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Background: The risk of microbial contamination of cellular products can be reduced when cultured in the presence of antibiotics. This however, may impact the sensitivity of microbiological tests. Given that the addition of antibiotics to cell/tissue products does not guarantee sterility but may just reduce the proliferation rate of microorganisms, microbiological testing of medicinal products remains obligatory. Thus, an appropriate method to test for microbial contamination of antibiotic-containing products has to be validated. Objectives: In the context of microbiological testing of a cellular advance therapy medicinal product, the method was validated and approved by German competent authorities for four different matrices with three matrices containing antibiotics. The paper shall provide help for establishing test methods for other investigational medicinal products which contain antibiotics. Methods: Matrices were spiked individually with Staphylococcus aureus, Bacillus subtilis, Pseudomonas aeruginosa, Streptococcus pyogenes, Escherichia coli, Clostridium sporogenes, Propionibacterium acnes, Candida albicans, and Aspergillus brasiliensis. Samples were pretreated with penicillinase for 1 h before inoculation and incubation in BacT/ALERT iFA Plus and iFN Plus culture bottles using 3D BacT/ALERT automates. Microorganisms within positive BacT/ALERT bottles were specified. The procedure was performed in two different laboratories to prove robustness of test. Results: All nine tested microorganisms were detected within 14 days of incubation in accordance with requirements of the European Pharmacopoiea in terms of sensitivity, specificity and robustness of the test. Penicillin and streptomycin did not have any influence on specifications defined within the investigational medicinal product dossier. Conclusions: Culturing cellular products in the presence of antibiotics can serve as an effective method to reduce contamination risk but only if the chosen antibiotics neither have any influence on specifications of the investigational medicinal product nor interfere with microbiological tests. Consequently, cells and tissues primarily contaminated with microorganisms, like placenta, may be considered as a source of cellular therapeutics when cultured for a sufficient time with antibiotics and tested with a validated method. The choice of microorganisms for the validation of the microbiological test should always consider all conceivable scenarios and should not be reduced to minimal criteria defined in European Pharmacopoiea, wrongfully believing to thus save time and effort.

Leukemic stem cells of acute myeloid leukemia patients carrying NPM1 mutation are candidates for targeted immunotherapy

The immune reconstitution after allogeneic hematopoietic stem cell transplantation comprises thymus-dependent and thymus-independent pathways. We wanted to improve the understanding of this complex process using two different measurements at definite checkpoints of T-cell neogenesis. We therefore assessed the thymus-dependent pathway by combining measurements of single joint T-cell receptor excision circles (sjTREC) and β T-cell receptor excision circles (βTREC) in an improved quantitative light-cycler hybridization polymerase chain reaction assay. In a subgroup of patients, we additionally assessed the proliferation kinetics of the CD31(+) thymic naïve cell population, which corresponds to recent thymic emigrants by six-color immunostaining. After the establishment of normal values in 22 healthy volunteers, we applied our polymerase chain reaction to 66 patients undergoing allogeneic hematopoietic stem cell transplantation at a median age of 44 years. It took more than 2 years after transplant to restore the pre-transplant thymic proliferation capacity. Only one third of the patients in our longitudinal study reached age-adjusted normal values for both sjTREC and βTREC at a median follow-up of 558 days, with acute graft-versus-host disease being the most prominent negative factor by univariate analysis. We observed several patterns of sjTREC and βTREC recovery suggesting different mechanisms of thymic damage in individual patients. In a comparison of CD31(+) thymic naïve cells between volunteers and patients after transplant we found a significantly higher peak proliferation rate within the latter population in the first year after transplantation. The combination of measurements of sjTREC and βTREC by our simplified polymerase chain reaction assay provides insight about the stage of T-cell development affected by different types of damage and may help to choose the correct therapeutic intervention. Besides the sole thymic T-cell neogenesis, proliferation within the CD31(+) thymic naïve cell compartment contributed to the replenishment of the naïve T-cell pool after transplantation.

BACKGROUND: Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic stem cell transplantation. For the clearance of CMV, CD8+ T cells are pivotal. STUDY DESIGN AND METHODS: Here, the novel streptamer technology was used at good manufacturing practice (GMP) level for adoptive transfer of CMV-specific T cells into acute leukemia patients with recurrent high CMV antigenemia after allogeneic stem cell transplantation. RESULTS: After a single transfusion, the frequency of CMV-specific CD8+CD45RA+CCR7– effector T cells increased dramatically from 0.0% to a maximum of 27.1% of all T cells. These T cells were clearly donor derived and did not stem from intrinsic reconstitution, as demonstrated by analysis of 1) donor chimerism through single-tandem repeats, 2) T-cell receptor excision circles, and 3) Vβ-chain typing by polymerase chain reaction. Clinically, the specific T-cell transfer resulted in a persistent clearance of the CMV antigenemia, which allowed the patients to discontinue toxic antiviral drug therapy without further high-level reactivation of CMV, demonstrating the power of the streptamer technology. CONCLUSION: Taken together, the streptamer technology offers the advantage of selecting virus-specific CD8+ T cells at GMP level for adoptive T-cell transfer, thus inducing long-lasting specific CD8+ T-cell responses without increasing the risk for graft-versus-host disease.

Background Recently, we demonstrated immunological and clinical responses to a RHAMM-R3 peptide vaccine in patients with acute myeloid leukemia, myelodysplastic syndrome and multiple myeloma. To improve the outcome of the vaccine, a second cohort was vaccinated with a higher dose of 1,000 μg peptide.Design and Methods Nine patients received four vaccinations subcutaneously at a biweekly interval. Immunomonitoring of cytotoxic CD8+ as well as regulatory CD4+ T cells was performed by flow cytometry as well as by enzyme-linked immunospot (ELISpot) assays. Parameters of clinical response were assessed.Results In 4 of 9 patients (44%) we detected positive immunological responses. These patients showed an increase of CD8+RHAMM-R3_tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells and an increase of R3-specific CD8+ T cells. Two of these patients showed a significant decrease of regulatory T cells (Tregs). In one patient without response Tregs frequency increased from 5 to 16%. Three patients showed clinical effects: one patient with myelodysplastic syndrome RAEB-1 showed a reduction of leukemic blasts in the bone marrow, another myelodysplastic syndrome patient an improvement of peripheral blood counts and one patient with multiple myeloma a reduction of free light chains. Clinical and immunological reactions were lower in this cohort than in the 300 μg cohort.Conclusions High-dose RHAMM-R3 peptide vaccination induced immunological responses and positive clinical effects. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematologic malignancies. However, higher doses of peptide did not improve the frequency and intensity of immune responses in this trial. (This study is registered at http://ISRCTN.org as ISRCTN32763606)

During the last years remission rates of more than 72% for arsenic(III)-oxide (As(2)O(3)) treatment in relapsed or refractory acute promyelocytic leukemia have been published. As(2)O(3) is under clinical investigation for therapy of leukemia and solid tumors. Due to the chemical affinity of arsenic and antimony, we analyzed the potency of antimony(III)-oxide (Sb(2)O(3)) to exert As(2)O(3)-like effects. Based on the same molar concentrations, lower efficacy in apoptosis induction and caspase-independent decrease of mitochondrial membrane potential was observed for Sb(2)O(3). No difference in sensitivity to As(2)O(3) or Sb(2)O(3) was detected in CEM cells when compared to their multiple drug resistant derivatives. Apoptosis was induced by combining sub-apoptotic concentrations of Sb(2)O(3) or As(2)O(3) with sub-apoptotic concentrations of DL: -buthionine-[S,R]-sulfoximine (BSO). Other modulators of the cellular redox system showed this effect to a lower extent and enhancement was not consistent for the different cell lines tested. Caspase inhibitors protected cell lines from Sb(2)O(3)- and As(2)O(3)-induced apoptosis. When BSO was added, the inhibitors lost their protective ability. The ability of modulators of the cellular redox system in clinically applicable concentrations to enhance the apoptotic effects of the two oxides in a synergistic way may be helpful to reduce their toxicity by optimizing their dose.

Antithymocyte globulin (ATG) and antilymphocyte globulin (ALG) are currently used successfully for immunosuppressive treatment of aplastic anemia. In this study we have investigated whether commercial ATG/ALG preparations contain antibodies against glycosylphosphatidyl-inositol anchored proteins (GPI-AP), which could be responsible for emergence of GPI-deficient populations in aplastic anemia after ATG/ALG therapy. We analyzed four commercial ATG/ALG preparations by competitive binding assays using flow cytometry. Quantification was achieved by calculating the concentration of ATG/ALG required to give 50% inhibition of binding the specific fluorochrome-labeled monoclonal antibody (EC50). High concentrations of antibodies against the GPI-anchored protein CD52 were found in all preparations (Lymphoglobulin Genzyme, Thymoglobulin Genzyme, ATGAM. Pharmacia & Upjohn, and ATG-Fresenius S Fresenius). Antibodies against the GPI-anchored protein CD48 are present in significant concentrations except in the preparation ATGAM. CD16 antibodies were found in lower concentrations. We could not detect significant concentrations of antibodies against the GPI-anchored proteins CD157 and CD14. Campath-1H, a monoclonal antibody against the GPI-anchored protein CD52, has been used as immunosuppressive tool for T-cell depletion. CD52 antibodies in ATG/ALG preparations might contribute in the same way to the immunosuppressive effects in treatment of aplastic anemia. It is known that in a substantial proportion of patients with aplastic anemia GPI-deficient cells are present in a low level at diagnosis or emerge after immunosuppressive therapy. GPI-anchored antibodies in ATG/ALG preparations might lead to a relative advantage for pre-existing GPI-deficient cells caused by an escape from the antibody-mediated attack.

