Rational and design of the T-STORHM Study: A prospective randomized trial comparing fresh whole blood to blood components for acutely bleeding trauma patients.

Massive hemorrhage remains the main cause of preventable death in combat settings and is also the main cause of year loss in developing countries. The management of these patients relies on blood transfusion and surgery. Time is a key factor, related to survival. Recent events highlight the need to be more efficient in the transfusion supply during terror attacks or mass casualties in civilian settings. Blood components therapy with a 1:1:1 ratio is associated with a decrease of mortality but encounters many logistic issues in those circumstances. Whole blood provides in one bag all the blood components in physiologic proportions with minimal amount of additive solution. Whole blood has been implemented in military as well as civilian settings worldwide. However, direct comparisons with component therapy in prospective clinical trials are scarce. Here we present the rational and the design of the T-STORHM (Trauma-Sang TOtal dans les Hémorragies Massives) trial. This prospective randomized multicentric clinical trial will test low titer group O whole blood to components therapy in the in-hospital management of trauma patients with massive hemorrhage. Sample size calculation, primary and secondary endpoints as trial blood products preparations are discussed. The trial is expected to start in 2019 in 6 civilians and military trauma centers. The French Military Health Service is promoting the study in collaboration with the French transfusion public service (Établissementfrançaisdusang).

In situ delivery of allogeneic natural killer cell (NK) combined with Cetuximab in liver metastases of gastrointestinal carcinoma: A phase I clinical trial.

Despite successful introduction of NK-based cellular therapy in the treatment of myeloid leukemia, the potential use of NK alloreactivity in solid malignancies is still elusive. We performed a phase I clinical trial to assess the safety and efficacy of in situ delivery of allogeneic NK cells combined with cetuximab in liver metastasis of gastrointestinal origin. The conditioning chemotherapy was administrated before the allogeneic NK cells injection via hepatic artery. Three escalating doses were tested (3.106, 8.106 and 12.106 NK cells/kg) following by a high-dose interleukin-2 (IL-2). Cetuximab was administered intravenously every week for 7 weeks. Nine patients with liver metastases of colorectal or pancreatic cancers were included, three per dose level. Hepatic artery injection was successfully performed in all patients with no report of dose-limiting toxicity. Two patients had febrile aplasia requiring a short-term antibiotherapy. Grade 3/4 anemia and thrombopenia were also observed related to the chemotherapy. Objective clinical responses were documented in 3 patients and among them 2 occurred in patients injected with cell products harboring two KIR ligand mismatches and one in a patient with one KIR ligand mismatch. Immune monitoring revealed that most patients presented an increase but transient of IL-15 and IL-7 cytokines levels one week after chemotherapy. Furthermore, a high expansion of FoxP3+regulatory T cells and PD-1+ T cells was observed in all patients, related to IL-2 administration. Our results demonstrated that combining allogeneic NK cells transfer via intra-hepatic artery, cetuximab and a high-dose IL-2 is feasible, well tolerated and may result in clinical responses.

Fifteen years of Nucleic Acid Testing in France: Results and lessons.

Of the 40 million donations screened with Nucleic acid testing (NAT) between July 2001 and December 2015 in France, 20 HIV-positive, 13 HCV-positive and 17 HBV (HBV-NAT was initiated in 2005 and extended to the whole country in 2010) donations were discarded thanks to NAT. The main benefit in terms of discarded donations is related to HBV with a yield of 0.88 per million donations, which is 12.5 and 1.8 times higher than for HCV and HIV respectively. The main risk factor found in these donors during the post donation interview was having sex with men for males (n=11, all repeat blood donors), having a partner HCV positive (n=6) or at-risk partner (originated from endemic area or HBV positive) for HBV (n=8) for HIV, HCV and HBV, respectively. Although the mean viral load was high for HIV (5.6 log copies/mL) and HCV (7 log IU/mL), HBV cases show low level of DNA (1.8 log IU/mL) demonstrating the need of a highly sensitive NAT assay. Overall, the clinical benefit for recipients remains those related to the prevention of HIV contaminations since HCV avoided transmissions are extremely rare (only one case in the last 5 years thanks to NAT) and the potential infectivity of HBV-NAT only positive cases is questionable due to the low level of HBV DNA and the presence of anti-HBs in more than a half of DNA positive/HBsAg and anti-HBc negative donors.

Case-control study of immediate and delayed vasovagal reactions in blood donors.

