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The public French Cord Blood Banks Network was established in 1999 with the objective of standardizing the practices governing umbilical cord blood (UCB) banking in France. The Network adopted a strategy to optimize its inventory and improve the quality of its banked units based on a quality improvement process using outcome data regularly provided by Eurocord. This study aimed to describe the results, over 10 years, of UCBT facilitated by a national network that used the same criteria of UCB collection and banking and to assess how modifications of banking criteria and unit selection might influence transplant outcomes. Nine hundred and ninety-nine units (593 single-unit and 203 double-unit grafts) were released by the Network to transplant 796 patients with malignant (83%) and non-malignant (17%) diseases. Median cell dose exceeded 3.5 × 107 TNC/kg in 86%. There was a trend to select units more recently collected and with higher cell dose. Neutrophil engraftment was 88.2% (85.7–90.7) and 79.3% (72.6–86.5) respectively for malignant and non-malignant diseases with a trend to faster recovery with higher cell doses. The respective 3-year transplant-related mortality were 31.1% (27.5–35.1) and 34.3% (27.0–43.5). OS was 49% ± 4 in malignant and 62% ± 4 in non-malignant disorders. In multivariate analysis, cell dose was the only unit-related factor associated with outcomes. Our results reflect the benefit on clinical outcomes of the strategy adopted by the Network to bank units with higher cell counts.

The development of Chimeric Antigen Receptor T cells therapy initiated by the United States and China is still currently led by these two countries with a high number of clinical trials, with Europe lagging in launching its first trials. In this systematic review, we wanted to establish an overview of the production of CAR-T cells in clinical trials around the world, and to understand the causes of this delay in Europe. We particularly focused on the academic centers that are at the heart of research and development of this therapy. We counted 1087 CAR-T cells clinical trials on ClinicalTrials.gov (Research registry ID: reviewregistry1542) on the date of 25 January 2023. We performed a global analysis, before analyzing the 58 European trials, 34 of which sponsored by academic centers. Collaboration between an academic and an industrial player seems to be necessary for the successful development and application for marketing authorization of a CAR-T cell, and this collaboration is still cruelly lacking in European trials, unlike in the leading countries. Europe, still far behind the two leading countries, is trying to establish measures to lighten the regulations surrounding ATMPs and to encourage, through the addition of fundings, clinical trials involving these treatments.

Mesenchymal stem/stromal cells (MSCs) are a promising tool for cell-based therapy thanks to their ability to secrete trophic factors and immunomodulatory potential. So far, these cells are used to treat a variety of inflammatory diseases like sepsis or severe graft-versus-host disease (GvHD). Considering the number of cells required for their use in cell therapy (between 1–3 × 106 cells/kg), a primary expansion is necessary. However, after an intravenous injection, few cells are found in tissue lesion or in bone marrow. One hypothesis is that chemotactic signals that guide MSCs to an appropriate environment would be altered during in vitro expansion. It is therefore essential to define better conditions of expansion, particularly with good manufacturing process, to obtain enough MSCs, while preserving their immunomodulatory properties and expression of migration molecules. The aim of this research project is to investigate the effect of cell confluence on migration potential and immunological properties of Wharton’s jelly (WJ) MSCs. This research is investigated with 3 levels confluency, at 50, 80 and 100% of confluence by using good manufacturing practice (GMP). We found a better expression of immunomodulatory molecules (PD-L1) and migratory molecules (CD44, membrane CXCR7) for cells that reached 50% confluence. Furthermore, co-stimulatory molecules appear to be less expressed when MSCs are at low confluency. In conclusion low confluence culture should be privileged to improve immunomodulatory and migration phenotype.

Viral infections are major complications of Hematopoietic Stem Cell Transplantation (HSCT). As efficacy of anti-viral drugs is limited in absence of immune reconstitution and often associated with severe side effects, infusion of Virus-Specific T cells (VSTs) becomes a promising alternative treatment for viral infections and diseases after HSCT. A lot of improvement in VST generation has been made since 1992, date of first attempts. Regarding stimulation antigen, pools of peptides from viral immunodominant proteins become the best choice compared to whole proteins or other types of antigens. In respect with generation methods, a huge improvement has been done both with cell culture thanks to faster protocols of expansion and with immunomagnetic isolation thanks to fully automated generation of VSTs with a close system. This latest kind of VST generation is fast (within 24 hours), compliant with GMP guidelines and allows a wide distribution among cell therapy laboratories. Furthermore, cell source is no longer limited to the HSCT donor. Third-party donors either related or unrelated are also sought. A promising perspective could be the generation of CART based on VSTs aiming both at targeting the malignant cells and controlling the viral infections simultaneously.

International audience

International audience; Monoclonal antibodies targeting tumors are one of the most important discoveries in the field of cancer. Although several effective antibodies have been developed, a relapse may occur. One of their mechanisms of action is Antibody Dependent Cell Cytotoxicity (ADCC), by engaging the Fc γ receptor CD16 expressing Natural Killer cells, innate lymphoid cells involved in cancer immunosurveillance and able to kill tumor cells. A lack of NK cells observed in many cancers may therefore be a cause of the low efficacy of antibodies observed in some clinical situations. Here we review clear evidences of the essential partnership between NK cells and antibodies showed in vitro, in vivo, and in clinical trials in different indications, describe the hurdles and ways to enhance ADCC and the evolution of monoclonal antibody therapy. NK cell adoptive immunotherapy combined with monoclonal antibodies may overcome the resistance to the treatment and enhance their efficacy

Sepsis is defined as life-threatening organ dysfunction caused by a deregulated immune host response to infection. The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted this multifactorial and complex syndrome. The absence of specific treatment neither against SARS-CoV-2 nor against acute respiratory distress syndrome (ARDS), the most serious stage of this infection, has emphasized the need to find alternative treatments. Several therapeutics are currently being tested, including mesenchymal stromal cells. These cells, already used in preclinical models of ARDS, sepsis, and septic shock and also in a few clinical trials, appear well-tolerated and promising, but many questions remain unanswered. Significance statement Sepsis is defined as life-threatening organ dysfunction caused by a deregulated immune host response to infection. The emergence of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted this multifactorial and complex syndrome. The absence of specific treatment neither against SARS-CoV-2 nor against acute respiratory distress syndrome (ARDS), the most serious stage of this infection, has emphasized the need to find alternative treatments. Several therapeutics are currently being tested, including, mesenchymal stromal cells. These cells, already used in preclinical models of ARDS, sepsis, and septic shock and also in a few clinical trials, appear well-tolerated and promising.

