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Background Naive T-cell-depleted grafts have been employed as an ex vivo T-cell depletion (TCD) platform to prevent graft-versus-host disease (GvHD) and improve immune reconstitution by providing rapid donor memory T-cell reconstitution after allogenic hematopoietic stem cell transplantation (allo-HSCT). CD45RA- memory T cells confer protection against viruses such as cytomegalovirus, Epstein-Barr virus, and adenovirus; however, reports have shown an unexpectedly high incidence of human herpesvirus (HHV)-6B encephalitis among pediatric allo-HSCT patients. Methods We report the first 18 consecutive allo-HSCT, 16 haplo-HSCT, and two human leukocyte antigen-matched related donors implanted with naive TCD grafts. All donors were administered three cell products: first, a CD34+ stem cell product; second, a CD45RA+ TCD graft, followed by an adoptive natural killer (NK) cell infusion within 10 days after HSCT. The study's primary endpoint was the incidence of HHV-6B encephalitis. Results Engraftment was achieved in 94.5% of cases; 2-year overall survival, event-free survival, and GvHD/relapse-free survival were 87.2% (95% CI 78.6-95.8), 67.3% (95% CI 53.1-81.5), and 64% (95% CI 50.5-78.1), respectively. HHV-6B reactivation occurred in 7 of the haplo-HSCT patients, six of who received a cell infusion with an NK/CD4 ratio Conclusions In this clinical study, we show that early adoptive NK cell infusion after a 45RA+ TCD allo-HSCT graft is safe and can prevent HHV-6B encephalitis. We recommend infusing adoptive NK cells after allo-HSCT using CD45RA+ TCD grafts.

Natural killer (NK) cells represent promising tools for cancer immunotherapy. We report the optimization of an NK cell activation–expansion process and its validation on clinical‐scale. Methods: RPMI‐1640, stem cell growth medium (SCGM), NK MACS and TexMACS were used as culture mediums. Activated and expanded NK cells (NKAE) were obtained by coculturing total peripheral blood mononuclear cells (PBMC) or CD45RA+ cells with irradiated K562mbIL15‐41BBL or K562mbIL21‐41BBL. Fold increase, NK cell purity, activation status, cytotoxicity and transcriptome profile were analyzed. Clinical‐grade NKAE cells were manufactured in CliniMACS Prodigy. Results: NK MACS and TexMACs achieved the highest NK cell purity and lowest T cell contamination. Obtaining NKAE cells from CD45RA+ cells was feasible although PBMC yielded higher total cell numbers and NK cell purity than CD45RA+ cells. The highest fold expansion and NK purity were achieved by using PBMC and K562mbIL21‐41BBL cells. However, no differences in activation and cytotoxicity were found when using either NK cell source or activating cell line. Transcriptome profile showed to be different between basal NK cells and NKAE cells expanded with K562mbIL21‐41BBL or K562mbIL15‐41BBL. Clinical‐grade manufactured NKAE cells complied with the specifications from the Spanish Regulatory Agency. Conclusions: GMP‐grade NK cells for clinical use can be obtained by using different starting cells and aAPC, This work was supported by the National Health Service of Spain, Instituto de Salud Carlos III (ISCIII), FONDOS FEDER grant (FIS) PI18/01301 to Pérez‐Martínez A, CRIS Foundation to Beat Cancer to Escudero A, Fernández A; Navarro A, Mirones I, and Fundación Mari Paz Jiménez Casado and La Sonrisa de Álex to Vela M.