CD4+CD25+ regulatory T cells (Tregs) can influence various immune responses. Little is known about the effects of the Abl/Src kinase inhibitor dasatinib on Tregs which regulate anti-tumor/leukaemia immune responses. The present study demonstrated that dasatinib inhibited proliferation of Tregs and CD4+CD25- T cells in a dose-dependent manner, which was associated with the decreased production of corresponding cytokines. Treatment of Tregs with dasatinib inhibited the suppressive capacity of Tregs. The mechanisms of this inhibition included arrest of cells in the G0/G1 phase of cell cycle, down-regulation of the transcription factor forkhead box P3, glucocorticoid-induced tumour necrosis factor receptor and the cytotoxic T lymphocyte associated protein 4 as well as inhibition of signaling events through Src and nuclear factor kappaB. Dasatinib showed an inhibitory effect on the proliferation and function of both Tregs and CD4+CD25- T cells at therapeutically relevant concentrations of the drug. Clinical administration of dasatinib might influence not only the graft-versus-leukaemia effect but also the graft-versus-host-disease in patients receiving dasatinib after allogeneic stem cell transplantation and/or donor lymphocytes infusion as the function of both Tregs and effector T cells are hampered in a similar way by dasatinib.

Adenosine deaminase (ADA) deficiency is a systemic metabolic disease that causes an autosomal recessive variant of severe combined immunodeficiency (SCID) and less consistently other complications including neurologic abnormalities. Hematopoietic stem cell transplantation (HSCT) is able to correct the immunodeficiency, whereas control of nonimmunologic complications has not been extensively explored. We applied HSCT in 15 ADA-deficient patients consecutively treated at our institutions since 1982 and analyzed long-term outcome. Seven patients received transplants without conditioning from HLA-matched family donors (MFDs); the other 8 patients received conditioning and were given transplants either from HLA-mismatched family donors (MMFDs; n = 6) or from matched unrelated donors (MUDs; n = 2). At a mean follow-up period of 12 years (range, 4-22 years), 12 patients are alive with stable and complete immune reconstitution (7 of 7 after MFD, 4 of 6 after MMFD, and 1 of 2 after MUD transplantation). Six of 12 surviving patients show marked neurologic abnormalities, which include mental retardation, motor dysfunction, and sensorineural hearing deficit. We were unable to identify disease or transplantation-related factors correlating with this divergent neurologic outcome. The high rate of neurologic abnormalities observed in long-term surviving patients with ADA deficiency indicates that HSCT commonly fails to control CNS complications in this metabolic disease.

Data regarding the role of mitochondria in arsenic trioxide (As2O3)-induced apoptosis are controversial. We investigated the contribution of caspases and mitochondrial depolarization to As2O3-induced apoptosis in the myeloid cell lines NB-4, HL-60 and U-937. Caspase inhibition reduced the amount of cells with As2O3 (20 micromol/l)-induced mitochondrial depolarization by about 50% in all cell lines. As2O3 also induced dose-dependent phosphatidylserine exposure in cells without depolarized mitochondria. We conclude that caspase activation is of similar importance in As2O3-induced apoptosis in myeloid cell lines as direct mitochondrial targeting and mitochondria are not necessary for caspase activation downstream of mitochondria.

SCID is a clinically defined group of monogenetic diseases characterized by life-threatening infections in early infancy. One variant of SCID is due to defects in the Zeta-chain-associated protein (ZAP-70; MIM+176947), which results in deficient signal transduction from the TCR to the nucleus and abnormal maturation and function of thymocytes. A characteristic finding is a complete lack of circulating CD8+ T cells, whereas CD4+ T cells are present, but without function.1 Although ZAP-70 is not expressed in B cells, humoral immunity is indirectly affected because of inadequate T-cell help. Currently, hematopoietic cell transplantation represents the only curative treatment.1

International audience; The aim of this study was to assess the immune modulatory properties of human mesenchymal stromal cells obtained from bone marrow (BM-MSCs), fat (ASCs) and cord blood (CB-MSCs) in the presence of a novel hydroxyapatite and tricalcium-phosphate (HA/TCP) biomaterial as scaffold for MSC delivery. In resting conditions, a short-term culture with HA/TCP did not modulate the anti-apoptotic and suppressive features of the various MSC types towards T, B and NK cells; in addition, when primed with inflammatory cytokines, MSC maintained or not on HA/TCP similarlyincreased their suppressive capacities. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype with up-regulation of OSTERIX and OSTEOCALCIN, similarly to what obtained with dexamethasone and, to a higher extent, BMP-4 treatment. MSC-derived osteoblasts did not trigger immune cell activation, but were less efficient than undifferentiated MSCs in inhibiting stimulated T and NK cells. Interestingly, their suppressive machinery included not only the activation of IDO, which plays a central role in T-cell inhibition, but also COX-2 that was not significantly involved in immune modulatory effect of human undifferentiated MSCs. COX-2 is significantly involved in bone healing, suggesting that its induction by HA/TCP could also contribute to the therapeutic activity of MSC for bone tissue engineering.

The receptor for hyaluronic acid-mediated motility (RHAMM) is a tumor-associated antigen in chronic lymphocytic leukemia (CLL). CD8(+) T cells primed with the RHAMM-derived epitope R3, which is restricted by human leukocyte antigen (HLA)-A2, effectively lyse RHAMM(+) CLL cells. Therefore, we initiated a phase I clinical trial of R3 peptide vaccination. Six HLA-A2(+) CLL patients were vaccinated four times at biweekly intervals with the R3 peptide (ILSLELMKL; 300 microg per dose) emulsified in incomplete Freund's adjuvant; granulocyte-macrophage colony stimulating factor (100 microg per dose) was administered concomitantly. Detailed immunological analyses were conducted throughout the course of peptide vaccination. No severe adverse events greater than CTC I degrees skin toxicity were observed. Four patients exhibited reduced white blood cell counts during vaccination. In five of six patients, R3-specific CD8(+) T cells were detected with the corresponding peptide/HLA-A2 tetrameric complex; these populations were verified functionally in four of five patients using enzyme-linked immunosorbent spot (ELISpot) assays. In patients with clinical responses, we found increased frequencies of R3-specific CD8(+) T cells that expressed high levels of CD107a and produced both interferon-gamma and granzyme B in response to antigen challenge. Interestingly, vaccination was also associated with the induction of regulatory T cells in four patients. Thus peptide vaccination in six CLL patients was safe and could elicit to some extent specific CD8(+) T-cell responses against the tumor antigen RHAMM.

SUMMARY: Research on expanded human stem cells has become an increasing field of interest during the last decade. The increasing interest in adult stem cells, especially mesenchymal stem and mesenchymal stromal cells, in hematology and regenerative medicine is also based on the simplicity of isolation and ex vivo expansion of these cells. These processes require an adequate quality control of source and product. In this review, we summarize various different attempts to characterize mesenchymal stem cells based on surface protein expression by flow cytometry and to define multipotent subpopulations of mesenchymal stem cells for prospective isolation. The importance of defining functional assays and a unique marker panel to characterize mesenchymal stem cells for clinical trials as well as the factors that can modulate the marker expression is discussed.

Objective To investigate the inhibitory effects of dasatinib on proliferation, function, and signaling events on CD8 + T cells. Materials and Methods Carboxyfluorescein diacetate succinimidyl ester and 5-bromo-2-deoxyuridine were used to detect proliferation and cell cycle of CD8 + T cells treated with dasatinib, respectively. Frequency and function of viral and leukemia-antigen–specific CD8 + T cells from healthy donors were measured by tetramer staining and ELISPOT assay. Western blotting analysis was performed to detect T-cell receptor (TCR), nuclear factor k appa B (NF-κB) and Src signaling events in T cells treated with dasatinib or imatinib. Results Dasatinib inhibited proliferation of CD8 + T cells in a dose-dependent manner, which was associated with lower secretion of interferon-gamma and granzyme B, as well as with arrest of CD8 + T cells in the G 0 /G 1 phase of cell cycle. Inhibition of CD8 + T cells was proven for blood samples from a patient under dasatinib medication when compared with their T-cell status without dasatinib. Western blotting confirmed that these effects were mediated through downregulation of the phosphorylation level of molecules from the TCR and the NF-κB signaling transduction cascade. Dasatinib proved to be more potent than imatinib on Src and TCR signaling events in Jurkat T cells. Conclusion Our study demonstrated that dasatinib impaired proliferation and function of CD8 + T cells via TCR and NF-κB signaling events without inducing apoptosis. Therefore, dasatinib might alter the graft-vs-leukemia effect and the graft-vs-host disease after allogeneic stem cell transplantation sustained by CD8 + T cells. Dasatinib might also be used as a novel immunosuppressant agent.