Background: Umbilical mesenchymal stem/stromal cells (MSCs), and especially those derived from Wharton’s jelly (WJ), are a promising engineering tool for tissue repair in an allogeneic context. This is due to their differentiation capacity and immunological properties, like their immunomodulatory potential and paracrine activity. Hence, these cells may be considered an Advanced Therapy Medicinal Product (ATMP). The purpose of this work was to differentiate MSCs from WJ (WJ-MSCs) into chondrocytes using a scaffold and to evaluate, in vitro, the immunomodulatory capacities of WJ-MSCs in an allogeneic and inflammatory context, mimicked by IFN-γ and TNF-α priming during the chondrogenic differentiation. Methods: Scaffolds were made from hydrogel composed by alginate enriched in hyaluronic acid (Alg/HA). Chondrogenic differentiation, immunological function, phenotype expression, but also secreted soluble factors were the different parameters followed during 28 days of culture. Results: During chondrocyte differentiation, even in an allogeneic context, WJ-MSCs remained unable to establish the immunological synapse or to induce T cell alloproliferation. Moreover, interestingly, paracrine activity and functional immunomodulation were maintained during cell differentiation. Conclusion: These results show that WJ-MSCs remained hypoimmunogenic and retained immunomodulatory properties even when they had undergone chondrocyte differentiation.

Viral infections represent one of the main causes of morbidity and mortality following hematopoietic stem cell transplantation. Antiviral treatment failure may be explained by the absence of specific immune reconstitution. In the 1990s, antiviral immunotherapy initially consisting in total donor lymphocyte infusion presented efficiency but was often associated with adverse events. Specific antiviral immunotherapy emerged and relied on the isolation of mono- or multivirus donor-derived-specific T cells with or without in vitro expansion. During the last 10 years, such an adoptive transfer has been proved feasible, and helpful in specific antiviral immune reconstitution, and rarely associated with adverse events. Two main evolutions contributed to allow a good reactivity to propose immunotherapy in case of antiviral treatment failure: the development of allogeneic cytotoxic T lymphocytes (CTL) banks and the improvement of CTL isolation methods using immunomagnetic technology, which presents the advantage to be fast.

Resume Objectif Revue des differentes publications concernant l’utilisation des cellules souches pour traiter la dysfonction erectile d’origine diabetique. Materiel et methodes Recherche bibliographique dans PUBMED realisee en utilisant les mots cles : stem cell therapy / diabetes associated erectile dysfunction . Parmi les 51 articles obtenus de la recherche PUBMED, nous avons retenu 16 articles pour leur specificite d’etude de la dysfonction erectile (DE) liee au diabete. Resultats Differents types de cellules souches ont ete etudies : mesenchymateuses adipeuses, ou issues de la moelle osseuse ainsi que des cellules endotheliales progenitrices. Les protocoles experimentaux sont assez similaires d’une etude a l’autre avec neanmoins quelques specificites concernant les cellules etudiees et le suivi de ces dernieres. La pression intracaverneuse (PIC) mesuree apres l’injection des cellules souches au sein des corps caverneux a toujours ete significativement superieure par rapport aux populations temoins. L’adjonction aux cellules souches de certains facteurs de croissance par transfection genique semble accroitre leur efficacite. Aucun marqueur ideal de suivi n’a pu etre identifie. Conclusion L’effet de l’injection de cellules souches sur la PIC semble etre lie non pas uniquement a l’effet cellulaire propre mais surtout a des effets paracrines qui restent a elucider.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a curative option for treatment of some malignant and non-malignant hematological diseases. However, post-HSCT patients are severely immunocompromised and susceptible to viral infections, which are a major cause of morbidity and mortality. Although antiviral agents are now available for most types of viral infections, they are not devoid of side effects and their efficacy is limited when there is no concomitant antiviral immune reconstitution. In recent decades, adoptive transfer of viral-specific T cells (VSTs) became an alternative treatment for viral infection after HSCT. However, two major issues are concerned in VST transfer: the risk of GVHD and antiviral efficacy. We report an exhaustive review of the published studies that focus on prophylactic and/or curative therapy by donor VST transfer for post-HSCT common viral infections. A low incidence of GVHD and a good antiviral efficacy was observed after adoptive transfer of VSTs from HSCT donor. Viral-specific T-cell transfer is a promising approach for a broad clinical application. Nevertheless, a randomized controlled study in a large cohort of patients comparing antiviral treatment alone to antiviral treatment combined with VSTs is still needed to demonstrate efficacy and safety.

