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Dirk H Busch

Publications and source records attributed to Dirk H Busch.

At least 19 recordsLinked to original sources
PubMed2006

A genetic screen for modifiers of the delta1-dependent notch signaling function in the mouse.

The Notch signaling pathway is an evolutionarily conserved transduction pathway involved in embryonic patterning and regulation of cell fates during development. Recent studies have demonstrated that this pathway is integral to a complex system of interactions, which are also involved in distinct human diseases. Delta1 is one of the known ligands of the Notch receptors. Mice homozygous for a loss-of-function allele of the Delta1 gene Dll1(lacZ/lacZ) die during embryonic development. Here, we present the results of two phenotype-driven modifier screens. Heterozygous Dll1(lacZ) knockout animals were crossed with ENU-mutagenized mice and screened for dysmorphological, clinical chemical, and immunological variants that are dependent on the Delta1 loss-of-function allele. First, we show that mutagenized heterozygous Dll1(lacZ) offspring have reduced body weight and altered specific clinical chemical parameters, including changes in metabolites and electrolytes relevant for kidney function. In our mutagenesis screen we have successfully generated 35 new mutant lines. Of major interest are 7 mutant lines that exhibit a Dll1(lacZ/+)-dependent phenotype. These mutant mouse lines provide excellent in vivo tools for studying the role of Notch signaling in kidney and liver function, cholesterol and iron metabolism, cell-fate decisions, and during maturation of T cells in the immune system.

Alleles↗
PubMed2006

CD8alpha+ dendritic cells are required for efficient entry of Listeria monocytogenes into the spleen.

In addition to their bridging function between innate and adaptive immunity, dendritic cells (DCs) may also contribute to primary resistance against infection. Here we analyzed the role of DCs during infection with Listeria monocytogenes by performing systemic in vivo depletion of these cells. We showed that CD8alpha(+) DCs were crucial for L. monocytogenes spreading and proliferation in the spleen. Efficient and rapid uptake of L. monocytogenes by CD8alpha(+) DCs required the small GTPase Rac1 and is a general characteristic of this DC subpopulation in filtering particles out of the blood. Thus, CD8alpha(+) DCs appear to play an important role for efficient bacterial entry into the spleen, which is of relevance for subsequent immune responses.

Animals↗
PubMed2006

Prophylactic anti-tumor immunity against a murine fibrosarcoma triggered by the Salmonella type III secretion system.

The potential of an attenuated Salmonella enterica serovar Typhimurium strain as a prophylactic anti-tumor vaccine against the murine fibrosarcoma WEHI 164 was evaluated. Tumor cells were transfected with the DNA sequence encoding the MHC class I-restricted peptide p60(217-225) from Listeria monocytogenes. BALB/c mice received a single orogastric immunization with Salmonella that translocates a chimeric p60 protein via its type III secretion system. Mice were subsequently challenged subcutaneously with p60(217-225)-expressing WEHI cells. In vivo protection studies revealed that 80% of these mice remained free of the fibrosarcoma after challenge, whereas all animals of the non-vaccinated control group did develop tumor growth. In further experiments, the distribution of tetramer-positive p60(217-225)-specific effector and memory CD8 T cells after Salmonella-based immunization and tumor application was analyzed. Costaining with CD62L and CD127 revealed a predominance of p60-specific central memory and effector memory CD8 T cells in spleens, whereas in blood samples the majority of p60-specific lymphocytes belonged to effector and effector memory CD8 T cell subsets. This is the first report demonstrating that a bacterial type III secretion system can be used for heterologous antigen delivery to induce cytotoxic effector and memory CD8 T cell responses resulting in an efficient prevention of tumor growth.

Animals↗
PubMed2006

Protective immunity towards intracellular pathogens.

Immunity towards intracellular pathogens is often dependent upon the generation of CD8(+) memory T cells, which provide long-lasting and effective protection. Over the past few years, we have gained novel insights into the heterogeneity of CD8(+) T cells, time points of lineage commitment, and lineage relationships between subpopulations. These studies suggest that memory CD8(+) T cells progressively develop from naïve cells early during the immune response and further differentiate unidirectionally into short-living effector cells. We have also learnt that different memory subsets play distinct roles in conferring protection: whereas effector memory T cells are able to provide immediate protection but are not necessarily maintained long-term, central memory T cells have the potential for constant self-renewal. Thus, neither subset really fulfills all criteria of memory. As protective effector memory cells can develop from central memory cells, vaccination strategies should focus on induction of a balanced ratio of the two memory T cell subsets.