The receptor for hyaluronic acid–mediated motility (RHAMM) is an antigen eliciting both humoral and cellular immune responses in patients with acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and multiple myeloma (MM). We initiated a phase 1 clinical trial vaccinating 10 patients with R3 (ILSLELMKL), a highly immunogenic CD8+ T-cell epitope peptide derived from RHAMM. In 7 of 10 patients, we detected an increase of CD8+/HLA-A2/RHAMM R3 tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells in accordance with an increase of R3-specific CD8+ T cells in enzyme linked immunospot (ELISpot) assays. In chromium release assays, a specific lysis of RHAMM-positive leukemic blasts was shown. Three of 6 patients with myeloid disorders (1/3 AML, 2/3 MDS) achieved clinical responses: one patient with AML and one with MDS showed a significant reduction of blasts in the bone marrow after the last vaccination. One patient with MDS no longer needed erythrocyte transfusions after 4 vaccinations. Two of 4 patients with MM showed a reduction of free light chain serum levels. Taken together, RHAMM-R3 peptide vaccination induced both immunologic and clinical responses, and therefore RHAMM constitutes a promising target for further immunotherapeutic approaches. This study is registered at http://ISRCTN.org as ISRCTN32763606 and is registered with EudraCT as 2005-001706-37.

The proteins of blood group systems are expressed on red blood cells (RBC) by definition. We searched nucleotide databases of human expressed sequence tags (EST) to collate the distribution of 22 distinct membrane proteins in cells and tissues other than RBC. The documented blood group genes are: MNS, Rh, Lutheran, Kell, Duffy, Kidd, Diego, Yt, Xg, Scianna, Dombrock, Colton, Landsteiner-Wiener, Kx, Gerbich, Cromer, Knops, Indian, Ok, Raph, John-Milton-Hagen and Gill. The genes were grouped according to their overall and their relative expression in embryo and adults. We describe the distribution of EST in cells, tissues and cell lines with a focus on non-RBC tissues.

Telomere length has been linked to disease stage and degree of (pan-)cytopenia in patients with bone marrow failure syndromes. The aim of the current study was to analyze the impact of replicative stress on telomere length in residual glycosylphosphatidylinositol-positive (GPI+) versus GPI– hematopoiesis in patients with paroxysmal nocturnal hemoglobinuria (PNH). Peripheral blood granulocytes from 16 patients and 22 healthy individuals were analyzed. For this purpose, we developed proaerolysin flow-FISH, a novel methodology that combines proaerolysin staining (for GPI expression) with flow-FISH (for telomere length measurement). We found significantly shortened telomeres in GPI– granulocytes (mean ± SE: 6.26 ± 0.27 telomere fluorescence units [TFU]), both compared with their GPI+ counterparts (6.88 ± 0.38 TFU; P = .03) as well as with age-matched healthy individuals (7.73 ± 0.23 TFU; P < .001). Our findings are in support of a selective growth advantage model of PNH assuming that damage to the GPI+ hematopoietic stem-cell (HSC) compartment leads to compensatory hyperproliferation of residual GPI–HSCs.

Arsenic trioxide (As2O3) has a long history of use in medicine. However, it was almost forgotten in Western medicine in the recent centuries. Prompted by reports from China about successful treatment of acute promyelocytic leukemia (APL) with As2O3, there was again increasing interest in this drug in the 1990s. This review summarizes the considerable knowledge about the mechanisms of action of As2O3 that was gained during the last 5-10 years. It is focused in particular on the effects of As2O3 in non-APL cells. Since As2O3 seems to induce apoptosis and inhibits growth in a large variety of cellular targets, it might become an alternative or adjunct drug to conventional chemotherapy. As2O3 can even be effective in cells resistant to conventional cytostatic agents. Insight into the cellular mechanisms, in particular the impact of the redox state on sensitivity towards As2O3 opens the possibility to enhance As2O3 effects by appropriate combination therapies.

Objective. Mitochondrial membrane potential (Δψ m ) and intracellular Ca 2+ play a crucial role in growth and differentiation in hemopoiesis. Some potassium channel openers such as diazoxide have the capacity to elevate cytosolic Ca 2+ and depolarize mitochondria in cardiomyocytes. To clarify if such substances have effects on hemopoietic cells we investigated the commonly used opener of the mitoK ATP channel, diazoxide, and the opener of BK channels, NS1619, for their potential to depolarize mitochondria, elevate cytosolic Ca 2+ , and induce apoptosis in the hemopoietic CD34 + cell line KG-1a. Methods. Fluorescent probes were used to investigate Δψ m , free Ca 2+ , and apoptosis (JC-1, fluo-3-AM and annexin V–FITC) by flow cytometry. To measure Δψ m with JC-1 in glycoprotein P + cells we used an improved dye loading technique with verapamil. Results. NS1619 induced stronger dose-dependent mitochondrial depolarizations than diazoxide. Depolarization was independent from caspase activation and could also be induced when the driving force for K + out of cells was near 0 mV. In Ca 2+ free solutions NS1619 induced stronger Ca 2+ elevations than diazoxide and elevated Ca 2+ also after Ca 2+ depletion of the endoplasmatic reticulum with caffeine. NS1619 did not enhance the Ca 2+ elevation induced by ionophores (CCCP, valinomycin) that depolarize mitochondria. Both agents were weak inducers of apoptosis. Conclusion. Diazoxide has similar effects in CD34 + cells as described for muscle or nerve cells. In accordance to the single channel conductance of mitoK ATP and BK channels, NS1619 is a more potent inducer of mitochondrial depolarization than diazoxide. NS1619 releases Ca 2+ from an intracellular pool that is insensitive to caffeine but depends strongly on Δψ m .

Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment, which makes them a critical target for further therapeutic options. Treatment for acute myeloid leukemia (AML) patients became more effective, yet relapse rates are still high. Immunotherapeutic approaches might overcome the immune escape mechanisms of LSC and might be a therapeutic option. Leukemia-associated antigens (LAA) represent targets that are recognized by cytotoxic T-lymphocytes (CTL) and might be useful for specific immunotherapy. We enriched the LSC containing fraction as CD34+CD38- cells (called LSC-EF) of 20 AML patients using FACS, analyzed expression patterns of LAA in LSC-EF and compared them to patterns in enriched HSC und bulk AML cells by microarray expression analysis and qRT-PCR. In immunoassays, we analysed the functional inhibition of LCS in colony forming assays (CFU) of AML patients by stimulated T cells. We detected a high expression of several LAA in LSC-EF. PRAME (p = 0.0085), RHAMM (p = 0.03), WT1 (p = 0.04) and Proteinase 3 (p = 0.04) showed significant differential expression in LSC-EF compared to HSC. Survivin (p = 0.7), HAGE (p = 0.5), SSXIP2 (p = 0.4) and Aurorakinase A (p = 0.7) did not indicate a significant differential expression. BAGE does not seem to be an ideal candidate as it is significantly higher expressed on normal HSC (p = 0.0056). Further, we evaluated the expression differences of LSC-EF compared to leukemic bulk. PRAME, RHAMM and WT1 qualified as interesting potential targets as they are lower expressed on leukemic bulk than on LSC-EF. In contrast, Proteinase 3 indicates a higher expression on leukemic bulk but lower in LSC-EF. qRT-PCR confirmed our array data. In immunoassays, T cells stimulated against LAA indicated a significant inhibition of CFUs in AML patient samples. PRAME, RHAMM and WT1 showed good immunogenic responses with CTL recognition rates in a range of 58-83%. PRAME hereby proved as most effective using two different patient samples, with a reduction of 83% colonies (p = 0.004). WT1 shows a significant reduction of 74% (p = 0.05) and RHAMM of 58% colonies (p = 0.2). Taken together, several LAA are significantly expressed on the LSC-EF, whereas PRAME, RHAMM and WT1 seemed most promising as these LAA are differentially expressed compared to normal stem cells and stimulation of cytotoxic T cells against these LAAs inhibit LSC in CFU assays. Disclosures No relevant conflicts of interest to declare.

