Clinical diversity and treatment approaches to blastic plasmacytoid dendritic cell neoplasm: a retrospective multicentre study.

BACKGROUND: Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare, aggressive type of haematologic precursor malignancy primarily often manifesting in the skin. We sought to provide a thorough clinical characterization and report our experience on therapeutic approaches to BPDCN. METHODS: In the present multicentric retrospective study, we collected all BPDCN cases occurring between 05/1999 and 03/2018 in 10 secondary care centres of the German-Swiss-Austrian cutaneous lymphoma working group. RESULTS: A total of 37 BPDCN cases were identified and included. Almost 90% of the patients had systemic manifestations (bone marrow, lymph nodes, peripheral blood) in addition to skin involvement. The latter presented with various types of cutaneous lesions: nodular (in more than 2/3) and bruise-like (in 1/3) skin lesions, but also maculopapular exanthema (in circa 1/6). Therapeutically, 22 patients received diverse combinations of chemotherapeutic regimens and/or radiotherapy. Despite initial responses, all of them ultimately relapsed and died from progressive disease. Eleven patients underwent haematopoietic stem cell transplantation (HSCT; autologous HSCT n = 3, allo-HSCT n = 8). The mortality rate among HSCT patients was only 33.33% with a median survival time of 60.5 months. CONCLUSION: Our study demonstrates the clinical diversity of cutaneous BPDCN manifestations and the positive development observed after the introduction of HSCT.

Exploitation of Langerhans cells for in vivo DNA vaccine delivery into the lymph nodes.

There is no clinically available cancer immunotherapy that exploits Langerhans cells (LCs), the epidermal precursors of dendritic cells (DCs) that are the natural agent of antigen delivery. We developed a DNA formulation with a polymer and obtained synthetic 'pathogen-like' nanoparticles that preferentially targeted LCs in epidermal cultures. These nanoparticles applied topically under a patch-elicited robust immune responses in human subjects. To demonstrate the mechanism of action of this novel vaccination strategy in live animals, we assembled a high-resolution two-photon laser scanning-microscope. Nanoparticles applied on the native skin poorly penetrated and poorly induced LC motility. The combination of nanoparticle administration and skin treatment was essential both for efficient loading the vaccine into the epidermis and for potent activation of the LCs to migrate into the lymph nodes. LCs in the epidermis picked up nanoparticles and accumulated them in the nuclear region demonstrating an effective nuclear DNA delivery in vivo. Tissue distribution studies revealed that the majority of the DNA was targeted to the lymph nodes. Preclinical toxicity of the LC-targeting DNA vaccine was limited to mild and transient local erythema caused by the skin treatment. This novel, clinically proven LC-targeting DNA vaccine platform technology broadens the options on DC-targeting vaccines to generate therapeutic immunity against cancer.

Targeting skin dendritic cells to improve intradermal vaccination.

Vaccinations in medicine are typically administered into the muscle beneath the skin or into the subcutaneous fat. As a consequence, the vaccine is immunologically processed by antigen-presenting cells of the skin or the muscle. Recent evidence suggests that the clinically seldom used intradermal route is effective and possibly even superior to the conventional subcutaneous or intramuscular route. Several types of professional antigen-presenting cells inhabit the healthy skin. Epidermal Langerhans cells (CD207/langerin(+)), dermal langerin(neg), and dermal langerin(+) dendritic cells (DC) have been described, the latter subset so far only in mouse skin. In human skin langerin(neg) dermal DC can be further classified based on their reciprocal expression of CD1a and CD14. The relative contributions of these subsets to the generation of immunity or tolerance are still unclear. Yet, specializations of these different populations have become apparent. Langerhans cells in human skin appear to be specialized for induction of cytotoxic T lymphocytes; human CD14(+) dermal DC can promote antibody production by B cells. It is currently attempted to rationally devise and improve vaccines by harnessing such specific properties of skin DC. This could be achieved by specifically targeting functionally diverse skin DC subsets. We discuss here advances in our knowledge on the immunological properties of skin DC and strategies to significantly improve the outcome of vaccinations by applying this knowledge.

A fusion inhibitor prevents spread of immunodeficiency viruses, but not activation of virus-specific T cells, by dendritic cells.