Animals↗
PubMed2006

Platelets secrete stromal cell-derived factor 1alpha and recruit bone marrow-derived progenitor cells to arterial thrombi in vivo.

The accumulation of smooth muscle and endothelial cells is essential for remodeling and repair of injured blood vessel walls. Bone marrow-derived progenitor cells have been implicated in vascular repair and remodeling; however, the mechanisms underlying their recruitment to the site of injury remain elusive. Here, using real-time in vivo fluorescence microscopy, we show that platelets provide the critical signal that recruits CD34+ bone marrow cells and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells to sites of vascular injury. Correspondingly, specific inhibition of platelet adhesion virtually abrogated the accumulation of both CD34+ and c-Kit+ Sca-1+ Lin- bone marrow-derived progenitor cells at sites of endothelial disruption. Binding of bone marrow cells to platelets involves both P-selectin and GPIIb integrin on platelets. Unexpectedly, we found that activated platelets secrete the chemokine SDF-1alpha, thereby supporting further primary adhesion and migration of progenitor cells. These findings establish the platelet as a major player in the initiation of vascular remodeling, a process of fundamental importance for vascular repair and pathological remodeling after vascular injury.

Animals↗
PubMed2006

Proteasomes shape the repertoire of T cells participating in antigen-specific immune responses.

Differences in the cleavage specificities of constitutive proteasomes and immunoproteasomes significantly affect the generation of MHC class I ligands and therefore the activation of CD8-positive T cells. Based on these findings, we investigated whether proteasomal specificity also influences CD8-positive T cells during thymic selection by peptides derived from self proteins. We find that one of the self peptides responsible for positive selection of ovalbumin-specific OT-1 T cells, which is derived from the f-actin capping protein (Cpalpha1), is efficiently generated only by immunoproteasomes. Furthermore, OT-1 mice backcrossed onto low molecular mass protein 7 (LMP7)-deficient mice show a 50% reduction of OT-1 cells. This deficiency is also observed after transfer of BM from OT-1 mice in LMP7-deficient mice and can be corrected by the injection of the Cpalpha1 peptide. Interestingly, WT and LMP7-deficient mice mount comparable immune responses to the ovalbumin-derived epitope SIINFEKL. However, their cytotoxic T lymphocytes (CTL) differ in the use of T cell receptor Vbeta genes. CTL derived from WT mice use Vbeta8 or Vbeta5 (the latter is also used by OT-1 cells), whereas SIINFEKL-specific CTL from LMP7-deficient mice are exclusively Vbeta8-positive. Taken together, our experiments provide strong evidence that proteasomal specificity shapes the repertoire of T cells participating in antigen-specific immune responses.

Actin Capping Proteins↗
PubMed2006

Systemic application of CpG-rich DNA suppresses adaptive T cell immunity via induction of IDO.

CpG-rich oligonucleotides (CpG-ODN) bind to Toll-like receptor 9 (TLR9) and are used as powerful adjuvants for vaccination. Here we report that CpG-ODN not only act as immune stimulatory agents but can also induce strong immune suppression depending on the anatomical location of application. In agreement with the adjuvant effect, subcutaneous application of antigen plus CpG-ODN resulted in antigen-specific T cell activation in local lymph nodes. In contrast, systemic application of CpG-ODN resulted in suppression of T cell expansion and CTL activity in the spleen. The suppressive effect was mediated by indoleamine 2,3-dioxygenase (IDO) as indicated by the observation that CpG-ODN induced IDO in the spleen and that T cell suppression could be abrogated by 1-methyl-tryptophan (1-MT), an inhibitor of IDO. No expression of IDO was observed in lymph nodes after injection of CpG-ODN, explaining why suppression was restricted to the spleen. Studies with a set of knockout mice demonstrated that the CpG-ODN-induced immune suppression is dependent on TLR9 stimulation and independent of type I and type II interferons. The present study shows that for the use of CpG-ODN as an adjuvant in vaccines, the route of application is crucial and needs to be considered. In addition, the results indicate that down-modulation of immune responses by CpG-ODN may be possible in certain pathological conditions.