Acquired somatic mutations of the PIG-A gene lead to deficient expression of glycosyl-phosphatidyl-inositol-anchored proteins (GPI-AP) by haematopoietic cells and play a causative role in the pathogenesis of paroxysmal nocturnal haemoglobinuria (PNH). However, PIG-A mutations do not explain how the defective PNH clone can expand. It was hypothesized that a selection process conferring a relative advantage to the GPI-AP-deficient population is required. Since GPI-AP-deficient cells are also detectable in a substantial proportion of patients with otherwise typical aplastic anaemia (AA), the mechanisms inducing bone marrow failure might selectively spare the GPI-deficient cells. In order to examine the growth characteristics of GPI-AP-deficient cells in more detail, we performed repeated analyses of GPI-AP expression on peripheral blood cells in 41 patients with AA. We observed four patterns of the course of GPI-AP-deficient populations: (1) 13 patients showed normal expression of GPI-AP in the first analysis and in at least two follow-up studies at a median time of 709 days after the first analysis. (2) Secondary evolution of a GPI-AP-deficient population was a rare event. Only 4 patients with initially normal GPI-AP expression developed a GPI-AP-deficient population during follow up after immunosuppressive treatment. (3) Persistence of GPI-AP-deficient cells was observed in 16 patients during a median follow-up time of 774 days. However, in some patients, the size of the GPI-AP-deficient population increased substantially. (4) Disappearance of a GPI-AP-deficient population was observed in 8 patients. The time course of GPI-AP expression in relation to the treatment suggests that therapeutic interventions might modulate the ratio of normal versus GPI-AP-deficient haematopoiesis. Overall, these data argue against an ‘absolute growth advantage’ of GPI-AP-deficient cells. Our data are consistent with the hypothesis that haematopoietic failure caused by damage to normal haematopoiesis allows the outgrowth of a GPI-AP-deficient population. Thus, in at least some patients GPI-AP-deficient cells might pre-exist at a very low percentage and replace haematopoiesis after an insult to the normal cells.

Treatment of acute myeloid leukemia (AML) patients became more effective, yet the relapse rate is still high and cure rate still low. Different therapeutic approaches, based on molecular targeting are offering new treatment strategies using markers like FLT3, NPM1, cKIT and DNMT3A. Many others are currently under investigation. Leukemic stem cells (LSC) might be the source for leukemic disease self-renewal and account for disease relapse after treatment. Therefore LSC represent a critical target for further therapeutic options. AML with mutated nucleophosmin (NPM1) gained attention because of its distinctive molecular, clinical, and prognostic features. NPM1 mutation is also an interesting target for specific immunotherapy (Greiner et al., Blood 2012), however LSC of this new entity have not been completely characterized. Differentially expressed genes might influence molecular and immunological relevant pathways which could have an impact on the better overall survival of the mutated patients. In this study, we were interested in expression differences of the LSC enriched cell fraction descendent of NPM1wt and NPM1mut primary AML patients. We suggest that expression differences between the patients groups could be a factor for the better overall survival of NPM1mut patients. In addition we aimed to study new targets on NPM1mut LSC for new therapeutical purposes. We enriched the CD34+CD38- fraction of primary AML peripheral blood mononuclear cells (PBMC), by Fluorescence-activated cell sorting (FACS) and Magnetic-activated cell sorting (MACS), comparing both methods in efficiency and feasibility. We chose FACS for further enrichment assays, as it resulted in a mean purity of 92% vs 88% using MACS, and a higher cell number after sorting (9% more) in the first pilot trials. We sorted 21 AML patient samples; 12 NPM1wt and 9 NPM1mut. We also enriched healthy donor samples for HSC purification using FACS, in order to compare expression levels. We showed that enriched CD34+CD38- cells in NPM1mut AML samples harbor cytoplasmic NPM1 via immunocytochemical staining, indicating that these cells belong to the leukemic clone. The cell number and RNA quality was sufficient for further Microarray studies in which we analysed the CD34+CD38- enriched compartments descendent from NPM1mut and NPM1wt patients. Those showed significant differences in gene expression patterns which noticeably are immunologically coined, for example: immunoglobulin superfamily, member 10 (p = 0.0003405; fold change: 6.22) and the interleukin 12 receptor, beta 1 (p = 0.000834, fold change: 1.87). This impression was confirmed by pathway analysis indicating deregulation of pathways like the NO2-dependent IL 12 Pathway in NK cells and the Th1/Th2 Differentiation and the Platelet Amyloid Precursor Protein Pathway. Functional assays, confirming the biological importance of these factors have been performed for example an assay to test the effect of IL12 on AML cellines. Furthermore we screened our data for new potential therapeutic target structures, specifically on enriched CD34+CD38- cells of NPM1mut patients, comparing the expression level of target genes to NPM1wt CD34+CD38- cells, and the expression level on HSC. Amongst others, our most promising candidates are SERPINA1 (p = 0.0062344, fold change: 14.32), OSCAR (p = 6.11E-05, fold change: 9.03) and several further interesting genes. These genes could be used in order to target CD34+CD38- cells of NPM1mut patients in a therapeutic manner. Taken together, we successfully enriched the CD34+CD38- fraction of NPM1mut and NPM1wt AML patients and performed Microarray analysis, unraveling gene expression differences between the two patients groups, which seem to be of an immunological nature. This could be a factor, amongst others, for the better overall survival of the NPM1mut patient group. Furthermore we described new potential targets structures on CD34+CD38- cells for NPM1mut patient’s treatment. Disclosures: No relevant conflicts of interest to declare.

Bone is one of the most frequently transplanted tissues, with allografts and autografts accounting for more than 80% of total grafts. Synthetic biomaterials in association with autologous or allogeneic mesenchymal stromal cells (MSCs) represent a valid alternative in orthopaedic and maxillofacial surgery. Aim of REBORNE consortium is to perform clinical trials using standardized protocols based on advanced biomaterials and MSCs. Aim of our Unit was to assess MSC immune modulatory properties in presence of a novel biomaterial consisting of hydroxyapatite and tricalcium-phosphate (HA/TCP, Biomatlante, France) as scaffold for MSC delivery. We assessed immune modulatory properties, in terms of immunophenotype, inhibitory and anti-apoptotic effects, of MSCs of different origin when associated with the HA/TCP biomaterial, including bone marrow-derived mesenchymal stromal cells (BM-MSCs), adipose-derived MSCs (ASCs) and cord blood-derived MSCs (CB-MSCs). The culture of all MSCs with HA/TCP, did not modulate the anti-apoptotic and suppressive features of all MSCs toward sorted-T, B and NK lymphocytes as compared to standard culture setting. When exposed to inflammatory cytokines, such as IFN-γ and TNF-α, all MSCs were induced to acquire the suppressive phenotype, characterized by MHC-1 and CAM molecules up-regulation, and de novo expression of HLA-DR molecules. The long-term culture of BM-MSCs with HA/TCP induced an osteoblast-like phenotype, as indicated by the up-regulation of osterix and osteocalcin transcription. The differentiation process is promoted in all MSC types, but especially in BM-MSCs, by dexamethasone and, to a higher extent, BMP-4 treatment. In view of using MSCs for advanced therapies in allogeneic setting, we evaluated the immunogenicity and immune modulatory features of pre-differentiated MSCs in association with HA/TCP towards both innate and adaptive immune cells, as well as the molecular pathways involved in MSC-mediated immune regulation. MSC-derived osteoblasts did not induce immune cell activation, as demonstrated by co-culture of unstimulated immune effector cells with pre-differentiated MSCs. We found a lower suppressive capability of pre-differentiated MSCs towards stimulated T and NK cells. However, some inhibitory effects could be exerted by BMP-4 treated-MSCs, and could be related to the presence of undifferentiated MSCs in culture, which could inhibit lymphocyte proliferation. The balance between the number of bone depositing osteocyte-like MSCs and immunosuppressive undifferentiated MSCs could be useful, in vivo, to promote bone healing and to reduce local inflammation, respectively. We then investigated in pre-differentiated MSCs the molecular mechanisms involved in the inhibition of T cell proliferation, by interfering with IDO and COX-2 activation. We found that pre-differentiated MSCs switched on their suppressive machinery by activating both IDO, which plays a central role in immune regulation, and COX-2, whose role is not significant in undifferentiated MSCs. COX-2 is produced rapidly as inflammation mediator after fracture, and recent data highlighted the role of COX-2 in improving fracture healing. In conclusion, this study increases the knowledge on MSC biology and gives pre-clinical data concerning the use of allogeneic MSC in combination with ceramic scaffolds to treat bone defects. Disclosures: No relevant conflicts of interest to declare.