BACKGROUND AND OBJECTIVE(S): Vasovagal reactions (VVRs) are the most common adverse events associated with blood donations. To assess the relative importance of VVR risk factors, a retrospective case-control study of severe immediate and delayed VVRs was performed. STUDY DESIGN: Vasovagal reactions were defined as immediate when occurring at the transfusion site and as delayed when occurring outside the transfusion site and within 24 h following donation. VVRs with probable or certain imputability and moderate to death severity were considered. One control/case was drawn randomly from among donors without VVR. Explanatory variables (sex, age, body mass index (BMI), donation status, type of phlebotomy) as well as the matching variables (donation region, date) and the interaction term (sex and BMI) were integrated into the multivariate model. RESULTS: In French hemovigilance data collected from 2011 to 2013, 8410 immediate and 833 delayed VVRs occurred among 8 834 214 donations. In multivariate analysis, occurrence of immediate VVR was strongly associated with first-time donation (OR 4·34; 95% CI: 3·93-4·79, P < 0·0001) and the 18-24 age group (OR 2·24; 95% CI: 2·00-2·45, P < 0·0001) and of delayed VVR with women with a normal BMI (OR 7·31; 95% CI: 4·96-10·77, P < 0·0001), overweight BMI (OR 7·89; 95% CI: 4·84-12·87, P < 0·0001) or obese BMI (OR 3·72; 95% CI: 1·42-9·74, P < 0·0001), and in men with an underweight BMI (OR 6·39; 95% CI: 1·56-26·13, P < 0·0001). Apheresis was a risk factor for occurrence of both immediate and delayed VVR. CONCLUSION: Our study highlights that first-time donation by a young person is particularly at risk of immediate VVR while a female donor is at risk of delayed VVR.

Exposure to hypomethylating agent, 5-azacytidine, may improve iCasp9 suicide gene therapy for treating GvHD in allografts.

Anti-tumor cellular immunotherapies that implement a suicide gene system can limit potential undesirable effects. In a haplo-identical bone marrow transplant clinical trial, over 90% of iCaspase-9-expressing cells were eradicated after AP1903 exposure, and signs of graft-versus-host disease disappeared. Nevertheless, low numbers of genetically modified T cells survived this treatment. We studied genetically modified cell lines (GMCL) that carried a dual iCaspase-9/ΔCD19 DNA construct (ΔCD19=truncated CD19). With AP1903 exposure, a low percentage of cells (1.47±0.67%; n=5 replications) persisted in vitro. Repeated exposures to increasing AP1903 doses generated low (GMCLLR) and high AP1903-responders (GMCLHR), which expressed different levels of surface ΔCD19 and intracellular iCaspase-9. Compared with GMCLHR, GMCLLR exhibited higher methylation of 5'-long-terminal repeat (LTR) promoters, both in the number of sequences with at least one methylated CpG (16 vs 51.5%, respectively) and in the number of CpG islands (1.2 vs 8.9%, respectively). Four days of 5-azacytidine exposure reduced methylation and increased ΔCD19 and iCaspase-9 expression. Interestingly, LTR demethylation restored GMCLLR sensitivity to AP1903 by 24.3-fold (1.8 vs 43.8%) without affecting GMCLHR. We showed that 5'-LTR-methylation inhibited transgene expression and caused AP1903 hypo-responsiveness. Treating with a hypomethylating agent restored AP1903 sensitivity. This approach can be applied in further clinical trials to improve iCaspase-9 response if low response is detected.

ATG-induced accelerated immune senescence: clinical implications in renal transplant recipients.

Persistent ATG-induced CD4(+) T cell lymphopenia is associated with serious clinical complications. We tested the hypothesis that ATG induces accelerated immune senescence in renal transplant recipients (RTR). Immune senescence biomarkers were analyzed at transplant and one-year later in 97 incident RTR -62 patients receiving ATG and 35 receiving anti-CD25 mAb (α-CD25). This consisted in: (i) thymic output; (ii) bone marrow renewal of CD34(+) hematopoietic progenitor cells (CD34(+) HPC) and lymphoid (l-HPC) and myeloid (m-HPC) progenitor ratio; (iii) T cell phenotype; and (iv) measurement of T cell relative telomere length (RTL) and telomerase activity (RTA). Clinical correlates were analyzed with a 3 year follow-up. Thymic output significantly decreased one-year posttransplant in ATG-treated patients. ATG was associated with a significant decrease in l-HPC/m-HPC ratio. Late stage differentiated CD57(+) /CD28(-) T cells increased in ATG-treated patients. One-year posttransplant T cell RTL and RTA were consequently lower in ATG-treated patients. ATG is associated with accelerated immune senescence. Increased frequency of late differentiated CD4(+) T cell frequency at transplantation tended to be predictive of a higher risk of subsequent opportunistic infections and of acute rejection only in ATG-treated patients but this needs confirmation. Considering pretransplant immune profile may help to select those patients who may benefit from ATG to prevent severe infections and acute rejection.

