Specific T-cell immune responses against colony-forming cells including leukemic progenitor cells of AML patients were increased by immune checkpoint inhibition.

The efficacy of immunotherapies in cancer treatment becomes more and more apparent not only in different solid tumors but also in hematological malignancies. However, in acute myeloid leukemia (AML), mechanisms to increase the efficacy of immunotherapeutic approaches have to be further elucidated. Targeting leukemic progenitor and stem cells (LPC/LSC) by specific CTL, for instance, in an adjuvant setting or in minimal residual disease, might be an option to prevent relapse of AML or to treat MRD. Therefore, we investigated the influence of immune checkpoint inhibitors on LAA-specific immune responses by CTL against leukemic myeloid blasts and colony-forming cells including leukemic progenitor cells (CFC/LPC). In functional immunoassays like CFU/CFI (colony-forming units/immunoassays) and ELISpot analysis, we detected specific LAA-directed immune responses against CFC/LPC that are postulated to be the source population of relapse of the disease. The addition of nivolumab (anti-PD-1) significantly increases LAA-directed immune responses against CFC/LPC, no effect is seen when ipilimumab (anti-CTLA-4) is added. The combination of ipilimumab and nivolumab does not improve the effect compared to nivolumab alone. The anti-PD1-directed immune response correlates to PD-L1 expression on progenitor cells. Our data suggest that immunotherapeutic approaches have the potential to target malignant CFC/LPC and anti-PD-1 antibodies could be an immunotherapeutic approach in AML. Moreover, combination with LAA-directed vaccination strategies might also open interesting application possibilities.

Donor lymphocyte infusion leads to diversity of specific T cell responses and reduces regulatory T cell frequency in clinical responders.

T cell responses against malignant cells play a major role in maintaining remission and prolonging overall survival in patients after allogeneic stem cell transplantation and donor lymphocyte infusion (DLI) due to graft-versus-leukemia effect. For better characterization of the T cell responses, we assessed frequency and diversity of leukemia-associated antigen (LAA)-specific cytotoxic T cells using ELISpot and pMHC multimer assays and analyzed the frequency of regulatory T cells (Treg) as well as cytokine profiles before/after DLI. The data were correlated to the clinical course of patients. Significantly more LAA-derived T cell epitopes (p = 0.02) were recognized in clinical responders (R) when compared to nonresponders (NR). In addition, pMHC multimer-based flow cytometry showed a significantly higher frequency of LAA-specific T cells in R versus NR. The frequency of Treg in R decreased significantly (p = 0.008) while keeping stable in NR. No differences in T cell subset analysis before/after DLI were revealed. Clinical responders were correlated to specific immune responses and all clinical responders showed an increase of specific immune responses after DLI. Cytokine assays using enzyme-linked immunosorbent assay showed a significant increase of IL-4 after DLI. Taken together, an increase of specific CTL responses against several LAA after DLI was detected. Moreover, this study suggests that enhanced LAA diversity in T cell responses as well as decreasing numbers of Treg contribute to clinical outcome of patients treated with DLI.

Translation of a standardized manufacturing protocol for mesenchymal stromal cells: A systematic comparison of validation and manufacturing data.