Background Mesenchymal stromal cells (MSC) have gained importance in tissue repair, tissue engineering and in immunosupressive therapy during the last years. Due to the limited availability of MSC in the bone marrow, ex vivo amplification prior to clinical application is requisite to obtain therapeutic applicable cell doses. Translation of preclinical into clinical-grade large-scale MSC expansion necessitates precise definition and standardization of all procedural parameters including cell seeding density, culture medium and cultivation devices. While xenogeneic additives such as fetal calf serum are still widely used for cell culture, its use in the clinical context is associated with many risks, such as prion and viral transmission or adverse immunological reactions against xenogeneic components. Methods and Findings We established animal-free expansion protocols using platelet lysate as medium supplement and thereby could confirm its safety and feasibility for large-scale MSC isolation and expansion. Five different GMP-compliant standardized protocols designed for the safe, reliable, efficient and economical isolation and expansion of MSC was performed and MSC obtained were analyzed for differentiation capacity by qPCR and histochemistry. Expression of standard MSC markers as defined by the International Society for Cellular Therapy as well as expression of additional MSC markers and of various chemokine and cytokine receptors was analysed by flow cytometry. Changes of metabolic markers and cytokines in the medium were addressed using the LUMINEX platform. Conclusions The five different systems for isolation and expansion of MSC described in this study are all suitable to produce at least 100 millions of MSC, which is commonly regarded as a single clinical dose. Final products are equal according to the minimal criteria for MSC defined by the ISCT. We showed that chemokine and integrin receptors analyzed had the same expression pattern, suggesting that MSC from either of the systems show equal characteristics of homing and adhesion.

Abstract 3671 Poster Board III-607 For effective elimination of malignant cells by specific T cells a co-activation of CD4- and CD8-positive T cells might be important. We performed two RHAMM-R3 peptide vaccination trials using 300μg and 1000μg for patients with AML, MDS and multiple myeloma overexpressing RHAMM. Similar mild toxicity of both cohorts was found, only mild drug-related adverse events were observed such as erythema and induration of the skin. In the 300μg cohort we detected in 7/10 (70 %) patients specific immune responses and also positive clinical effects in 5/10 (50 %) patients. In the high dose peptide vaccination trial (1000μg peptide) 4/9 (44 %) patients showed positive immune responses. These patients showed an increase of CD8+RHAMM-R3 tetramer+/CD45RA+/CCR7-/CD27-/CD28- effector T cells and an increase of R3-specific CD8+ T cells. In the higher peptide dose cohort three patients showed positive clinical effects. However, higher doses of peptide do not improve the frequency and intensity of immune responses in this clinical trial and might induce immune tolerance. In this work, we investigated the co-existence of serological immune responses against RHAMM detected by a RHAMM-specific ELISA of patients with AML, MDS and multiple myeloma treated in these two peptide vaccination trials. We correlated these results to specific T cell responses of CD8-positive T cells measured by ELISpot assays for interferon gamma and Granzyme B, tetramer staining and chromium release assays. Moreover, these results were compared to the frequency of regulatory T cells. 4/19 patients have a positive serological immune response in ELISA assay, all of these patients developed also strong specific CD8-positive T cell responses during peptide vaccination detected by ELISpot assays and tetramer staining. As expected, peptide vaccination did not result in the induction of humoral immune responses. In further ELISA assays we measured IL-2 and IL-10 levels in the sera of the patients before and three weeks after four vaccinations. While IL-10 levels remained at a rather low level over the time of vaccination, we detected an increase of IL-2 up to the five-fold of the initial levels in four of ten patients. Moreover, we performed a proteome array to detect cytokine and chemokine regulation in sera of patients vaccinated in these two trials during and after RHAMM-R3 peptide vaccination. 36 cytokines, chemokines and acute phase proteins were measured and both cohorts vaccinated with different peptide doses were compared. Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Co-existence of immune responses of CD4-positive T cells against the target RHAMM seems to be important for an induction of strong immune responses of CD8-positive T cells. Disclosures: No relevant conflicts of interest to declare.

Introduction: Eculizumab is a humanized monoclonal antibody inhibiting the cleavage of complement factor C5 and preventing terminal complement activation. Therapy with eculizumab leads to a highly significant reduction of complement-mediated intravascular hemolysis and related morbidities including thromboembolic events in Paroxysmal Nocturnal Hemoglobinuria (PNH). It is a new targeted, disease-modifying treatment of PNH. In a placebo-controlled, double-blind randomized study of eculizumab more than 50% of patients (pts) achieved transfusion independence with the remaining pts showing a 44% reduction in the number of red blood cell (RBC) units transfused. In this study we analyzed the proportion of cells with deficiency of GPI-anchored proteins, i.e. PNH cell populations, during eculizumab therapy. Furthermore, we examined a potential mechanism to explain residual hemolysis in some pts despite complete blockade of the terminal complement cascade. Methods: We performed flow cytometric analysis of GPI-anchored proteins, measured levels of hemolysis (LDH), direct bilirubin, hemoglobin and ferritin, and carried out a direct antiglobulin test in samples from 15 (7 female, 8 male) PNH pts during eculizumab therapy. To identify PNH cells, flow cytometric analyses of CD58/CD59 on reticulocytes and erythrocytes, CD66b/CD24/CD16 on granulocytes as well as CD14/CD48 on monocytes were performed. For comparison, the direct antiglobulin test was also performed in 22 (11 female, 11 male) PNH pts without eculizumab therapy. Results: Flow cytometric results of the 15 PNH pts before eculizumab (baseline) and at the time of latest follow-up during eculizumab were compared: GPI-deficient erythrocyte population doubled (29.3% +/−19.3% vs. 56.9% +/−24.0%). The proportion of GPI-deficient reticulocytes (79.1% +/−6.8% vs. 84.6% +/−8.7%), granulocytes (87.5% +/−9.7% vs. 85.5% +/−12.4%) and monocytes (79.0% +/−20.6% versus 73.6%+/−16.2%) did not change significantly. We observed a mean decrease in LDH from 2802 U/l +/− 1015 U/l to 240 U/l +/− 62 U/l; while total bilirubin was stable (47.2 +/− 33.3 μmol/l versus 45.0 +/− 27.8 μmol/l), direct bilirubin showed a threefold increase (3.2 +/− 3.2 μmol/l versus 9.6 +/− 2.4 μmol/l). An increase of ferritin levels was also observed (270 +/− 609 ng/ml versus 651 +/− 652 ng/ml). We performed a direct antiglobulin test in the control group of 22 pts not receiving eculizumab therapy and in 12 pts that received eculizumab. 10 of the 12 pts were tested before and during eculizumab therapy. Only three of all 34 pt who had not received eculizumab at time of antiglobulin test showed a positive antiglobulin test with polyspecific antiglobulin serum, two of them with monospecific anti-IgG and one of them was positive with monospecific anti-C3d. In contrast, all but one of the 12 pts during eculizumab therapy reacted positive with polyspecific antiglobulin serum and monospecific anti-IgG and anti-C3d; in addition, one pt was positive with anti-C3c. The only patient with a negative antiglobulin test during eculizumab therapy was treated for PNH-related thromboembolic events without having transfusion dependent hemolytic anemia. Two of the 12 pts had anti-erythrocytic allo-antibodies (anti-D, -C, -E, -Lua, and -Kpa) before initiation of eculizumab therapy and one of them had additional warm auto-antibodies. Conclusions: 1) A positive direct antiglobulin test (due to C3d loading of PNH RBC) seems to be a consistent finding during eculizumab therapy, which has therefore to be taken into account as a new differential diagnosis of a positive direct antiglobulin test. 2) Eculizumab does not block the early stages of complement activation and therefore C3 cleavage products may occur and bind to CD55/CD59-deficient RBC. The inhibition of terminal membrane attack complex formation by eculizumab allows the survival of these cells which otherwise would undergo a rapid intravascular hemolysis due to C5b-9 formation. 3) The endogenous population of GPI–anchored protein deficient RBC increases during eculizumab therapy. 4) Our data are preliminary and need to be confirmed in larger patient groups: For this purpose, we recommend the monitoring of signs of extravascular hemolysis and ferritin during eculizumab therapy.

