Alexander Scheffold - Academia.edu (original) (raw)

Papers by Alexander Scheffold

European Journal of Immunology

Upon primary activation, T helper (Th) cell populations express different cytokines transiently a... more Upon primary activation, T helper (Th) cell populations express different cytokines transiently and with different kinetics. Stimulation of naive murine splenic Th cells with the bacterial superantigen Staphylococcus aureus enterotoxin B (SEB) in vitro results in expression of IL-2, IFN-gamma and IL-10 with fast, intermediate and slow kinetics, respectively. This first report of a functional analysis of cells separated alive according to cytokine expression shows that these cytokines are not produced by different Th cell subpopulations, but can be expressed sequentially by individual Th cells. Th cells, activated with SEB for 1 day and isolated according to expression of IL-2, using the cellular affinity matrix technology, upon continued stimulation with SEB later secrete most of the IFN-gamma and IL-10. Likewise, after 2 days of SEB culture, cells expressing IFN-gamma, separated according to specific surface-associated IFN-gamma as detected by magnetofluorescent liposomes, 1 day later secrete IL-10. Thus, individual Th1 cells can contribute to the control of their own IFN-gamma expression by sequential expression of first IL-2, supporting their proliferation, and later IL-10, down-regulating the production of IFN-gamma-inducing monokines and limiting the pro-inflammatory effects of IFN-gamma.

European cytokine network

ABSTRACT

Cytotherapy, 2015

Evidence of the criticality of the adaptive immune response for controlling invasive aspergillosi... more Evidence of the criticality of the adaptive immune response for controlling invasive aspergillosis has been provided. This observation is supported by the fact that invasive aspergillosis, a grave complication of allogeneic stem cell transplantation, occurs long after myeloid reconstitution in patients with low T-cell engraftment and/or on immunosuppressants. Adoptive T-cell transfer might be beneficial, but idiosyncrasies of Aspergillus fumigatus and the anti-Aspergillus immune response render established selection technologies ineffective. We developed a Good Manufacturing Practice (GMP)-compliant protocol for preparation of A. fumigatus-specific CD4+ cells by sequentially depleting regulatory and cytotoxic T cells, activating A. fumigatus-specific T-helper cells with GMP-grade A. fumigatus lysate, and immuno-magnetically isolating them via the transiently up-regulated activation marker, CD137. In 13 full-scale runs, we demonstrate robustness and feasibility of the approach. From ...

We analyzed for the first time the expression of chemokines in subpopulations of the murine immun... more We analyzed for the first time the expression of chemokines in subpopulations of the murine immune system at the single-cell level. We demonstrate in vitro and in a model of murine listeriosis that macrophage inflammatory protein (MIP)-1␣, MIP-1␤, regulated on activation normal T cell expressed and secreted (RANTES), and activation-induced, T cell-derived, and chemokine-related cytokine (ATAC)͞lymphotactin are cosecreted to a high degree with IFN-␥ by activated individual natural killer (NK), CD8 ؉ T, and CD4 ؉ T helper 1 (Th1) cells. Functionally, ATAC and the CC chemokines cooperate with IFN-␥ in the up-regulation of CD40, IL-12, and tumor necrosis factor-␣, molecules playing a central role in the effector phase of macrophages. Our data indicate that (i) MIP-1␣, MIP-1␤,

PLOS ONE, 2015

Leukocyte adhesion and transmigration are central features governing immune surveillance and infl... more Leukocyte adhesion and transmigration are central features governing immune surveillance and inflammatory reactions in body tissues. Within the liver sinusoids, chemokines initiate the first crucial step of T-cell migration into the hepatic tissue. We studied molecular mechanisms involved in endothelial chemokine supply during hepatic immune surveillance and liver inflammation and their impact on the recruitment of CD4 + T cells into the liver. In the murine model of Concanavalin A-induced T cell-mediated hepatitis, we showed that hepatic expression of the inflammatory CXC chemokine ligands (CXCL)9 and CXCL10 strongly increased whereas homeostatic CXCL12 significantly decreased. Consistently, CD4 + T cells expressing the CXC chemokine receptor (CXCR)3 accumulated within the inflamed liver tissue. In histology, CXCL9 was associated with liver sinusoidal endothelial cells (LSEC) which represent the first contact site for T-cell immigration into the liver. LSEC actively transferred basolaterally internalized CXCL12, CXCL9 and CXCL10 via clathrin-coated vesicles to CD4 + T cells leading to enhanced transmigration of CXCR4 + total CD4 + T cells and CXCR3 + effector/memory CD4 + T cells, respectively in vitro. LSEC-expressed CXCR4 mediated CXCL12 transport and blockage of endothelial CXCR4 inhibited CXCL12-dependent CD4 + T-cell transmigration. In contrast, CXCR3 was not involved in the endothelial transport of its ligands CXCL9 and CXCL10. The clathrin-specific inhibitor chlorpromazine blocked endothelial chemokine internalization and CD4 + T-cell transmigration in vitro as well as migration of CD4 + T cells into the inflamed liver in vivo. Moreover, hepatic accumulation of CXCR3 + CD4 + T cells during T cell-mediated hepatitis was strongly reduced after administration of chlorpromazine. These data demonstrate that LSEC actively provide perivascularly expressed homeostatic and inflammatory chemokines by CXCR4-and clathrin-PLOS ONE |