BACKGROUND: Many data are available on expansion protocols for mesenchymal stromal cells (MSCs) for both experimental settings and manufacturing for clinical trials. However, there is a lack of information on translation of established protocols for Good Manufacturing Practice (GMP) from validation to manufacturing for clinical application. We present the validation and translation of a standardized pre-clinical protocol for isolation and expansion of MSCs for a clinical trial for reconstitution of alveolar bone. METHODS: Key parameters of 22 large-scale expansions of MSCs from bone marrow (BM) for validation were compared with 11 expansions manufactured for the clinical trial "Jaw bone reconstruction using a combination of autologous mesenchymal stromal cells and biomaterial prior to dental implant placement (MAXILLO1)" aimed at reconstruction of alveolar bone. RESULTS: Despite variations of the starting material, the robust protocol led to stable performance characteristics of expanded MSCs. Manufacturing of the autologous advanced therapy medicinal product MAXILLO-1-MSC was possible, requiring 21 days for each product. Transport of BM aspirates and MSCs within 24 h was guaranteed. MSCs fulfilled quality criteria requested by the national competent authority. In one case, the delivered MSCs developed a mosaic in chromosomal finding, showing no abnormality in differentiation capacity, growth behavior or surface marker expression during long-term culture. The proportion of cells with the mosaic decreased in long-term culture and cells stopped growth after 38.4 population doublings. CONCLUSIONS: Clinical use of freshly prepared MSCs, manufactured according to a standardized and validated protocol, is feasible for bone regeneration, even if there was a long local distance between manufacturing center and clinical site. Several parameters, such as colony forming units fibroblasts (CFU-F), percentage of CD34+ cells, cell count of mononuclear cells (MNCs) and white blood cells (WBCs), of the BM may serve as a predictive tool for the yield of MSCs and may help to avoid unnecessary costs for MSC manufacturing due to insufficient cell expansion rates.

Gender, cholinesterase, platelet count and red cell count are main predictors of peripheral blood stem cell mobilization in healthy donors.

BACKGROUND AND OBJECTIVES: Mobilization of CD34+ cells by stimulation with G-CSF shows considerable variation across stem cell donors. Upfront prediction of CD34+ cell counts in peripheral blood based on easily available steady-state parameters would be helpful for the planning of apheresis and stem cell transplantation. Commonly accepted steady-state predictors for the mobilization are gender, body mass index and platelet count. The aim of the study was the identification of novel predictors that might influence mobilization efficacy and to create a model for the prediction of stem cell mobilization. METHODS: A total of 333 healthy stem cell donors who donated peripheral stem cells in our institution were retrospectively analysed. All available data before stem cell mobilization with G-CSF were included in the database. Primary end-point was CD34+ cell count before the first apheresis. RESULTS: In this cohort cholinesterase, differential blood cell counts including platelets, gender and body mass index were significantly correlated with CD34+ cell count. G-CSF dose per lean body weight showed a significant correlation with mobilization efficacy in women but not in men. A multivariate analysis identified gender, cholinesterase and platelet and red cell count as main predictors of mobilization. Red cell count showed a strong gender dependence, with higher predictive value in females. CONCLUSION: The counts of eosinophils, platelets, red cells, cholinesterase and gender are the most important predictors of CD34+ cell mobilization in our deduced models. The red cell count as a predictor for mobilization showed a differential gender dependence.

Healthy donor hematopoietic stem cell mobilization with biosimilar granulocyte-colony-stimulating factor: safety, efficacy, and graft performance.

BACKGROUND: Biosimilar granulocyte-colony-stimulating factors (G-CSFs) have been available in the European Union since 2008, and Sandoz' biosimilar filgrastim was approved in the United States in March 2015 for all of the reference product's indications except acute radiation syndrome. Biosimilar G-CSFs have been largely embraced by the medical community, except for some reservations about healthy-donor stem cell mobilization, for which use outside of clinical studies was cautioned against by some members of the scientific community. STUDY DESIGN AND METHODS: In a two-center safety surveillance study (National Clinical Trial NCT01766934), 245 healthy volunteer stem cell donors were enrolled. Of 244 donors who began mobilization with twice-daily Sandoz biosimilar filgrastim, 242 received a full (n = 241) or partial (n = 1) course of G-CSF and underwent apheresis. Efficacy and safety were assessed and are reported here. RESULTS: Biosimilar filgrastim was accompanied by the typical G-CSF class-related adverse effects of expected frequency and severity. Median mobilization for CD34-positive stem cells was 97/µL (range, 20-347/µL); after one apheresis (91%) or two aphereses (9%) from all but three donors (1.2%), cell doses in excess of the typical 4 × 106 CD34-positive cells/kg of the recipient had been collected (range, 3-52 × 106 /kg). Biochemical and hematologic alterations were consistent with previous reports; all had normalized by the first follow-up 1 month after mobilization. Stem cell products engrafted with typical probability and kinetics for G-CSF-mobilized stem cell products. CONCLUSION: These data support the use of biosimilar filgrastim for healthy-donor stem cell mobilization as safe and effective.

