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The 1990s saw rapid growth in international activity in hematopoietic cell transplantation. As national donor registries were established and international collaboration increased, a need to transfer cellular therapy products across national borders emerged. A lack of international standards for identification, terminology and labeling resulted in significant challenges for import and export. Twenty years of effort by a large group of experts supported by professional societies and accreditation bodies has today achieved a high degree of standardization. This review highlights the main landmarks in this journey and serves as a reminder of the importance of taking the "long view" when working toward international standardization. It demonstrates the need for continual maintenance and enhancement of standards to meet the changing needs of the cell therapy industry and highlights recent developments in ISBT 128.

Donor lymphocyte infusions (DLI) can induce durable remissions in multiple myeloma (MM) patients, but this occurs rather infrequently. As the graft-versus-tumor (GvT) effect of DLI depends on the presence of host-dendritic cells (DCs), we tested in a phase I/II trial whether the efficacy of DLI could be improved by simultaneous vaccination with host-DCs. We also analyzed the possibility of further improving the GvT effect by loading the DCs with peptides of mismatched hematopoietic cell-specific minor histocompatibility antigens (mHags). Fifteen MM patients not responding to a first DLI were included. Eleven patients could be treated with a second equivalent dose DLI combined with DC vaccinations, generated from host monocytes (moDC). For four patients, the DC products did not meet the quality criteria. In four of the treated patients the DCs were loaded with host mHag peptides. Toxicity was limited and no acute GvHD occurred. Most patients developed objective anti-host T-cell responses and in one patient a distinct mHag-specific T-cell response accompanied a temporary clinical response. These findings confirm that DLI combined with host-DC vaccination, either unloaded or loaded with mHag peptides, is feasible, safe and capable of inducing host-specific T-cell responses. The limited clinical effects may be improved by developing more immunogenic DC products or by combining this therapy with immune potentiating modalities like checkpoint inhibitors.Bone Marrow Transplantation advance online publication, 14 November 2016; doi:10.1038/bmt.2016.250.

Advances in stem cell biology and immunology raise hopes for new developments in regenerative medicine as well as cell-based immune intervention, with potential applications in many medical disciplines. Emerging from the medical practices of organ, tissue and cell transplantations over several decades, the development of cellular therapies nowadays enter a new era in which the industry may take cell manufacturing activities away from academic facilities, using complex procedures for cell engineering and large-scale production at centralized facilities. Recent changes in regulations push towards this historical transition; however, a pragmatic analysis of met and unmet clinical needs, as well as of specific procedural and organizational aspects that govern human cell procurement and processing suggest that a significant role will remain for academic facilities in the near future. Taking advantage of the large experience in the field of haematopoietic stem cell transplantation, we here review actual and potential consequences of regulatory changes in these medical practices, with a focus on the unique system for quality management that has been established in Europe and the United States of America to address specific risks associated with these therapeutic procedures.

Background aims: Bone marrow (BM) transplantations are an accepted therapeutic strategy for hematologic conditions. In the past decades, interest for BM-derived cell therapy has extended toward the field of regenerative medicine. Irrespective of the treatment strategy, its success depends on the amount of cells available for transplantation. Both patient and procedural factors have been shown to influence the cell density of the BM aspirate. In the present study, the influence of core diameter of the BM aspiration device on cell density of the BM aspirate is studied. Methods: BM harvesting procedures performed in a clinical trial investigating the effect of BM cell therapy in patients with severe peripheral artery disease were retrospectively studied (clinicaltrials.gov NCT00371371). Patients underwent BM harvesting through the use of either a 15-gauge (n = 85) or an 8-gauge (n = 75) needle. The numbers of harvested white blood cells (WBC) and CD34+ hematopoietic cells (HPC) were quantified. Results: The amount of WBC per milliliter of BM aspirate was significantly higher when the 8-gauge needle (27.8 × 106 WBC/mL [95%CI 25.4-30.5 × 106]) was used compared with the smaller 15-gauge core needle (20.1 × 106 WBC/mL [95% confidence interval (CI), 18.7-21.7 × 106], P < 0.001). For the amount of CD34+ HPC, a similar pattern was observed (185 × 103 HPC/mL [95% CI, 161-213 × 103]; 114 × 103 HPC/mL [95% CI, 96-134 × 103]; P < 0.001). Conclusions: The application of a BM aspiration device with a larger core diameter is associated with an increased cell density of the BM aspiration product in patients with severe peripheral artery disease.