Dendritic cells (DCs) play a key role in innate immune responses, and their interactions with T cells are critical for the induction of adaptive immunity. However, immunodeficiency viruses are efficiently captured by DCs and can be transmitted to and amplified in CD4(+) T cells, with potentially deleterious effects on the induction of immune responses. In DC-T-cell cocultures, contact with CD4(+), not CD8(+), T cells preferentially facilitated virus movement to and release at immature and mature DC-T-cell contact sites. This occurred within 5 min of DC-T-cell contact. While the fusion inhibitor T-1249 did not prevent virus capture by DCs or the release of viruses at the DC-T-cell contact points, it readily blocked virus transfer to and amplification in CD4(+) T cells. Higher doses of T-1249 were needed to block the more robust replication driven by mature DCs. Virus accumulated in DCs within T-1249-treated cocultures but these DCs were actually less infectious than DCs isolated from untreated cocultures. Importantly, T-1249 did not interfere with the stimulation of virus-specific CD4(+) and CD8(+) T-cell responses when present during virus-loading of DCs or for the time of the DC-T-cell coculture. These results provide clues to identifying strategies to prevent DC-driven virus amplification in CD4(+) T cells while maintaining virus-specific immunity, an objective critical in the development of microbicides and therapeutic vaccines.

DC-sign+ CD163+ macrophages expressing hyaluronan receptor LYVE-1 are located within chorion villi of the placenta.

The purpose of this study was to investigate with immunohistochemical methods antigen presenting cells and their relationship to blood and lymphatic vessels in human term placenta. Fetal placental antigen presenting cells, historically also known as Hofbauer cells, were located in the chorionic villi below the syncytiotrophoblast and in the vicinity of fetal capillaries. DC-SIGN/CD209 expression was observed on CD163+, CD68+, CD45+, HLA-A,B,C+, DC-LAMP/CD208-, CD86-, Langerin/CD207-, FXIIIa-, CD1a- cells consistent with the macrophage nature of these cells. These fetal DC-SIGN+ cells lack HLA-DR, -DP, -DQ expression. Moreover, we show for the first time that they co-express the hyaluronan receptor LYVE-1. In contrast, no LYVE-1+ vessel structures, i.e. lymphatic vessels, were detected. Human term decidua hosted a variety of CD45+ cells, further phenotyped as CD163+, DC-SIGN+, CD68+, HLA-DR+, HLA-A,B,C+. Mature dendritic cells were never observed in human term placenta. In summary, human term placenta is an immunoprivileged organ without lymphatic drainage and with numerous DC-SIGN+ macrophages within the chorionic villi. We hypothesize that these cells may fulfil a function in innate responses against pathogens as well as be involved in the homeostasis of hyaluronan metabolism in the rapidly differentiating placenta.

Who is really in control of skin immunity under physiological circumstances - lymphocytes, dendritic cells or keratinocytes?

Our views of the skin immunity theatre are undergoing constant change. These not only reflect paradigm shifts in general immunology and skin biology, but also have profound clinical implications, which call for strategic changes in dermatological therapy. Nowhere can this be witnessed at a greater level of instructiveness and fascination than when addressing the question posed by this new Controversies feature. Thus, after a very long period of dominance by T cells and Langerhans cells as 'lead actors' on the skin immunity stage, the lowly keratinocyte has recently made an astounding theatrical appearance as a key protagonist of the innate skin immunity system, which may control even acquired skin immune responses. Further enhancing dramatic complexity and tension, the mast cell has entered as an additional actor claiming centre stage, and the epidermal Langerhans cell has slipped in a surprise appearance as the chief agent of immunotolerance. May you, esteemed reader, enjoy the spectacle offered here by selected immunodermatology authorities who double as 'stage managers' pushing their respective favourite actors into the limelight. You get everything you may expect from a good performance - complete with the impresario's overture that lures you into the theatre and sets the stage, competing divas, recently discovered new talents and even the critic's digest while the performance is still ongoing. By the time the curtain drops, you will have reached your own, independent conclusions on how to answer the title question of this play - at least for the time being...

CADASIL-an unusual manifestation with prominent cutaneous involvement.

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leucencephalopathy (CADASIL) is a rare vascular disorder affecting mainly the central nervous system with transient ischaemic attacks, strokes, psychiatric symptoms and dementia. It is a progressive familial disease owing to mutations in the Notch3 gene. Clinically apparent skin involvement is usually absent. Electron microscopy of seemingly uninvolved skin reveals characteristic granular deposits in the basal lamina of vessels and adnexals. We report on a case of CADASIL with generalized haemorrhagic macules and patches. Typical neurological symptoms as well as classical findings in histopathology and electron microscopy confirmed the diagnosis. Immunofluorescence showed an increased number of vessels with walls markedly thickened by deposits of fibrin, complement and immunoglobulins. This method could serve as an additional method for accurate diagnosis of CADASIL.