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.

Sex and body mass index but not CXCL12 801 G/A polymorphism determine the efficacy of hematopoietic cell mobilization: a study in healthy volunteer donors.

Analyses of healthy donors of granulocyte colony-stimulating factor (G-CSF) mobilized hematopoietic stem and progenitor cells (HSPCs) and of patients undergoing autologous stem cell transplantation have suggested that individuals harboring the CXCL12-A allele mobilize a higher number of CD34 + HSPCs after G-CSF administration. We typed 463 healthy unrelated donors (376 men and 87 women) who had received daily subcutaneous injections at a mean dose of 7.36 ± 1.71 µg/kg G-CSF for 5 days for CXCL12 801 G/A using a real-time PCR assay. Interestingly, the median concentration of mobilized CD34 + cells on day 5 was almost identical in donors with the A-allele (79/µL; range, 11 to 249/µL) and the G/G-group (82/µL; range, 15 to 268/µL). In addition, the allelic distribution was not different in donors (n = 11) who mobilized less than 20/µL CD34 + cells. No difference in the overall yield of CD34 + cells in the apheresis product and in the number of CD34 + cells/kg recipient could be detected between both groups. In a multivariate regression model for the endpoint CD34 + cells/µL at day 5, only male sex (regression coefficient, 11.5; 95% confidence interval, 1.7 to 21.2, P = .021) and body mass index as continuous variables (regression coefficient, 3.5; 95% confidence interval, 2.5 to 4.5, P = .0001) but not age, smoking status, or CXCL12 allelic status represented independent variables. Our data derived from a large well-controlled cohort contradict previous analyses suggesting an association between CXCL12 allelic status and the yield of CD34 + HSPC after G-CSF mobilization. Concentration of CD34 + cells in the peripheral blood, the most objective parameter, could not be predicted by CXCL12 genotype.

Donor Deferral Rates after the Implementation of a New German Blood Donor Questionnaire.

BACKGROUND: The implementation of a new national German blood donor questionnaire was proposed to improve donor and recipient safety. METHODS: We compared deferral/exclusion rates of whole blood donors before (May 2010, n = 64,735) and after (May 2011, n = 71,687) the implementation of a new blood donor questionnaire. Considering seasonal variations, analysis was performed with respect to collection site (mobile vs. fixed), sex, donor status (first-time vs. repeat), age, and the frequencies of sexual risk behavior and other reasons for deferral. RESULTS: We observed a statistically significant increase (p < 0.001) of the overall deferral/exclusion rate from 6.2 to 8.1%, irrespective of type of collection site (fixed: from 6.0 to 8.5%; mobile: from 6.2 to 8.0%), sex (females: from 7.5 to 9.9%; males: from 5.1 to 6.6%), donor status (first-time donors: from 19.7 to 24.7%; repeat donors: from 4.6 to 6.3%) or age (18-29 years: from 9.1 to 11.7%; 60-71 years: from 5.1 to 6.6%). Confidential self-exclusion increased from 0.08 to 0.14% (p < 0.001). Besides risk behavior, various medical reasons could be identified that explain this increase. CONCLUSIONS: The new blood donor questionnaire resulted in an increased deferral/exclusion of all donor groups. Thus the impact on future blood supply must be considered carefully, and long-term studies and investigation of donor acceptance will be needed.

Results of a multicenter phase I/II trial of TCRαß and CD19-depleted haploidentical hematopoietic stem cell transplantation for adult and pediatric patients.