Contains fulltext : 182504.pdf (Publisher’s version ) (Closed access) Allogeneic stem cell transplantation (allo-SCT) with or without donor lymphocyte infusions (DLI) is the only curative option for several hematological malignancies. Unfortunately, allo-SCT is often associated with GvHD, and patients often relapse. We therefore aim to improve the graft-versus-tumor effect, without increasing the risk of GvHD, by targeting hematopoietic lineage-restricted and tumor-associated minor histocompatibility antigens using peptide-loaded dendritic cell (DC) vaccinations. In the present multicenter study, we report the feasibility, safety and efficacy of this concept. We treated nine multiple myeloma patients with persistent or relapsed disease after allo-SCT and a previous DLI, with donor monocyte-derived mHag-peptide-loaded DC vaccinations combined with a second DLI. Vaccinations were well tolerated and no occurrence of GvHD was observed. In five out of nine patients, we were able to show the induction of mHag-specific CD8+ T cells in peripheral blood. Five out of nine patients, of which four developed mHag-specific T cells, showed stable disease (SD) for 3.5-10 months. This study shows that mHag-based donor monocyte-derived DC vaccination combined with DLI is safe, feasible and capable of inducing objective mHag-specific T-cell responses. Future research should focus on further improvement of the vaccination strategy, toward translating the observed T-cell responses into robust clinical responses.

Item does not contain fulltext BACKGROUND: Immunomagnetic selection of CD34(+) hematopoietic progenitor cells (HPC) using CliniMACS CD34 selection technology is widely used to provide high-purity HPC grafts. However, the number of nucleated cells and CD34+ cells recommended by the manufacturer for processing in a single procedure or with 1 vial of CD34 reagent is limited. METHODS: In this retrospective evaluation of 643 CliniMACS CD34-selection procedures, we validated the capacity of CliniMACS tubing sets and CD34 reagent. Endpoints of this study were the recovery and purity of CD34+ cells, T-cell depletion efficiency and recovery of colony-forming units-granulocyte-macrophage (CFU-GM). RESULTS: Overloading normal or large-scale tubing sets with excess numbers of total nucleated cells, without exceeding the maximum number of CD34+ cells, had no significant effect on the recovery and purity of CD34+ cells. In contrast, overloading normal or large-scale tubing sets with excess numbers of CD34+ cells resulted in a significantly lower recovery of CD34+ cells. Furthermore, the separation capacity of 1 vial of CD34 reagent could be increased safely from 600 x 10(6) CD34+ cells to 1000 x 10(6) CD34+ cells with similar recovery of CD34(+) cells. Finally, T-cell depletion efficiency and the fraction of CD34+ cells that formed CFU-GM colonies were not affected by out-of-specification procedures. DISCUSSION: Our validated increase of the capacity of CliniMACS tubing sets and CD34 reagent will reduce the number of selection procedures and thereby processing time for large HPC products. In addition, it results in a significant cost reduction for these procedures.

The publication of new standards for terminology and labeling marks an important step in ensuring consistency and traceability of cellular therapies at the global level. However, it is only with the widespread implementation of the standard that the benefits can be truly realized. This paper provides guidance on the practical aspects of adopting these new standards for organizations with differing current levels of computerization. It discusses project management, equipment, licensing, and validation topics. J. Clin. Apheresis, 2007. © 2007 Internal Cellular Therapy Coding and Labeling Advisory Group.

Background aims Methods for processing products used for hematopoietic progenitor cell (HPC) transplantation must ensure their safety and efficacy. Personnel training and ongoing competency assessment is critical to this goal. Here we present results from a global survey of methods used by a diverse array of cell processing facilities for the initial training and ongoing competency assessment of key personnel. Methods The Alliance for Harmonisation of Cellular Therapy Accreditation (AHCTA) created a survey to identify facility type, location, activity, personnel, and methods used for training and competency. A survey link was disseminated through organizations represented in AHCTA to processing facilities worldwide. Responses were tabulated and analyzed as a percentage of total responses and as a percentage of response by region group. Results Most facilities were based at academic medical centers or hospitals. Facilities with a broad range of activity, product sources and processing procedures were represented. Facilities reported using a combination of training and competency methods. However, some methods predominated. Cellular sources for training differed for training versus competency and also differed based on frequency of procedures performed. Most facilities had responsibilities for procedures in addition to processing for which training and competency methods differed. Although regional variation was observed, training and competency requirements were generally consistent. Conclusions Survey data showed the use of a variety of training and competency methods but some methods predominated, suggesting their utility. These results could help new and established facilities in making decisions for their own training and competency programs.