Background Acute myeloid leukemia (AML) accounts for approximately 20% of pediatric leukemia cases; 30% of these patients experience relapse. The antileukemia properties of natural killer (NK) cells and their safety profile have been reported in AML therapy. We proposed a phase 2, open, prospective, multicenter, nonrandomized clinical trial for the adoptive infusion of haploidentical K562-mb15-41BBL–activated and expanded NK (NKAE) cells as a consolidation strategy for children with favorable and intermediate risk AML in first complete remission after chemotherapy (NCT02763475). Patients and Methods Before the NKAE cell infusion, patients underwent a lymphodepleting regimen. After the NKAE cell infusion, patients were administered low doses (1 × 106/IU/m2) of subcutaneous interleukin-2. The primary study endpoint was AML relapse-free survival. We needed to include 35 patients to demonstrate a 50% reduction in relapses. Results Seven patients (median age, 7.4 years; range, 0.78-15.98 years) were administered 13 infusions of NKAE cells, with a median of 36.44 × 106 cells/kg (range, 6.92 × 106 to 193.2 × 106 cells/kg). We observed chimerism in 4 patients (median chimerism, 0.065%; range, 0.05-0.27%). After a median follow-up of 33 months, the disease of 6 patients (85.7%) remained in complete remission. The 3-year overall survival was 83.3% (95% confidence interval, 68.1-98.5), and the cumulative 3-year relapse rate was 28.6% (95% confidence interval, 11.5-45.7). The study was terminated early because of low patient recruitment. Conclusion This study emphasizes the difficulties in recruiting patients for cell therapy trials, though NKAE cell infusion is safe and feasible. However, we cannot draw any conclusions regarding efficacy because of the small number of included patients and insufficient biological markers.

Background. Acute myeloid leukemia (AML) accounts for approximately 25% of pediatric leukemia. The long term overall survival rate now approaches 70%, but up 30% relapse. The anti-leukemia properties of Natural Killer (NK) cells and its safety profile has been reported previously at different phases of AML treatment. We proposed a phase II open, a prospective multicenter, non-randomized clinical trial for adoptive infusion of haploidentical K562-mb15-41BBL activated and expanded Natural Killer (NKAE) cells as a consolidation strategy in children with favorable and intermediate-risk AML who were in first complete remission after chemotherapy (NCT02763475). Previous to NKAE cell infusion, patients received a lymphodepleting regimen. After NKAE cell infusion, patients received low doses (1×106/IU/m2) of IL-2 subcutaneously every 48 hours for 2 weeks. Procedure. Seven patients, median age 7.4 years (range, 0.78–15.98), received 13 infusions of NKAE cells, with a median of 36.44×106 NKAE cells/kg (range, 6.92–193.2×106 cells/kg). Results. Three pair donor-recipient were KIR–HLA-mismatched. Donor KIR haplotype score was better in two cases, and neutral in the rest of the cases. Chimerism was observed in 4 patients median chimerism 0.065%, (range 0.05-0.27%). With a median of follow up of 33 moths, 6 (85.7%) patients remain alive and in complete remission. The 3-year overall survival was 83.3% (95% confidence interval 68.1-98.5), and the 3-year relapse cumulative incidence was 28.6% (95% confidence interval 11.5-45.7). Conclusions. This study shows that NKAE cell infusion as a consolidation strategy was feasible and safe but could not improve the pediatric AML relapse rate in this small cohort.

IntroductionAcute myeloblastic leukaemia (AML) constitutes the second most common haematological malignancy in the paediatric population. Current treatment regimens are based on the administration of polychemotherapy, combining high doses of cytarabine with anthracyclines and topoisomerase inhibitors. Allogeneic haematopoietic stem cell transplantation (HSCT) is an option for high-risk patients with AML (and for intermediate-risk patients if a sibling donor is available). With this strategy, AML survival has increased substantially; however, it has remained stagnant at approximately 60%, with relapse being the principal culprit. The predominant role of the immune system and natural killer (NK) cells in controlling paediatric AML has gained importance within the context of HSCT. In this protocol, we propose incorporating this cell therapy as an adjuvant treatment through the infusion of activated and expanded haploidentical NK (NKAE) cells in paediatric patients with AML who are in cytological remission after completing consolidation therapy, and with no indication for HSCT.Methods and analysisPatients up to 30 years of age, diagnosed with AML, in their first cytological remission, who have completed both the induction and the consolidation phases of chemotherapy and do not meet the criteria for allogeneic HSCT are eligible. The patients will receive two doses of NKAE cells once a week, using a GMP K562-mbIL15-41BBL stimulus from a haploidentical donor and interleukin 2 subcutaneously. The patients will then be followed up for 36 months to assess the primary endpoint, which is the probability of relapse after NK cell infusion.Ethics and disseminationThis clinical trial was approved by the Clinical Research Ethics Committee of La Paz University Hospital and The Spanish Agency of Medicines and Medical Devices. Findings will be disseminated through peer-reviewed publications, conference presentations and community reporting.Trial registration numberEudraCT code: 2015-001901-15, ClinicalTrials.gov Identifier:NCT02763475.