Prevention of hepatitis C virus infection by adoptive allogeneic immunotherapy using suicide gene-modified lymphocytes: an in vitro proof-of-concept.

Hepatitis C virus (HCV)-induced, end-stage liver disease is a major indication for liver transplantation, but systematic graft reinfection accelerates liver disease recurrence. Transplantation recipients may be ineligible for direct-acting antivirals, owing to toxicity, resistance or advanced liver disease. Adoptive immunotherapy with liver graft-derived, ex vivo-activated lymphocytes was previously shown to prevent HCV-induced graft reinfections. Alternatively, the applicability and therapeutic efficacy of adoptive immunotherapy may be enhanced by 'ready for use' suicide gene-modified lymphocytes from healthy blood donors; moreover, conditional, prodrug-induced cell suicide may prevent potential side effects. Here, we demonstrate that allogeneic suicide gene-modified lymphocytes (SGMLs) could potently, dose- and time-dependently, inhibit viral replication. The effect occurs at effector:target cell ratios that exhibits no concomitant cytotoxicity toward virus-infected target cells. The effect, mediated mostly by CD56+ lymphocytes, is interleukin-2-dependent, IFN-γ-mediated and, importantly, resistant to calcineurin inhibitors. Thus, post-transplant immunosuppression may not interfere with this adoptive cell immunotherapy approach. Furthermore, these cells are indeed amenable to conditional cell suicide; in particular, the inducible caspase 9 suicide gene is superior to the herpes simplex virus thymidine kinase suicide gene. Our data provide in vitro proof-of-concept that allogeneic, third-party, SGMLs may prevent HCV-induced liver graft reinfection.

[Hepatitis E virus: Blood transfusion implications]. / Virus de l'hépatite E, implications en transfusion sanguine.

Hepatitis E virus (HEV) is a non-enveloped RNA virus transmitted by the fecal-oral route. Autochthonous hepatitis E occurring in developed countries is caused by genotypes 3 and 4 and is a zoonotic infection. Humans are infected mostly after ingestion of undercooked meat from infected animals. Most HEV 3 and 4 infections are clinically inapparent. However, genotype 3 (HEV 3) can lead to chronic hepatitis in immuno-compromised patients such as organ-transplant recipients and patients with haematological malignancies. In Europe, HEV 3 is implicated in transfusion-transmitted HEV infection. In France, as observed in several European countries, prevalence of HEV RNA and specific IgG antibodies are high indicating that viral circulation is important. The systematic HEV NAT screening of blood donations used for preparation of solvent detergent plasma indicate that 1 to 2218 donation is infected by HEV RNA. The need or implementation's impacts of safety measures to prevent HEV transmission by blood transfusion are under reflexion by French's health authorities. The HEV NAT screening is the only available tool of prevention. Alternative strategies are under investigation including individual or mini pool NAT testing all or part of blood donations.

IL-22 deficiency in donor T cells attenuates murine acute graft-versus-host disease mortality while sparing the graft-versus-leukemia effect.

Acute graft-versus-host disease (aGVHD) remains a major complication following allogeneic hematopoietic cell transplantation (allo-HCT), limiting the success of this therapy. Many proinflammatory cytokines secreted following the conditioning regimen have been linked to aGVHD initiation. Interleukin-22 (IL-22) is a cytokine related to IL-10 for its structure and is secreted by T helper type 17 (TH17) cells and innate immune cells. Given the paradoxical role of IL-22 in inflammation with both protective or proinflammatory functions, we investigated whether IL-22 could have a role in aGVHD pathophysiology in a mouse allo-HCT model. In this study, we show that IL-22 deficiency in donor T cells can decrease the severity of aGVHD, while limiting systemic and local inflammation in aGVHD target organs. In addition, we found that Foxp3+ regulatory T cells (Treg cells) were increased in recipient mice that received IL-22-deficient T cells, suggesting that Treg were involved in the reduced severity of GVHD. Finally, we found that the graft-versus-leukemia (GVL) effect mediated by donor T cells was preserved in the absence of IL-22. Overall, these data suggest that targeting of IL-22 may represent a valid approach towards decreasing aGVHD severity after allo-HCT while preserving the GVL effect.

