The role of EBI-3 in a murine model of lung metastases of melanoma

In dieser Arbeit wurde die Rolle des Epstein-Barr Virus induzierten Gens 3 in einem Mausmodel des durch B16-F10 Zellen hervorgerufenen metastasierenden Melanoms untersucht. Das von aktivierten antigenpräsentierenden Zellen exprimierte EBI-3 gehört zur Familie der löslichen Typ 1 Zytokinrezeptoren, weist eine hohe Homologie zur p40 Untereinheit des IL-12 auf und bildet zusammen mit p28 das IL-27. Die intravenöse Injektion der B16-F10 Zelllinie führte zu einer signifikanten Erniedrigung der Tumormetastasen in den EBI-3 defizienten Lungen sowie zu einer höheren Lebenserwartung dieser Mäuse im Vergleich zu den B6 Wildtypen. Darüber hinaus habe ich in den EBI-3 defizienten Mäusen eine verminderte VCAM-1 Expression auf den Endothelzellen der Lunge gefunden während Änderungen in der VEGF Expression nicht detektiert wurden. Der immunologische Hintergrund, der diesen therapeutischen Effekt hervorrief, konnte durch die T-Zellaktivierung durch die kürzlich neu beschriebene DC Population, welche Interferon-produzierende Killer Dendritische Zellen genannt werden (IK-DC), die zusätzlich von aktivierten und maturierten klassischen DCs unterstützt wurden, erklärt werden. IK-DCs von EBI-3 defizienten Mäusen produzierten höhere Mengen an IFN-g während die klassischen DCs MHC und co-stimulatorische Moleküle exprimierten, welche die Sekretion von IL-12 initiierten. Das Zusammenspiel der genannten Faktoren induzierte eine verstärkte CD4 und CD8 T-Zellantwort in den Lungen dieser Mäuse. Dies wiederum resultierte im TNF- und TRAIL abhängigen programmierten Zelltod der B16-F10 Melanomzellen in den Lungen der EBI-3 defizienten Mäuse, wohingegen sowohl weitere anti-apoptotische Mechanismen als auch T regulatorische Zellen keinen Einfluss auf die in den EBI-3 defizienten Mäusen beobachtete Tumorabwehr zu spielen scheint. Schlussendlich konnten EBI-3 defiziente CD8+ T-Zellen, welche zuvor mit Tumorantigen geprimed wurden, adoptiv in B6 Wildtypmäuse transferiert werden, was zeigte, dass diese Zellen in der Lage sind, die Tumormasse in den Empfängermäusen signifikant zu verringern. Zusammengefasst, demonstrieren diese Daten, dass das Blockieren von EBI-3 im metastasierenden Melanom ein vielversprechender Angriffspunkt in der Tumortherapie darstellt.

Tonsilar metastases of melanoma in an older person

We presented a rare case of metastasis of melanoma in palatine tonsils alerting healthcare professionals to this diagnose in black oral lesions. © The Author 2013. Published by Oxford University Press on behalf of the British Geriatrics Society. All rights reserved.

Intraepidermal Epidermotropic Metastatic Melanoma: a Clinical and Histopathological Mimicker of Melanoma in Situ Occurring in Multiplicity

The distinction between primary melanoma and melanoma metastatic to the skin has major prognostic implications. We report a case of a 67-year-old male with a diagnosis of a superficial spreading melanoma (stage IB) rendered 6 years earlier who presented clinically with an atypical nevus on his left thigh. Histopathological examination showed an intraepidermal melanocytic proliferation that was interpreted as melanoma in situ. Subsequently, 45 additional pigmented macules appeared in crops over a 9-month period. Clinically and dermoscopically, these lesions were extremely polymorphic. Histopathological findings were compatible with melanoma in situ, as each lesion consisted of a wholly intraepidermal proliferation of markedly atypical melanocytes arranged singly and in nests. A complete gastrointestinal study showed multiple pigmented metastatic lesions throughout the stomach and small bowel, which supported a diagnosis of metastatic melanoma with gastrointestinal and epidermotropic skin involvement. Monosomy of chromosome 9 and a BRAF V600E mutation were detected in the primary tumor sample and in macro-dissected secondary lesions. No CDKN2A or CDK4 germline mutations were found. Intraepidermal epidermotropic metastases of melanoma have been rarely described in literature. In this case, histopathology alone was insufficient to distinguish metastatic melanoma from multiple in situ melanomas. The recognition of epidermotropic metastases should be based on the correlation between clinical, dermoscopic, histopathological and molecular findings.