Purpose: Engineering T cells with receptors to redirect the immune system against cancer has most recently been described as a scientific breakthrough. However, a main challenge remains the GMP-grade purification of immune cells selectively expressing the introduced receptor in order to reduce potential side effects due to poorly or nonengineered cells. Experimental Design: In order to test a novel purification strategy, we took advantage of a model γδT cell receptor (TCR), naturally interfering with endogenous TCR expression and designed the optimal retroviral expression cassette to achieve maximal interference with endogenous TCR chains. Following retroviral transduction, nonengineered and poorly engineered immune cells characterized by a high endogenous αβTCR expression were efficiently depleted with GMP-grade anti-αβTCR beads. Next, the engineered immune cells were validated for TCR expression, function against a panel of tumor cell lines and primary tumors and potential allo-reactivity. Engineered immune cells were further validated in two humanized mouse tumor models. Results: The untouched enrichment of engineered immune cells translated into highly purified receptor-engineered cells with strong antitumor reactivity both in vitro and in vivo. Importantly, this approach eliminated residual allo-reactivity of engineered immune cells. Our data demonstrate that even with long-term suboptimal interference with endogenous TCR chains such as in resting cells, allo-reactivity remained absent and tumor control preserved. Conclusions: We present a novel enrichment method for the production of untouched engineered immune cells, ready to be translated into a GMP-grade method and potentially applicable to all receptor-modified cells even if interference with endogenous TCR chains is far from complete. Clin Cancer Res; 21(17); 3957–68. ©2015 AACR.

The evolution of articular cartilage repair procedures has resulted in a variety of cell-based therapies that use both autologous and allogeneic mesenchymal stromal cells (MSCs). As these cells are increasingly available and show promising results both in vitro and in vivo, cell-based strategies, which aim to improve ease of use and cost-effectiveness, are progressively explored. The use of MSCs in cartilage repair makes it possible to develop single-stage cell-based therapies. However, true single-stage procedures rely on one intervention, which will limit cell sources to fraction concentrates containing autologous MSCs or culture-expanded allogeneic MSCs. So far, it seems both autologous and allogeneic cells can safely be applied, but clinical studies are still ongoing and little information on clinical outcome is available. Further development of cell-based therapies may lead to clinical-grade, standardized, off-the-shelf products with easy handling for orthopedic surgeons. Although as of yet no preclinical or clinical studies are ongoing which explore the use of induced pluripotent stem cells for cartilage repair, a good manufacturing practice-grade induced pluripotent stem cell line might become the basis for such a product in the future, providing that cell fate can be controlled. The use of stem cells in clinical trials brings along new ethical issues, such as proper controls and selecting primary outcome measures. More clinical trials are needed to estimate detailed risk-benefit ratios and trials must be carefully designed to minimize risks and burdens for patients while choosing outcome measures that allow for adequate comparison with results from similar trials. In this review, we discuss the different aspects of new stem cell-based treatments, including safety and ethical issues, as well as provide an overview of current clinical trials exploring these approaches and future perspectives.

Peripheral blood progenitor cells (PBPC) have been established as an alternative source of hematopoietic stem cells for allogeneic transplantation, but an increased incidence of both acute and chronic graft-vs-host disease (GVHD) has become apparent. We performed a prospective randomized trial comparing bone marrow transplantation (BMT) vs PBPC transplantation (PBPCT) using CD34(+) selection for T-cell depletion (TCD) in both study arms.Between January 1996 and October 2000, 120 patients with a diagnosis of acute leukemia, myelodysplasia, multiple myeloma, or lymphoma were randomized to receive either filgrastim-mobilized PBPC or BM from HLA-identical sibling donors after standard high-dose chemoradiotherapy. Patient characteristics did not differ between study arms.Recipients of PBPC received more CD3(+) T cells (median: 3.0 vs 2.0 x 10(5)/kg, p0.0001) and more CD34(+) cells (median: 3.6 vs 0.9 x 10(6)/kg, p0.0001). Neutrophil and platelet recoveries occurred significantly faster after PBPCT. The cumulative incidence of acute GVHD grades II-IV was 37% after BMT vs 52% after PBPCT and was most significantly (p=0.007) affected by the number of CD3(+) T cells in the graft. Acute GVHD appeared strongly associated with increased treatment-related mortality (TRM) in a time-dependent analysis. Higher numbers of CD34(+) cells were associated with less TRM. With a median follow-up of 37 months (range: 12-75), overall survival at 4 years from transplantation was 60% after BMT and 34% for recipients of PBPCT (p=0.04), which difference was largely due to increased GVHD and TRM in PBPC recipients receiving T-cell dosages greater than 2 x 10(5)/kg.Outcome following T cell-depleted PBPCT critically depends on the number of CD3(+) T cells, whereby high T-cell numbers may blunt a favorable effect of higher CD34(+) cell numbers.