Extensive chronic GVHD is associated with donor blood CD34+ cell count after G-CSF mobilization in non-myeloablative allogeneic PBSC transplantation.

The correlation between the incidence of GVHD and the number of infused CD34(+) cells remains controversial for PBSC transplantation after a reduced-intensity-conditioning (RIC) regimen. We evaluated 99 patients transplanted with an HLA-identical sibling after the same RIC (2-Gy-TBI/fludarabine). Donor and recipient characteristics, donor's blood G-CSF-mobilized CD34(+) cell count, and number of infused CD34(+) and CD3(+) cells were analyzed as risk factors for acute and chronic GVHD There was a trend for an increased incidence of extensive chronic GVHD in the quartile of patients receiving more than 10 × 10(6) CD34(+) cells/kg (P = 0.05). Interestingly, the number of donor's blood CD34(+) cells at day 5 of G-CSF mobilization was closely associated with the incidence of extensive chronic GVHD, that is, 48% (95% CI: 28-68) at 24-months in the quartile of patients whose donors had the highest CD34(+) cell counts versus 24.3% (95% CI: 14-34) in the other patients (P = 0.007). In multivariate analysis, the only factor correlating with extensive chronic GVHD (cGVHD) was the donor's blood CD34(+) cell count after G-CSF (HR 2.49; 95% CI: 1.16-5.35, P = 0.019). This study shows that the incidence of cGVHD is more strongly associated with the donor's ability to mobilize CD34(+) cells than with the number of infused CD34(+) cells.

Plasmacytoid dendritic cells and Th17 immune response contribution in gastrointestinal acute graft-versus-host disease.

The contribution of Th17 cells in acute graft-versus-host disease (aGVHD) has been demonstrated in aGVHD mouse models. However, their contribution in human gastrointestinal aGVHD remains unclear. We evaluated Th17 cells in a cohort of 23 patients at diagnosis of aGVHD. In this study, we have shown that the absolute number of Th17 cells using the CCR6 and CD161 markers were significantly higher in the intestinal mucosa of patients with aGVHD compared with intestinal mucosa of patients without aGVHD. Moreover, in keeping with the increase of CCR6+ and CD161+ T cells, RORγt the key transcription factor that orchestrates the differentiation of Th17 cells, was significantly increased in the intestinal mucosa of patients with aGVHD compared with intestinal mucosa of patients without aGVHD (P=0.01). Since plasmacytoid dendritic cells (PDCs) have been reported to drive the differentiation of the Th17 subset, we quantified PDCs in these patients. PDC CD123+ cells were increased in the intestinal mucosa of patients with aGVHD. Furthermore, the number of CD123+ PDCs paralleled the histological grade of aGVHD, providing evidence for a role of Th17-mediated responses and a potential new pathophysiological link between PDCs and Th17 in human aGVHD.

Relevance of an academic GMP Pan-European vector infra-structure (PEVI).

In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.

[Extracorporeal photochemotherapy]. / La photochimiothérapie extracorporelle.

Photopheresis or extracorporeal photochemotherapy (ECP) is a cellular therapy which combines a leukapheresis followed by ex vivo treatment using psoralen and ultraviolet A irradiation before reinfusion into the patient. Its mechanisms of action remain unclear and selective photodestruction of leukocytes cannot explain the long-lasting immunomodulatory effects. Recent studies demonstrated that ECP down regulates the immune response and induces tolerance through the maturation of dendritic cells and the production of regulatory T cells. Based on these effects, ECP is mainly used for treatment of Sezary syndrome, graft-versus-host disease, organ graft rejection and autoimmune diseases. However, it is still not clear how ECP both activates tumor immunity against cutaneous T-cell lymphoma and induces tolerance in autoreactive disorders. In addition, the use of adjuvant therapies, the long-term effects and various treatment protocols remain to be investigated along with the specific indications.

Evidence for anti-tumour effect of allogeneic haematopoietic SCT in cases without sustained donor engraftment.

Remissions of haematological malignancies have been reported after allo-SCT, despite donor cell rejection, suggesting that sustained allogeneic engraftment is not mandatory to obtain a lasting anti-tumour effect. To evaluate the potential benefit from transient post-allo-SCT alloreactivity, we took advantage of the Société Française de Greffe de Moëlle et Thérapie Cellulaire (SFGM-TC) registry to colligate 14 patients with an efficient and long-lasting allogeneic (GVL) effect after allo-SCT for haematological malignancies, despite transient or absent engraftment. None received a second allogeneic graft after autologous recovery. The median duration of remission after autologous reconstitution was 118 (12-252) months. Although we cannot exclude the possibility that some patients were cured before allo-SCT, this retrospective analysis does strongly suggest that an efficient GVL effect can be observed without sustained donor engraftment, and that the transient presence of donor T cells might be sufficient to induce a powerful GVL effect.

