Biomedical subjects

Eckhart Kämpgen

Publications and source records attributed to Eckhart Kämpgen.

15 recordsLinked to original sources

PubMed2006

An improved method for RNA isolation and removal of melanin contamination from melanoma tissue: implications for tumor antigen detection and amplification.

Several kits are available to isolate RNA(1) from tissues. However, melanoma tissue is often rich in melanin that co-purifies with DNA/RNA and inhibits subsequent PCR reactions, hampering tumor antigen detection and amplification. This problem has not yet been addressed systematically. Here we generated a photometric protocol to determine both the melanin and RNA concentration by correcting the latter for the absorption coefficient of melanin at 260 nm. Subsequently, different combinations of silica-based RNA-binding, size-exclusion, and ion-exchange columns were used in 8 protocols for isolation of RNA from melanoma tissue to compare efficacy of melanin removal and yield of RNA. Furthermore, the capability of the different RNA preparations to function as template in RT-PCRs with products of different length, i.e. GAP-DH, tyrosinase, and gp100, was tested. We found that the combination of silica-based RNA-binding and size-exclusion columns was not sufficient to remove melanin from highly contaminated tumor samples, and subsequent RT-PCR failed to give larger products. However, protocols including ion-exchange columns resulted in efficient removal of melanin, while retaining reasonable RNA yields in samples from highly pigmented melanomas. Efficient RT-PCR of larger products turned out to be inversely correlated to the melanin contamination. This RNA-purification method will help scientists to isolate polynucleotides from melanin-containing tumor samples, which subsequently can be used in antigen detection assays and vaccination strategies using amplified total tumor RNA.

Antigens, Neoplasm↗

PubMed2006

Mature monocyte-derived dendritic cells respond more strongly to CCL19 than to CXCL12: consequences for directional migration.

The chemokine receptor CCR7 is crucial for migration of mature dendritic cells (DC) directed toward secondary lymphoid organs; however, there is little knowledge about the function of the homeostatic chemokine receptor CXCR4 in DC and its contribution to directional migration of DC during inflammation. By comparing the impact of chemokine receptor engagement on mature DC we found that the CCR7 ligand CCL19 holds a stronger chemotactic potency than the CXCR4 ligand CXCL12. Moreover, CCL19 elicited rapid, steep and long-lasting mobilization of intracellular calcium in individual cells and induced intense phosphorylation of extracellular signal-regulated kinase 1/2 and protein kinase B, while the intracellular signals elicited by CXCL12 were in part distinct and significantly weaker. Analysis of chemokine receptor expression revealed that although CCR7 and CXCR4 were expressed by a similar percentage of DC, the mean fluorescence intensity of CCR7 was up to six times higher, suggesting a higher receptor density. Based on these correlations we propose that the type of chemokine signal in conjunction with the expression and functional activity of the respective chemokine receptor is also determining the migration rate and potency of a chemotactic response in mature DC. In conclusion, our data support the fundamental role of CCR7 for rapidly guiding DC toward secondary lymphoid organs at an extra- and intracellular molecular level and on the contrary render CXCR4 a weaker contributor to directional migration of DC during inflammation.

Calcium↗

PubMed2005

A new way to generate cytolytic tumor-specific T cells: electroporation of RNA coding for a T cell receptor into T lymphocytes.

Effective T cell receptor (TCR) transfer until now required stable retroviral transduction. However, retroviral transduction poses the threat of irreversible genetic manipulation of autologous cells. We, therefore, used optimized RNA transfection for transient manipulation. The transfection efficiency, using EGFP RNA, was >90%. The electroporation of primary T cells, isolated from blood, with TCR-coding RNA resulted in functional cytotoxic T lymphocytes (CTLs) (>60% killing at an effector to target ratio of 20:1) with the same HLA-A2/gp100-specificity as the parental CTL clone. The TCR-transfected T cells specifically recognized peptide-pulsed T2 cells, or dendritic cells electroporated with gp100-coding RNA, in an IFNgamma-secretion assay and retained this ability, even after cryopreservation, over 3 days. Most importantly, we show here for the first time that the electroporated T cells also displayed cytotoxicity, and specifically lysed peptide-loaded T2 cells and HLA-A2+/gp100+ melanoma cells over a period of at least 72 h. Peptide-titration studies showed that the lytic efficiency of the RNA-transfected T cells was similar to that of retrovirally transduced T cells, and approximated that of the parental CTL clone. Functional TCR transfer by RNA electroporation is now possible without the disadvantages of retroviral transduction, and forms a new strategy for the immunotherapy of cancer.