Human herpesvirus-8 infection of umbilical cord-blood-derived CD34+ stem cells enhances the immunostimulatory function of their dendritic cell progeny.

CD34(+) progenitor cells carrying human herpesvirus-8, Kaposi's sarcoma-associated herpesvirus (HHV-8/KSHV), have been described in the peripheral blood of AIDS patients suffering from Kaposi's sarcoma (KS). In this study, we investigated the influence of HHV-8 on the differentiation of CD34(+) progenitor cells. Native CD34(+) cells derived from cord blood could be infected by a laboratory strain of HHV-8, as shown by immunofluorescence staining and polymerase chain reaction, but no significant initial maturation/differentiation effects were observed. In addition, these infected cells were differentiated into immature and mature dendritic cells (DCs) using cytokine induction with recombinant human granulocyte-macrophage colony-stimulating factor (rhGm-CSF), recombinant human tumor necrosis factor (rhTNF-alpha) and recombinant human stem cell factor (rhSCF). Double immunofluorescence and flow cytometry studies demonstrated that virus infection did not impair the development of immature and mature DC populations. Subsequently, the immunostimulating capacity of DC populations was tested in a mixed lymphocyte reaction using allogeneic T-cells. The HHV-8-infected CD34(+) progenitor cell-derived mature DC population showed a significantly enhanced antigen-presenting capacity, compared to non-infected DCs, which was not observed with the immature DCs. This suggests stimulation of DC function by HHV-8 infection. Because there are only a small percentage of HHV-8-positive DCs in the preparations and because it is not clear whether infection is abortive or productive to some extent, this seems to be most likely due to an indirect viral effect.

Trichothiodystrophy: quantification of cysteine in human hair and nails by application of sodium azide-dependent oxidation to cysteic acid.

The term "trichothiodystrophy" (TTD) covers several autosomal recessive diseases whose diagnostic hallmark is short, brittle hair low in sulfur and cystine because of impaired synthesis of high-sulfur matrix protein. Clinical symptoms associated with TTD represent a variable range of abnormalities in organs derived from ectoderm and neuroectoderm. Important laboratory tests of the hair for the diagnosis of TTD comprise polarizing microscopy ("tiger-tail" pattern), electron microscopy, and amino acids analysis of hydrolyzed hair with a special focus on cystine. However, only very few institutions determine the amino acid composition of human hair and nail clippings, which requires special sample preparation including hydrolysis. If no special precautions are taken, quantification of cysteine and cystine becomes inaccurate because of decomposition of these residues during hydrolysis. We therefore performed the sample work-up with azide-dependent oxidation which we have for the first time adapted for analysis of hair and nail clippings. With our control and parent data resembling published data on hair and nail samples, we obtained a decreased proportion of cysteine (half cystine, determined as cysteic acid) in materials obtained from a boy with TTD. Clearly, the method for the quantification of cysteine following sodium azide-dependent oxidation is a suitable and rather convenient approach to the quantification of cyst(e)ine and other amino acids in hair and nail proteins, and is a valuable contribution to the diagnosis of TTD.

Expression of the C-C chemokine MIP-3 alpha/CCL20 in human epidermis with impaired permeability barrier function.

External assault to the skin is followed by an epidermal response including synthesis of DNA, lipids, cytokines and migration of antigen presenting cells. MIP-3 alpha (CCL20, LARC, Exodus-1, Scya20) is a recently described C-C chemokine, predominantly expressed in extralymphoid tissue, which is known to direct migration of dendritic cell precursors and memory lymphocytes to sites of antigen invasion. We assessed the expression of MIP-3 alpha in human skin using semi-quantitative polymerase chain reaction. In vivo, MIP-3 alpha mRNA was constitutively expressed at low levels in untreated human epidermis. After acute disruption of the epidermal permeability barrier MIP-3 alpha mRNA was upregulated in the epidermal fraction, whereas dermal MIP-3 alpha mRNA levels remained unchanged. In vitro, MIP-3 alpha was increased in cultured keratinocytes treated with IL-1 alpha and TNF-alpha and was present in immature and mature dendritic cells, THP-1 monocytic cells and activated T cells. Finally, skin biopsies from patients with psoriasis, contact dermatitis and mycosis fungoides showed abundant expression. In biopsies from atopic dermatitis and graft vs. host disease a weak signal was present, whereas no expression was found in scleroderma and toxic epidermal necrolysis. We conclude that regulation of MIP-3 alpha mRNA is part of the epidermal response to external assault. Its upregulation may represent a danger signal for increased immunosurveillance in barrier disrupted skin and inflammatory skin conditions with impaired barrier function to counteract potential antigen invasion.