Intranasal treatment with C57BL/6 MSCs reduces lesion volume and improves motor and cognitive behavior in the neonatal hypoxic-ischemic (HI) mouse model. In this study, we investigated the potential of human MSCs (hMSCs) to treat HI brain injury in the neonatal mouse. Assessing the regenerative capacity of hMSCs is crucial for translation of our knowledge to the clinic. We determined the neuroregenerative potential of hMSCs in vitro and in vivo by intranasal administration 10 d post-HI in neonatal mice. HI was induced in P9 mouse pups. 1×10(6) or 2×10(6) hMSCs were administered intranasally 10 d post-HI. Motor behavior and lesion volume were measured 28 d post-HI. The in vitro capacity of hMSCs to induce differentiation of mouse neural stem cell (mNSC) was determined using a transwell co-culture differentiation assay. To determine which chemotactic factors may play a role in mediating migration of MSCs to the lesion, we performed a PCR array on 84 chemotactic factors 10 days following sham-operation, and at 10 and 17 days post-HI. Our results show that 2×10(6) hMSCs decrease lesion volume, improve motor behavior, and reduce scar formation and microglia activity. Moreover, we demonstrate that the differentiation assay reflects the neuroregenerative potential of hMSCs in vivo, as hMSCs induce mNSCs to differentiate into neurons in vitro. We also provide evidence that the chemotactic factor CXCL10 may play an important role in hMSC migration to the lesion site. This is suggested by our finding that CXCL10 is significantly upregulated at 10 days following HI, but not at 17 days after HI, a time when MSCs no longer reach the lesion when given intranasally. The results described in this work also tempt us to contemplate hMSCs not only as a potential treatment option for neonatal encephalopathy, but also for a plethora of degenerative and traumatic injuries of the nervous system.

The Joint Accreditation Committee of ISCT Europe and EBMT (JACIE) started in Europe at the end of the 1990s, with the support of the European group for Blood and Marrow Transplantation (EBMT) and the European branch of the International Society for Cell Therapy (ISCT). JACIE pursues the same goals as the Foundation for the Accreditation of Cell Therapy (FACT), the accreditation program for hematopoietic stem cell transplantation (HSCT) activities in North America: both aim at promoting excellence (1), harmonization and improving clinical results. FACT – JACIE standards (the fi fth version is currently being prepared) establish criteria for a comprehensive quality management program that covers the three critical domains in HSCT: clinical, collection and processing, and their interactions with ancillary activities.

In tissue engineering, strategies are being developed to repair large bone defects by combining biomaterials and bone marrow-derived multipotent mesenchymal stromal cells (MSCs). For expansion of MSCs under good manufacturing practice conditions, human platelet lysate (PL) can serve as substitute for fetal bovine serum (FBS) in culture media. We compared the in vivo bone-forming capacity of passage 3 MSCs cultured with either PL or FBS for nine different human donors. We also tested the growth kinetics, antigen expression profile, and the multilineage differentiation capacity in vitro of these MSCs. The in vivo bone-forming capacity was determined by seeding culture-expanded MSCs onto biphasic calcium phosphate scaffolds. Hybrid constructs were implanted subcutaneously in nude mice, retrieved after 6 weeks, and analyzed using histomorphometry. PL-supplemented cultures resulted in significantly larger colonies, shorter culture time period, and higher population doublings between P1 and P3 compared to FBS-containing cultures. No differences were observed in antigen expression profiles or differentiation capacities into the osteoblastic, chondrogenic, and adipogenic lineages, qualitatively. In vivo bone formation with PL-supplemented cultures of MSCs was demonstrated in 9/9 donors versus 6/9 for FBS-supplemented cultures. These results warrant the use of PL for ex vivo expansion of human MSCs for bone tissue engineering applications.