Apoptosis↗

PubMed2005

Generation of an optimized polyvalent monocyte-derived dendritic cell vaccine by transfecting defined RNAs after rather than before maturation.

Transfection with RNA is an attractive method of Ag delivery to dendritic cells (DCs), but has not yet been standardized. We describe in this study the methods to efficiently generate an optimized mature monocyte-derived DC vaccine at clinical scale based on the electroporation of several RNAs either into immature DC followed by maturation or, alternatively, directly into mature DCs, which has not been possible so far with such high efficiency. Electroporation of DCs resulted in high yield, high transfection efficiency (>90%), and high migration capacity. Intracellular staining allowed the study of the expression kinetics of Ags encoded by the transfected RNAs (MelanA, MAGE-3, and survivin) and a validation of the vaccine (>/=90% transfection efficiency). Expression of all three Ags peaked 3-4 h after electroporation in DC transfected either before or after maturation, but decreased differently. The DC vaccine can also be cryopreserved and nevertheless retains its viability, stimulatory capacity as well as migratory activity. In addition, we uncover that DC transfected after rather than before maturation appear to be preferable vaccines not only from a production point of view but also because they appear to be immunologically superior for CTL induction in sharp contrast to common belief. DCs transfected after maturation not only more effectively generate and present the Mage-3.A1 and MelanA.A2.1 epitopes to T cell clones, but they even are superior in priming to the standard proteasome-dependent MelanA.A2.1 wild-type prototype tumor epitope, both in terms of T cell expansion and effector function on a per cell basis.

Antigen Presentation↗

PubMed2005

Lack of toxicity of therapy-induced T cell responses against the universal tumour antigen survivin.

Prognosis of disseminated melanoma remains gloomy as neither chemotherapeutic nor unspecific immune modulatory approaches were able to improve the overall survival of these patients. Hence, specific immunotherapy has received increasing attention. Disappointing clinical results, however, indicate that the choice of suitable antigens is of special importance. To this end, the inhibitor of apoptosis (IAP) protein survivin, which is over-expressed in several tumours but is largely undetectable in adult tissues, appears to be a promising target for vaccination purposes, since down-regulation or loss of expression is associated with impaired tumour progression. Consequently, five heavily pretreated stage IV melanoma patients were vaccinated with the HLA-A2 restricted survivin(96-104) epitope presented by autologous dendritic cells (DCs) in a compassionate use setting. Four of these patients mounted strong T cell responses to this epitope as measured by ELISPOT assay. Furthermore, in situ peptide/HLA-A2 multimer staining confirmed that these survivin reactive cells infiltrated both visceral and soft tissue metastases.

Adult↗

PubMed2004

Shift from systemic to site-specific memory by tumor-targeted IL-2.

IL-2 has been approved for treatment of patients with cancer. Moreover, it has been used as a component of vaccines against cancer. In this regard, we have recently demonstrated that dendritic cell-based peptide vaccination in mice required IL-2 to mount an effective immune response against established melanoma metastases. In this study, we confirm this observation by use of tumor-targeted IL-2. However, the development of a protective systemic memory was substantially impaired by this measure, i.e., mice, which successfully rejected s.c. tumors of B16 melanoma after vaccination with dendritic cells pulsed with tyrosinase-related protein 2-derived peptides plus a boost with targeted IL-2, failed to reject a rechallenge with experimental pulmonary metastases. Detailed analysis revealed a change in the distribution of the tumor-reactive T cell population: although targeted IL-2 expanded the local effector population, tyrosinase-related protein 2-reactive T cells were almost completely depleted from lymphatic tissues.

Animals↗

PubMed2003

Production of IL-12 and IL-18 in human dendritic cells upon infection by Listeria monocytogenes.

Dendritic cells (DCs) are major antigen-presenting cells of the immune system, which need to be activated in order to initiate an immune response. Here, we describe the immunostimulatory effects on human monocyte-derived DCs observed upon infection with Listeria monocytogenes or after treatment with listerial lipoteichoic acid (LTA) and lipopolysaccharide (LPS), respectively. All stimuli caused upregulation of costimulatory molecules, induced T-cell proliferative responses and secretion of cytokines in vitro. Infection of DCs with L. monocytogenes induced release of interleukin (IL)-12 and IL-18. In contrast treatment with purified listerial LTA yielded high levels of IL-18 release, but only minimal IL-12 production. Treatment of DCs with LPS conversely induced significant amounts of IL-12 production, but no IL-18. The release of both stimulating cytokines IL-12 and IL-18 upon infection with entire bacteria suggests that attenuated strains of L. monocytogenes may be a valuable tool for subunit vaccine delivery.