Proliferation in monocyte-derived dendritic cell cultures is caused by progenitor cells capable of myeloid differentiation

Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes. DC were generated from myeloid-enriched PB non-T cells or sorted monocytes. DC generated from either population functioned as potent antigen-presenting cells. Uptake of [H-3]-thymidine was observed in DC cultured from myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor necrosis factor-alpha led to maturation of the DC, but did not inhibit proliferation. Ki67(+) cells were observed in cytospins of these DC, and by double staining were CD3(-)CD19(-)CD11c(-)CD40(-) and myeloperoxidase(+), suggesting that they were myeloid progenitor cells. Analysis of the starting population by flow cytometry demonstrated small numbers of CD34(+)CD33(-)CD14(-) progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays. Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF. Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes. (C) 1998 by The American Society of Hematology.

Proliferation in monocyte-derived dendritic cell cultures is due to cells capable of myeloid differentiation

Dendritic cells (DC) can be generated by culture of adherent peripheral blood (PB) cells in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4). There is controversy as to whether these DC arise from proliferating precursors or simply from differentiation of monocytes. DC were generated from myeloid-enriched PB non-T cells or sorted monocytes. DC generated from either population functioned as potent antigen-presenting cells. Uptake of [H-3]-thymidine was observed in DC cultured from myeloid-enriched non-T cells. Addition of lipopolysaccharide or tumor necrosis factor-alpha led to maturation of the DC, but did not inhibit proliferation. Ki67(+) cells were observed in cytospins of these DC, and by double staining were CD3(-)CD19(-)CD11c(-)CD40(-) and myeloperoxidase(+), suggesting that they were myeloid progenitor cells. Analysis of the starting population by flow cytometry demonstrated small numbers of CD34(+)CD33(-)CD14(-) progenitor cells, and numerous granulocyte-macrophage colony-forming units were generated in standard assays. Thus, production of DC in vitro from adherent PB cells also enriches for progenitor cells that are capable of proliferation after exposure to GM-CSF. Of clinical importance, the yield of DC derived in the presence of GM-CSF and IL-4 cannot be expanded beyond the number of starting monocytes. (C) 1998 by The American Society of Hematology.

Increased alpha-defensins 1-3 production by dendritic cells in HIV-infected individuals is associated with slower disease progression.

Defensins are natural endogenous antimicrobial peptides with potent anti-HIV activity and immuno-modulatory effects. We recently demonstrated that immature dendritic cells (DC) produce α-defensins1-3 and that α-defensins1-3 modulate DC generation and maturation. Since DC-HIV interaction plays a critical role during the first steps of HIV infection, we investigated the possible impact of α-defensins1-3 production by DC on disease progression.

Increased alpha-defensins 1-3 production by dendritic cells in HIV-infected individuals is associated with slower disease progression.

Defensins are natural endogenous antimicrobial peptides with potent anti-HIV activity and immuno-modulatory effects. We recently demonstrated that immature dendritic cells (DC) produce α-defensins1-3 and that α-defensins1-3 modulate DC generation and maturation. Since DC-HIV interaction plays a critical role during the first steps of HIV infection, we investigated the possible impact of α-defensins1-3 production by DC on disease progression.

MHC class II expression is regulated in dendritic cells independently of invariant chain degradation

We have investigated the mechanisms that control MHC class II (MHC II) expression in immature and activated dendritic cells (DC) grown from spleen and bone marrow precursors. Degradation of the MHC II chaperone invariant chain (li), acquisition of peptide cargo by MHC II, and delivery of MHC II-peptide complexes to the cell surface proceeded similarly in both immature and activated DC. However, immature DC reendocytosed and then degraded the MHC II-peptide complexes much faster than the activated DC. MHC II expression in DC is therefore not controlled by the activity of the protease(s) that degrade Ii, but by the rate of endocytosis of peptide-loaded MHC II. Late after activation, DC downregulated MHC II synthesis both in vitro and in vivo.

Lentiviral vector-mediated tyro sinase-related protein 2 gene transfer to dendritic cells for the therapy of melanoma

Dendritic cells (DCs) are the most potent professional antigen-presenting cells (APCs), which play a vital role in primary immune responses. Introducing genes into DCs will allow constitutive expression of the encoded proteins and thus prolong the presentation of the antigens derived therefrom. In addition, multiple and unidentified epitopes encoded by the entire tumor-associated antigen (TAA) gene may enhance T cell activation. This study demonstrated that an HIV-1-based lentiviral vector conferred efficient gene transfer to DCs. The transgene, murine tyrosinase-related protein 2 (mTRP-2), encodes a clinically relevant melanoma-associated antigen (MAA), which has been found to be a tumor rejection antigen for B16 melanoma. The transfer and proper processing of mTRP-2 in DCs, in terms of RNA transcription activity and protein expression, were verified by RT-PCR and specific antibody, respectively. Administration of mTRP-2 gene-modified DCs (DC-HR'CmT2) to C57BL/6 mice evoked strong protection against tumor challenge, for which the presence of CD4(+) and CD8(+) cells during both the priming and challenge phase was essential. In a therapy model, our results showed that four of seven mice with preestablished tumor remained tumor free for 80 days after therapeutic vaccination. Given the results shown in this study, mTRP-2 gene transfer to DCs provides a potential therapeutic strategy for the management of melanoma, especially in the early stage of the disease.