T hese standards have been developed through the co-operative endeavor of the following organizations: AABB, American Society for Blood and Marrow Transplantation (ASBMT), American Society for Apheresis (ASFA), European Group for Blood and Marrow Transplantation (EBMT), Foundation for the Accreditation of Cellular Therapy (FACT), ICCBBA, International Society of Blood Transfusion (ISBT), International Society for Cellular Therapy (ISCT), ISCT Europe, Joint Accreditation Committee of ISCT and EBMT (JACIE), National Marrow Donor Program (NMDP), and the World Marrow Donor Association (WMDA). The use of standard terminology will help to ensure a common understanding of product definitions. The labeling standard, supported by the ISBT 128 information system, will ensure unique global identification of cellular therapy products, an international reference table for product descriptions, and label design that is consistent worldwide. The organizations supporting these standards believe that their adoption will significantly improve the quality, safety, and traceability of cellular therapy products. In developing the terminology standard the Advisory Group recognized that the frequent movement of cellular therapy products between countries (and continents) required a consistent use of terminology between ISBT 128 and other published standards (e.g., Circular of Information, AABB, FACT, JACIE, Netcord). The Advisory Group has attempted to achieve these aims by removing complexity and redundancy from the coding system wherever possible and, by wide consultation, agreeing to terminology acceptable to all for inclusion in future publication of their standards and guidance. TERMINOLOGY STANDARD

Item does not contain fulltext BACKGROUND: Microvascular endothelial cells (MVEC) derived from s.c. fat are seeded on vascular grafts to prevent early occlusion. We have demonstrated the presence of contaminating cells contributing to MVEC seeding-related intimal hyperplasia in MVEC isolates from fat tissue. We found that cell isolates additionally purified after the isolation process, were associated with a reduced thrombogenicity and development of intimal hyperplasia in vitro. A combination of 11Fibrau (F11)- and CD14-coated Dynabeads was used to deplete the contaminating cells, fibroblasts, and monocytes/macrophages. Unfortunately, clinical-grade F11 is not available, and thus cannot be used for clinical practice. CD34 selection with clinical-grade products is widely used for the isolation of hematopoietic progenitors, and endothelial cells (EC) express CD34 on their surfaces. The aims of this study were to test the effectiveness of two different CD34-selection techniques for purification of MVEC, and to compare the results with those of the F11/CD14-method. METHODS: Liposuction fat was enzymatically digested and centrifuged twice to remove adipocytes and collagenase. CD34 selection was performed using the commercially available methods from Nexell or Miltenyi. Both techniques were modified for our use. The purity after isolation and culture, and recovery were determined by flow-cytometry (CD31-expression) and compared with that of cells purified with the F11/CD14-method. RESULTS: Besides MVEC, the contaminating fibroblasts and macrophages/monocytes weakly expressed the CD34 Ag. Enrichment of MVEC was not successful with the Miltenyi method. Variations in neither the dose of Ab nor the use of direct selection and different separation programs improved the results. With the Nexell method, MVEC were enriched to 86%, a comparable purity to that obtained with the F11/CD14-method. However, a lower recovery was achieved with the Nexell method. CONCLUSION: Enrichment of MVEC could be achieved with a modified protocol of the clinical grade CD34(+) selection method from Nexell, but not with the CD34 method from Miltenyi.

