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Broadley S.P.,TU Munich | Broadley S.P.,University of Lübeck | Plaumann A.,TU Munich | Coletti R.,Ludwig Maximilians University of Munich | And 14 more authors.
Cell Host and Microbe | Year: 2016

Efficient clearance of bacteremia prevents life-threatening disease. Platelet binding to intravascular bacteria, a process involving platelet glycoprotein GPIb and bacterial opsonization with activated complement C3, influences blood clearance and anti-infective immunity. Using intravital microscopy of the bloodstream of mice infected with Listeria monocytogenes, we show that bacterial clearance is not a uniform process but a “dual-track” mechanism consisting of parallel “fast” and “slow” pathways. “Slow clearance” is regulated by time-dependent bacterial opsonization, stochastic platelet binding, and capture of bacteria-platelet-complexes via the complement receptor of the immunoglobulin superfamily, CRIg. The mechanism spares some bacteria from “fast clearance” and rapid destruction in the liver via Kupffer cell scavenger receptors, keeping them available for adaptive immunity induction by splenic CD8α+ dendritic cells. We consistently find “fast” and “slow” clearance patterns for a broad panel of other Gram+ and Gram− bacteria. Thus, dual-track clearance balances rapid restoration of blood sterility with induction of specific antibacterial immunity. © 2016 Elsevier Inc.


Dossinger G.,TU Munich | Bunse M.,Max Delbrück Center for Molecular Medicine | Bet J.,TU Munich | Albrecht J.,Helmholtz Center Munich | And 13 more authors.
PLoS ONE | Year: 2013

Adoptive therapy using T cells redirected to target tumor- or infection-associated antigens is a promising strategy that has curative potential and broad applicability. In order to accelerate the screening process for suitable antigen-specific T cell receptors (TCRs), we developed a new approach circumventing conventional in vitro expansion-based strategies. Direct isolation of paired full-length TCR sequences from non-expanded antigen-specific T cells was achieved by the establishment of a highly sensitive PCR-based T cell receptor single cell analysis method (TCR-SCAN). Using MHC multimer-labeled and single cell-sorted HCMV-specific T cells we demonstrate a high efficacy (approximately 25%) and target specificity of TCR-SCAN receptor identification. In combination with MHC-multimer based pre-enrichment steps, we were able to isolate TCRs specific for the oncogenes Her2/neu and WT1 even from very small populations (original precursor frequencies of down to 0.00005% of CD3+ T cells) without any cell culture step involved. Genetic re-expression of isolated receptors demonstrates their functionality and target specificity. We believe that this new strategy of TCR identification may provide broad access to specific TCRs for therapeutically relevant T cell epitopes. © 2013 Dössinger et al.


Nauerth M.,TU Munich | Weissbrich B.,TU Munich | Knall R.,TU Munich | Franz T.,TU Munich | And 23 more authors.
Science Translational Medicine | Year: 2013

Adoptive immunotherapy is a promising therapeutic approach for the treatment of chronic infections and cancer. T cells within a certain range of high avidity for their cognate ligand are believed to be most effective. T cell receptor (TCR) transfer experiments indicate that a major part of avidity is hardwired within the structure of the TCR. Unfortunately, rapid measurement of structural avidity of TCRs is difficult on living T cells. We developed a technology where dissociation (koff rate) of truly monomeric peptide-major histocompatibility complex (pMHC) molecules bound to surface-expressed TCRs can be monitored by real-time microscopy in a highly reliable manner. A first evaluation of this method on distinct human cytomegalovirus (CMV)-specific T cell populations revealed unexpected differences in the koff rates. CMV-specific T cells are currently being evaluated in clinical trials for efficacy in adoptive immunotherapy; therefore, determination of koff rates could guide selection of the most effective donor cells. Indeed, in two different murine infection models, we demonstrate that T cell populations with lower koff rates confer significantly better protection than populations with fast koff rates. These data indicate that koff rate measurements can improve the predictability of adoptive immunotherapy and provide diagnostic information on the in vivo quality of T cells. Copyright 2013 by the American Association for the Advancement of Science; all rights reserved.


Buchholz V.R.,TU Munich | Buchholz V.R.,Helmholtz Center Munich | Buchholz V.R.,National Center for Infection Research | Graf P.,TU Munich | Busch D.H.,TU Munich
Cellular and Molecular Life Sciences | Year: 2012

During the past two decades of research in T cell biology, an increasing number of distinct T cell subsets arising during the transition from naý̈ve to antigen-experienced T cells have been identified. Recently, it has been appreciated that, in different experimental settings, distinct T cell subsets can be generated in parallel within the same immune response. While signals driving a single ''lineage'' path of T cell differentiation are becoming increasingly clear, it remains largely enigmatic how the phenotypic and functional diversification creating a multi-faceted T cell response is achieved. Here, we review current literature indicating that diversification is a stable trait of CD8 + T cell responses. We showcase novel technologies providing deeper insights into the process of diversification among the descendants of individual T cells, and introduce two models that emphasize either intrinsic noise or extrinsic signals as driving forces behind the diversification of single cellderived T cell progeny populations in vivo. © 2011 Springer Basel AG.


Weissbrich B.,TU Munich | Nauerth M.,TU Munich | Busch D.H.,TU Munich | Busch D.H.,Helmholtz Center Munich | Busch D.H.,National Center for Infection Research
OncoImmunology | Year: 2013

T cells expressing high avidity T-cell receptors (TCRs) have been shown to mediate superior therapeutic effects. A novel koff-rate assay allows for the quantitative and reproducible assessment of the avidity of TCRs for their ligands directly on living T cells, ex vivo. This assay might facilitate the selection of T cells with an optimal avidity for their target, hence favoring the development of adoptive immunotherapeutic regimens. © 2013 Landes Bioscience.


