Ramin Lotfi

Safe adoptive transfer of virus‐specific T‐cell immunity for the treatment of systemic adenovirus infection after allogeneic stem cell transplantation

During periods of immunosuppression, such as postallogeneic stem cell transplantation (SCT), patients are at significant risk for severe viral infections. Human adenovirus (HAdV) infection is a serious complication post‐SCT, especially in children. Virus‐specific T cells are essential for the clearance of HAdV, as antiviral chemotherapy has revealed limited success. We present feasibility data for a new treatment option using virus‐specific donor T cells for adoptive transfer of immunity to patients with HAdV‐infection/reactivation. Virus‐specific donor T cells were isolated and infused into nine children with systemic HAdV infection after SCT. Isolation was based on γ‐interferon (IFN‐γ) secretion after short in vitro stimulation with viral antigen, resulting in a combination of CD4+ and CD8+ T cells. 1·2–50 × 103/kg T cells were infused for adoptive transfer. Isolated cells showed high specificity and markedly reduced alloreactivity in vitro. Adoptive transfer of HAdV‐specific immunity was successful in five of six evaluable patients, documented by a dose‐independent and sustained in vivo expansion of HAdV‐specific T cells, associated with a durable clearance/decrease of viral copies. T‐cell infusion was well tolerated in all nine patients, except one case with graft‐versus‐host disease II of the skin. In conclusion, induction of a specific T‐cell response through adoptive transfer was feasible and effective. When performed early in the course of infection, adoptive T‐cell transfer may protect from HAdV‐related complications.

Human mesenchymal stem cells respond to native but not oxidized damage associated molecular pattern molecules from necrotic (tumor) material.

Necrosis is a characteristic feature of advanced solid tumors. Released necrotic factors, also referred to as damage associated molecular patterns (DAMPs), are known to critically impact the tumor microenvironment by enhancing angiogenesis or influencing the immune response. We have recently shown that DAMPs can act as chemoattractants and activators of granulocytes. We demonstrate that necrotic material from both normal and tumor cells promotes proliferation and trafficking of human mesenchymal stem cells (MSCs). We characterize the protein high mobility group box 1 (HMGB1) as a crucial member of DAMPs within necrotic material. In addition, we show that DAMPs interfere with expression of indoleamine 2, 3‐dioxygenase (IDO) in MSCs. The biological activity of necrotic material toward MSCs is abolished once these DAMPs are oxidized. MSCs found within tumor tissue can act as immunoregulatory cells and are able to promote tumor metastasis, thus playing a crucial role within the tumor microenvironment. Here, we reveal DAMPs to be crucial factors in the setting of MSC biology within the tumor microenvironment. The tumor microenvironment is characterized by reducing and hypoxic conditions that protect DAMPs from oxidation. Based on our results, oxidizing conditions should be considered for therapeutic approaches that target the tumor microenvironment.

S100A4 and Uric Acid Promote Mesenchymal Stromal Cell Induction of IL-10+/IDO+ Lymphocytes

Simple stress or necrotic cell death with subsequent release of damage-associated molecular patterns (DAMPs) is a characteristic feature of most advanced tumors. DAMPs within the tumor microenvironment stimulate tumor-associated cells, including dendritic cells and mesenchymal stromal cells (MSCs). The presence of tumor-infiltrating MSCs is associated with tumor progression and metastasis. Oxidized necrotic material loses its stimulatory capacity for MSCs. As a DAMP, S100A4 is sensitive to oxidation whereas uric acid (UA) acts primarily as an antioxidant. We tested these two biologic moieties separately and in combination for their activity on MSCs. Similar to necrotic tumor material, S100A4 and UA both dose-dependently induced chemotaxis of MSCs with synergistic effects when combined. Substituting for UA, alternative antioxidants (vitamin C, DTT, and N-acetylcysteine) also enhanced the chemotactic activity of S100A4 in a synergistic manner. This emphasizes the reducing potential of UA being, at least in part, responsible for the observed synergy. With regard to MSC proliferation, both S100A4 and UA inhibited MSCs without altering survival or inducing differentiation toward adipo-, osteo-, or chondrocytes. In the presence of S100A4 or UA, MSCs gained an immunosuppressive capability and stably induced IL-10– and IDO-expressing lymphocytes that maintained their phenotype following proliferation. We have thus demonstrated that both S100A4 and UA act as DAMPs and, as such, may play a critical role in promoting some aspects of MSC-associated immunoregulation. Our findings have implications for therapeutic approaches targeting the tumor microenvironment and addressing the immunosuppressive nature of unscheduled cell death within the tumor microenvironment.