Rationale αβT- cell depleted allogeneic haematopoietic stem cell transplantations (allo-SCT) have so far been reported in haploidentical stem cell transplantations. First results have been promising with a good engraftment and low incidence of GVHD. However, high rates of life-threatening GVHD are still observed within the context of matched related (MRD) and unrelated (MUD) donors. Therefore we aimed to assess the feasibility of αβT-cell depletion in the context of MUD and MRD and aimed to establish a transplantation regimen for patients with MRD and MUD undergoing an αβT- cell depleted allo-SCT. Objective To test feasibility of the generation of αβT- cell depleted grafts from MRD and MUD and assess the engraftment in the context of different conditioning regimens after allo-SCT in patients with poor risk leukemia. Method Initial proof runs for the generation of αβT-cell and CD19 depleted grafts have been performed in 4 healthy donors. Grafts for transplantation of the first 5 patients (cohort I) have been depleted with GMP-grade anti-αβTCR and anti-CD19 antibodies. The subsequent grafts for patients in cohort II (n=4) and III (n=2) have been selectively depleted with GMP-grade anti-αβTCRs antibodies. Three conditioning regimens have been investigated (I): fludarabine 120 mg/m2 + cyclophosfamide 4800 mg/m2, (II): fludarabine 120 mg/m2 + busilvex AUC=90 and (III): ATG (Genzyme®) 4 mg/m2 + fludarabine 120 mg/m2 + busilvex AUC=90 followed by αβT- cell depleted grafts from matched related or unrelated donors. No additional immune suppression was given after allo-SCT. Main study parameters/endpoints: (1) Feasibility to generate an αβT-cell depleted graft form MRD and MUD. (2) Engraftment and reconstitution of T-cells within the context of different transplantation regimens. Results Products for 11 patients have been successfully processed and used for αβT-cell depleted allo-SCT between 2011 and 2013. A ∼4 log depletion of αβT-cells has been observed in the product with a recovery of ∼75% of CD34+ cells. In cohort I, primary engraftment (chimerism > 95%) was 40%. Engrafted patients showed a rapid reconstitution of γδT-cells and αβT-cells with a broad αβT-cell repertoire as determined by spectratyping. Therefore the next cohort was dose-intensified and the CD19-depletion omitted (cohort II). 75% of cohort II showed a swift engraftment. Again a dominance of γδT-cells was observed which associated with a rapidly reconstituting αβT-cell repertoire. Omitting CD19-depletion did not result in severe EBV-reactivations. One patient had an EBV reactivation under a short course of prednisone; however EBV was rapidly cleared after tapering steroids. In order to further increase engraftment cohort III was additionally treated with an early application of ATG (day -10/-9) and no graft failure has been observed so far. Conclusion αβT-cell depletion is feasible in the context of MRD and MUD. An intensified conditioning with additional host T-cell depletion seems to be beneficial for a profound engraftment. αβT-cell depletion associates with a swift and dominant reconstitution of γδT-cells as well as a rapidly restoring αβT-cell repertoire which is able to control viral re-activations. Disclosures: Kuball: Miltenyi: GMP product development Other.

Abstract 736 Introduction Despite multiple improvements in the last decade in the field of HSCT, acute graft versus host disease (aGVHD) remains a life-threatening complication and reduces substantially efficacy of HSCT. In particular, the outcome of patients with severe steroid-resistant aGVHD is very poor. Therefore, it remains important to search for new therapeutic strategies for the treatment of aGVHD. Objective Feasibility of the generation and efficacy of mesenchymal stroma cells (MSCs) generated with fetal calf serum (FCS) has been suggested recently. However, FCS is a putative source of prions and virus transmission. Therefore, the feasibility of the generation of MSCs expanded with human plasma and platelet lysate (hPPL) was tested as well as the feasibility and safety of the application of hPPL-MSCs in patients with steroid-refractory aGVHD. Furthermore multiple immunological changes after infusion of MSC were characterized, in vitro. However, truly active mechanisms in human are poorly understood as well as whether infusion of MSC selectively impairs GVHD-inducing immune cells or also anti-virus and anti-leukemia reactive T-cells. Therefore, phenotypical and functional changes in immunological cell types and cytokine levels were investigated. Method In an open-label, non-randomized prospective phase I/II study MSCs were extracted from the bone marrow of healthy volunteers, expanded with hPPL, and stored. Patients with steroid-refractory aGVHD grade II to IV were treated with hPPL-MSC. 50 patients were included and received up to four infusions. Response rate, transplantation-related deaths, and other adverse events were assessed for up to 12 months after the last infusion of the cells. In addition, a comprehensive phenotypical and functional analysis was performed with PBMCs and serum isolated from all patients before, during, and after infusion of MSC. Results Between January 2009 and July 2012, 50 patients were included, 2 patients dropped out, 5 patients are so far incompletely documented. Thus 43 patients were so far available for analysis, 6 children and 37 adults. Median age was 51,5 years (1.3–65.9). Organs involved in aGVHD were the skin (56%), the gastro-intestinal tract (86%) and the liver (33%). Overall grade was II for 11 (26%), III for 28 (65%), and IV for 3 (7%) patients. Mean number of infusion were 3 (1–4). No severe side effects were observed. Median follow-up was 4.06 months (range 0.43–12). Complete overall response was observed in 24 patients (56%) after a median of 53 days (range 3–116 days). The overall survival was significantly better in responders when compared to non-responders (p Conclusion Generation and infusion of hPPL-MSCs in steroid-resistant aGVHD grade II- IV is feasible, safe and appears to be effective. In addition, also patients who initially responded to hPPL-MSCs but develop later a relapse of aGVHD during tapering or cessation of immunosuppressive drugs become again sensitive to the treatment with steroids. Infusion of MSC did not impair anti-virus and anti-leukemia reactive T-cells. Identified biomarkers predict very early a usually late clinical resolution of GVHD, thus might be useful to early guide clinical decisions. Disclosures: No relevant conflicts of interest to declare.

