Eva Mischak-Weissinger

Lenalidomide maintenance after allogeneic HSCT seems to trigger acute graft- versus -host disease in patients with high-risk myelodysplastic syndromes or acute myeloid leukemia and del(5q): results of the LENAMAINT trial

We read with interest the recent article by Mollgard et al. [1][1] concerning the efficacy of single agent lenalidomide (LEN) in patients with chromosome 5 abnormalities and advanced myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). In general, allogeneic stem cell transplantation (

Tetramer monitoring to assess risk factors for recurrent cytomegalovirus reactivation and reconstitution of antiviral immunity post allogeneic hematopoietic stem cell transplantation

S. Borchers, S. Luther, U. Lips, N. Hahn, J. Kontsendorn, M. Stadler, S. Buchholz, H. Diedrich, M. Eder, U. Koehl, A. Ganser, E. Mischak‐ Weissinger. Tetramer monitoring to assess risk factors for recurrent cytomegalovirus reactivation and reconstitution of antiviral immunity post allogeneic hematopoietic stem cell transplantation.
Transpl Infect Dis 2011: 13: 222–236. All rights reserved

Regulation of advanced therapy medicinal products in Europe and the role of academia

BACKGROUND AIMS Advanced therapy medicinal products (ATMP) are gene therapy, somatic cell therapy or tissue-engineered products regulated under (EC) No. 1394/2007 to ensure their free movement within the European Union while guaranteeing the highest level of health protection for patients. Academic good manufacturing practice (GMP) centers are major contributors in the development of ATMPs and this study assessed the impact of regulations on them. METHODS European academic and non-industrial facilities (n = 747) were contacted, and a representative sample of 50 replied to a detailed questionnaire. Experienced centres were further selected in every Member State (MS) for semi-structured interviews. Indicators of ATMP production and development success were statistically assessed, and opinions about directive implementation were documented. RESULTS Facilities experienced in manufacturing cell therapy transplant products are the most successful in developing ATMPs. New centres lacking this background struggle to enter the field, and there remains a shortage of facilities in academia participating in translational research. This is compounded by heterogeneous implementation of the regulations across MS. CONCLUSIONS GMP facilities successfully developing ATMPs are present in all MS. However, the implementation of regulations is heterogeneous between MS, with substantial differences in the definition of ATMPs and in the approved manufacturing environment. The cost of GMP compliance is underestimated by research funding bodies. This is detrimental to development of new ATMPs and commercialization of any that are successful in early clinical trials. Academic GMP practitioners should strengthen their political visibility and contribute to the development of functional and effective European Union legislation in this field.

Elevated frequencies of leukemic myeloid and plasmacytoid dendritic cells in acute myeloid leukemia with the FLT3 internal tandem duplication

Some 30% of acute myeloid leukemia (AML) patients display an internal tandem duplication (ITD) mutation in the FMS-like tyrosine kinase 3 (FLT3) gene. FLT3-ITDs are known to drive hematopoietic stem cells towards FLT3 ligand independent growth, but the effects on dendritic cell (DC) differentiation during leukemogenesis are not clear. We compared the frequency of cells with immunophenotype of myeloid DC (mDC: Lin−, HLA-DR+, CD11c+, CD86+) and plasmacytoid DC (pDC: Lin−, HLA-DR+, CD123+, CD86+) in diagnostic samples of 47 FLT3-ITD− and 40 FLT3-ITD+ AML patients. The majority of ITD+ AML samples showed high frequencies of mDCs or pDCs, with significantly decreased HLA-DR expression compared with DCs detectable in ITD− AML samples. Interestingly, mDCs and pDCs sorted out from ITD+ AML samples contained the ITD insert revealing their leukemic origin and, upon ex vivo culture with cytokines, they acquired DC morphology. Notably, mDC/pDCs were detectable concurrently with single lineage mDCs and pDCs in all ITD+ AML (n = 11) and ITD− AML (n = 12) samples analyzed for mixed lineage DCs (Lin−, HLA-DR+, CD11c+, CD123+). ITD+ AML mDCs/pDCs could be only partially activated with CD40L and CpG for production of IFN-α, TNF-α, and IL-1α, which may affect the anti-leukemia immune surveillance in the course of disease progression.

