenotype when APC maturation signals were blocked, we used the same approach in the C3H.SW→B6 model to study the roles of IL12 and type I IFNs, which are produced as a consequence of DC maturation. B6 p35−/− and B6 wt mice developed similar GVHD, excluding an essential role for host IL12. Type I IFN receptor-deficient (IFNIR−/−) mice developed similar GVHD as did wt controls, except that IFNIR−/− CD8 recipients lost more weight but had less histologic liver GVHD (P=0.0024). To focus on the activity of Type I IFNs on host hematopoietic cells, we used as recipients IFNIR−/− → B6 chimeras. Both clinical and histologic GVHD were similar in IFNIR−/−→B6 and wt→B6 control chimeras, suggesting that the differences in weight loss and hepatic GVHD observed in IFNIR−/− hosts, were due to parenchymal expression of IFNIR−/−. In sum, these data indicate that DC maturation signals in GVHD are at a minimum redundant, and independent of TLR ligands such as LPS, which are certainly available to the host immune system. This may be a consequence of the ubiquitous expression of alloantigen, such that any maturation signal will create APCs primed to activate donor T cells. If so, strategies for preventing GVHD by blocking APC maturation may need to target final common APC maturation pathways. Alternatively, instructive APC maturation may not be required for alloreactive T cell activation in GVHD.
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Mochizuki, Kazuhiro, Lijun Meng, Izumi Mochizuki, Qing Tong, Shan He, Yongnian Liu, Janaki Purushe, et al. "Programming of Donor T Cells Using Allogeneic Delta-like Ligand 4-Positive Dendritic Cells to Reduce Gvhd but Retain GVL Activity." Blood 126, no. 23 (December 3, 2015): 233. http://dx.doi.org/10.1182/blood.v126.23.233.233.
Full textAbstract:
Abstract Host antigen-presenting cells (APCs) are critical for inducing a potent graft-versus-leukemia (GVL) response after allogeneic hematopoietic stem-cell transplantation (allo-HSCT). In this setting, host APCs activate donor T cells to become effector T cells that recognize and react to antigens in malignant cells. However, alloreactive T cells also mediate graft-versus-host disease (GVHD), which causes significant morbidity and mortality after allo-HSCT. Many studies suggest that if alloreactive T cells have reduced capacity to expand in local tissues, they will be unable to trigger severe GVHD. Thus, it is possible that host APC induction of qualitative changes in donor T cells can potentially modify their anti-host toxicities while retaining the GVL effect. Here we report the establishment of a cellular programming approach that reduces the GVHD toxicity of donor T cells using host dendritic cells (DCs) that express high levels of Dll4 (named Dll4hi DCs). We have previously identified inflammatory Dll4hi DCs. They occurred in HSCT mice early during GVHD induction and had a greater ability than Dll4-negative DCs to induce IFN-γ and IL-17 in alloantigen-activated T cells. However, only approximately 0.03 X 105 Dll4hi DCs were recovered from one HSCT mouse. To provide adequate numbers of Dll4hi DCs for therapeutic translation, we developed a novel culture system capable of producing large number of Dll4hi DCs (about 100.0 X 105) from the bone marrow (BM) of one mouse using Flt3L and the TLR agonists lipopolysaccharide (LPS) and R848, which activate TLR4 and TLR7/8, respectively. Dll4hi DCs showed significantly different phenotype as compared to conventional DCs derived from GM-CSF-stimulated BM cells (named GM-DCs), as evidenced by expressing higher levels of Dll4, Ifnb, Il4, Il6 and Ido, and producing lower levels of iNOS and arginase I. When cultured with C57BL/6 (B6) mouse CD4+ T cells (H2b) at a T cell: DC ratio of 4:1 for 5 days, BALB/c mouse Dll4hi DCs (H2d) induced 3- to 5-fold more in frequency of alloreactive effector T cells producing high levels of IFN-γ and IL-17 compared to GM-DCs. Following transfer, allogeneic Dll4hi DC-induced CD4+ T cells were unable to mediate severe GVHD in BALB/c recipients, with all of them surviving 60 days after allo-HSCT. In contrast, both unstimulated B6 CD4+ T cells and allogeneic GM-DC-induced B6 CD4+ T cells caused lethal GVHD in all BALB/c recipients, indicating that GM-DCs could not be used for reducing the GVHD toxicity of donor CD4+ T cells. Mechanistic analysis showed that Dll4hi DC-induced CD4+ T cell recipients showed 2- to 6-fold less donor CD4+ T cells in the spleen, liver, and intestine 12 days after transplantation compared to unstimulated CD4+ T cell recipients. This reduction of Dll4hi DC-induced CD4+ T cells was associated with markedly increased apoptosis in recipient mice. IFN-γ production by Dll4hi DC-induced CD4+ T cells was essential for their anti-GVHD effects. Absence of T cell IFN-γ led to improved survival and expansion of Dll4hi DC-induced CD4+ T cells in transplant recipients and caused lethal GVHD. Finally, we demonstrated that Dll4hi DC-induced alloreactive T cells had acquired the ability to kill A20 leukemic cells in BALB/c recipients and control growth of P815 mastocytoma cells in the second model of BDF1 recipients, leading to significantly improved survival of mice receiving allo-HSCT. Furthermore, in the third mouse model of GVHD directed against minor histocompatibility antigens, B6 Dll4hi DC-induced C3H.SW CD8+ T cells produced high levels of IFN-γ, had reduced capacity to mediate GVHD in B6 recipients, but preserved GVL activity against C1498 myeloid leukemic cells. In summary, our findings demonstrate that in vitro Dll4hi DC programming represents a novel and effective platform to reduce toxicities of donor T cells. This strategy has several potential advantages compared to current and developing methods for the modification of donor T cells to reduce GVHD, including a relatively short period of culture, no requirement for T cell subset selection and no need of viral transduction. Importantly, this method may lead to new strategies that can produce large amount of leukemic cell-reactive donor T cells with decrease capability of causing severe GVHD. Disclosures No relevant conflicts of interest to declare.