Hematopoietic stem cell transplantation (HSCT) from haploidentical donors is a viable option for patients lacking HLA-matched donors. Here we report the results of a prospective multicenter phase I/II trial of transplantation of TCRαß and CD19-depleted peripheral blood stem cells from haploidentical family donors after a reduced-intensity conditioning with fludarabine, thiotepa, and melphalan. Thirty pediatric and 30 adult patients with acute leukemia (n = 43), myelodysplastic or myeloproliferative syndrome (n = 6), multiple myeloma (n = 1), solid tumors (n = 6), and non-malignant disorders (n = 4) were enrolled. TCR αß/CD19-depleted grafts prepared decentrally at six manufacturing sites contained a median of 12.1 × 106 CD34+ cells/kg and 14.2 × 103 TCRαß+ T-cells/kg. None of the patients developed grade lll/IV acute graft-versus-host disease (GVHD) and only six patients (10%) had grade II acute GVHD. With a median follow-up of 733 days 36/60 patients are alive. The cumulative incidence of non-relapse mortality at day 100, 1 and 2 years after HSCT was 5%, 15%, and 17% for all patients, respectively. Estimated probabilities of overall and disease-free survival at 2 years were 63% and 50%, respectively. Based on these promising results in a high-risk patient cohort, haploidentical HSCT using TCRαß/CD19-depleted grafts represents a viable treatment option.

Automatic interface-controlled apheresis collection of stem/progenitor cells: results from an autologous donor validation trial of a novel stem cell apheresis device.

BACKGROUND: Cryopreserved hematopoietic progenitor cells collected by apheresis from granulocyte-colony-stimulating factor with or without chemotherapy-mobilized patients have become the preferred type of autograft to support treatment of diseases amenable to high-dose chemotherapy. A novel apheresis system, the Spectra Optia v.5.0 (CaridianBCT), was constructed to meet certain shortcomings of manual apheresis systems such as the COBE Spectra MNC (CaridianBCT), including the need for continuous optical or manual monitoring and readjustment of buffy coat position and sensitivity to inconsistent blood flow. By use of optical sensors, which provide real-time automatic interface (buffy coat) and collection line control, the Spectra Optia promises to automatically guide apheresis procedures, potentially freeing up operator time and reducing variability in collection efficiency (CE2). STUDY DESIGN AND METHODS: In a two-center clinical trial, 35 autologous stem cell donors were subjected to apheresis with the Spectra Optia to validate feasibility and effectiveness of apheresis procedures. Results were compared to data from 80 autologous apheresis procedures with the COBE Spectra MNC. RESULTS: Usability and function of the automatic interface management were excellent. CD34+ cell quality, assessed by viability staining, colony-forming unit-culture frequency, and engraftment kinetics, was equally good with both systems. CE2 of the Spectra Optia, calculated as CD34+ contents in the product divided by the number of CD34+ cells presented to the collection port, exceeded that of the COBE Spectra MNC. Spectra Optia product volumes were significantly smaller. Very high white blood cell and platelet counts modestly reduced CE2 with the Spectra Optia. CONCLUSION: The Spectra Optia is a novel automatic apheresis system supporting autologous stem cell collection with at least equal efficiency and superior user-friendliness compared to the COBE Spectra MNC.

Adoptive transfer and selective reconstitution of streptamer-selected cytomegalovirus-specific CD8+ T cells leads to virus clearance in patients after allogeneic peripheral blood stem cell 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) Vß-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.

Role of Donor Clonal Hematopoiesis in Allogeneic Hematopoietic Stem-Cell Transplantation.