Hematopoietic stem cell transplantation is a well-established efficient therapy for hematological diseases, but Graft-Versus-Host Disease (GVHD) is a major and frequent complication encumbering its outcome despite the administration of calcineurin inhibitor based GvHD-prophylaxis. Corticosteroids represent the worldwide first line treatment, however in case of steroid refractory acute GVHD there is no consensus about a subsequent treatment although Ruxolitinib is subject to a phase III trial. Multiple molecules have been tried, most of them immunosuppressive, increasing the risk of deadly infections and transplantation-related mortality (TRM). Recent studies reported that mesenchymal stromal cells (MSC) infusion which have immune modulatory abilities might be effective and harmless in steroid or treatment-refractory GVHD (R-GVHD). In France, MSCs are considered as an Advanced Therapy Medicinal Product. For 4 years, its administration as a compassionate use is subject to approval by an expert committee from SFGM-TC before its validation by the French regulatory agency (ANSM). We retrospectively analyzed the demands for MSC use in France since 2011 for patients suffering from R-GVHD. We evaluated the response at day 28 (range 23-28) and at the last follow-up and its safety. Eleven demands were validated by both expert committee and ANSM, 8 patients (pts) received ex-vivo expanded MSCs, 1 pt refused the therapy, 1 infusion was postponed due to COVID-19 related sanitary crisis and the last 1 didn't receive MSCs due to relapse. Among pts who received MSCs, median age was 6 years (2-69), sex ratio was 0,6. All pts underwent their first HSCT for either malignant disease (62,5%) or non-malignant disease (37,5%). Four pts were transplanted from sibling donor, 2 pts from mismatched unrelated donors and 2 pts from haplo-identical donors. Stem cells source was bone marrow for 4 pts, peripheral blood stem cells for 3 and cord blood for 1. Donors median age was 28,8 years (0-49,5), 1 male had a female donor. Six pts got a myeloablative conditioning regimen (TBI-based for 2). All pts received a ciclosporin-based (CSA) GVHD prophylaxis (CSA alone, n=1; CSA + Mycophenolate Mofetil (MMF) or Methotrexate, n=7). Five pts had ATG. Six pts were suffering from acute GVHD, while 2 from extensive chronic GVHD (cGVHD). All 6 pts with acute GVHD presented a grade III or IV, refractory to corticosteroids and at least 2 other lines of GVHD therapy. All but one had a multipolar GVHD with at least 2 affected organs. Five pts were still taking corticosteroids, and six were taking additional immunosuppressive molecules (Tacrolimus, Ruxolitinib, Etanercetp, Inolimomab, MMF) at time of MSC infusion. Five pts received German commercialized MSCs (Obnitixâ, MEDAC, Germany; see Bader et al, 2018), 2 get mother's derived MSCs (not the initial donor), and 1 from a third-party donor. A median of 4 infusions were administered (1-4), once a week for 4 weeks. Mean single dose of MSCs was 1.23.10e6/kg (range: 0,86 - 3). No toxicity was reported except for 1 pt who experienced anaphylactic reaction within minutes, leading to the interruption of infusion (mother's derived MSCs prepared with fetal bovine serum where all other preparations were performed with platelet lysate). The median time from GVHD onset to first MSC infusion was 135 days (63-457). Overall response rate was 86% (6/7) at the first and at the last evaluation with 1 complete response (CR) and 5 partial responses (reduction of at least one grade of at least one affected organ). One pt did not respond and the last 1 was not evaluable due to anaphylactic reaction. Both were suffering from cGVHD. Among the seven pts who received complete MSC infusions, median follow-up was 1,5 months (1,1-18,5) due to premature TRM, overall survival (OS) at six months was 33,3%. Five pts died, all of them from a transplantation-related cause: GVHD n=2, severe infections n=3. Literature reported better outcomes lately, Bader and al, 2019 reported a 64% OS at 6 months and 51% of CR at last follow up. Those disparities might be explained by a delayed treatment after GVHD onset (135 days versus 28 days) and a median of 3 (2-10) therapies after receiving corticosteroids before MSC infusion due to difficulty to obtain MSCs in France. Besides, we included patient suffering from R-cGVHD. Regarding those results, MSC efficacy and safety should be confirmed in a proper clinical trial. Figure Disclosures Rubio: Neovii: Research Funding; Novartis: Honoraria; MSD: Honoraria; Gilead: Honoraria; Medac: Consultancy. Dalle:Jazz Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; AbbVie Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bellicum: Consultancy, Honoraria; Medac: Consultancy, Honoraria; Orchard: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees.

International audience; BACKGROUND: The use of mesenchymal stem cells (MSCs) is being extensively studied in clinical trials in the setting of various diseases including diabetes, stroke, and progressive multiple sclerosis. The unique immunomodulatory properties of MSCs also point them as a possible therapeutic tool during sepsis and septic shock, a devastating syndrome associated with 30-35% mortality. However, MSCs are not equal regarding their activity, depending on their tissue origin. Here, we aimed at comparing the in vivo properties of MSCs according to their tissue source (bone marrow (BM) versus Wharton's jelly (WJ)) in a murine cecal ligation and puncture (CLP) model of sepsis that mimics a human peritonitis. We hypothesized that MSC properties may vary depending on their tissue source in the setting of sepsis.METHODS: CLP, adult, male, C57BL/6 mice were randomized in 3 groups receiving respectively 0.25 × 106 BM-MSCs, 0.25 × 106 WJ-MSCs, or 150 μL phosphate-buffered saline (PBS) intravenously 24 h after the CLP procedure.RESULTS: We observed that both types of MSCs regulated leukocyte trafficking and reduced organ dysfunction, while only WJ-MSCs were able to improve bacterial clearance and survival.CONCLUSION: This study highlights the importance to determine the most appropriate source of MSCs for a given therapeutic indication and suggests a better profile for WJ-MSCs during sepsis.

International audience; Mesenchymal stem cells (MSC) have emerged as alternative sources of stem cells for regenerative medicine because of their multipotency and strong immune-regulatory properties. Also, human leukocyte antigen G (HLA-G) is an important mediator of MSC-mediated immunomodulation. However, it is unclear whether MSC retain their immune-privileged potential after differentiation. As promising candidates for cartilage tissue engineering, the immunogenic and immunomodulatory properties of chondro-differentiated MSC (chondro-MSC) require in-depth exploration. In the present study, we used the alginate/hyaluronic acid (Alg/HA) hydrogel scaffold and induced both bone marrow- and adipose tissue-derived MSC into chondrocytes in three-dimensional condition. Then, MSC before and after chondrocyte differentiation were treated or not with interferon γ and tumor necrosis factor α mimicking inflammatory conditions and were compared side by side using flow cytometry, mixed lymphocyte reaction, and immunostaining assays. Results showed that chondro-MSC were hypoimmunogenic and could exert immunosuppression on HLA-mismatched peripheral blood mononuclear cells as well as undifferentiated MSC did. This alloproliferation inhibition mediated by MSC or chondro-MSC was dose dependent. Meanwhile, chondro-MSC exerted inhibition on natural killer cell-mediated cytolysis. Also, we showed that HLA-G expression was upregulated in chondro-MSC under hypoxia context and could be boosted in allogenic settings. Besides, the Alg/HA hydrogel scaffold was hypoimmunogenic and its addition for supporting MSC chondrocyte differentiation did not modify the immune properties of MSC. Finally, considering their chondro-regenerative potential and their retained immunosuppressive capacity, MSC constitute promising allogenic sources of stem cells for cartilage repair.