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Francis, Lanie, Judy Anderson, Michael Timm, Noriyoshi Kurihara, Ujjal Singha, Yazan Alsayed, Alissa Huston, Suzanne Lentzsch, G. David Roodman, and Irene M. Ghobrial. "The Effect of Rapamycin, 17-AAG and the Combination on the Bone Marrow Microenvironment in Multiple Myeloma (MM)." Blood 106, no. 11 (November 16, 2005): 3476. http://dx.doi.org/10.1182/blood.v106.11.3476.3476.
Full textAbstract:
Abstract The bone marrow microenvironment in MM is characterized by the presence of upregulated osteoclast activity (OCL) and increased angiogenesis. We have recently demonstrated that the HSP90 inhibitor 17-AAG (provided by the NCI) and the mTOR inhibitor rapamycin (LC Laboratories, MA) have synergistic inhibitory activity on MM cells. The objective of this study was to determine the effect of rapamycin, 17-AAG and the combination on OCL formation and angiogenesis. Rapamycin (0.01–100nM), 17-AAG (10–1000nM) and the combination was tested using an in vitro human OCL formation assay and a human angiogenesis assay (AngioKit, TCS Cellworks, UK). Nonadherent human marrow mononuclear cells (1 x 105/100 μL) were plated in 96-well plates in the presence or absence of DMSO, rapamycin, 17-AAG or the combination. RANKL (100ng/ml) and MCSF (20ng/ml) were added to all wells except control media and MCSF. After 3 weeks, cells were fixed, and the number of OCL-like multinucleated cells were scored. To test the effect of the agents on early OCL precursors, we added the inhibitory agents on days 1, 7 or 14 of the culture. The AngioKit is comprised of human endothelial cells in a 24 well plate. The endothelial cells proliferate and then migrate through the matrix to form tubular structures and anastomosing tubules by 2 weeks. Two control wells were treated with VEGF (+ve control) and two with suramin (−ve control). The optimized medium and test samples were replaced on days 4, 7, and 9 after initial treatment. On day 11, cultures were fixed and stained with antibodies to CD31 to detect vessel formation. The degree of tubule formation was quantitated using computerized image analysis (Angiosys, TCS Cellworks, UK). Single agent rapamycin (20–100nM) inhibited OCL formation by 35% as compared to control in all tested doses indicating that PI3K/mTOR is an important regulator of OCL formation. The effect was similar on day 7 and day 14 indicating that this pathway is important for early and late OCL formation. 17-AAG 100–600nM significantly inhibited OCL formation with 100nM 17-AAG inducing 12% OCL formation as compared to control, while 300 and 600nM completely abrogated OCLs (0% OCLs). This effect was similar at day 7. However, when 17-AAG was added on day 14, it only induced 50–60% reduction in OCL formation indicating that 17-AAG affects early OCL formation. The combination of the two agents completely inhibited OCL formation on day 1 and 7 and led to a 65% reduction in OCLs when added on day 14 of the culture. Rapamycin showed a marked decrease in angiogenesis (similar to the negative control suramin), even at the lowest level tested of 0.01nM. 17-AAG demonstrated some inhibition of angiogenesis at 10 nM, and completely abrogated angiogenesis at 500–1000nM. In summary, rapamycin and 17-AAG inhibit OCL formation and angiogenesis. The effect of 17-AAG was on early OCL formation while rapamycin was on both early and late OCL. These results are contradictory to previous data indicating that 17-AAG increases OCL activity in MM. Rapamycin had a significant inhibitory effect on angiogenesis even at low doses. These results demonstrate that the use of rapamycin analogues and 17-AAG in clinical trials may have a therapeutic effect, not only on MM cells, but also on the bone marrow microenvironment. Supported in part by an ASH Scholar Award and an MMRF grant.