PURPOSE: Clonal hematopoiesis of indeterminate potential (CHIP) occurs in the blood of approximately 20% of older persons. CHIP is linked to an increased risk of hematologic malignancies and of all-cause mortality; thus, the eligibility of stem-cell donors with CHIP is questionable. We comprehensively investigated how donor CHIP affects outcome of allogeneic hematopoietic stem-cell transplantation (HSCT). METHODS: We collected blood samples from 500 healthy, related HSCT donors (age ≥ 55 years) at the time of stem-cell donation for targeted sequencing with a 66-gene panel. The effect of donor CHIP was assessed on recipient outcomes, including graft-versus-host disease (GVHD), cumulative incidence of relapse/progression (CIR/P), and overall survival (OS). RESULTS: A total of 92 clonal mutations with a median variant allele frequency of 5.9% were identified in 80 (16.0%) of 500 donors. CHIP prevalence was higher in donors related to patients with myeloid compared with lymphoid malignancies (19.2% v 6.3%; P ≤ .001). In recipients allografted with donor CHIP, we found a high cumulative incidence of chronic GVHD (cGVHD; hazard ratio [HR], 1.73; 95% CI, 1.21 to 2.49; P = .003) and lower CIR/P (univariate: HR, 0.62; 95% CI, 0.40 to 0.97; P = .027; multivariate: HR, 0.63; 95% CI, 0.41 to 0.98; P = .042) but no effect on nonrelapse mortality. Serial quantification of 25 mutations showed engraftment of 24 of 25 clones and disproportionate expansion in half of them. Donor-cell leukemia was observed in two recipients. OS was not affected by donor CHIP status (HR, 0.88; 95% CI, 0.65 to 1.321; P = .434). CONCLUSION: Allogeneic HSCT from donors with CHIP seems safe and results in similar survival in the setting of older, related donors. Future studies in younger and unrelated donors are warranted to extend these results. Confirmatory studies and mechanistic experiments are warranted to challenge the hypothesis that donor CHIP might foster cGVHD development and reduce relapse/progression risk.

Multimer staining of cytomegalovirus phosphoprotein 65-specific T cells for diagnosis and therapeutic purposes: a comparative study.

BACKGROUND: Cytomegalovirus (CMV) disease represents a serious complication after allogeneic peripheral blood stem cell (PBSC) transplantation. If possible, stem cell donors for transplantation are selected on the basis of their CMV serostatus. However, the cytomegalovirus-specific immune status can be further characterized by measuring CMV phosphoprotein 65-specific CD8(+) T cell frequencies using tetramers, pentamers, and streptamers. We therefore investigated the specificity and sensitivity of all 3 methods and compared the results to patient serostatus. METHODS: Twenty-three samples from CMV-seropositive healthy volunteers and 15 samples from CMV-seropositive patients before and after allogeneic PBSC transplantation were stained with tetramers, pentamers, or streptamers and analyzed by flow cytometry. RESULTS: Similar frequencies of CD8(+) and multimer(+) T cells could be measured by all 3 multimer technologies. The lowest background signals (< or =0.02%) were obtained using tetramer technology. Frequencies of 0.19%-2.48% of CMV phosphoprotein 65 495-503-specific CD8(+) T cells were detected in healthy volunteers. Antigen-specific T cells were detected in only 11 (48%) of 23 seropositive healthy volunteers. CMV antigenemia before day 100 after allogeneic PBSC transplantation occurred in 2 of 3 patients without any specific T cells. CONCLUSION: These findings demonstrate the power of multimer staining and a certain limitation of serologic testing to define appropriate donors for transplantation. Therefore, whenever possible, CMV-seropositive donors of transplants to seropositive recipients should be screened for their CD8(+) T cell frequency. All 3 multimer technologies can be used, yielding similar results. The streptamer technology additionally offers the advantage of selecting CMV phosphoprotein 65-specific CD8(+) T cells at the good manufacturing practice level for adoptive T cell transfer.

Carboxylated superparamagnetic iron oxide particles label cells intracellularly without transfection agents.

