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Casati A.,Miltenyi Biotec GmbH | Varghaei-Nahvi A.,U.S. National Cancer Institute | Feldman S.A.,U.S. National Cancer Institute | Assenmacher M.,Miltenyi Biotec GmbH | And 4 more authors.
Cancer Immunology, Immunotherapy | Year: 2013

The adoptive transfer of lymphocytes genetically engineered to express tumor-specific antigen receptors is a potent strategy to treat cancer patients. T lymphocyte subsets, such as naïve or central memory T cells, selected in vitro prior to genetic engineering have been extensively investigated in preclinical mouse models, where they demonstrated improved therapeutic efficacy. However, so far, this is challenging to realize in the clinical setting, since good manufacturing practices (GMP) procedures for complex cell sorting and genetic manipulation are limited. To be able to directly compare the immunological attributes and therapeutic efficacy of naïve (TN) and central memory (TCM) CD8+ T cells, we investigated clinical-scale procedures for their parallel selection and in vitro manipulation. We also evaluated currently available GMP-grade reagents for stimulation of T cell subsets, including a new type of anti-CD3/anti-CD28 nanomatrix. An optimized protocol was established for the isolation of both CD8+ TN cells (CD4-CD62L+CD45RA +) and CD8+ TCM (CD4-CD62L +CD45RA-) from a single patient. The highly enriched T cell subsets can be efficiently transduced and expanded to large cell numbers, sufficient for clinical applications and equivalent to or better than current cell and gene therapy approaches with unselected lymphocyte populations. The GMP protocols for selection of TN and TCM we reported here will be the basis for clinical trials analyzing safety, in vivo persistence and clinical efficacy in cancer patients and will help to generate a more reliable and efficacious cellular product. © 2013 Springer-Verlag Berlin Heidelberg.

Van Der Heijde D.,Leiden University | Van Der Helm-Van Mil A.H.M.,Leiden University | Aletaha D.,Medical University of Vienna | Bingham C.O.,Johns Hopkins University | And 15 more authors.
Annals of the Rheumatic Diseases | Year: 2013

The aim of this report was to propose a definition for erosive disease in the context of inflammatory arthritis in light of the 2010 American College of Rheumatology (ACR)/European League Against Rheumatism (EULAR) rheumatoid arthritis (RA) criteria for use in clinical practice and studies. A EULAR task force was formed including 16 rheumatologists and one rheumatology fellow. The process was both evidence based and consensus based, and included, between March 2010 and April 2012, analyses of data from two cohorts, two face-to-face meetings, one online voting and one teleconference. The Leiden Early Arthritis Cohort and the French ESPOIR cohort were used for the evidence-based part. The outcome measures, which were initiation of methotrexate therapy, or any disease-modifying antirheumatic drug therapy within the first year of disease and arthritis persistency over 5 years, were studied with the aim to give the best definition of erosive disease. A decision was made to select a definition with a high specificity and focus on patients who did not otherwise fulfil the 2010 ACR/EULAR RA criteria (<6 points). By a unanimous vote the following definition was selected: erosive disease for use in the 2010 ACR/EULAR RA classification criteria is defined when an erosion (defined as a cortical break) is seen in at least three separate joints at any of the following sites: the proximal interphalangeal, the metacarpophalangeal, the wrist (counted as one joint) and the metatarsophalangeal joints on radiographs of both hands and feet. A highly specific definition for erosive disease has thus been formulated.

Matic J.,Max Planck Institute for Intelligent Systems (Stuttgart) | Matic J.,University of Heidelberg | Deeg J.,Max Planck Institute for Intelligent Systems (Stuttgart) | Deeg J.,University of Heidelberg | And 6 more authors.
Nano Letters | Year: 2013

Anti-CD3 (aCD3) nanoarrays fabricated by self-assembled nanopatterning combined with site-directed protein immobilization techniques represent a novel T cell stimulatory platform that allows tight control over ligand orientation and surface density. Here, we show that activation of primary human CD4+ T cells, defined by CD69 upregulation, IL-2 production and cell proliferation, correlates with aCD3 density on nanoarrays. Immobilization of aCD3 through nanopatterning had two effects: cell activation was significantly higher on these surfaces than on aCD3-coated plastics and allowed unprecedented fine-tuning of T cell response. © 2013 American Chemical Society.

Bacher P.,Charite - Medical University of Berlin | Kniemeyer O.,Leibniz Institute for Natural Product Research and Infection Biology | Kniemeyer O.,University Hospital | Schonbrunn A.,Charite - Medical University of Berlin | And 9 more authors.
Mucosal Immunology | Year: 2014

Foxp3 + regulatory T cells (Treg) have a central role for keeping the balance between pro- and anti-inflammatory immune responses against chronically encountered antigens at mucosal sites. However, their antigen specificity especially in humans is largely unknown. Here we used a sensitive enrichment technology for antigen-reactive T cells to directly compare the conventional vs. regulatory CD4 + T-cell response directed against two ubiquitous mucosal fungi, Aspergillus fumigatus and Candida albicans. In healthy humans, fungus-specific CD4 + CD25 + CD127 - Foxp3 + Treg are strongly expanded in peripheral blood and possess phenotypic, epigenetic and functional features of thymus-derived Treg. Intriguingly, for A. fumigatus, the strong Treg response contrasts with minimal conventional T-cell memory, indicating selective Treg expansion as an effective mechanism to prevent inappropriate immune activation in healthy individuals. By contrast, in subjects with A. fumigatus allergies, specific Th2 cells were strongly expanded despite the presence of specific Treg. Taken together, we demonstrate a largely expanded Treg population specific for mucosal fungi as part of the physiological human T-cell repertoire and identify a unique capacity of A. fumigatus to selectively generate Treg responses as a potentially important mechanism for the prevention of allergic reactions. © 2014 Society for Mucosal Immunology.

Bacher P.,Charite - Medical University of Berlin | Kniemeyer O.,Leibniz Institute for Natural Product Research and Infection Biology | Kniemeyer O.,Friedrich - Schiller University of Jena | Kniemeyer O.,Jena University Hospital | And 20 more authors.
Journal of Immunology | Year: 2014

CD4+T cells orchestrate immune responses against fungi, such as Aspergillus fumigatus, a major fungal pathogen in humans. The complexity of the fungal genome and lifestyle questions the existence of one or a few immune-dominant Ags and complicates systematic screening for immunogenic Ags useful for immunotherapy or diagnostics. In this study, we used a recently developed flow cytometric assay for the direct ex vivo characterization of A. fumigatus-specific CD4+T cells for rapid identification of physiological T cell targets in healthy donors. We show that the T cell response is primarily directed against metabolically active A. fumigatus morphotypes and is stronger against membrane protein fractions compared with cell wall or cytosolic proteins. Further analysis of 15 selected single A. fumigatus proteins revealed a highly diverse reactivity pattern that was donor and protein dependent. Importantly, the parallel assessment of T cell frequency, phenotype, and function allowed us to differentiate between proteins that elicit strong memory T cell responses in vivo versus Ags that induce T cell exhaustion or no reactivity in vivo. The regulatory T cell (Treg) response mirrors the conventional T cell response in terms of numbers and target specificity. Thus, our data reveal that the fungal T cell immunome is complex, but the ex vivo characterization of reactive T cells allows us to classify Ags and to predict potential immunogenic targets. A. fumigatus-specific conventional T cell responses are counterbalanced by a strong Treg response, suggesting that Treg-depletion strategies may be helpful in improving antifungal immunity. © 2014 by The American Association of Immunologists, Inc.

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