Adult↗

PubMed2003

Unique appearance of proliferating antigen-presenting cells expressing DC-SIGN (CD209) in the decidua of early human pregnancy.

Intact human pregnancy can be regarded as an immunological paradox in that the maternal immune system accepts the allogeneic embryo without general immunosuppression. Because dendritic cell (DC) subsets could be involved in peripheral tolerance, the uterine mucosa (decidua) was investigated for DC populations. Here we describe the detailed immunohistochemical and functional characterization of HLA-DR-positive antigen-presenting cells (APCs) in early pregnancy decidua. In contrast to classical macrophages and CD83(+) DCs, which were found in comparable numbers in decidua and nonpregnant endometrium, only decidua harbored a significant population of HLA-DR(+)/DC-SIGN(+) APCs further phenotyped as CD14(+)/CD4(+)/CD68(+/-)/CD83(-)/CD25(-). These cells exhibited a remarkable proliferation rate (9.2 to 9.8% of all CD209(+) cells) by double staining with Ki67 and proliferating cell nuclear antigen. Unique within the DC-family, the majority of DC-SIGN(+) decidual APCs were observed in situ to have intimate contact with CD56(+)/CD16(-)/ICAM-3(+) decidual natural killer cells, another pregnancy-restricted cell population. In vitro, freshly isolated CD14(+)/DC-SIGN(+) decidual cells efficiently took up antigen, but could not stimulate naive allogeneic T cells at all. Treatment with an inflammatory cytokine cocktail resulted in down-regulation of antigen uptake capacity and evolving capacity to effectively stimulate resting T cells. Fluorescence-activated cell sorting analysis confirmed the maturation of CD14(+)/DC-SIGN(+) decidual cells into CD25(+)/CD83(+) mature DCs. In summary, this is the first identification of a uterine immature DC population expressing DC-SIGN, that appears only in pregnancy-associated tissue, has a high proliferation rate, and a conspicuous association with a natural killer subset.

Abortion, Therapeutic↗

PubMed2003

Visualization and characterization of migratory Langerhans cells in murine skin and lymph nodes by antibodies against Langerin/CD207.

Dendritic cells are professional antigen-presenting cells that initiate primary immunity. Migration from sites of antigen uptake to lymphoid organs is crucial for the generation of immune responses. We investigated the migratory pathways specifically of epidermal Langerhans cells by tracing them from the epidermis to the draining lymph nodes. This was possible with a new monoclonal antibody, directed against murine Langerin/CD207, a type II lectin specific for Langerhans cells. In situ, resident, and activated Langerhans cells express Langerin in the epidermis and on their way through dermal lymphatic vessels. Both emigrated and trypsinization-derived Langerhans cells expressed high levels of Langerin intracellularly but reduced it upon prolonged culture periods. Sizeable numbers of Langerin+ cells were found in skin draining lymph nodes but not in mesenteric nodes. Langerin+ cells localized to the T cells areas and rarely to B cell zones. Numbers of Langerin-expressing cells increased after application of a contact sensitizer. In the steady state, Langerhans cells in the skin-draining nodes expressed maturation markers, such as 2A1 and costimulatory molecules CD86 and CD40. These molecules, CD86 and CD40, were further upregulated upon inflammatory stimuli such as contact sensitization. Thus, the novel anti-Langerin monoclonal antibody permits the unequivocal visualization of migratory Langerhans cells in the lymph nodes for the first time and thereby allows to dissect the relative immunogenic or tolerogenic contributions of Langerhans cells and other types of dendritic cells.

Animals↗

PubMed2003

MHC class II-mediated apoptosis in dendritic cells: a role for membrane-associated and mitochondrial signaling pathways.