Activation of Innate and Adaptive Immunity by a Recombinant Human Cytomegalovirus Strain Expressing an NKG2D Ligand

Development of an effective vaccine against human cytomegalovirus (HCMV) is a need of utmost medical importance. Generally, it is believed that a live attenuated vaccine would best provide protective immunity against this tenacious pathogen. Here, we propose a strategy for an HCMV vaccine that aims at the simultaneous activation of innate and adaptive immune responses. An HCMV strain expressing the host ligand ULBP2 for the NKG2D receptor was found to be susceptible to control by natural killer (NK) cells, and preserved the ability to stimulate HCMV-specific T cells. Infection with the ULBP2-expressing HCMV strain caused diminished cell surface levels of MHC class I molecules. While expression of the NKG2D ligand increased the cytolytic activity of NK cells, NKG2D engagement in CD8+ T cells provided co-stimulation and compensated for lower MHC class I expression. Altogether, our data indicate that triggering of both arms of the immune system is a promising approach applicable to the generation of a live attenuated HCMV vaccine.

Characterization of human cytomegalovirus genome diversity in immunocompromised hosts by whole genomic sequencing directly from clinical specimens.

Background Advances in next-generation sequencing (NGS) technologies allow comprehensive studies of genetic diversity over the entire genome of human cytomegalovirus (HCMV), a significant pathogen for immunocompromised individuals. Methods Next-generation sequencing was performed on target enriched sequence libraries prepared directly from a variety of clinical specimens (blood, urine, breast milk, respiratory samples, biopsies, and vitreous humor) obtained longitudinally or from different anatomical compartments from 20 HCMV-infected patients (renal transplant recipients, stem cell transplant recipients, and congenitally infected children). Results De novo-assembled HCMV genome sequences were obtained for 57 of 68 sequenced samples. Analysis of longitudinal or compartmental HCMV diversity revealed various patterns: no major differences were detected among longitudinal, intraindividual blood samples from 9 of 15 patients and in most of the patients with compartmental samples, whereas a switch of the major HCMV population was observed in 6 individuals with sequential blood samples and upon compartmental analysis of 1 patient with HCMV retinitis. Variant analysis revealed additional aspects of minor virus population dynamics and antiviral-resistance mutations. Conclusions In immunosuppressed patients, HCMV can remain relatively stable or undergo drastic genomic changes that are suggestive of the emergence of minor resident strains or de novo infection.

Cytomegalovirus-Specific T Cells Isolated by IFN-γ Secretion Assay Do Not Induce Significant Graft-Versus-Host Reactions In Vitro.

Background Graft-versus-host (GvH) disease (GvHD) remains a serious concern for patients undergoing antiviral cellular therapy. Despite the major improvements in cellular immunotherapy, the immunogenicity of virus-specific T cells has not yet been fully defined. This present study aims to examine how cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTLs) respond to allogeneic antigen stimulation and whether they give rise to GvHD target tissue damage. Methods Cytomegalovirus-specific CTLs were isolated by the IFN-&ggr; secretion assay (gamma-catch) from healthy seropositive volunteers and expanded in vitro. The levels of intracellular IFN-&ggr;, cytotoxic activity, IFN-&ggr; and granzyme B secretion, and CD25 expression were measured using flow cytometry (fluorescence-activated cell sorting). The ability of CMV-CTLs to induce GvHD target tissue damage was evaluated using the human in vitro skin explant assay (skin explant assay). Results Cytomegalovirus-specific CTLs responded specifically to CMV-phosphoprotein 65 stimulation by secreting IFN-&ggr; and killing virus peptide loaded autologous phytohemagglutinin (PHA) blasts. Compared with unselected peripheral blood mononuclear cells, CMV-CTLs induced significantly less severe cutaneous GvH tissue damage. This observation coincided with low levels of CD25 expression, as well as IFN-&ggr; and granzyme B secretion after allogeneic antigen stimulation in both the mixed lymphocyte reaction and in the skin explant assay. Conclusions Cytomegalovirus-specific CTLs isolated by the IFN-&ggr; secretion assay from HLA-unmatched healthy donors exhibited a high level of anti-CMV potency without inducing significant cutaneous GvH tissue damage in vitro. This finding provides novel evidence supporting the safe use of in vitro expanded CMV-CTLs as an antiviral therapy in transplant patients with refractory CMV infections.