Despite being a rare disease, cancer is the first cause of mortality due to disease during the paediatric age in the developed countries. The current, great increase in new treatments, such as immunotherapy, constitutes a new clinical and regulatory paradigm. Cellular immunotherapy is one of these types of immunotherapy. In particular, the advanced therapy drugs with chimeric antigen receptors in the T-lymphocytes (CAR-T), and particularly the CAR-T19 cells, has opened up a new scenario in the approach to haematology tumours like acute lymphoblastic leukaemia and the B-Cell lymphomas. The approval of tisagenlecleucel and axicabtagene ciloleucel by the regulatory authorities has led to the setting up of the National Plan for Advanced Therapies-CAR-T drugs in Spain. There is evidence of, not only the advantage of identifying the centres most suitable for their administration, but also the need for these to undergo a profound change in order that their healthcare activity is extended, in some cases, to the ability for the in-house manufacture of these types of therapies. The hospitals specialised in paediatric haematology-oncology thus have the challenge of progressing towards a healthcare model that integrates cellular immunotherapy, having the appropriate capacity to manage all aspects relative to their use, manufacture, and administration of these new treatments.

Background: Mesenchymal stem cells may offer therapeutic potential for asthma due to their immunomodulatory properties and host tolerability, yet prior evidence suggests that bloodborne progenitor cells may participate in airway remodeling. Here, we tested whether mesenchymal stem cells administered as anti-inflammatory therapy may favor airway remodeling and therefore be detrimental. Methods: Adipose tissue-derived mesenchymal stem cells were retrovirally transduced to express green fluorescent protein and intravenously injected into mice with established experimental asthma induced by repeat intranasal house dust mite extract. Controls were house dust mite-instilled animals receiving intravenous vehicle or phosphate-buffered saline-instilled animals receiving mesenchymal stem cells. Data on lung function, airway inflammation, and remodeling were collected at 72 h after injection or after 2 weeks of additional intranasal challenge. Results: The mesenchymal stem cells homed to the lungs and rapidly downregulated airway inflammation in association with raised T-helper-1 lung cytokines, but such effect declined under sustained allergen challenge despite a persistent presence of mesenchymal stem cells. Conversely, airway hyperresponsiveness and contractile tissue underwent a late reduction regardless of continuous pathogenic stimuli and inflammatory rebound. Tracking of green fluorescent protein did not show mesenchymal stem cell integration or differentiation in airway wall tissues. Conclusions: Therapeutic mesenchymal stem cell infusion in murine experimental asthma is free of unwanted pro-remodeling effects and ameliorates airway hyper-responsiveness and contractile tissue remodeling. These outcomes support furthering the development of mesenchymal stem cell-based asthma therapies, although caution and solid preclinical data building are warranted. This work was supported by the Fondo de Investigación Sanitaria of Spain (FIS Operating Grants PI05/2478, PI05/2217, PI08/1822, PI08/0029, and PI11/01001; Ministry of Science and Innovation and European Regional Development Fund), GlaxoSmithKline (investigator-driven Collaborative Research Trial), the Sociedad Española de Neumología y Cirugía Torácica (SEPAR) and the Fundació Catalana de Pneumología (FUCAP/Vifor Pharma grant). LMP is the recipient of an Ángeles Alvariño postdoctoral fellowship of Xunta de Galicia, Spain. IMA is supported by Grant PLE2009-0115 of the Ministry of Science and Innovation of Spain. OAC and RFI are recipients of FIS postgraduate fellowships FI07/00399 and FI05/00171, respectively. IC is supported by FIS Fund CA10/01315. BLC is supported by Xunta de Galicia Research Unit Consolidation and Structuring Fund INCITE07PXI916208ES. DRB was for part of this work supported by FIS Miguel Servet investigator salary grant CP04/00313. Sí