Cytotoxic elimination of dendritic cells (DC) in lymphoid tissue represents an important pathway of immune regulation. However, the mechanism of DC removal is still controversial since mature DC are insensitive to death receptor-mediated killing and other surface or soluble molecules mediating DC death in vivo have yet to be characterized. Class II ligation is the only known signal that induces rapid cell death in mature DC, thus our studies have now focused on the requirements for this cell death using the advantages of tools available for both the mouse and human systems. Anti-class II mAb could be grouped into (i) mAb that both bound to class II and caused class II-mediated cell death as well as (ii) those that bound to class II, but did not cause apoptosis. mAb binding stable class II dimers as well as those mAb recognizing either the alpha or beta chains of class II were found in both groups. Whereas class II-mediated death was enhanced by DC-DC homotypic interactions, DC clustering itself was insufficient to induce apoptosis. Although DC death could be inhibited by uncoupling actin filament bundling, the inhibition of various proteases, including the caspases, and protein transport mediators failed to inhibit class II-mediated cell death. Neither Bid, poly-ADP-ribose polymerase, caspases-3, -7 and -8 nor FLICE-inhibitory protein were found to be cleaved during class II apoptosis. Lastly, although class II mAb induced a rapid mitochondrial membrane depolarization in DC, cell death was not inhibited by Bcl-2 over-expression in DC. The independence of this form of apoptosis from protein or RNA synthesis, coupled to the rapidity of the mitochondrial depolarization and the lack of protection by Bcl-2, suggests that mature DC express pre-formed pro-apoptotic molecules that are involved in class II-mediated death.

Amino Acid Chloromethyl Ketones↗

PubMed2002

Anatomic location and T-cell stimulatory functions of mouse dendritic cell subsets defined by CD4 and CD8 expression.

Mouse spleen contains CD4+, CD8alpha+, and CD4-/CD8alpha- dendritic cells (DCs) in a 2:1:1 ratio. An analysis of 70 surface and cytoplasmic antigens revealed several differences in antigen expression between the 3 subsets. Notably, the Birbeck granule-associated Langerin antigen, as well as CD103 (the mouse homologue of the rat DC marker OX62), were specifically expressed by the CD8alpha+ DC subset. All DC types were apparent in the T-cell areas as well as in the splenic marginal zones and showed similar migratory capacity in collagen lattices. The 3 DC subtypes stimulated allogeneic CD4+ T cells comparably. However, CD8alpha+ DCs were very weak stimulators of resting or activated allogeneic CD8+ T cells, even at high stimulator-to-responder ratios, although this defect could be overcome under optimal DC/T cell ratios and peptide concentrations using CD8+ F5 T-cell receptor (TCR)-transgenic T cells. CD8alpha- or CD8alpha+ DCs presented alloantigens with the same efficiency for lysis by cytotoxic T lymphocytes (CTLs), and their turnover rate of class I-peptide complexes was similar, thus neither an inability to present, nor rapid loss of antigenic complexes from CD8alpha DCs was responsible for the low allostimulatory capacity of CD8alpha+ DCs in vitro. Surprisingly, both CD8alpha+ DCs and CD4-/CD8- DCs efficiently primed minor histocompatibility (H-Y male antigen) cytotoxicity following intravenous injection, whereas CD4+ DCs were weak inducers of CTLs. Thus, the inability of CD8alpha+ DCs to stimulate CD8+ T cells is limited to certain in vitro assays that must lack certain enhancing signals present during in vivo interaction between CD8alpha+ DCs and CD8+ T cells.

Adoptive Transfer↗

PubMed2002

Analysis of maturation states of rat bone marrow-derived dendritic cells using an improved culture technique.

In this study, we examined in more detail the development of rat bone marrow-derived dendritic cells (BMDC). A two-stage culture system was used to propagate BMDC from rat bone marrow precursors. BMDC developed within clusters of proliferating cells after repetitive addition of rat granulocyte/macrophage colony-stimulating factor and rat interleukin (IL)-4 at a concentration of 5 ng/ml to the cultures. Fluorescence-activated cell sorter analysis performed at an early stage of development (day 6) revealed an immature phenotype with intermediate levels of major histocompatibility complex (MHC) class II expression and low levels of the costimulator molecules CD80 and CD86. Upon further culture, a strong upregulation of MHC class II, costimulatory and adhesion molecules could be observed, whereas macrophage marker antigens were downregulated. Late-stage BMDC (day 10) showed a high expression of MHC class I and II, ICAM-1, Ox62 and CD11c, and revealed a split pattern of B7-1 and B7-2. The cell yield was about 40% of the initially plated bone marrow cells with 80% MHC class II-high and less than 20% MHC class II-low positive cells. Full maturation of rat BMDC (day 12) with an almost uniform expression of B7 was achieved by subsequent subculture and further stimulation with rat tumour necrosis factor alpha (TNF-alpha), lipopolysaccharide (LPS) or soluble CD40 ligand (CD40L). Analysis of the cell supernatant revealed a strong IL-12 production after LPS or CD40L, and to a lesser extent after TNF-alpha stimulation. Additionally, LPS-treated, but not CD40L-treated BMDC secreted TNF-alpha into the supernatant. Early-stage BMDC sufficiently triggered a T cell receptor (TCR) downregulation, but did not stimulate naive T cells in an allogeneic mixed leukocyte reaction (MLR) and revealed a low stimulatory capacity in an antigen-specific T cell assay. In contrast, late-stage BMDC and especially fully mature BMDC strongly induced TCR internalisation, elicited high T cell responses in the allogeneic MLR similar to those obtained by mature rat spleen dendritic cells and efficiently activated antigen-specific T cells. In conclusion, this protocol allows easy access to large numbers of rat BMDC at defined maturation stages and selective studies for the manipulation of immune responses in rat models.