Feasibility and safety of treating non-unions in tibia, femur and humerus with autologous, expanded, bone marrow-derived mesenchymal stromal cells associated with biphasic calcium phosphate biomaterials in a multicentric, non-comparative trial

BACKGROUND ORTHO-1 is a European, multicentric, first in human clinical trial to prove safety and feasibility after surgical implantation of commercially available biphasic calcium phosphate bioceramic granules associated during surgery with autologous mesenchymal stromal cells expanded from bone marrow (BM-hMSC) under good manufacturing practices, in patients with long bone pseudarthrosis. METHODS Twenty-eight patients with femur, tibia or humerus diaphyseal or metaphyso-diaphyseal non-unions were recruited and surgically treated in France, Germany, Italy and Spain with 100 or 200 million BM-hMSC/mL associated with 5-10 cc of bioceramic granules. Patients were followed up during one year. The investigational advanced therapy medicinal product (ATMP) was expanded under the same protocol in all four countries, and approved by each National Competent Authority. FINDINGS With safety as primary end-point, no severe adverse event was reported as related to the BM-hMSC. With feasibility as secondary end-point, the participating production centres manufactured the BM-hMSC as planned. The ATMP combined to the bioceramic was surgically delivered to the non-unions, and 26/28 treated patients were found radiologically healed at one year (3 out of 4 cortices with bone bridging). INTERPRETATION Safety and feasibility were clinically proven for surgical implantation of expanded autologous BM-hMSC with bioceramic. FUNDING EU-FP7-HEALTH-2009, REBORNE Project (GA: 241876).

Identification of Elite Neutralizers With Broad and Potent Neutralizing Activity Against Human Cytomegalovirus (HCMV) in a Population of HCMV-Seropositive Blood Donors

To improve the potency of anti-human cytomegalovirus (HCMV) immunoglobulin preparations, we intended to find elite neutralizers among 9000 HCMV-seropositive blood donors. We identified the top 2.6% neutralizers by use of high-throughput screening and further analyzed the 80 neutralizers with the most effective plasma for strain-independent activity. Of those, 58 had broad neutralizing activity against various HCMV strains and hence were regarded as elite neutralizers. All elite neutralizers were then analyzed to determine their effect on individual virus particles during entry. Most had plasma specimens that preferentially inhibited viral penetration, whereas 2 had exceptional plasma specimens that prevented adsorption of virus to cells. Furthermore, the neutralizing capacity of plasma samples from 3 randomly chosen elite neutralizers was up to 10-fold higher than that for commercial immunoglobulins. In a retrospective analysis of 6 selected donors, anti-HCMV neutralization titers in repeated donations were constantly high over 5 years. In conclusion, plasma samples from elite-neutralizing donors can be considered to improve antibody-based treatment of HCMV infections.

Increased requirement for platelet transfusions concurrent with enhanced bleeding during romiplostim treatment in a patient with thrombocytopenia due to bone marrow failure

Dear Editor, Romiplostim is a thrombopoietic agent. It induces increases in platelet counts in both healthy adults and patients with idiopathic thrombocytopenia (ITP) [1]. At the age of 46 years (Nov. 2003), a female patient was diagnosed with aplastic anemia (AA) showing distinct trilinear bone marrow hypoplasia, specifically of megakaryocytic lineage. Allosensitization against HLA A3, A24, B7, B27 was already present at diagnosis of AA. Six months after triple therapy with cyclosporine A (CSA), corticosteroids, and antithymocyte globulin (ATG) [2], she achieved partial remission with no need for further transfusions for 5 years. CSA medication was stopped after 3 years. In August 2008, she became again thrombocytopenic with requirement for transfusional support. At this time, she was diagnosed with amegakaryocytic thrombocytopenia. HLAmatched platelet concentrates (PCs) were given every 10 days with a very good increment of platelet counts (Fig. 1a). Additional alloantibodies against HPA 1b, HLA-A1,3,11,23, 24,66, and HLA-B 7,8,13,27,35,37,38,39,41,44,45,47, 49,50,52,53,55,56,60,61,62,63,73,75,76 occurred in the course of time. The patient was reluctant to other immunosuppressive regimens. Isolated thrombocytopenia (March 2010, WBC 8.1 K/μL, Hb 14.1 g/dL, platelets 6 K/μL) was not considered as clear-cut indication for allogeneic transplantation. Therefore, we tried an off-label use of romiplastim after informed consent. Romiplastim (Nplate, AMGEN GmbH Germany) was administered in escalating doses subcutaneously once a week for 4 weeks. Eighteen days after start of romiplostim, she developed refractoriness to HLAand HPA-matched PCs with distinct signs of bleeding in terms of generalized petechiae combined with mucosal hemorrhage leading to pharyngeal congestion (Fig. 1b) and macrohematuria (Fig. 1a and c). Treatment with ε-aminocapronate was of very temporary limited value. She needed matched PCs every 24 to 48 h due to active bleeding. Post-transfusional increment of platelet count dropped back to levels around 10 K/μL within 2 h after transfusion. Besides significantly shortened survival of transfused platelets (Fig. 1a and c), their functionality was strongly impaired given that before romiplostim treatment, the patient had presented much less clinical signs of bleeding with similar pre-transfusional platelet counts. Due to thrombocytopenia, it was not possible to prove impaired platelet functionality in vitro. R. Lotfi (*) :H. Schrezenmeier Institute for Transfusion Medicine, University of Ulm, Helmholtzstraße 10, 89081 Ulm, Germany e-mail: r.lotfi@blutspende.de

ATP promotes immunosuppressive capacities of mesenchymal stromal cells by enhancing the expression of indoleamine dioxygenase

MSCs are often found within tumors, promote cancer progression and enhance metastasis. MSCs can act as immuosuppressive cells, partially due to the expression of the enzyme indoleamine dioxygenase (IDO) which converts tryptophan to kynurenine. Decreased concentration of tryptophan and increased kynurenine, both interfere with effective immune response. Damage associated molecular patterns (DAMPs) including ATP are found within the tumor microenvironment, attract MSCs, and influence their biology.