Graft failure occurs in 3-5% of hematopoietic stem cell transplantations (HSCT) and raises up to 10% for mismatched HSCT. It is a severe complication leading to lethal infections or bleedings. Several studies reported that engraftment could be improved by mesenchymal stromal cells (MSCs) infusion. Those cells are multipotent cells capable of differentiation in at least 3 lineages (adipocytes, chondrocytes and osteocytes) and of supportive effects on hematopoiesis. This treatment is considered as an Advanced Therapy Medicinal Product in France and there is currently no authorization for its use outside of clinical trials. We retrospectively analyzed the demands from HSCT French centers to the expert committee of the SFGMTC and the French regulatory agency (ANSM), since 2014, in order to ask for MSCs exceptional recourse for patients with graft failure. Nine requests for MSCs were made. One patient did not receive any MSCs because MSCs bag was contaminated, another one had an haploidentical transplantation without MSCs instead. Finally, 7 patients received 1 or 2 infusions of MSCs (Table). Median age was of 6 years (4-23), sex ratio was of 2,5. Two patients (29%) had an acute lymphoblastic leukemia (ALL) and 5 patients (71%) had an aplastic anemia (idiopathic n=4, congenital n=1). Five patients had received one transplantation, and 2 patients had received two transplantations, followed by a primary graft failure (n=6) and a secondary graft failure (n=1). Only 2 patients had received a myeloablative conditioning regimen (MAC) for the first procedure, one chemo- and one TBI- based. The other 5 patients had received a reduced-intensity conditioning (RIC) regimen, 4 of them with 2 grays TBI. Two patients had received a cord blood unit and 5 bone marrows from 1 sibling, 3 haploidentical and 1 mismatched donors. Only 2 patients had received antithymoglobulins in their previous regimen. Engraftments failed despite a median richness of 5,14.106 CD34 cells/kg/bone marrow graft (1,84-9,5) and of 1,25.105CD34 cells/kg/cord blood unit (1,2-1,3). Median delay between first HSCT with graft failure and MSCs infusion was of 2 months (1-32). To prepare last HSCT,six patients received a reduced intensity conditioning regimen with 2 grays TBI (Baltimore), and one patient received a myeloablative, chemo-based, regimen, with antithymoglobulins. Three patients received a graft from the same previous donor. Six patients received a haploidentical graft (5 bone marrows, 1 peripheral blood stem cells), and 1 received bone marrow from a mismatched donor. Median number of CD34+ cells was of 9,02.106/kg (1,61-12,43). All MSCs were from 8 pooled donors (OBNITIX®) except for one patient who received MSCs from a relative donor (which was different from the HSCT donor). All infusions were well tolerated and made on the same day as HSCT, apart from one patient who received MSCs for a secondary graft failure on day 228 post-transplantation, followed by an infusion of CD34+ cells on day 231, and a second infusion of MSCs on day 259. Mean dose of MSCs was of 1,72.106/kg (1,25-3,1). Median time for neutrophil recovery (neutrophils over 0.5 G/L) and platelet recovery (platelets over 50 G/L) on 3 consecutive days was respectively of 23 days (10-34) and 35 days (26-238). At day 30, 5 patients (71%) had a full donor chimerism. Only one patient presented a graft failure leading to a third HSCT. No secondary graft failure occurred after a median follow-up of 13 months (4-104). At last follow-up, none of the patients who achieved platelet and neutrophil recovery required neither blood or platelets transfusion nor thrombopoietin receptor analogs. Infusion of MSCs at the time of HSCT to prevent graft failure was safe and effective for 6/7 patients. Larger prospective trials are necessary to confirm MSCs impact on engraftment and graft function. Table Disclosures Dalle: AbbVie Pharmacyclics: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Orchard: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Medac: Consultancy, Honoraria; Bellicum: Consultancy, Honoraria; Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rubio:Medac: Consultancy; Gilead: Honoraria; MSD: Honoraria; Novartis: Honoraria; Neovii: Research Funding.

International audience; Hematopoietic stem cell transplantation (HSCT) is the only curative therapy for patients with chemotherapy-resistant hematologic malignancies that are usually fatal in absence of treatment. Hematopoietic stem cell transplantation is associated with significant early and late morbidity and mortality. Graft-versus-host disease, infections, and relapse are the most important causes of mortality after HSCT. Until now, these complications have been managed mainly with pharmacological drugs, but in some situations, this approach clearly shows its limit. As such, there is a significant need for novel therapies for the treatment of complications after allogeneic HSCT. In this review, the currently available adoptive immunotherapies offering an alternative in case of treatment failure of HSCT complications will be described. The results of the main clinical trials based on immune cell infusion will be discussed and the strategies aiming at maximizing cytotoxic T-lymphocyte, regulatory T-cell, natural killer cell, cytokine-induced killer cell, and gamma delta T-cell efficacies in the context of immunotherapy approaches after allogeneic HSCT in patients with hematologic malignancies will be gathered.

International audience; S-Nitrosoglutathione (GSNO) is a good candidate for nitric oxide (NO(•)) delivery, and its nanoformulation improves NO(•) stability and bioavailability. We have compared the effect of empty Eudragit nanoparticles (eENP), GSNO-loaded ENP (gENP), and free GSNO on THP-1 human monocytic cell line. We investigated cellular viability and growth by WST-1 and trypan blue tests. ENP uptake was studied using transmission electron microscopy, confocal microscopy, and flow cytometry. Transcriptomic profiles were obtained using microarray. ENP entered cells by clathrin- and caveolae-mediated endocytosis. Exposure to either free GSNO or gENP induced an activation of genes from the same clusters, in favor of intracellular delivery of GSNO by ENP. GSNO nanoformulation might be a therapeutic option for NO(•) delivery.