Single-photon emission computed tomography (SPECT) offers the possibility to study the biodistribution of 111In-labeled human mesenchymal stem cells (hMSCs). This functional information can be complemented by magnetic resonance imaging (MRI) that provides the anatomical context to the SPECT image. However, acquiring such studies with separate SPECT/MRI systems requires a multimodality registration in order to fuse both images. In this work, a special bed was designed based on three non-coplanar glass capillary tubes filled with a mixture of 99mTc and CuSO4, visible in both modalities. Moreover, it was necessary to design a semi-automated rigid registration procedure based on matching these rods by minimizing the distance between corresponding lines. The maximum target registration error achieved was 0.9 mm, in the range of SPECT resolution.

Homing is an inherent, complex, multistep process performed by cells such as human bone marrow mesenchymal stem cells (hMSCs) to travel from a distant location to inflamed or damaged tissue and tumors. This ability of hMSCs has been exploited as a tumor-targeting strategy in cell-based cancer therapy. The purpose of this study was to investigate the applicability of 111In-oxine for tracking hMSCs in vivo by combining single-photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). 111In-labeled hMSCs (106 cells) were infused intraperitoneally in neuroblastoma-bearing mice, whereas a control group received a dose of free 111In-oxine. SPECT and MRI studies were performed 24 and 48 hours afterwards. Initially, the images showed similar activity in the abdomen in both controls and hMSC-injected animals. In general, abdominal activity decreases at 48 hours. hMSC-injected animals showed increased uptake in the tumor area at 48 hours, whereas the control group showed a low level of activity at 24 hours, which decreased at 48 hours. In conclusion, tracking 111In-labeled hMSCs combining SPECT and MRI is feasible and may be transferable to clinical research. The multimodal combination is essential to ensure appropriate interpretation of the images. Financial disclosure of authors: This work was funded in part by grants from Ministerio de Economía y Competitividad (PLE2009-0115), Red Tematica de Investigacion Cooperativa en Cancer (RTICC/ISCIII; RD12/0036/0027), the Madrid Regional Government (S-BIO-0204-2006–MesenCAM and P2010/BMD-2420-CellCAM), and the Ministerio de Ciencia e Innovación (CEN-20101014 and TEC-2010-21619-C04-01). Sí

Tumor-homing is a complex, multistep process used by many cells, like mesenchymal stem cells (MSCs), to travel from a distant location to a tumor. The purpose of this study is to investigate the applicability of 111In-oxine for tracking human MSCs in vivo with single photon emission computed tomography (SPECT) and magnetic resonance imaging (MRI). 111In labeled hMSCs (106 cells) were infused intraperitoneally in neuroblastoma tumor-bearing mice, and SPECT/MRI images were performed 24 and 48 hour afterwards. High initial hMSC localization occurred in the abdomen cavity and 48 hour later hMSC-injected animals showed tumor uptake. MRI information was essential to properly define Regions of Interest on the images. Tracking 111In labeled hMSC combining SPECT and MRI is feasible and may be transferable to human studies.