Adenoviridae↗
PubMed2006

Unidirectional development of CD8+ central memory T cells into protective Listeria-specific effector memory T cells.

Three distinct subsets of antigen-experienced CD8(+) T cells have been identified so far: short-living effector T cells (T(EC)) and two long-living subsets, described as central (T(CM)) and effector memory (T(EM)) T cells. The lineage relationships of these subpopulations as well as their involvement in protection have not yet been conclusively determined. We recently described a novel marker combination (CD127 and CD62L) to identify all three major CD8(+) T cell subsets in mice infected with Listeria monocytogenes (L.m.). Extensive lineage relationship analyses on highly purified subpopulations after in vitro and in vivo stimulation demonstrated that T(CM) can develop into T(EM) or T(EC), whereas T(EM) can only progress to T(EC) cells. Short-living T(EC) never regained a T(EM) or T(CM) phenotype. These data strongly suggest a hierarchical and unidirectional order of developmental stages. In vivo priming protocols that preferentially induced one of the different CD8(+) T cell subsets demonstrated that predominance of T(EM) (CD40 stimulation) correlated best with effective protection against L.m., whereas generation of neither T(CM) (by immunization with heat-killed L.m.) nor T(EC) (by systemic co-administration of CpG during primary infection) conferred substantial long-term protective immunity. These findings have important implications for the design of more effective T cell-based vaccines.

Animals↗
PubMed2006

Generation and characterization of dickkopf3 mutant mice.

dickkopf (dkk) genes encode a small family of secreted Wnt antagonists, except for dkk3, which is divergent and whose function is poorly understood. Here, we describe the generation and characterization of dkk3 mutant mice. dkk3-deficient mice are viable and fertile. Phenotypic analysis shows no major alterations in organ morphology, physiology, and most clinical chemistry parameters. Since Dkk3 was proposed to function as thyroid hormone binding protein, we have analyzed deiodinase activities, as well as thyroid hormone levels. Mutant mice are euthyroid, and the data do not support a relationship of dkk3 with thyroid hormone metabolism. Altered phenotypes in dkk3 mutant mice were observed in the frequency of NK cells, immunoglobulin M, hemoglobin, and hematocrit levels, as well as lung ventilation. Furthermore, dkk3-deficient mice display hyperactivity.

Adaptor Proteins, Signal Transducing↗
PubMed2005

Chemokine receptor Ccr2 deficiency reduces renal disease and prolongs survival in MRL/lpr lupus-prone mice.

MRL/MpJ-Fas(lpr)/J (MRL/lpr) mice represent a well-established mouse model of human systemic lupus erythematosus. MRL/lpr mice homozygous for the spontaneous lymphoproliferation mutation (lpr) are characterized by systemic autoimmunity, massive lymphadenopathy associated with proliferation of aberrant T cells, splenomegaly, hypergammaglobulinemia, arthritis, and fatal immune complex-mediated glomerulonephritis. It was reported previously that steady-state mRNA levels for the chemokine (C-C motif) receptor 2 (Ccr2) continuously increase in kidneys of MRL/lpr mice. For examining the role of Ccr2 for development and progression of immune complex-mediated glomerulonephritis, Ccr2-deficient mice were generated and backcrossed onto the MRL/lpr genetic background. Ccr2-deficient MRL/lpr mice developed less lymphadenopathy, had less proteinuria, had reduced lesion scores, and had less infiltration by T cells and macrophages in the glomerular and tubulointerstitial compartment. Ccr2-deficient MRL/lpr mice survived significantly longer than MRL/lpr wild-type mice despite similar levels of circulating immunoglobulins and comparable immune complex depositions in the glomeruli of both groups. Anti-dsDNA antibody levels, however, were reduced in the absence of Ccr2. The frequency of CD8+ T cells in peripheral blood was significantly lower in Ccr2-deficient MRL/lpr mice. Thus Ccr2 deficiency influenced not only monocyte/macrophage and T cell infiltration in the kidney but also the systemic T cell response in MRL/lpr mice. These data suggest an important role for Ccr2 both in the general development of autoimmunity and in the renal involvement of the lupus-like disease. These results identify Ccr2 as an additional possible target for the treatment of lupus nephritis.