Sepsis and septic shock are the leading cause of admission and mortality in non-coronary intensive care units. Currently, however, no specific treatments are available for this syndrome. Due to the failure of conventional treatments in recent years, research is focusing on innovative therapeutic agents, including cell therapy. One particular type of cell, mesenchymal stromal/stem cells (MSCs), has raised hopes for the treatment of sepsis. Indeed, their immunomodulatory properties, antimicrobial activity and capacity of protection against organ failure confer MSCs with a major advantage to treat the immune and inflammatory dysfunctions associated with sepsis and septic shock. After a brief description of the pathophysiology of sepsis and septic shock, the latest advances in the use of MSCs to treat sepsis will be presented.

International audience; The ability of natural killer (NK) cells to kill tumor cells without antigen recognition makes them appealing as an adoptive immunotherapy. However, NK cells are not routinely used in the context of leukemic relapse after hematopoietic stem cell transplantation. Patients who experience relapse can be treated with donor lymphocyte infusions (DLI) based on small-cell fractions frozen at the time of transplantation. Since peripheral blood stem cells (PBSCs) are increasingly used as a stem cell source and as a source of cells for DLI, we aimed to evaluate the impact of G-SCF mobilization on NK cell phenotype, subset repartition, and functionality. Immunomagnetically isolated NK cells from healthy donor blood, donor PBSCs, and patient PBSCs were expanded for 14 days with IL-15. The expansion capacity, phenotype, and functions (cytokine secretion and cytotoxicity) of NK cell subsets based on CD56 and CD16 expression were then evaluated. Mobilized sources showed a significant decrease of CD56brightCD16+ NK cells (28 versus 74%), whereas a significant increase (64 versus 15%) of CD56brightCD16- NK cells was observed in comparison with peripheral blood. Patient-mobilized NK cells showed a significantly decreased cytotoxicity, and antibody-dependent cell cytototoxicity (ADCC) was also observed to a lesser extent in NK cells from healthy donor PBSC. G-CSF-mobilized NK cell TNF-α and IFN-γ secretion was impaired at day 0 compared to healthy donors but was progressively restored after culture. In conclusion, expansion of NK cells from G-CSF-mobilized sources may progressively improve their functionality.

International audience; Virus-specific T-cell (VST) infusion becomes a promising alternative treatment for refractory viral infections after hematopoietic stem cell transplantation (HSCT). However, VSTs are often infused during an immunosuppressive treatment course, especially corticosteroids, which are a first-line curative treatment of graft-versus-host disease (GVHD). We were interested in whether corticosteroids could affect adenovirus (ADV)-VST functions. After interferon (IFN)-γ based immunomagnetic selection, ADV-VSTs were in vitro expanded according to three different culture conditions: without methylprednisolone (MP; n = 7), with a final concentration of MP 1 µg/mL (n = 7) or MP 2 µg/mL (n = 7) during 28 ± 11 days. Efficacy and alloreactivity of expanded ADV-VSTs was controlled in vitro. MP transitorily inhibited ADV-VST early expansion. No impairment of specific IFN-γ secretion capacity and cytotoxicity of ADV-VSTs was observed in the presence of MP. However, specific proliferation and alloreactivity of ADV-VSTs were decreased in the presence of MP. Altogether, these results and the preliminary encouraging clinical experiences of co-administration of MP 1 mg/kg and ADV-VSTs will contribute to safe and efficient use of anti-viral immunotherapy.

Background Allogeneic hematopoietic stem cell transplantation (HSCT), the most widely used potentially curable cellular immunotherapeutic approach in the treatment of hematological malignancies, is limited by life-threatening complications: graft versus host disease (GVHD) and infections especially viral infections refractory to antiviral drugs. Adoptive transfer of virus-specific T cells is becoming an alternative treatment for infections following HSCT. We report here the results of a phase I/II multicenter study which includes a series of adenovirus-specific T cell (ADV-VST) infusion either from the HSCT donor or from a third party haploidentical donor for patients transplanted with umbilical cord blood (UCB). Methods Fourteen patients were eligible and 11 patients received infusions of ADV-VST generated by interferon (IFN)-γ-based immunomagnetic isolation from a leukapheresis from their original donor (42.9%) or a third party haploidentical donor (57.1%). One patient resolved ADV infection before infusion, and ADV-VST could not reach release or infusion criteria for two patients. Two patients received cellular immunotherapy alone without antiviral drugs as a pre-emptive treatment. Results One patient with adenovirus infection and ten with adenovirus disease were infused with ADV-VST (mean 5.83 ± 8.23 × 103 CD3+IFN-γ+ cells/kg) up to 9 months after transplantation. The 11 patients showed in vivo expansion of specific T cells up to 60 days post-infusion, associated with adenovirus load clearance in ten of the patients (91%). Neither de novo GVHD nor side effects were observed during the first month post-infusion, but GVHD reactivations occurred in three patients, irrespective of the type of leukapheresis donor. For two of these patients, GVHD reactivation was controlled by immunosuppressive treatment. Four patients died during follow-up, one due to refractory ADV disease. Conclusions Adoptive transfer of rapidly isolated ADV-VST is an effective therapeutic option for achieving in vivo expansion of specific T cells and clearance of viral load, even as a pre-emptive treatment. Our study highlights that third party haploidentical donors are of great interest for ADV-VST generation in the context of UCB transplantation. (N° Clinical trial.gov: NCT02851576, retrospectively registered). Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0469-0) contains supplementary material, which is available to authorized users.

Many clinical trials report mesenchymal stem/stromal cells (MSCs) efficacy in various indications. Therefore, standardization of MSC production becomes necessary. MSC properties are impacted by tissue origin, especially if they are from extraembryonic tissue or adult sources. For this reason, we evaluated the impact of MSC tissue origin on production. Methods: Three productions of MSC from Wharton’s Jelly (WJ) or from bone marrow (BM) were performed according to good manufacturing practice. The identity (phenotype, differentiation, and clonogenic capacities), safety (karyotype, telomerase activity, sterility, and donor qualification), and functionality (viability, mixed lymphocyte reaction) of each cell batch were analyzed. Results: Slight differences between MSC sources were observed for phenotype, telomerase activity, and clonogenic capacities. Conclusion: Both sources have made it possible to quickly and easily obtain clinical grade MSC. However, as availability of the source is thought to be essential, WJ seems more advantageous than BM.