Human CD8+ CD57- TEMRA cells: Too young to be called "old"

End-stage differentiation of antigen-specific T-cells may precede loss of immune responses against e.g. viral infections after allogeneic stem cell transplantation (SCT). Antigen-specific CD8+ T-cells detected by HLA/peptide multimers largely comprise CD45RA-/CCR7- effector memory (TEM) and CD45RA+/CCR7- TEMRA subsets. A majority of terminally differentiated T-cells is considered to be part of the heterogeneous TEMRA subset. The senescence marker CD57 has been functionally described in memory T-cells mainly composed of central memory (TCM) and TEM cells. However, its role specifically in TEMRA cells remained undefined. Here, we investigated the relevance of CD57 to separate human CD8+ TEMRA cells into functionally distinct subsets. CD57- CD8+ TEMRA cells isolated from healthy donors had considerably longer telomeres and showed significantly more BrdU uptake and IFN-γ release upon stimulation compared to the CD57+ counterpart. Cytomegalovirus (CMV) specific T-cells isolated from patients after allogeneic SCT were purified into CD57+ and CD57- TEMRA subsets. CMV specific CD57- TEMRA cells had longer telomeres and a considerably higher CMV peptide sensitivity in BrdU uptake and IFN-γ release assays compared to CD57+ TEMRA cells. In contrast, CD57+ and CD57- TEMRA cells showed comparable peptide specific cytotoxicity. Finally, CD57- CD8+ TEMRA cells partially changed phenotypically into TEM cells and gained CD57 expression, while CD57+ CD8+ TEMRA cells hardly changed phenotypically and showed considerable cell death after in vitro stimulation. To the best of our knowledge, these data show for the first time that CD57 separates CD8+ TEMRA cells into a terminally differentiated CD57+ population and a so far functionally undescribed “young” CD57- TEMRA subset with high proliferative capacity and differentiation plasticity.

Proteomic screening applied to the diagnosis of chronic graft-versus-host disease

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is a curative treatment for many hematologic malignancies or hematopoietic dysfunction syndromes, but the application is still limited due to major complications, such as severe graft versus host disease (GvHD) and infectious complications. Diagnosis chronic GvHD is based on clinical features and biopsies, a non invasive, unbiased laboratory test does not exist. We used the urine collected from 20 patients (10 with limited cGvHD, 10 with extensive cGvHD) to establish a proteomic pattern that allowed the diagnosis of cGvHD development and tested the resulting set of polypeptide markers (27 differentially excreted peptides) on more than 200 patients prospectively and blinded for the correct classification of cGvHD samples. The majority of the patients included were transplanted for hematological malignancies (n=209), 6 for hematopoietic failure syndromes. Conditioning regimens included dose reduced conditioning regimens (FLAMSA and ClaraC for the majority of the patients of MHH), as well as standard conditioning regimens (TBI+Cy or Busulfan+Cy) for about 35% of the patients, with GvHDprophylaxis including cyclosporine A and mycophenolate (MMF) or metothrexate (MTX) as appropriate. Eighty percent of the patients received ATG (antithymocyte globulin) prior to HSCT. A peptide pattern of 27 peptides, differentiating chronic from acute GvHD was developed. Controls were patients at least 100 days post HSCT, with no GvHD in the history, no infections and without relapse at the time of sampling. Prospective and blinded evaluation of the patients revealed the correct classification of patients developing cGvHD with a sensitivity of 85% and specificity of 95% .Further evaluation of the cGvHD patterns specific for particular organ manifestations of cGvHD are currently ongoing. Interestingly, the cGvHD pattern seems to be predictive for GvHD developing post DLI, while the aGvHD-specific proteomic pattern only predicts GvHD of the intestine, which may be more similar to “late acute GvHD”. Figure 1: On-line coupling of capillary electrophoresis and mass spectrometer