Abstract 2875 Poster Board II-851 Allogeneic stem cell transplantation followed by donor lymphocyte infusions (DLI) induces a clinical significant graft versus myeloma (GvM) effect in 30-50% of multiple myeloma (MM) patients. Since this effect can be dependent on the presence of host dendritic cells (DC) at the time of DLI, combining DLI with infusion of host DCs may improve GvM effect. To evaluate this we conducted a phase I/II trial in which DLI- non responding MM patients received host monocyte derived DC vaccinations in combination with a second DLI. Patients received 3 DC vaccinations both i.v. and i.d. with two-week intervals. The first vaccination was combined with DLI. Half of the i.d. DCs was loaded with KLH for immunomonitoring purposes. Specific anti-host and anti-KLH T cell responses were monitored using IFN-gamma release and proliferation assays. To date six patients completed all 3 DC vaccinations, one patient received only one vaccination. Exacerbation of mild pre-existing graft versus host disease occurred in two patients after the first and second vaccination respectively; no other adverse events were seen. Starting from the second vaccination patients developed positive skin reactions against KLH-DCs (n=5/6) and against DCs (n=2/5). The delayed type hypersensivity (DTH) skin tests performed after the last vaccination showed positive indurations against host DCs (n=5/5) and against KLH (n=4/5). All patients (n=4/4) developed significant but variable proliferative and IFN-gamma responses against KLH and host DCs. While 5 patients developed no clinical GvM effect, in one patient disease was stabilized for 29 weeks and the cellular responses against host DCs and KLH were still present 20 weeks after the first vaccination. Another patient, who entered the study with minimal residual disease, is clinically stable for 18 weeks now, however he has not yet been evaluated further. We conclude that administration of host DCs combined with DLI is safe and feasible, and that the vaccinations result in enhanced T cell responses. However, clinical GvM effects are limited. This GvM effect may be increased after vaccination with DCs loaded with hematopoietic restricted minor H antigens. Disclosures: Off Label Use: Rituximab, a monoclonal anti-CD20 antibody, is used to deplete B cells in the treatment of chronic GVHD.

The factors determining the predominantly erythroid direction of human fetal liver hematopoiesis are unknown. We compared the capacities of human fetal liver and bone marrow stromas to sustain fetal and adult hematopoiesis in long-term cultures. In various marrow-fetal liver combinations of stroma and recharge, the maintenance of erythroid (BFU- e) and myeloid (CFU-GM) precursors in the nonadherent phase was determined. The morphology of the fetal liver nucleated cells during culture was also examined. This study shows that fetal liver stromas efficiently support fetal BFU-e for 6 to 7 weeks in vitro. Bone marrow stromas were not able to maintain fetal BFU-e beyond 4 weeks. Significant numbers of marrow BFU-e were not sustained in vitro on either source of stroma. On the other hand, the stroma layers of fetal liver and marrow origin were equally effective in maintaining fetal CFU- GM and adult CFU-GM in long-term culture. These findings show that the human embryonic liver stroma is a preferential site for stimulating fetal erythropoiesis. They do not demonstrate differences in stroma function to explain the relative paucity of myelopoiesis in the fetal liver.

Additionally, we show that MIF is able to upregulate the expression of cytokines described as chemoattractants for MSC (IL8, IL6 and CCL2), reinforcing the role of this molecule as the first trigger for several downstream signalling pathways. Importantly we show that knock down (using lentiviral shRNAs) of either CXCR4 (in MSCs) or MIF (in cancer cells) decreases significantly MSC homing to tumors in an in vivo pulmonary metastasis model. Interestingly, MIF has been shown by others to be over-expressed in a large variety of human cancers and closely correlates with tumor aggressiveness and metastatic potential. Therefore, MSC homing to tumors triggered by MIF could be a general mechanism for a variety of cancers. This improved understanding of MSC tumor tropism will further enable development of novel cellular therapies for cancers by increasing the homing potential of these cells.