Cryopreservation as a way to maintain extracorporeal photopheresis regimen for GvHD treatment while circumventing patient temporary inability to undergo apheresis

International audience

International audience; Adoptive antiviral cellular immunotherapy by infusion of virus-specific T cells (VSTs) is becoming an alternative treatment for viral infection after hematopoietic stem cell transplantation. The T memory stem cell (TSCM) subset was recently described as exhibiting self-renewal and multipotency properties which are required for sustained efficacy in vivo. We wondered if such a crucial subset for immunotherapy was present in VSTs. We identified, by flow cytometry, TSCM in adenovirus (ADV)-specific interferon (IFN)-γ+ T cells before and after IFN-γ-based immunomagnetic selection, and analyzed the distribution of the main T-cell subsets in VSTs: naive T cells (TN), TSCM, T central memory cells (TCM), T effector memory cell (TEM), and effector T cells (TEFF). In this study all of the different T-cell subsets were observed in the blood sample from healthy donor ADV-VSTs, both before and after IFN-γ-based immunomagnetic selection. As the IFN-γ-based immunomagnetic selection system sorts mainly the most differentiated T-cell subsets, we observed that TEM was always the major T-cell subset of ADV-specific T cells after immunomagnetic isolation and especially after expansion in vitro. Comparing T-cell subpopulation profiles before and after in vitro expansion, we observed that in vitro cell culture with interleukin-2 resulted in a significant expansion of TN-like, TCM, TEM, and TEFF subsets in CD4IFN-γ T cells and of TCM and TEM subsets only in CD8IFN-γ T cells. We demonstrated the presence of all T-cell subsets in IFN-γ VSTs including the TSCM subpopulation, although this was weakly selected by the IFN-γ-based immunomagnetic selection system.

Resume Les infections virales sont associees a une morbidite et une mortalite importantes apres allogreffe de cellules souches hematopoietiques (CSH) du fait de la faible efficacite des traitements medicamenteux antiviraux en l’absence de reconstitution immunitaire concomitante. Une alternative therapeutique aux antiviraux permettant de restaurer la reponse immunitaire specifique est proposee par plusieurs equipes. L’immunotherapie adoptive consiste a transferer une immunite specifique issue du donneur par l’administration de lymphocytes T cytotoxiques (CTL) diriges contre un ou plusieurs virus, isoles au prealable in vitro et eventuellement amplifies. Les progres realises, durant cette decennie, ont montre que cette approche est un atout essentiel dans la lutte contre les infections virales chez les receveurs d’allogreffes de CSH. Les techniques de production par selection immunomagnetique permettent une meilleure reactivite et une disponibilite rapide des CTL au moment ou le patient en a besoin.

Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-β) treatment under the 3-dimensional (3-D) culture condition. In addition to these molecular and biochemical methods, the mechanical regulation of differentiation and matrix formation by MSCs is only starting to be considered. Recently, mechanical loading has been shown to induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of a calibrated agitation on the chondrogenesis of human bone MSCs (MSCs) in a 3-D alginate culture (day 28) and on the maintenance of chondrogenic phenotypes. Biomechanical stimulation of MSCs increased: (i) types 1 and 2 collagen formation; (ii) the expression of chondrogenic markers such as COMP and SOX9; and (iii) the capacity to maintain the chondrogenic phenotypes. Notably, these effects were shown without TGF-β treatment. These results suggest that a mechanical stimulation could be an efficient method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering in a 3-D environment. Additionally, it appears that MSCs and chondrocyte responses to mechanical stimulation are not identical.

This article is focused on the current European and French regulations from a tissue and cell therapy perspective. The first part covers the different Directives of the European Parliament such as the 2004/23/CE and the 2006/17/CE that are applied in France through different Laws (2011-814 Bioethics), Decrees and Orders. The French 2007-1220 Decree sets a framework for science-oriented research as opposed to the 2008-968 Decree that applies to therapy-oriented organizations. The French good manufacturing practices that apply to tissue and cells were published in October 2010, they have been applicable for all tissue and cellular therapy product processing facilities. The sole purpose of all these regulations is to promote good clinical care by increasing safety and control at every single stage of the tissue and cell therapy lifecycle.

Human tissues don't regenerate spontaneously, explaining why regenerative medicine and cell therapy represent a promising alternative treatment (autologous cells or stem cells of different origins). The principle is simple: cells are collected, expanded and introduced with or without modification into injured tissues or organs. Among middle-term therapeutic applications, cartilage defects, bone repair, cardiac insufficiency, burns, liver or bladder, neurodegenerative disorders could be considered.

Objective Natural killer (NK) cells are a lymphocyte subset that, in a hematopoietic stem cell transplantation setting, mediates a graft-vs-leukemia effect without any graft-vs-host disease. We aimed to evaluate an isolation method that can be used with Good Manufacturing Practices−grade reagents and to compare three cytokines for expansion in order to design future clinical protocols based on donor NK-cell infusions to cure relapse after allograft. Materials and Methods NK cells were enriched using a CD3/CD19 depletion method and expanded for 13 days in the presence of 2, 10, and 50 ng/mL interleukin (IL)-2, IL-15, or IL-7. NK-cell cytotoxicity was evaluated after isolation and culture. Expression of NKG2D, KIR2DL2, and KIR2DL1 was monitored during expansion. Results Highly T- and B-cell−depleted NK cells were obtained and enriched 2.6-fold. The optimal cytokine concentration for expansion was 10 ng/mL for IL-2 or 50 ng/mL for IL-15. NK-cell cytotoxicity was significantly improved after an overnight incubation with 10 or 50 ng/mL IL-2 or with 2, 10, or 50 ng/mL IL-15, and after 13 days with 50 ng/mL IL-15. The use of a combination of IL-2 and IL-15 showed no additional benefit and negative results were obtained with IL-7. The three NK cell receptors were significantly upregulated after culture, mainly with IL-2 or IL-15. Conclusion In our study, 10 ng/mL IL-2 or 50 ng/mL IL-15 were the optimal concentrations for expansion and were equivalent in significantly enhancing cytotoxicity and modifying NK-cell receptor expression patterns.