Ca2+-activated K+ channels in rat otic ganglion cells: Role of Ca2+ entry via Ca2+ channels and nicotinic receptors

1. Intracellular recordings were made from neurones in the rat otic ganglion in vitro in order to investigate their morphological, physiological and synaptic properties. We took advantage of the simple structure of these cells to test for a possible role of calcium influx via nicotinic acetylcholine receptors during synaptic transmission. 2. Cells filled with biocytin comprised a homogeneous population with ovoid somata and sparse dendritic trees. Neurones had resting membrane potentials of -53 +/- 0.7 mV (n = 69), input resistances of 112 + 7 M Omega, and membrane time constants of 14 +/- 0.9 ms (n = 60). Upon depolarization, all cells fired overshooting action potentials which mere followed by an apamin-sensitive after-hyperpolarization (AHP). In response to a prolonged current injection, all neurones fired tonically. 3. The repolarization phase of action potentials had a calcium component which was mediated by N-type calcium channels. Application of omega-conotoxin abolished both the repolarizing hump and the after-hgrperpolarization suggesting that calcium influx via N-type channels activates SK-type calcium-activated potassium channels which underlie the AHP. 4. The majority (70%) of neurones received innervation from a single preganglionic fibre which generated a suprathreshold excitatory postsynaptic potential mediated by nicotinic acetylcholine receptors. The other 30% of neurones also had one or more subthreshold nicotinic inputs. 5. Calcium influx via synaptic nicotinic receptors contributed to the AHP current, indicating that this calcium has access to the calcium-activated potassium channels and therefore plays a role in regulating cell excitability.

Dendritic branching patterns of pyramidal cells in the visual cortex of the new world marmoset monkey, with comparative notes on the old world macaque monkey

The basal dendritic arbors of 442 supragranular pyramidal cells in visual cortex of the marmoset monkey were compared by fractal analyses. As detailed in a previous study,(1) individual cells were injected with Lucifer Yellow and processed for a DAB reaction product. The basal dendritic arbors were drawn, in the tangential plane, and the fractal dimension (D) determined by the dilation method. The fractal dimensions were compared between cells in ten cortical areas containing cells involved in visual processing, including the primary visual area (Vi), the second visual area (V2), the dorsoanterior area (DA), the dorsomedial area (DM), the dorsolateral. area (DL), the middle temporal area (MT), the posterior parietal area (PP), the fundus of the superior temporal area (FST) and the caudal and rostral subdivisions of inferotemporal cortex (ITc and ITr, respectively). Of 45 pairwise interareal comparisons of the fractal dimension of neurones, 20 were significantly different. Moreover, comparison of data according to previously published visual processing pathways revealed a trend for cells with greater fractal dimensions in higher cortical areas. Comparison of the present results with those in homologous cortical areas in the macaque monkey(2) revealed some similarities between the two species. The similarity in the trends of D values of cells in both species may reflect developmental features which, result in different functional attributes.

Complex interplay between LPS and IL-10 in dendritic cells: uncoupling of inflammatory chemokine receptors and positive effects on B cell chemokines

Abstract: Protective immune responses against pathogen invasion and transformed cells requires the coordinated action of distinct leukocyte subsets and soluble factors, overall termed immunological network. Among antigen-presenting cells (APC), a crucial role is played by dendritic cells (DC), which initiate, amplify and determine the outcome of the immune response. Micro-environmental conditions profoundly influence DC in such ways that the resulting immune response ranges from successful immune stimulation to abortive response or immune suppression. For instance, the presence in the milieu of anti-inflammatory cytokine interleukin-10 (IL-10) reverts most of the effects mediated on DC by even strong pro-inflammatory agents such as bacterial Lipopolysaccharide (LPS), in terms of differentiation, activation and functions. In an environment containing both LPS and IL-10, uncoupling of receptors for inflammatory chemokines already occurs after a few hours and in a reversible manner on DC, allowing scavenging of chemokines and, consequently, attenuation of the inflammatory process which could be deleterious to the organism. By studying the effects on DC of concomitant stimulation by LPS and IL-10 from the gene expression point of view, we were able to define four distinct transcriptional programs: A. the inhibition of inflammation and immunity, B. the regulation of tissue remodeling, C. the tuning of cytokine/growth factor receptors and G protein-coupled receptors, D. the stimulation of B cell function and lymphoid tissue neogenesis. Among the latter genes, we further demonstrated that IL-10 synergizes with Toll-like receptor ligands for the production of functionally active B cell attracting chemokine CXCL13. Our data provide evidence that the combined exposure of APC to LPS and IL-10, via the production of CXCL13, involves humoral immunity by attracting antibody-producing cells. It is well known that the persistent release of CXCL13 leads to the development of ectopic lymphoid tissue aggregates and production of high levels of antibodies, thus favoring the induction of auto-immunity. Our findings suggest that the IL-10 produced in chronic inflammatory conditions may promote lymphoid tissue neogenesis through increased release of CXCL13. IL-10 is an anti-inflammatory cytokine inhibiting cellular-mediated TH 1-polarized immune responses. In this study we demonstrate that IL- 10 strongly supports the development of humoral immunity. IL-10 and CXCL13 can thus be targets for specific therapies in auto-immune diseases.