Surrogate light chain expression during B lineage differentiation was examined by using indicator... more Surrogate light chain expression during B lineage differentiation was examined by using indicator fluorochrome-filled lipo- somes in an enhanced immunofluores- cence assay. Pro-B cells bearing surro- gate light chain components were found in mice, but not in humans. A limited subpopulation of relatively large pre-B cells in both species expressed pre-B cell receptors. These cells had reduced ex- pression of

American journal of respiratory and critical care medicine, 2015

Journal of immunology (Baltimore, Md. : 1950), 2014

CD4(+) T cells orchestrate immune responses against fungi, such as Aspergillus fumigatus, a major... more 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 u...

Nature medicine, 2000

... The expression vectors for IFN-and IL-10 were obtained from P. Lollini 12 and G. Richter 13 a... more ... The expression vectors for IFN-and IL-10 were obtained from P. Lollini 12 and G. Richter 13 as indicated, and used without modification. ... 3, the plasmids pIM11 (IL-4−His) and pIM 12 (IFN-−His) were obtained by cloning PCR-amplified fragments with mouse IL-4 and IFN-cDNA ...

Proceedings of the National Academy of Sciences, 2008

Immunotechnology, 1995

Immunofluorescence and immunomagnetism are important technologies for analysis and sorting of cel... more Immunofluorescence and immunomagnetism are important technologies for analysis and sorting of cells according to specific marker molecules. Due to the limited sensitivity at least several thousands of antigens per cell are required for optical detection. Molecules expressed at low copy numbers cannot be analysed, although they may be of considerable functional importance. Development of a magnetic and fluorescent staining reagent for analysis and sorting of cells according to antigens expressed at low number. To this end, uniformly sized, antibody-conjugated liposomes loaded with large amounts of dye molecules and small magnetic particles were generated. A method for the preparation of homogeneously sized large unilamellar liposomes which contain carboxyfluorescein, magnetic particles and surface-bound antibodies had to be developed. These liposomes were then tested for their ability to enhance immunofluorescence compared to conventional staining in a model system and by staining of CD25 on resting B and T cells. Large unilamellar liposomes, homogeneous in size and loaded with fluorescein and magnetic beads can be prepared by combining membrane extrusion and magnetic filtration. Hapten-specific antibodies conjugated to their surface make them a universal tool for immunofluorescence. With such liposomes, intensity of fluorescent staining can be increased 100-1000-fold without increased background fluorescence, compared to conventional fluorochrome-conjugated antibodies. Due to the simultaneous magnetic labelling, stained cells can easily be isolated by MACS. The magnetofluorescent liposomes proved to be useful for improvement of sensitivity of detection and physical separation in general and to visualize and sort cells according to antigens expressed at low levels. The high affinity IL2 receptor CD25 is expressed in low copy number on a significant fraction of resting B and T lymphocytes in human peripheral blood, as can be shown exclusively by the magnetofluorescent liposomes.

Japan Arthritis Res Ther 2003, 5(Suppl 3):1 (DOI 10.1186/ar800) Apoptosis is a principal mechanis... more Japan Arthritis Res Ther 2003, 5(Suppl 3):1 (DOI 10.1186/ar800) Apoptosis is a principal mechanism in metazoans by which superfluous or potentially harmful cells are eliminated. Deregulation of this process leads to a variety of diseases such as cancer and autoimmune diseases. Stimuli that can induce apoptosis are relatively diverse, and include the death factors (Fas ligand, tumor necrosis factor and TRAIL), DNA damage, and oxidative stress. Regardless of the origin of the apoptotic stimulus, commitment to apoptosis leads to activation of caspases, a family of cysteine proteases. Cleavage of a select group of cellular substrates by caspases is responsible for the morphological and biochemical changes that characterize apoptotic cell death. The degradation of nuclear DNA into nucleosomal units is one of the features of apoptotic cell death, and is mediated by a caspase-activated DNase (CAD). Cells deficient in CAD undergo cell death without the DNA fragmentation, but CAD-null mice did not show any adverse phenotypes. A close examination of the apoptotic cells in these mice indicated that apoptotic cells are always in macrophages. It seems that at an early stage of apoptosis, the dying cells present an 'eat me signal' on their surface. This signal is recognized by macrophages for engulfment, and DNase II in the lysosomes of macrophages degrades DNA of apoptotic cells. Mice deficient in both CAD and DNase II genes were established, and the development of various organs was found to be severely impaired in these mutant mice. The mice accumulated a large amount of undigested DNA in macrophages in various tissues during development. This accumulation of DNA in macrophages activated the innate immunity to induce the expression of the interferon β gene. The interferon thus produced seems to be responsible for the impaired tissue development. These results indicate that the degradation of DNA during apoptotic cell death is an essential step of apoptosis to maintain mammalian homeostasis.