Tissue engineering is a multidisciplinary field that applies the principles of engineering, life sciences, cell and molecular biology toward the development of biological substitutes that restore, maintain, and improve tissue function. In Western Countries, tissues or cells management for clinical uses is a medical activity governed by different laws. Three general components are involved in tissue engineering: (1) reparative cells that can form a functional matrix; (2) an appropriate scaffold for transplantation and support; and (3) bioreactive molecules, such as cytokines and growth factors that will support and choreograph formation of the desired tissue. These three components may be used individually or in combination to regenerate organs or tissues. Thus the growing development of tissue engineering needs to solve four main problems: cells, engineering development, grafting and safety studies.

Cartilage tissue engineering gives the ability to product adaptable neocartilage to lesion with autologous cells. Our work aimed to develop a stratified scaffold with a simple and progressive spraying build-up to mimic articular cartilage environment. An Alginate/Hyaluronic Acid (Alg/HA) hydrogel seeded with human Mesenchymal Stem Cells (hMSC) was construct by spray. First, cells repartition and actin organization were study with confocal microscopy. Then, we analyzed cells viability and finally, metabolic activity. Our results indicated a homogenous cells repartition in the hydrogel and a pericellular actin repartition. After 3 days of culture, we observed about 52% of viable cells in the scaffold. Then, from day 7 until the end of culture (D28), the proportion of living cells and their metabolic activity increased, what indicates that culture conditions are not harmful for the cells. We report here that sprayed method allowed to product a scaffold with hMSCs that confer a favorable environment for neocartilage construction: 3D conformation and ability of cells to increase their metabolic activity, therefore with few impact on hMSCs.

Dimethylsulfoxide (DMSO) is a cryoprotective substance often used to allow long term storage of stem cells or tissue grafts. However, a high frequency of adverse events is associated with the infusion of thawed cells. These events are in part due to DMSO, leading many cell therapy facilities to introduce a washing step before the delivery of the grafts. The lack of method for evaluating the residual quantities of this substance in the reinfused cells led us to develop a technique, based on capillary zone electrophoresis for assaying DMSO. The cryoprotectant was measured in 55 hematopoietic stem cell grafts, 6 parathyroids and 5 blood vessels immediately after thawing and after washing or bathing in a saline solution. The results showed that DMSO reduction in stem cell grafts reached more than 90% after the washing procedure. Furthermore, this study has shown that 2 washing steps significantly improved DMSO elimination as compared to 1 washing step. For parathyroids and blood vessels, bathing the tissues after thawing in a saline solution allowed more than 95% DMSO reduction. This study demonstrated that the technique of DMSO measurement used here, is simple and feasible on complex matrices such as protein samples after dilution. It is an appropriate method for residual quantification of the cryoprotectant before graft.

International audience; Leukemia, one of the most aggressive hematopoietic malignancies, is characterized by excessive proliferation, survival, and impaired differentiation of hematopoietic stem cells. Interleukin 15, a proinflammatory cytokine, induces proliferation and promotes cell survival of human T and B lymphocytes, as well as natural killer cells. However, it may also play a detrimental role in the onset of leukemia. This review provided an overview of the aberrant expression of Interleukin 15 and its role in the development and progression of this hematological malignancy. Also, we critically explored the potential therapeutic opportunities involved in targeting the disruption of interleukin-15 signaling as well as in interleukin-15-mediated enhancement of antitumor immunity.

5th China-France International Symposium, Kunming, PEOPLES R CHINA, DEC 12-14, 2013; International audience; INTRODUCTION: Stem cells for autologous and allogenic transplantation are obtained from several sources including bone marrow, peripheral blood or cord blood. Accurate enumeration of viable CD34+ hematopoietic stem cells (HSC) is routinely used in clinical settings, especially to monitor progenitor cell mobilization and apheresis. The number of viable CD34+ HSC has also been shown to be the most critical factor in haematopoietic engraftment. The International Society for Cellular Therapy actually recommends the use of single-platform flow cytometry system using 7-AAD as a viability dye. AIM: In a way to move routine analysis from a BD FACSCalibur (TM) instrument to a BD FACSCanto (TM) II, according to ISO 15189 standard guidelines, we define laboratory performance data of the BD (TM) Stem Cell Enumeration (SCE) kit on a CE-IVD system including a BD FACSCanto II flow cytometer and the BD FACSCanto (TM) Clinical Software. InterQC (TM) software, a real time internet laboratory QC management system developed by Vitro (TM) and distributed by Becton Dickinson (TM), was also tested to monitor daily QC data, to define the internal laboratory statistics and to compare them to external laboratories. METHODS: Precision was evaluated with BD (TM) Stem Cell Control (high and low) results and the InterQC software, an internet laboratory QC management system by Vitro. This last one drew Levey-Jennings curves and generated numeral statistical parameters allowing detection of potential changes in the system performances as well as interlaboratory comparisons. Repeatability, linearity and lower limits of detection were obtained with routine samples from different origins. Agreement evaluation between BD FACSCanto II system versus BD FACSCalibur system was tested on fresh peripheral blood, freeze-thawed apheresis, fresh bone marrow and fresh cord blood samples. RESULTS: Instrument's measure and staining repeatability clearly evidenced acceptable variability on the different samples tested. Intra-and inter-laboratory CV in CD34+ cell absolute count are consistent and reproducible. Linearity analysis, established between 2 and 329 cells/mu l showed a linear relation between expected counts and measured counts (R-2 = 0.97). Linear regression and Bland-Altman representations showed an excellent correlation on samples from different sources between the two systems and allowed the transfer of routine analysis from BD FACSCalibur to BD FACSCanto II. CONCLUSIONS: The BD SCE kit provides an accurate measure of the CD34 HSC, and can be used in daily routine to optimize the enumeration of hematopoietic CD34+ stem cells by flow cytometry. Moreover, the InterQC system seems to be a very useful tool for laboratory daily quality monitoring and thus for accreditation.