Animals↗

PubMed2002

Specific peptide-mediated immunity against established melanoma tumors with dendritic cells requires IL-2 and fetal calf serum-free cell culture.

Melanoma, despite its aggressive growth characteristics, is an antigenic tumor expressing several characterized neo- and differentiation antigens. Dendritic cells (DC) when pulsed with defined peptides have been shown to effectively induce melanoma-specific T cell responses in humans and mice. These protect animals from challenge with melanoma, but so far have failed to induce significant tumor regressions. To study the efficacy of DC-based anti-tumor vaccinations, we set up a therapeutic model using C57BL/6J mice with established pulmonary and subcutaneous metastases induced by the B16-melanoma cell line B78-D14. Mice were vaccinated twice with 20,000 antigen-presenting cells, either bone marrow-derived DC or epidermal Langerhans cells (LC), which were loaded with the tyrosinase-related protein 2 (TRP2) peptide. Generally, DC cultured with fetal calf serum (FCS) induced a dominant unspecific response. This was not seen using LC cultured without serum; however, vaccination with TRP2-loaded FCS-free LC alone failed to influence the growth of established B16 tumors. A reproducible reduction of tumor size and weight was only obtained if LC vaccinations with TRP2 were followed by a 5-day treatment of mice with 200,000 IU IL-2 intraperitoneally twice/daily. Omitting the TRP2 peptide abolished the efficacy of this combined treatment, demonstrating the crucial role of priming a melanoma-specific T cell response. Microcytotoxic assays performed with spleen-derived T cells and melanoma as well as congenic fibroblast lines as targets confirmed the TRP2-dependent specificity of LC-induced immune responses. Thus, despite the fact that tumor-specific T cells were primed, an additional IL-2-dependent stimulus was needed to translate this immune response into a therapeutic effect against established tumors.

Animals↗

PubMed2002

Aggregation of antigen-specific T cells at the inoculation site of mature dendritic cells.

Cellular immune responses are initiated by direct interaction of naive T cells with professional antigen-presenting cells, i.e., dendritic cells. In general, this interaction takes place in secondary lymphoid organs to which both naive T cells and mature dendritic cells preferentially home. This physiologic scenario differs substantially, however, from therapeutic dendritic-cell-based vaccinations used to treat human cancer. In fact, only a small fraction of intradermally injected dendritic cells migrate to the draining lymph node and the majority of cells remain at the site of inoculation. These sites are characterized by a distinct oligoclonal T cell infiltrate comprising both L-Selectin+/CD45RA+ and L-Selectin+/CD45RA- cells. Blood vessels expressing peripheral lymph node addressin represent possible entry channels for such naive and central memory T cells, the former probably attracted by dendritic cell-CK1 produced by the injected dendritic cells. In situ staining with multimeric peptide/major histocompatibility complex class I complexes revealed that infiltrating T cells specifically recognize peptide epitopes presented by the injected dendritic cells. Thus, the fraction of dendritic cells not migrating to secondary lymphatic tissue after therapeutic inoculation nevertheless seem to be involved in a specific immune modulation.

Antigens, Neoplasm↗

PubMed2002

Increased interleukin 4 (IL-4) receptor expression and IL-4-induced decrease in IL-12 production by Langerhans cells infected with Leishmania major.

Langerhans cells (LC) take up Leishmania major and are critical for the induction of the parasite-specific T-cell response. Their functional activities are regulated by cytokines. We analyzed whether infection of LC with L. major modulates the expression of their cytokine receptors. The expression of the interleukin 4 (IL-4) receptor was increased on infected LC from susceptible mice but not on those from resistant mice. Moreover, IL-4 treatment strongly decreased the lipopolysaccharide-induced IL-12 response of infected LC from susceptible mice. This modulation of IL-4 receptor expression and IL-12 production by infection of LC with Leishmania may contribute to the development of Th2 cells and to susceptibility to infection.

Animals↗