Background: Cytomegalovirus (CMV) disease constitutes a serious complication after allogeneic peripheral blood stem cell transplantation (allo-PBSCT). For the clearance of CMV, CD8+ T cells are pivotal. Patients after allo-PBSCT with recurrent CMV reactivation usually lack such CMV specific T cells. Conventional antiviral therapy of CMV reactivation characteristically results in myelosuppression and further suppression of CMV specific T cells. Adoptive transfer of CMV specific T cells may help to overcome this problem. A novel technology designated “streptamers” allows the selection of CMVpp65 specific CD8+ T cell up to 98% purity without altering the functional properties of the selected T cells and without requiring cumbersome and time consuming T cell cultures. Materials and Methods: Here, the novel streptamer technology was used for adoptive transfer of CMV specific T cells into two acute leukemia patients with recurrent high CMV antigenemia after allo-PBSCT. Standard peripheral blood mononuclear cell apheresis was performed on the former stem cell donors of two patients with acute leukemia. Isolation of CMV specific donor lymphocytes was performed using a Good Manufacturing Product (GMP)-grade Streptamer selection kit on a CliniMacs™ device. Briefly, MHC-Streptamers (CMVpp65/HLA-B7 for patient 1; CMVpp65/HLA-A2 for patient 2) were labeled with beads overnight to obtain MHC-streptamer-bead complexes. Subsequently CMV specific T-lymphocytes were immunomagnetically labeled by incubating mononuclear cells with MHC-Streptamer-bead complexes. Cells were run on a CliniMacs™ device. The positive fraction was then incubated with biotin to detach the steptamers from the T cells. Results: A single specific donor lymphocyte infusion (sDLI) of 0.4 or 2.2 ×105 CMVpp65 specific T cells per kg body weight was performed in an AML or ALL patient respectively, after allogeneic PBSCT developing a CMVpp65 antigenemia with a maximum of 959 or 716 CMVpp65 positive/500,000 cells and treatment with foscarnet, ganciclovir and valganciclovir. After sDLI, the CMV antigenemia was cleared and remained persistently controlled even after discontinuation of valganciclovir therapy in both patients. No acute or chronic toxic side effect, particularly no aggravation of graft-versus-host disease (GvHD) was observed. A strong and sustained increase of the absolute count of CMV-specific CD8+ T cells in concordance with the increase of CD3+CD8+ T cells up to 440/μl was detected. CMV-specific CD8+ T cells showed no significant expression of CCR7, CD62L or CD107, but stained increasingly positive for CD45RA, indicating a preferential effector T cell phenotype. Results from stimulation experiments of CD3+ T cells with HLA-B7 versus HLA-A2 restricted CMVpp65 derived peptides demonstrate late reconstitution of HLA-A2-restricted CMV-specific T cells, whereas the adoptively transferred HLA-B7-restricted CMV-specific T-cell response augmented very early und was maintained over time. The chimerism analysis of the in vivo expanded CMV-specific CD8+ T cells demonstrated a 100% donor chimerism. T cell receptor excision circle (sjTRECs) analysis revealed a frequency of sjTRECs two logs lower than expected, indicating peripheral expansion rather than thymic proliferation of CMV specific CD8+ T cells. cDNA generated from FACS-purified donor-derived CMV B7 pp65-specific CD8+ T cells was probed with the indicated 5′ Vß14-specific and 3′ CDR3-specific primers for the presence of clonotypic T cells. The respective CDR3 region sequence was identical for both donor T cells and CMVpp65 specific T cells in the patients at different time points after the adoptive T cell transfer, thus clearly indicating that the expanded CMV specific T cell were of clonogenic donor origin. Conclusion: Streptamer technology offers the advantage of selecting CMV specific CD8+ T cells at GMP level for adoptive T cell transfer. Two CMVpp65 specific T cell transfers resulted in a marked increase of CMV-specific CD8+ T cells and induced long-lasting CD8+ T cell responses, which allowed the patients to discontinue toxic antiviral drug therapy without further high level reactivation of CMV.

There is an accumulation of in vivo (graft-versus-leukemia effect) and in vitro (spontaneous remissions after infections) data providing evidence that CLL might be effectively targeted by T-cell based immunotherapy. Earlier, we characterized the receptor for hyaluronic acid mediated motility (RHAMM) as antigen associated with proliferation and negative prognosis in CLL. We also demonstrated that RHAMM-derived epitope(R3)- primed T cells were able to lyse RHAMM+ target CLL cells. Therefore, we initiated a small phase I/II clinical trial with R3 peptide vaccination for patients with CLL. Six CLL patients in Binet stage 0 of the disease were vaccinated four times at a biweekly interval with HLA-A2 restricted RHAMM-derived epitope R3 (ILSLELMKL, 300μg/dose on day 3) emulsified in incomplete Freund’s adjuvant (IFA) with concomitant administration of GM-CSF (100μg/dose, days 1–5). R3-specific T-cell responses were assessed by tetramer staining and ELISPOT assays. T-cell subsets which play a role in regulation of immune responses including CD3+CD4+CD25hiCD127loFOXP3+ T regs, Th17, CD8+CD137+, CD8+CD103+ and IL-17 producing CD8+ T cells (CD8+IL-17+) were evaluated by flow cytometry. No severe adverse events greater than CTC Io skin toxicity could be observed. Four of six patients showed a reduction of WBC during vaccination. Although these WBC changes did not meet the NCI response criteria, we described these favorable hematological changes achieved in short period of immunotherapy as hematological improvement (defined as at least 20% reduction of WBC during vaccination). The immune responses were found in 5/6 patients as assessed by tetramer-staining (positive response defined as an increase of R3-specific CD8+ T cell frequency by more than 100% after vaccination) and confirmed in 4/5 as assessed by ELISPOT assay. Patients included in this study showed median Tregs frequency of 4.2%, range: 2.5–8%. There was no significant difference of Tregs percentages between patients who improved clinically when compared with non-responders (median 6.1% vs. 3.7%). Vaccination induced Tregs in 4 patients (2 non-responders and 2 responders). Two other patients who improved hematologically did not significantly change frequency of Tregs or even reduced it during vaccination (Figure 1). Median expression of CD103 on CD8+ T cells was 1.84%, range: 0.41–5.63%. In one non-responder, we observed an increase in frequency of CD103+CD8+ T-cells during vaccination from 1.46% to 2.56%. During vaccination, changes in CD8+CD103+ T cell subset did not correlate with the frequency of Tregs, nonetheless we could find an inverse correlation with inflammatory Th17 T cells (r2=−0.5, p In conclusion, peptide vaccination in CLL patients is safe and feasible to mount immune responses against the tumor antigen RHAMM. Most of patients benefited hematologically from vaccination. Although in some patients we observed an induction of tumor-specific T cells without induction of Tregs there is a rationale to add novel active agents against Tregs in future vaccination trials. Figure 1. Peptide vaccination induced changes in WBC, percentages of regulatory T cells (Tregs) as well as R3 specific tetramer ‘CD’ T cells (tetra) of A CLL patients. Patients B, C, E and F improved hematologically during vaccination. Figure 1. Peptide vaccination induced changes in WBC, percentages of regulatory T cells (Tregs) as well as R3 specific tetramer ‘CD’ T cells (tetra) of A CLL patients. Patients B, C, E and F improved hematologically during vaccination.

We have demonstrated immunological responses and positive clinical effects of a peptide vaccination for patients with AML, MDS, MM and CLL with a limited tumor load or a minimal residual disease over-expressing RHAMM using 300 μg RHAMM-R3 peptide (Schmitt et al., Blood 2008; Giannopoulos et al., abstract submitted). To date, 26 patients were enrolled in this clinical peptide vaccination trial. Here, we report on the second cohort of nine patients with AML, MDS and MM vaccinated with a higher peptide dose (1000 μg RHAMM-R3 peptide). The vaccine was given four times at a biweekly interval and GM-CSF was added for five days each vaccination. Similar to the patients vaccinated with 300 μg peptide only mild drug-related adverse events were observed such as erythema and induration of the skin. Immunomonitoring was performed using ELISpot assays for Interferon gamma and Granzyme B, tetramer-based flow cytometry and chromium release assays. Moreover, the frequency of regulatory T cells was quantified at different time points of vaccination. In this second cohort of patients treated with 1,000 μg peptide we detected specific immune responses in a lower frequency (4/9 patients) in contrast to patients in the 300 μg cohort (7/10 patients). In these patients with immune responses we found an increase of CD8+/HLA-A2/RHAMM-R3 tetramer+/CD45RA+/CCR7−/CD27−/CD28− effector T cells in flow cytometry in accordance with an increase of R3-specific CD8+ T cells in ELISpot assays. Two patients with positive immune responses showed a significant decrease of regulatory T cells. One patient without positive immune and clinical effects showed an increase of the frequency of regulatory T cells (5.03% to 15.9%). Three out of nine patients treated with 1,000 μg showed positive clinical effects: One patient with MDS RAEB-2 showed a reduction of leukemic blasts in the bone morrow to lower than 5%, one MDS patient achieved a normalization of the peripheral blood counts and one patient with multiple myeloma experienced a reduction of light chain in serum. The patients in the 300 μg cohort showed also a higher frequency of positive clinical effects (5 out of 10 patients). Taken together, RHAMM-R3 peptide vaccination induced both immunological and clinical responses. Therefore, RHAMM constitutes a promising structure for further targeted immunotherapies in patients with different hematological malignancies. However, higher doses of peptide do not improve the frequency and intensity of immune responses in this clinical trial.