5th China-France International Symposium, Kunming, PEOPLES R CHINA, DEC 12-14, 2013; International audience; Since the 1960s and the therapeutic use of hematopoietic stem cells of bone marrow origin, there has been increasing interest in the study of undifferentiated progenitors that have ability to proliferate and differentiate in different tissues. Different stem cells (SC) with different potential can be isolated and characterised. Despite the promise of embryonic stem cells, in many cases, adult stem cells provide a more interesting approach to clinical applications. It is undeniable that mesenchymal stem cells (MSC) from bone marrow, adipose tissue or MSC of Wharton Jelly, which have limited potential, are of interest for clinical applications in regenerative medicine because they are easily separated and prepared and no ethical problems are involved in their use. During the last 10 years, these multipotent cells have generated considerable interest and in particular have been shown to escape allogeneic immune response and be capable of immunomodulatory activity. These properties may be of a great interest for regenerative medicine. Different clinical applications are under study (cardiac insufficiency, atherosclerosis, stroke, bone, cartilage, diabetes, ophthalmology, urology, liver, organ's reconstruction ...).

We describe two brothers who suffered from hyper-IgM syndrome (HIGM1) with similar clinical features: recurrent infections, especially cryptosporidium gastroenteritis with cholangitis. Their activated T cells did not express CD40L. Nucleotide sequencing revealed a mutation in both boys with respect to intron 4 and exon 5 boundaries of the CD40L gene in Xq26. They underwent successful bone marrow transplantation (BMT) from HLA-geno-identical siblings. The Cryptosporidium infection and cholangitis resolved thereafter. At 6 months after BMT, expression of CD40L on activated T lymphocytes was normal. After 1 year, both boys are well, and immune reconstitution has improved. Based on these two successful experiences, BMT with a genoidentical sibling seems a reasonable therapeutic approach for HIGM1, if Cryptosporidium infection occurs.

BACKGROUND: The implementation of a quality-assurance program is a major requirement to ensure quality and safety of the final PBPC components intended for clinical use. It is not clear whether the quantification of CFU-GM and CD34+ cells should be done on fresh components and after cryopreservation, which better represents the actual composition of the graft. STUDY DESIGN AND METHODS: Correlation between prefreeze and postthaw MNCs, CD34+ cells, and CFU-GM collected from 126 patients undergoing BMT (n=43) or PBPC (n =83) transplantation were evaluated. The statistical incidence of prefreeze and postthaw parameters as well as patient characteristics and conditioning regimens on hematologic recovery were analyzed. RESULTS: By multivariate analysis, prefreeze and postthaw CD34+ cells were the only two variables significantly and independently correlated to hematologic recovery. Low prefreeze and postthaw CD34+ cell numbers associated to a low CD34+ yield characterize PBPC grafts from patients who have the slowest hematologic recovery. The postthaw PBPC CD34+ cell number can be estimated before conditioning regimen by thawing a small aliquot of the graft. CONCLUSION: In association to prefreeze CD34+ cell number and to CD34+ yield, postthaw CD34+ cell number may be useful in monitoring cell loss during processing and identifying patients at risk of slow PBPC engraftment.

Summary. Complete DiGeorge syndrome (cDGS) is a congenital disorder characterized by typical facies, thymic aplasia, susceptibility to infections, hypoparathyroidism and conotruncal cardiac defect. Fetal thymus or post-natal thymus tissue transplantations and human leucocyte antigen (HLA)-genoidentical bone marrow transplantations were followed in a few cases by immune reconstitution. More recently, a peripheral blood mononuclear cell transplantation (PBMCT) was performed with an HLA-genoidentical donor and followed by a partial T-cell engraftment and immune reconstitution. We report a boy with cDGS, without cardiac defect, who suffered recurrent severe infections. At the age of 4 years, he underwent PBMCT from his HLA-genoidentical sister. He received no conditioning regimen, but graft-versus-host disease (GVHD) prophylaxis was with oral cyclosporin A and mycophenolate mofetil. Toxicity was mild, with grade I acute GVHD. The patient is currently 2·5 years post-PBMCT with excellent clinical performances. Mixed chimaerism can only be observed on the T-cell population (50% donor T cells). T-lymphocyte count fluctuated (CD3 more than 400 × 106/l at d 84 and CD4 more than 200 × 106/l at d 46). Exclusive memory phenotype T cells and absence of new thymic emigrants suggest expansion of infused T cells. T-cell mitogen and tetanus antigen responses normalized a few months after transplantation. After immunizations, specific antibodies were produced. PBMCT from an HLA identical sibling could be an efficient treatment of immune deficiency in cDGS.

5th China-France International Symposium, Kunming, PEOPLES R CHINA, DEC 12-14, 2013; International audience; BACKGROUND: Non-union of long bones is still a current problem in traumatology. Although corticocancellous bone autograft remains the usual procedure for the treatment of non-union, innovative therapies such as, percutaneous autologous concentrated bone marrow grafting (PABMG), are now appearing. MATERIAL AND METHODS: Over a period of 8 years, 45 non-union of long bones were treated by PABMG in the Department of Orthopaedic and Traumatologic Surgery (University Hospital of Nancy, France): 26 tibiae, 16 femurs, 3 humeri. Efficiency was evaluated by clinical criteria: full weight-bearing without pain, absence of motion at non-union site, and radiological criteria: healing of 3 corticales out of 4. RESULTS: Eighteen out of 28 non-unions at the tibia were healed (69%), 10 at the femur (63%), but none was noticed at the humerus. Some pejorative prognosis factors were noted such as: tobacco, alcohol abuse, diabetes and history of infection at the fracture site. An earlier grafting improved the success rate. The number of CFU-F (Colony Forming Unit Fibroblastic) affected the healing time more than the healing rate. CONCLUSION: The procedure, even though a little invasive, enables the healing of non-union in two out of three cases with less morbidity than conventional procedures. This procedure fits perfectly into the therapeutic arsenal of non-union.