Stemberger C.,TU Munich | Dreher S.,TU Munich | Tschulik C.,Cell Therapeutics | Piossek C.,Cell Therapeutics | And 17 more authors.
PLoS ONE | Year: 2012

A general obstacle for clinical cell preparations is limited purity, which causes variability in the quality and potency of cell products and might be responsible for negative side effects due to unwanted contaminants. Highly pure populations can be obtained best using positive selection techniques. However, in many cases target cell populations need to be segregated from other cells by combinations of multiple markers, which is still difficult to achieve - especially for clinical cell products. Therefore, we have generated low-affinity antibody-derived Fab-fragments, which stain like parental antibodies when multimerized via Strep-tag and Strep-Tactin, but can subsequently be removed entirely from the target cell population. Such reagents can be generated for virtually any antigen and can be used for sequential positive enrichment steps via paramagnetic beads. First protocols for multiparameter enrichment of two clinically relevant cell populations, CD4 high/CD25 high/CD45RA high 'regulatory T cells' and CD8 high/CD62L high/CD45RA neg 'central memory T cells', have been established to determine quality and efficacy parameters of this novel technology, which should have broad applicability for clinical cell sorting as well as basic research. © 2012 Stemberger et al.


Buchholz V.R.,TU Munich | Graf P.,TU Munich | Busch D.H.,TU Munich | Busch D.H.,Helmholtz Center Munich | Busch D.H.,National Center for Infection Research
Frontiers in Immunology | Year: 2013

CD8+ T cell immune responses provide immediate protection against primary infection and durable memory capable of rapidly fighting off re-infection. Immediate protection and lasting memory are implemented by phenotypically and functionally distinct T cell subsets. While it is now widely accepted that these diverge from a common source of naïve T cells (Tn), the developmental relation and succession of effector and memory T cell subsets is still under intense debate. Recently, a distinct memory T cell subset has been suggested to possess stem cell-like features, sparking the hope to harness its capacity for self-renewal and diversification for successful therapy of chronic infections or malignant diseases. In this review we highlight current developmental models of memory generation, T cell subset diversification and T cell stemness. We discuss the importance of single cell monitoring techniques for adequately mapping these developmental processes and take a brief look at signaling components active in the putative stem cell-like memory T cell compartment. © 2013 Buchholz, Gräf and Busch.


Busch D.H.,TU Munich | Busch D.H.,National Center for Infection Research | Frassle S.P.,TU Munich | Sommermeyer D.,Fred Hutchinson Cancer Research Center | And 5 more authors.
Seminars in Immunology | Year: 2016

Adoptive transfer of primary (unmodified) or genetically engineered antigen-specific T cells has demonstrated astonishing clinical results in the treatment of infections and some malignancies. Besides the definition of optimal targets and antigen receptors, the differentiation status of transferred T cells is emerging as a crucial parameter for generating cell products with optimal efficacy and safety profiles.Long-living memory T cells subdivide into phenotypically as well as functionally different subsets (e.g. central memory, effector memory, tissue-resident memory T cells). This diversification process is crucial for effective immune protection, with probably distinct dependencies on the presence of individual subsets dependent on the disease to which the immune response is directed as well as its organ location.Adoptive T cell therapy intends to therapeutically transfer defined T cell immunity into patients. Efficacy of this approach often requires long-term maintenance of transferred cells, which depends on the presence and persistence of memory T cells. However, engraftment and survival of highly differentiated memory T cell subsets upon adoptive transfer is still difficult to achieve. Therefore, the recent observation that a distinct subset of weakly differentiated memory T cells shows all characteristics of adult tissue stem cells and can reconstitute all types of effector and memory T cell subsets, became highly relevant. We here review our current understanding of memory subset formation and T cell subset purification, and its implications for adoptive immunotherapy. © 2016 Elsevier Ltd.


Nauerth M.,TU Munich | Stemberger C.,TU Munich | Stemberger C.,Juno Therapeutics | Mohr F.,TU Munich | And 5 more authors.
Cytometry Part A | Year: 2016

High epitope-specific sensitivity of CD8+ T cells is required for optimal immune protection against intracellular pathogens as well as certain malignancies. The quality of antigen recognition of CD8+ T cells is usually described as “avidity” to its cognate peptide MHCI complex. T cell avidity is mainly dependent on the structural qualities of the T cell receptor (TCR), as convincingly demonstrated by recombinant TCR re-expression experiments. Based on reversible MHCI multimer staining and koff-rate measurements of monomeric peptide MHCI complexes, we recently established a microscopic assay for determining the structural avidity of individual CD8+ T cells. Here we demonstrate that this assay can be adapted for rapid flow-cytometric avidity screening of epitope-specific T cell populations. Furthermore, we show that—in combination with conventional nonreversible MHCI multimer staining—even very small epitope-specific CD8+ T cell populations can be analyzed directly ex vivo without the need for previous TCR cloning or T cell sorting. This simplified approach provides highly accurate mean TCR-ligand koff-rate values for poly- or oligoclonal T cell populations and is ideally suited for high-throughput applications in basic research as well as clinical settings. © 2016 International Society for Advancement of Cytometry. © 2016 International Society for Advancement of Cytometry

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