Cell labeling by superparamagnetic iron oxide particles (SPIO) has emerged as a potentially powerful tool to monitor trafficking of transplanted cells by magnetic resonance tomography, e.g., in studies for tissue repair. However, intracellular labeling is mostly achieved by transfection agents not approved for clinical use. In this work, the feasibility and efficiency of labeling human mesenchymal stem cells (MSC) and HeLa cells with two commercially available SPIOs (Resovist and Feridex) without transfection agents was evaluated. In both cell types, Resovist without a transfection agent was more efficiently taken up than Feridex. Increasing the concentration of Resovist can yield similar amounts of iron in cells as SPIOs with transfection agents. This offers the opportunity to omit transfection agents from the labeling protocol when Resovist is used. Intracellular localization of the contrast agents is found by light microscopy and confirmed by electron microscopy. Coagulation of the SPIO nanoparticles, which is problematic for the quantification of the intracellular iron content, was observed and analyzed with a fluorescent activated cell sorter. As Resovist consists of a carboxydextran shell in contrast to Feridex which is composed of a dextran shell, we synthesized fluorescent polymeric nanoparticles as model systems with different amounts of carboxyl groups on the surface by the miniemulsion process. A steady increase in uptake of nanoparticles was detected with a higher density of carboxyl groups showing the relevance of charged groups as in the case of Resovist. Aggregation of these polymeric nanoparticles was not found.

Chronic myeloid leukemia cells express tumor-associated antigens eliciting specific CD8+ T-cell responses and are lacking costimulatory molecules.

Specific immunotherapies for patients with chronic myeloid leukemia (CML) might eliminate residual CML cells after therapy with imatinib or chemotherapy and might enhance a specific graft-versus-leukemia effect after allogeneic stem cell transplantation. Here, we investigated the mRNA expression and T-cell recognition of tumor-associated antigens or leukemia-associated antigens (LAAs) in 34 patients with CML. Several LAAs are expressed in CML and therefore are candidate structures for specific immunotherapies: bcr-abl (100%), G250 (24%), hTERT (53%), MPP11 (91%), NEWREN60 (94%), PRAME (62%), Proteinase3 (71%), RHAMM/CD168 (83%), and WT1 (53%), but not BAGE, MAGE-A1, SSX2, or NY-ESO-1. The frequency of mRNA expression of RHAMM/CD168, Proteinase3, and PRAME was higher in acceleration phase and blast crisis. In flow cytometry, CD34+ progenitor cells typed positive for HLA molecules but were deficient for CD40, CD80, CD83, and CD86. However, RHAMM/CD168 R3-peptide (ILSLELMKL)-specific T-cell responses in CML patients were demonstrated by ELISPOT analysis and specific lysis of RHAMM/CD168 R3-pulsed T2 cells and CD34+ CML cells in chromium-51 release assays. RHAMM-R3-specific T cells could be phenotyped as CD8+R3*tetramer+CD45RA+CCR7-CD27- early effector T cells by tetramer staining. Therefore, vaccination strategies inducing such RHAMM-R3-directed effector T cells might be a promising approach to enhance specific immune responses against CML cells.

Peptide vaccination in the presence of adjuvants in patients after hematopoietic stem cell transplantation with CD4+ T cell reconstitution elicits consistent CD8+ T cell responses.

Rationale: Patients receiving an allogeneic stem cell graft from cytomegalovirus (CMV) seronegative donors are particularly prone to CMV reactivation with a high risk of disease and mortality. Therefore we developed and manufactured a novel vaccine and initiated a clinical phase I trial with a CMV phosphoprotein 65 (CMVpp65)-derived peptide. Methods: Ten patients after allogeneic stem cell transplantation received four vaccinations at a biweekly interval. All patients were monitored for CMVpp65 antigenemia. Flow cytometry for CMV-specific CD8+ and γδ T cells as well as neutralizing anti-CMV antibodies were correlated to clinical parameters. Results: The vaccination was well tolerated. Seven of nine patients cleared CMVpp65 antigenemia after four vaccinations and are still free from antigenemia to this day. Two patients with CMV reactivation showed persisting CMV antigenemia. One patient received prophylactic vaccination and did not develop antigenemia. An increase of up to six-fold in frequency of both CMV-specific CD8+ T cells and/or Vδ2negative γδ T cells was detected. Titers of neutralizing antibodies increased up to the tenfold. Humoral and cellular immune responses correlated with clearance of CMV. Conclusion: In summary, CMVpp65 peptide vaccination for patients after allogeneic stem cell transplantation at high risk for CMV reactivation was safe, well tolerated and clinically encouraging. A study in solid-organ transplant patients is ongoing.