Exhaustion of tumor-specific CD8⁺ T cells in metastases from melanoma patients.

In chronic viral infections, CD8⁺ T cells become functionally deficient and display multiple molecular alterations. In contrast, only little is known of self- and tumor-specific CD8⁺ T cells from mice and humans. Here we determined molecular profiles of tumor-specific CD8⁺ T cells from melanoma patients. In peripheral blood from patients vaccinated with CpG and the melanoma antigen Melan-A/MART-1 peptide, we found functional effector T cell populations, with only small but nevertheless significant differences in T cells specific for persistent herpesviruses (EBV and CMV). In contrast, Melan-A/MART-1-specific T cells isolated from metastases from patients with melanoma expressed a large variety of genes associated with T cell exhaustion. The identified exhaustion profile revealed extended molecular alterations. Our data demonstrate a remarkable coexistence of effector cells in circulation and exhausted cells in the tumor environment. Functional T cell impairment is mediated by inhibitory receptors and further molecular pathways, which represent potential targets for cancer therapy.

PD-1 and Tim-3 regulate the expansion of tumor antigen-specific CD8⁺ T cells induced by melanoma vaccines.

Although melanoma vaccines stimulate tumor antigen-specific CD8(+) T cells, objective clinical responses are rarely observed. To investigate this discrepancy, we evaluated the character of vaccine-induced CD8(+) T cells with regard to the inhibitory T-cell coreceptors PD-1 and Tim-3 in patients with metastatic melanoma who were administered tumor vaccines. The vaccines included incomplete Freund's adjuvant, CpG oligodeoxynucleotide (CpG), and the HLA-A2-restricted analog peptide NY-ESO-1 157-165V, either by itself or in combination with the pan-DR epitope NY-ESO-1 119-143. Both vaccines stimulated rapid tumor antigen-specific CD8(+) T-cell responses detected ex vivo, however, tumor antigen-specific CD8(+) T cells produced more IFN-γ and exhibited higher lytic function upon immunization with MHC class I and class II epitopes. Notably, the vast majority of vaccine-induced CD8(+) T cells upregulated PD-1 and a minority also upregulated Tim-3. Levels of PD-1 and Tim-3 expression by vaccine-induced CD8(+) T cells at the time of vaccine administration correlated inversely with their expansion in vivo. Dual blockade of PD-1 and Tim-3 enhanced the expansion and cytokine production of vaccine-induced CD8(+) T cells in vitro. Collectively, our findings support the use of PD-1 and Tim-3 blockades with cancer vaccines to stimulate potent antitumor T-cell responses and increase the likelihood of clinical responses in patients with advanced melanoma.

Functional education of invariant NKT cells by dendritic cell tuning of SHP-1.

Invariant NKT (iNKT) cells play key roles in host defense by recognizing lipid Ags presented by CD1d. iNKT cells are activated by bacterial-derived lipids and are also strongly autoreactive toward self-lipids. iNKT cell responsiveness must be regulated to maintain effective host defense while preventing uncontrolled stimulation and potential autoimmunity. CD1d-expressing thymocytes support iNKT cell development, but thymocyte-restricted expression of CD1d gives rise to Ag hyperresponsive iNKT cells. We hypothesized that iNKT cells require functional education by CD1d(+) cells other than thymocytes to set their correct responsiveness. In mice that expressed CD1d only on thymocytes, hyperresponsive iNKT cells in the periphery expressed significantly reduced levels of tyrosine phosphatase SHP-1, a negative regulator of TCR signaling. Accordingly, heterozygous SHP-1 mutant mice displaying reduced SHP-1 expression developed a comparable population of Ag hyperresponsive iNKT cells. Restoring nonthymocyte CD1d expression in transgenic mice normalized SHP-1 expression and iNKT cell reactivity. Radiation chimeras revealed that CD1d(+) dendritic cells supported iNKT cell upregulation of SHP-1 and decreased responsiveness after thymic emigration. Hence, dendritic cells functionally educate iNKT cells by tuning SHP-1 expression to limit reactivity.