Animals↗
PubMed2005

NKG2D-independent suppression of T cell proliferation by H60 and MICA.

The activating receptor NKG2D recognizes a wide range of different ligands, some of which are primarily expressed in "stressed" tissues or on tumor cells. Until now, similar stimulatory effects on natural killer and CD8+ T cells have been described for all NKG2D ligands, and the NKG2D receptor/ligand system has therefore been interpreted as a sensor system involved in tumor immune surveillance and activation of immune responses. We show here that the NKG2D ligands H60 and MIC class 1 chain-related protein A (MICA) can also mediate strong suppressive effects on T cell proliferation. Responsiveness to H60- and MICA-mediated suppression requires IL-10 and involves a receptor other than NKG2D. These findings might provide explanations for the observation that strong in vivo NKG2D ligand expression, such as that on tumor cells, sometimes fails to support effective immune responses and links this observation to a distinct subgroup of NKG2D ligands.

Animals↗
PubMed2005

Importance of integrin LFA-1 deactivation for the generation of immune responses.

The dynamic regulation of ligand binding is considered crucial for integrin function. However, the importance of activity regulation for integrin function in vivo is largely unknown. Here, we have applied gene targeting to delete the GFFKR sequence of the lymphocyte function-associated antigen-1 (LFA-1) alpha(L) subunit cytoplasmic domain in mouse germline. Lymphocytes from Lfa-1(d/d) mutant mice showed constitutive activation of LFA-1-mediated cell adhesion and impaired de-adhesion from intercellular adhesion molecule-1 that resulted in defective cell migration. In contrast, signaling through LFA-1 was not affected in Lfa-1(d/d) cells. T cell activation by superantigen-loaded and allogeneic APCs, cytotoxic T cell activity, T-dependent humoral immune responses, and neutrophil recruitment during aseptic peritonitis were impaired in Lfa-1(d/d) mice. Thus, deactivation of LFA-1 and disassembly of LFA-1-mediated cell contacts seem to be vital for the generation of normal immune responses.

Animals↗
PubMed2005

Protective CD8 T cell immunity triggered by CpG-protein conjugates competes with the efficacy of live vaccines.

In contrast to infectious (live) vaccines are those based on subunit Ag that are notoriously poor in eliciting protective CD8 T cell responses, presumably because subunit Ags become insufficiently cross-presented by dendritic cells (DCs) and because the latter need to be activated to acquire competence for cross-priming. In this study, we show that CpG-Ag complexes overcome these limitations. OVA covalently linked to CpG-DNA (CpG-OVA complex), once it is efficiently internalized by DCs via DNA receptor-mediated endocytosis, is translocated to lysosomal-associated membrane protein 1 (LAMP-1)-positive endosomal-lysosomal compartments recently shown to display competence for cross-presentation. In parallel, CpG-OVA complex loaded DCs become activated and acquire characteristics of professional APCs. In vivo, a single s.c. dose of CpG-OVA complex (10 mug of protein) induces primary and secondary clonal expansion/contraction of Ag-specific CD8 T cells similar in kinetics to live vaccines; examples including Listeria monocytogenes genetically engineered to produce OVA (LM-OVA) and two viral vector-based OVA vaccines analyzed. Interestingly, CpG-OVA complex induced almost equal percentages of Ag-specific memory CD8 T cells as did infection with LM-OVA. A single dose vaccination with CpG-OVA complex protected mice against lethal doses of LM-OVA. These data underscore that the synergy imparted by CpG-OVA complex-mediated combined triggering of innate and specific immunity might be key to initiate CD8 T cell-based immunoprotection by synthetic vaccines based on subunit Ag.

Animals↗
PubMed2005

DC-NK cell cross talk as a novel CD4+ T-cell-independent pathway for antitumor CTL induction.