Caspase-8L expression protects CD34+ hematopoietic progenitor cells and leukemic cells from CD95-mediated apoptosis.

Regulation of sensitivity or resistance for apoptosis by death receptor ligand systems is a key control mechanism in the hematopoietic system. Dysfunctional or deregulated apoptosis can potentially contribute to the development of immune deficiencies, autoimmune diseases, and leukemia. Control of homeostasis starts at the level of hematopoietic stem cells (HSC). To this end, we found that CD34+ hematopoietic progenitor cells are constitutively resistant to CD95-mediated apoptosis and cannot be sensitized during short-term culture to death receptor-mediated apoptosis by cytokines. Detailed analysis of the death machinery revealed that CD34+ cells do not express caspase-8a/b, a crucial constituent of the death-inducing signaling complex (DISC) of death receptors. Instead, we found a smaller splice variant termed caspase-8L to be present in HSC. Forced expression of caspase-8L using a recombinant lentiviral vector was able to protect hematopoietic cells from death receptor-induced apoptosis even in the presence of caspase-8a/b. Furthermore, we found that caspase-8L is recruited to the DISC after CD95 triggering, thereby preventing CD95 from connecting to the caspase cascade. These results demonstrate an antiapoptotic function of caspase-8L and suggest a critical role as apoptosis regulator in HSC. Similar to CD34+ HSC, stem cell-derived leukemic blasts from AML(M0) patients only expressed caspase-8L. Additionally we found, caspase-8L expression in several AML and ALL samples. Thus, caspase-8L expression might explain constitutive resistance to CD95-mediated apoptosis in CD34+ progenitor cells and might participate in the development of stem cell-derived and other leukemias by providing protection from regulatory apoptosis.

Bone marrow transplantation nephropathy after an intensified conditioning regimen with radioimmunotherapy and allogeneic stem cell transplantation.

UNLABELLED: Intensification of the conditioning regimen with a radioactively labeled anti-CD66 antibody is feasible before allogeneic stem cell transplantation. The use of radioimmunotherapy may deliver a significant dose of radiation to the kidneys. We therefore studied the incidence and clinical picture of bone marrow transplantation (BMT) nephropathy in our patients receiving radioimmunotherapy before allogeneic stem cell transplantation. METHODS: This study was a clinical trial of 114 consecutive patients who received conditioning with a radiolabeled anti-CD66 antibody-188Re (n = 93) or 90Y (n = 21)-between 1998 and 2003. RESULTS: Although BMT nephropathy has developed in none of the patients in the [90Y]anti-CD66 group, 6 of 93 patients receiving [188Re]anti-CD66 presented with signs of BMT nephropathy at a median of 11.5 mo after stem cell transplantation. The absorbed renal dose was significantly lower in the 90Y group (4 vs. 7 Gy, P < 0.0001). Of the patients receiving [188Re]anti-CD66 who are alive, BMT nephropathy developed in 19% (6/32). Five of 6 patients with BMT nephropathy received total-body irradiation. The patients presented with elevated serum creatinine, proteinuria, anemia, hypertension, and signs of microangiopathy. All 6 patients in whom BMT nephropathy has developed are alive at a median follow-up of 58 mo after stem cell transplantation, and 1 patient has entered a dialysis program. CONCLUSION: BMT nephropathy appears to be a significant problem after allogeneic stem cell transplantation with intensified conditioning using the 188Re-labeled anti-CD66 applied in this study, particularly when combined with total-body irradiation.