As the nervous system exerts direct and indirect effects on stem cells mobilization and catecholamines mobilize hematopoietic stem cells, we hypothesized that dopamine might induce mesenchymal progenitor cells (MPCs) mobilization. We show that dopamine induced in vitro MPCs migration through D2-class receptors, and their alternative phosphoinositide 3-kinase/Akt pathways. Also, administration of catecholamines induced in vivo mobilization of colony-forming unit-fibroblast in mice. In contrast, in vitro and in vivo MPCs migration was suppressed by D2-class receptors antagonists and blocking antibodies, consistent with dopamine signaling pathway implication. In humans, patients treated with L-dopa or catecholaminergic agonists showed a significant increase of a MPC-like population (CD45-CD31-CD34-CD105+) in their peripheral blood. These findings reveal a new link between catecholamines and MPCs mobilization and suggest the potential use of D2-class receptors agonists for mobilization of MPCs in clinical settings. We thank Iván Gutierrez, Ander Abarrategi, Manuel Masip, Mar Arriero, and Daniel Pérez for his assistance in several techniques. This work was supported by grants from the Fondo de Investigaciones Sanitarias (FIS; PI05/2217 and PI08/0029), the Madrid Regional Government (S‐BIO‐0204–2006, MesenCAM; P2010/BMD‐2420, CellCAM) in Spain, and Consejería de Salud de la Junta de Andalucía (0027/2006) to J.G.‐C. The experiments were approved by the appropriate committees. Sí

Over the last decade, genetic and cell biology studies have indicated that tumour growth is not only determined by malignant cancer cells themselves, but also by the tumour microenvironment. Cells present in the tumour microenvironment include fibroblasts, vascular, smooth muscle, adipocytes, immune cells and mesenchymal stem cells (MSC). The nature of the relationship between MSC and tumour cells appears dual and whether MSC are pro- or anti-tumorigenic is a subject of controversial reports. This review is focused on the role of MSC and bone marrow (BM) niches in cancer.

The development of a new vascular network is essential for the onset and progression of many pathophysiologic processes. Cyclooxygenase-2 displays a proangiogenic activity in in vitro and in vivo models, mediated principally through its metabolite prostaglandin E2 (PGE2). Here, we provide evidence for a novel signaling route through which PGE2 activates the Alk5-Smad3 pathway in endothelial cells. PGE2 induces Alk5-dependent Smad3 nuclear translocation and DNA binding, and the activation of this pathway involves the release of active TGFβ from its latent form through a process mediated by the metalloproteinase MT1-MMP, whose membrane clustering is promoted by PGE2. MT1-MMP–dependent transforming growth factor β (TGFβ) signaling through Alk5 is also required for PGE2-induced endothelial cord formation in vitro, and Alk5 kinase activity is required for PGE2-induced neovascularization in vivo. These findings identify a novel signaling pathway linking PGE2 and TGFβ, 2 effectors involved in tumor growth and angiogenesis, and reveal potential targets for the treatment of angiogenesis-related disorders.

Background Rasmussen encephalitis is a devastating pediatric syndrome of unknown etiology that is characterized by progressive loss of neurological function and intractable focal epilepsy. Cytotoxic T lymphocytes have an active role in the pathogenic process of Rasmussen encephalitis. We studied the implication of CXCL10-CXCR3, a chemotactic axis involved in the pathogenesis of several cases of immune encephalitis. Methods We analyzed surgical specimens of children with Rasmussen encephalitis, and performed functional in vitro assays to test the implications of the pathological findings. Results We found that cytotoxic T lymphocytes infiltrating the damaged areas of primary biopsies expressed CXCR3, whereas neurons and astrocytes in the same areas expressed CXCL10. The in vitro assays demonstrated we found that astrocytes upregulated the expression of CXCL10 messenger RNA and the release of CXCL10 to the supernatants on stimulation with polyinosinic-polycyticylic acid, a synthetic double-stranded RNA that mimics infections with either RNA or DNA viruses. Activated T lymphocytes responded to the production of CXCL10 by astrocytes by increasing their migration in a transwell assay. Finally, the chemotaxis induced by the stimulated astrocytes was completely abrogated in the presence of a small molecule antagonist of CXCR3. Conclusions Our results suggest that the CXCR3-CXCL10 axis has a role in recruiting pathogenic T lymphocytes into the brains of patients with Rasmussen encephalitis. This chemotactic mechanism may be targeted pharmacologically.