It is generally accepted that priming of antitumor CD8+ cytotoxic T lymphocytes (CTLs) needs help that can be provided by CD4+ T cells. We show that interactions between dendritic cells (DCs) and natural killer (NK) cells can bypass the T helper arm in CTL induction. Bone marrow-derived DCs caused rejection of the A20 lymphoma and induced tumor-specific long-term memory, although they were not loaded with tumor-derived antigen. Experiments using CD40(-) knock-out mice and cell depletion showed that this effect did not require CD4+ cells. Both primary rejection and long-term CTL memory were the result of NK cell activation by DCs. NK cytotoxicity, which was necessary for primary rejection, was dependent on expression of natural killer group 2 D (NKG2D) ligands on tumor cells. Blocking of these ligands using NKG2D tetramers abrogated tumor killing in vitro and in vivo. The long-term response was due to CTLs directed against antigen(s) expressed on A20 and in vitro-differentiated DCs. The mechanism leading to CD4+ helper cell-independent CTL responses was elucidated as a cascade that was initiated by NK cell activation. This pathway was dependent on inter-feron-gamma expression and involved priming endogenous DCs for interleukin-12 production. Our data suggest a novel pathway linking innate and adaptive immunity.

Animals↗
PubMed2005

NK cell activation through the NKG2D ligand MULT-1 is selectively prevented by the glycoprotein encoded by mouse cytomegalovirus gene m145.

The NK cell-activating receptor NKG2D interacts with three different cellular ligands, all of which are regulated by mouse cytomegalovirus (MCMV). We set out to define the viral gene product regulating murine UL16-binding protein-like transcript (MULT)-1, a newly described NKG2D ligand. We show that MCMV infection strongly induces MULT-1 gene expression, but surface expression of this glycoprotein is nevertheless completely abolished by the virus. Screening a panel of MCMV deletion mutants defined the gene m145 as the viral regulator of MULT-1. The MCMV m145-encoded glycoprotein turned out to be necessary and sufficient to regulate MULT-1 by preventing plasma membrane residence of MULT-1. The importance of MULT-1 in NK cell regulation in vivo was confirmed by the attenuating effect of the m145 deletion that was lifted after NK cell depletion. Our findings underline the significance of escaping MULT-1/NKG2D signaling for viral survival and maintenance.

Animals↗
PubMed2005

Sex-dependent susceptibility to Listeria monocytogenes infection is mediated by differential interleukin-10 production.

It is well documented that sex-dependent factors affect susceptibility to infection, with most mouse models demonstrating higher resistance in females. We made the unexpected observation that infection with the intracellular bacterium Listeria monocytogenes showed an opposite pattern in several commonly used inbred mouse strains: female C57BL/6J, BALB/c, C3H/HeN, and CBA/J mice were significantly more susceptible to Listeria infection. The pronounced sensitivity of females to Listeria, which was revealed by significantly higher lethality rates, correlated also with increased bacterial numbers in organ tissues (spleen and liver) and several immunological changes in peripheral blood samples. Surprisingly, increased severity of infection in females was associated with elevated interleukin-10 (IL-10) levels in plasma. Experiments using Il10 knockout mice, for which no differences between the susceptibilities of males and females to Listeria infection could be detected, confirmed the important role of this immunosuppressive cytokine for the outcome of disease. Our findings are likely to have clinical relevance, since similar sex differences with regard to infection with Listeria monocytogenes and other intracellular pathogens have been reported for humans.

Animals↗
PubMed2005

Isolation and expansion of tumor-reactive cytotoxic T-cell clones for adoptive immunotherapy.

Attempts to treat patients with tumor-reactive cytotoxic T-lymphocytes (CTL) have been limited. This is due to the difficulty of isolating and expanding functionally active T-cells, which are present at extremely low frequencies in the peripheral blood. Recently developed multimers of the HLA-peptide complex mimic the natural ligand of the T-cell receptor and, therefore, fluorochrome-labeled multimers allow visualization and isolation of rare T-cells with defined specificity. Multimer-guided T-cell sorting permits the in vitro culture of antigen-specific T-cells as lines or clones. Cytolytic T-cells capable of recognizing HLA-peptide complexes endogenously processed by tumor cells are selected for further expansion, since lysis of tumor cells in vitro is a prerequisite for effective tumor elimination in vivo. The expansion of tumor-reactive CD8+ T-cells yields cell numbers sufficient for adoptive transfer. Tumor-reactive T-cells retain the functional activity in terms of cytolysis after expansion, encouraging their use in the immunotherapy of cancer patients.

Antigens↗