[Banking and indications for cord blood grafts]. / Conservation et indications des greffons de sang placentaire.

A cord blood graft is now frequently used in the context of allogeneic hematopoietic transplantation. In 2007, 27% of unrelated allotransplantations performed in France used a cord blood graft. In comparison to other sources of hematopoietic grafts, cord blood has a number of advantages (immediate availability of the graft, possibility of a lesser HLA compatibility between donor and recipient...) and inconveniences (a limited number of stem cells, increased risk of infectious complications...). Recently, the possibility of combining two cord blood grafts has been demonstrated. In 2007, 55% of unrelated cord blood grafts transplantation were performed as such. Results of multicentric studies published as of now, as well as considerable potential associated with cord blood transplantation requires important efforts to increase both the quantity and quality of cord blood grafts made available for transplantation.

[Chagas disease screening in the blood donor population]. / Dépistage de la maladie de Chagas chez les donneurs de sang.

In May 2007, the French Blood Service (Etablissement français du sang, EFS) introduced systematic screening of at-risk blood donors for anti-Trypanosoma cruzi antibodies. This concerned donors originating from an endemic area, donors with mothers originating from such an area and individuals who had lived in or travelled to endemic areas, whatever the length of their stay. Five samples out of 163,740 were positive, all from individuals originating from an endemic area. One thousand three hundred seventy-four blood donations were considered as equivocal because they had discordant results on the two Elisa tests used in screening. The authors discuss difficulties presented by routine screening of travellers and residents as well as the advantages and drawbacks of the strategy used. They present arguments in favour of its simplification.

[Towards the use of intravenous apoptotic leukocyte infusion as a cell-based therapy approach?]. / Vers l'utilisation thérapeutique de l'administration intraveineuse de leucocytes apoptotiques du donneur en thérapie cellulaire ?

In the past few years, significant advances have been performed in the field of cell-based therapies. This concerns mainly regenerative medicine with the use of stem cells, as well as the modulation of immune responses. In order to modulate allogeneic immune responses after transplantation, we have developed a cell therapy approach based on the immunomodulatory properties of intravenous donor apoptotic cell infusion. In allogeneic hematopoietic cell transplantation settings, we reported that intravenous apoptotic leukocyte infusion, simultaneously to allogeneic bone marrow grafts, favors hematopoietic engraftment, prevents alloimmunization and delays graft-versus-host disease. Here, we review the different factors and cells implicated in the immunomodulatory properties of apoptotic cells. Then, we discuss the potential significance of such observations in transfusion practice.

Alloreactivity of ex vivo-expanded T cells is correlated with expansion and CD4/CD8 ratio.

Background We have demonstrated previously that retroviral-mediated transfer of a suicide gene into bone marrow (BM) donor T cells allows an efficient control of graft-versus-host disease (GvHD) after allogeneic BM transplantation. However, the 12 days of ex vivo culture required for the production of gene-modified cells (GMC), including soluble CD3 monoclonal antibody (MAb)-mediated activation and expansion with interleukin (IL)-2, induced a decrease of GMC alloreactivity and a reversal of their CD4/CD8 ratio. Improving the culture protocol in order to maintain the highest alloreactivity is of critical importance in obtaining an optimal graft-versus-leukemia (GvL) effect. Methods Peripheral blood mononuclear cells were activated with soluble CD3 MAb or CD3 and CD28 MAb co-immobilized on beads and expanded for 12 days in the presence of IL-2, IL-7 or IL-15 before analysis of alloreactivity and phenotype. Results Replacing the CD3 MAb by CD3/CD28 beads led to similar in vitro alloreactivity but improved the expansion and in vivo alloreactivity of GMC. Replacing the IL-2 with IL-7, but not IL-15, or decreasing IL-2 or IL-7 concentrations, improved the in vitro alloreactivity of expanded cells but was associated with lower expansion. Indeed, the alloreactivity of expanded cells was negatively correlated with cell expansion and positively correlated with CD4/CD8 ratio and CD8 expression level. Discussion Quantitative (i.e. low CD4/CD8 ratio) and qualitative (e.g. low CD8 expression) defects may account for the decreased alloreactivity of GMC. Using CD3/CD28 beads and/or IL-7 is more beneficial than CD3 MAb and IL-2 for preventing perturbations of the alloreactivity and phenotype of GMC.