Cytomegalovirus (CMV) reactivation constitutes a serious complication after allogeneic peripheral blood stem cell transplantation (PBSCT). The frequency of CMVpp65 specific CD8+ T cells is pivotal for the clearance of CMV. CMVpp65 specific CD8+ T cell frequencies can be measured using tetra-, penta- and streptamer technologies, streptamers can also be applied therapeutically. In donors, these frequencies might allow us to define the best available donor in addition to the mere serostatus. In the present study we investigated the specificity and sensitivity of all three methods and compared the results to the serostatus. A therapeutical application, i.e. an adoptive transfer of CMV specific CD8+ T cells selected by streptamer technology to a patient with acute lymphatic leukemia suffering from life-threatening CMV antigenemia after allogeneic PBSCT was performed. 23 samples from CMV seropositive healthy volunteers (HV) and 10 samples from CMV seropositive patients before and after allogeneic stem cell transplantation (all HLA-A2 or -B7 positive) were analyzed with tetra-, penta- or streptamer conjugated to PE by flow cytometry. Our lab took part in an inter-lab CMV multimer assay including 20 European countries in the framework of www.kimt.de. For the adoptive T cell transfer a donor leukapheresis was performed followed by an HLA-B7 CMVpp65 streptamer positive selection. The patient received 2×10E5 CMV specific CD8+ T cells per kg body weight as a single transfusion. Optimal amounts of HLA-A2 multimers to stain a pellet of 10E6 cells were 0.44 mcg tetramer, 0.15 mcg pentamer and 0.2 mcg MHC/0.3 mcg streptactin complex. Surprisingly, only in 48% (11/23) seropositive HV CD8+ multimer+ T cells could be detected. The ALL patient developed a foscarvir resistant CMV antigenemia with a maximum of 959/500,000 CMVpp65 positive cells. After a switch to ganciclovir/valganciclovir and an adoptive transfer of CMV specific T cells, the antigenemia was cleared. Valganciclovir was discontinued, but CMV antigenemia remained controlled. The frequency of CMVpp65 specific CD8+ T cells increased dramatically from 0.0% till 19.8%. All of these T cells were donor derived as demonstrated by small tandem repeat (STR) analysis. The patient did not develop signs of CMV disease at any time point. This study demonstrates the power of multimer staining to define appropriate donors for transplantation. Donors should be screened for their CMVpp65 specific CD8+ T cell frequency. All three multimer technologies can be used yielding similar results. The streptamer technology additionally offers the advantage to select CMVpp65 specific CD8+ T cells at the GMP level for adoptive T cell transfer and can induce long-lasting CD8+ T cell responses effectively clearing even a high virus load.

Background: For CML/ALL patients with refractory disease or at relapse after allogeneic stem cell transplantation (allo-PBSCT), one might administer tyrosine kinase inhibitors (TKIs) like imatinib (Glivec), nilotinib (Tasigna) and dasatinib (Sprycel), or donor lymphocyte infusions (DLIs). TKIs will inhibit the proliferation of CML progenitor cells, but might also hamper the graft-versus-leukemia (GVL) effect considered to be crucial for the eradication of the disease. Moreover, CD8+ T cells specific for cytomegalovirus (CMVpp65) might be impaired. Methods: Imatinib, nilotinib and dasatinib were added at concentrations of 0–20 μM, 0–4 μM and 0–50 nM respectively to proliferation assays of Tregs and CD8+ T cells. Mixed lymphocyte peptide cultures (MLPCs) were performed with peptides derived from influenza matrix protein (IMP), CMVpp65 as a viral antigen and the receptor for hyaluronic acid mediated motility (RHAMM-R3) as a leukemia-associated antigen. CD8+ T cells from these MLPCs from healthy donors and patients with CML after allo-PSCT by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays, as well as Tregs after 3 days of culture with anti-CD3 and anti-CD28. Activity of T cells from the peripheral blood of patients under dasatinib and after withdrawal of dasatinib. Western blots (WBs) for T cell receptor (TCR) related molecules and NFkB were performed. Results: The release of interferon gamma and granzyme B by CD8+ HLA-A2/tetramer+effector T cells specific for IMP, CMVpp65 and RHAMM-R3 was inhibited by all TKIs in a dose-dependent fashion and correlating to the time of TKI exposure. The inhibition was reversible after removal of the drugs from the MLPC. The proliferation and function of Tregs were also significantly inhibited by TKIs in a dose-related fashion. In WBs, TKIs decreased the expression of ZAP70, Lck and Akt, as well as NFκB p65/p100/p105 and c-Rel. T cell activity was impaired when TKIs were clinically administered. The potency of T cell inhibition was imatinib: nilotinib: dasatinib = 1:2:40 under therapeutical serum respectively culture medium levels (2 μM:1 μM: 25 nM). Conclusion: When administering TKIs to patients after allo-PBSCT, the inhibition of both CD8+ and Tregs must be taken into consideration with respect to GVL and anti-viral T cell response. Inhibition of CD8+ T lymphocytes was partially reversible after removal of TKIs from the cultures. Using western blotting analysis, we found that these effects are mediated at least in part by down-regulating the levels of phosphorylation of TCR and NF-κB family members.

Most patients with AML, MDS, MM and chronic lymphatic leukemia (CLL) express the receptor for hyaluronic acid mediated motility (RHAMM/CD168) on their tumor cells. We characterized RHAMM/CD168 as a leukemia-associated antigen (LAA) eliciting both humoral and cellular immune responses in patients with different hematological malignancies. CD8 positive T cells primed with the RHAMM/CD168-derived peptide R3 (ILSLELMKL) were able to lyse autologous AML blasts expressing this LAA. Therefore, we initiated a phase I/II R3 peptide vaccination to induce immunological and hematological responses for patients with AML, MDS or MM overexpressing RHAMM/CD168. Patients were included with positive RHAMM/CD168 expression but with a limited tumor load. At a biweekly interval, RHAMM R3 peptide (300 mcg for the first 12 patients and 1000 mcg for patients 13–24) emulsified with the incomplete Freund’s adjuvant (day 3) and GM-CSF (100 mcg, days 1–5) was administrated four times subcutaneously. The primary aim of the study is safety and feasibility of this peptide vaccination, secondary aims the evaluation of a specific T cell immune response to RHAMM/CD168 R3 peptide and the assessment of the influence of the R3 peptide vaccination on the remission status. Since January 2005, 14 patients have been enrolled in the study. The first ten patients (2 AML, 4 MDS, 4 MM) have completed the course of four vaccinations and have been completely evaluated. Therapy related adverse events observed under R3-peptide vaccination were erythema and induration of the skin at the site of injection (CTC I°). In 5/10 patients, we detected in the peripheral blood a significant increase of specific CD8+ T cells recognizing the R3 peptide in ELISPOT analysis and seven-color flow cytometry including tetramer staining. As expected, after vaccination with the HLA class I peptide R3 no significant increase of IgG titers against the antigen RHAMM could be detected. Clinical responses have been assessed by the examination of peripheral blood and bone-marrow samples before and after vaccination. Patients showed a reduction of the tumor-specific expressed antigen RHAMM/CD168 in real-time RT-PCR analysis after vaccination. 3/6 patients with myeloid disorders (1 AML, 2 MDS/RAEB1) showed a reduction of CD33+ cells in FACS analysis of the bone-marrow after four vaccinations from 10 and 7 % to 1–2 and

The method of gene transfer into progenitor cells is critical as viral vector transduction involves the risk of tumor induction by non-specific genomic integration. Non-viral transfection systems often fail due to low transfection efficiency. However, gene transfer into human CD34+ hematopoietic progenitor (HPC) and mesenchymal stem cells (MSC) is an essential tool for in vitro- and in vivo-applications and therapeutic strategies such as tissue engineering and gene therapy. We recently reported an transient genetic labelling of human CD34+ HPC with deltaLNGFR-plasmid-DNA for in vivo application: Transient transfection was efficient for both, CD34+ HSC (41% ± 2%) and leukemia cell lines (55% ± 4.9%) using the method of nucleofection. Moreover, mature myeloid cells (CD66b+) derived from transfected human CD34+ HPC and leukemia cells maintained deltaLNGFR expression at a high percentage (70% ± 1.6% and 58% ± 2% respectively). In this work, we investigated labelling of CD34+ HPC with mRNA. Human CD34+ HPC and human MSC were transfected with in vitro-transcribed mRNA for deltaLNGFR, a marker gene approved for human in vivo-application, using nucleofection. EGFP was used as a control. 24h after nucleofection, FACS-analysis showed a higher transfection efficiency compared to plasmid transfected CD34+ HPC and MSC: A high transfection frequency was found for mRNA-transfected HPCs using deltaLNGFR (82.4±9.7%) and EGFP (88.7±2.6%). We found also a high transfection rate for MSC using the marker genes deltaLNGFR (92.4±3.6%) and EGFP (83.3±4.1%). Cell viability was not affected by mRNA-transfection. Moreover, differentiation assays of deltaLNGFR-selected MSC after transfection, showed that differentiation of MSC into mesenchymal cells like chondrocytes, adipocytes and osteoblasts was not affected by mRNA nucleofection. Taken together, mRNA based nucleofection is a powerful, highly efficient and non-toxic approach for transient labelling of human progenitor cells or, via transfection of selective proteins, for transient manipulation of stem cell function. It may thus be useful to transiently manipulate stem cell characteristics and combine principles of gene therapy and tissue engineering.