Ernst Schering Research Foundation Workshop, 2005

... hyperreactivity and inflammation. J Clin Invest 105:61ą70 Hardung F, Kunkel D, Simon J, Petro... more ... hyperreactivity and inflammation. J Clin Invest 105:61ą70 Hardung F, Kunkel D, Simon J, Petrow P, Braeuer R, Krenn V, et al. (2001) Defining the functional properties of arthritogenic T cells in an animal model of arthritis. Arthritis Rheum ...

Annals of the Rheumatic Diseases, 2014

ABSTRACT Background Ustekinumab blocks the action of IL-12 and IL-23 by interfering with binding ... more ABSTRACT Background Ustekinumab blocks the action of IL-12 and IL-23 by interfering with binding of the p40 subunit to the IL-12Rβ1 subunit used by IL-12 and IL-23 receptors. IL-12 and IL-23 are important cytokines that promote Th1 and Th17 responses. They are produced by monocytes in response to bacterial products such as LPS. Objectives This study aimed to clarify if therapeutic blockade of p40 modulates frequencies of Th1 and Th17 effector cells and/or if it affects innate responses to LPS. Methods 20 AS Patients were treated with 90 mg Ustekinumab s.c. at baseline, week 4 and week 16 [1]. Heparinized peripheral venous blood was collected at baseline and at week 24 from these patients and from 20 healthy donors. To determine T effector cell frequencies, we performed in vitro stimulation of whole blood with either phorbol myristate acetate/ionomycin (PMA/I) or with the superantigen SEB for 6 hours with Brefeldin A added after 2 hours. IFNg and TNFa production by CD4+ T cells was determined after intracellular staining and detection by FACS. The frequency of Th17 cells was determined by FACS after enrichment of CD40L+ T cells directly after stimulation according to the method of Bacher et al. [2]. To analyse innate cytokine responses to bacterial stimuli whole blood was mixed with media and incubated with or without LPS for 20 hours. IL-23 and IL-22 (a cytokine induced by IL-23) was detected in the supernatants by ELISA. Results Ustekinumab treatment did not result in changes of the frequency of Th1 cells according to IFNg or TNFa secretion or in Th17 cells (Table 1). In 20h whole blood stimulations we observed elevated spontaneous (w.o. LPS) IL-22 secretion in AS patients at baseline compared to healthy controls. This elevated spontaneous IL-22 secretion decreased upon Ustekinumab treatment (Table 2). Also the LPS-induced IL-23 levels were lower after treatment with Ustekinumab at week 24 compared to baseline (p<0.05). Conclusions Ustekinumab treatment did not affect Th1 or Th17 effector cell frequencies in AS patients. However, reduction of IL-22 and IL-23 production upon Ustekinumab treatment may point to effects rather on innate immune functions. References Acknowledgements This study was supported by an unrestricted research grant from Janssen-Cilag. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4729

Cytometry, 2003

The presentation of antigenic peptides to specific T cells is one of the key events for the induc... more The presentation of antigenic peptides to specific T cells is one of the key events for the induction of a T-cell-dependent immune response. The nature of the antigen-presenting cells which present distinct peptides has been difficult to analyze so far due to the low number of peptides presented in vivo by a single antigen-presenting cell.

LaboratoriumsMedizin, 2004

... Immunity 1998;8:167–75. 9 Murali-Krishna K, Altman JD, Suresh M, Sourdive DJD, Zajac AJ, Mill... more ... Immunity 1998;8:167–75. 9 Murali-Krishna K, Altman JD, Suresh M, Sourdive DJD, Zajac AJ, Miller JD, Slansky J, Ahmed R. Counting antigen-specific CD8 T cells: A reevaluation of bystander activation during viral infection. ... 45 Lu L, Kaliyaperumal A, Boumpas DT, Datta SK. ...