Imatinib mesylate (IM, STI571, Gleevec®) is highly effective in the treatment of patients with chronic myelogenous leukemia (CML). In patients with residual disease or relapse after allogeneic stem cell transplantation (allo-SCT), the optimal strategy is a matter of debate: donor lymphocyte infusions (DLIs) alone, IM alone or a combination of both have been administered to CML patients in this clinical situation. As an impairment of anti-viral CD8+ T lymphocyte function by IM has been described, we questioned whether IM also affects specific anti-leukemic CD8+ T lymphocytes. Moreover, we asked to which extent CD4+CD25hi T regulatory cells might be affected by IM thus changing the balance between cytotoxic and tolerogenic T cells. After mixed lymphocyte peptide culture (MLPC), we assessed CD8+ T cells from healthy donors and patients with CML after allo-SCT by tetramer staining/multi-color flow cytometry and enzyme linked immunosorbent spot (ELISPOT) assays, as well as CD4+CD25hicells after 3 days of culture with anti-CD3 and anti-CD28. The release of interferon gamma and granzyme B by CD8+ T lymphocytes specific for R3 (ILSLELMKL), a T cell epitope peptide derived from the leukemia-associated antigen designated RHAMM/CD168 (receptor for hyaluronic acid mediated motility) was inhibited by IM in a clearly dose-dependent fashion in a range from 1 to 25 μM. These T cells were further characterized as CD8+ HLA-A2/R3_tetramer+ WT1_tetra-CD45RA+CD27-CD28-CCR7- effector T cells with the ability to lyse R3 peptide labeled T2 cells and CD34+ CML progenitor cells. The inhibition of CD8+ T lymphocytes was a function of the time of exposure to IM and reversible after removal of IM from the MLPC. On the other hand, the proliferation and function of CD4+CD25hiFoxP3+GITR+TGFß1+ CD69+CD152+ T regulatory cells were also significantly inhibited by IM in a dose-related fashion. These findings suggest that the clinical impact IM must not necessarily result in a reduced efficacy of the graft-versus-leukemia effect observed after allo-SCT and/or DLIs but might depend rather on the ratio of CD8+ cytotoxic T cells and CD4+CD25hi T regulatory cells.

Objective: Replication-dependent telomere shortening can result in cellular senescence or genetic instability. Telomere length both reflects and limits the replicative potential of normal somatic cells. Shortened telomeres have been linked to disease stage, degree of cytopenia as well as to response to treatment in patients with bone marrow failure syndromes. Paroxysmal nocturnal haemoglobinuria (PNH) is caused by a somatic mutation in the X-linked PIG-A gene resulting in a deficiency of GPI-linked proteins on the cell surface. Pancytopenia in PNH is thought to result from an auto-immune mediated depletion of the GPI+ stem cell compartment. In the current study, we aimed to develop a methodology that allows the selective analysis of telomere length in granulocytes from patients with PNH dependent on GPI expression. The aim was to find out whether replicative stress posed on the residual GPI− HSC is reflected in shortened telomere length compared to their GPI+ counterparts. Methods: The telomere length of GPI+ and GPI− granulocytes of 12 patients with PNH and 22 healthy individuals was analysed. For this purpose, we developed Proearolysine Multi-color flow FISH which is based on the crosslinking of a fluorescence-labeled non-toxic derivative of the bacterial toxine Aerolysine (that selectively binds to the GPI anchor of cells) in combination with fluorescence in situ hybridization and flow cytometry. Results: We found significantly (p Conclusion: Autoimmune-mediated damage to the GPI+ HSC compartment leads to compensatory hyperproliferation of predominantly residual GPI− HSC. In line with this hypothesis, coexisting GPI-negative HSC clones showed even more accelerated telomere shortening as compared to their GPI+ counterparts. Potentially, replicative exhaustion of individual HSC clones could contribute to disease progression in PNH. Whether acquired genetic instability due to progressive telomere shortening also accounts for an increased incidence of secondary clonal disorders will need to be evaluated in future prospective studies.

Flow cytometric analysis of GPI-anchored proteins (GPI-AP) is the gold standard for diagnosis of paroxysmal nocturnal hemoglobinuria (PNH). Due to therapy options and the relevance of GPI-deficient clones for prognosis in aplastic anaemia detection of PNH is gaining importance. However, no generally accepted standard has been established. This study analysed the usefulness of a flow cytometric panel with CD58, CD59 on reticulocytes and erythrocytes, CD24/CD66b and CD16, FLAER on granulocytes and CD14, and CD48 on monocytes. Actual cut-off (mean + 2 SD) for GPI-deficient cells was established in healthy blood donors. We studied 1,296 flow cytometric results of 803 patients. Serial monitoring was analysed during a median follow-up of 1,039 days in 155 patients. Of all, 22% and 48% of 155 follow-up patients. showed significant GPI-AP-deficiency at time of initial analyses. During follow-up in 9%, a new PNH diagnosis, and in 28%, a significant change of size or lineage involvement was demonstrated. Highly significant correlations for GPI-AP deficiency were found within one cell lineage (r2 = 0.61–0.95, p

Abstract 337 Background: MSC cells can differentiate into different tissues and exhibit non-HLA-restricted immunosuppressive properties. They are promising candidates for cellular therapy. Therapeutic use requires large-scale GMP-grade expansion of MSC. Several protocols have been published. Here we systematically compare different expansion procedures with particular emphasis on role of cytokines/chemokines in the expansion medium. Materials and Methods: Bone marrow (BM) was obtained by aspiration from the iliac crest of healthy donors after informed consent and IRB approval. BM aspirate (anticoagulated with heparin (500 U/ml)) was incubated without manipulation in 5-chamber stacks (CellStacks; Corning) in a medium free of animal components (α-MEM (Lonza) with 10% human platelet lysate (hPL)). In the single-step protocol 1.2×104 MNC/ cm2 were seeded. Non-adherent cells were washed off after 72–96 hrs. Partial medium exchange (40%) was performed twice a week (wk). After 11 days MSC were harvested by incubation with recombinant trypsin (TrypZean, Lonza). In the two-step protocol 5×104 leukocytes/cm2 were seeded in 2-chamber stacks. Non-adherent cells were removed after 72–96 hours and complete medium exchange was performed twice/wk. Cells were harvested after 10 days and the harvest was seeded in a 2nd culture at a density of 0.4×104 MSC/cm2. This 2nd culture was harvested after 5 days. Cytokines/chemokines in hPL and in culture medium during the course of expansion was measured by Milliplex MAP Kit (Millipore Corp). Surface marker expression was measured on FACSAria and FACScan. Results: Higher number of MSC could be achieved in cultures with hPL compared to fetal calf serum. hPL was equally effective in supporting MSC proliferation if prepared from apheresis platelet concentrates (PC), buffy coat-derived pooled PC in plasma or pooled PC in additive solution. hPL contained large amounts of PDGF-AB/BB (790 ng/ml; mean of 3 batches of hPL from buffy coat-derived pooled PC), PDGF-AA (266 ng/ml), RANTES (2706 ng/ml), sCD40L (27 ng/ml), GRO (11 ng/ml), sVCAM (2511 ng/ml), sICAM (188 ng/ml). During culture, sCD40L declined rapidly to very low levels. Concentration of PDGF-AA, RANTES and sICAM remained almost stable. In contrast, PDGF-AB/BB declined to low levels ( BM samples from healthy donors (n=4) were split in order to perform paired comparison of single-step vs. two-step expansion protocol. In the single-step protocol 16.3×103±5.8×103 MSC/μl BM seeded were harvested after 11±0 days. In the two-step protocol 12.0×103±4.4×103 MSC/μl BM were harvested after 10 days at the end of passage 0 and 104.0×103±60.4×103 after 5±1 days at the end of passage 1. The overall consumption of medium in the single-step protocol was substantially higher than in the two-step protocol. Phenotype of MSC from the two culture systems did not significantly differ regarding standard markers (positive for CD73, CD90, CD105, HLA-class I; neg. for CD45, CD3, CD34, HLA-DR). However, in passaging experiments we could demonstrate that proportion of MSC positive for CD49a, CD71, CCR4/CD194, CD349 and MSCA-1 decreased whereas proportion of cells positive for c-kit/CD117, CCR3/CD193, CXCR4 and CD200 increased. Conclusion: hPL-based system allows efficient expansion of MSC up to a total number >1×109 MSC from a 15 ml BM aspirate in 2–3 wks with only one passaging step. hPL is a rich source of cytokines, some of which (PDGF-AB/BB) seem to be consumed during expansion and arrive at very low concentrations at the end of the expansion culture. A two-step system provides higher number of MSC per BM cells seeded and requires less medium/culture vessels. Phenotype and differentiation capacity does not differ between single- or two-step culture. However, further passaging goes along with substantial changes of the phenotype. Previous conflicting results regarding chemokine expression of MSC might be due to differences in ex-vivo culture period. Given that chemokine receptor expression affects in-vivo behaviour of cells, MSC harvested after initial expansion (passage 0 or 1) substantially differ from older cells, emphasizing the need to highly standardize all parameters of expansion. (Supported by EU 7th Framework Programme, Projects CASCADE and REBORNE). Disclosures: Fekete: Institute of Clinical Transfusion Medicine and Immunogenetics: Employment. Fuerst:Institute of Clinical Transfusion Medicine and Immunogenetics: Employment. Schrezenmeier:Institute of Clinical Transfusion Medicine and Immunogenetics: Employment.