Infectious and whole inactivated simian immunodeficiency viruses interact similarly with primate dendritic cells (DCs): differential intracellular fate of virions in mature and immature DCs.

As potential targets for human immunodeficiency virus type 1 and simian immunodeficiency virus (HIV-1 and SIV), dendritic cells (DCs) likely play a significant role in the onset and spread of infection as well as in the induction of antiviral immunity. Using the SIV-macaque system to study the very early events in DC-virus interactions, we compared chemically inactivated SIV having conformationally and functionally intact envelope glycoproteins (2,2'-dithiodipyridine [AT-2] SIV) to infectious and heat-treated SIV. Both human and macaque DCs interact similarly with SIV without detectable effects on DC viability, phenotype, or endocytic function. As assessed by measuring cell-associated viral RNA, considerable amounts of virus are captured by the DCs and this is reduced when the virus is heat treated or derived from a strain that expresses low levels of envelope glycoprotein. Immunostaining for SIV proteins and electron microscopy indicated that few intact virus particles are retained at the periphery of the endocytically active, immature DCs. This contrasts with a perinuclear localization of numerous virions in large vesicular compartments deeper within mature DCs (in which macropinocytosis is down-regulated). Both immature and mature DCs are capable of clathrin-coated pit-mediated uptake of SIV, supporting the notion that the receptor-mediated uptake of virus can occur readily in mature DCs. While large numbers of whole viruses were preferentially found in mature DCs, both immature and mature DCs contained similar amounts of viral RNA, suggesting that different uptake/virus entry mechanisms are active in immature and mature DCs. These findings have significant implications for cell-to-cell transmission of HIV-1 and SIV and support the use of AT-2 SIV, an authentic but noninfectious form of virus, as a useful tool for studies of processing and presentation of AT-2 SIV antigens by DCs.

Dendritic cell vaccines for cancer therapy.

Dendritic cells (DC) are professional antigen-presenting cells whose primary function is the initiation of immune response. Based on the finding that the immune system usually fails to identify and kill cancer cells, DC have been recently used as vaccines for stimulation of tumour-specific immunity. This review focuses on pitfalls related to DC-based vaccination against solid tumours and on improvement of this immunotherapeutic approach for routine treatment of cancer disease.

Granulomatous slack skin: a distinct disorder or a variant of mycosis fungoides?

About 75% of cutaneous lymphomas belong to the group of T-cell lymphomas. Mycosis fungoides is the most common entity in this group. Granulomatous slack skin is a rare form of cutaneous T-cell lymphoma closely related to mycosis fungoides. We present here a patient with areas of lax skin for several years who developed a generalized erythroderma with associated immunoactivation and a deterioration in his general condition. This report discusses clinically and histologically the differential diagnoses, namely granulomatous slack skin and granulomatous mycosis fungoides, and suggests that these 2 disorders are only variants in the broad spectrum of a single disease.

Migration of dendritic cells into lymphatics-the Langerhans cell example: routes, regulation, and relevance.

Dendritic cells are leukocytes of bone marrow origin. They are central to the control of the immune response. Dendritic cells are highly specialized in processing and presenting antigens (microbes, proteins) to helper T lymphocytes. Thereby, they critically regulate further downstream processes such as the development of cytotoxic T lymphocytes, the production of antibodies by B lymphocytes, or the activation of macrophages. A new field of dendritic cell biology is the study of their potential role in inducing peripheral tolerance. The immunogenic/tolerogenic potential of dendritic cells is increasingly being utilized in immunotherapy, particularly for the elicitation of antitumor responses. One very important specialization of dendritic cells is their outstanding capacity to migrate from sites of antigen uptake to lymphoid organs. Much has been learned about this process from studying one particular type of dendritic cell, namely, the Langerhans cell of the epidermis. Therefore, the migratory properties of Langerhans cells are reviewed. Knowledge about this "prototype dendritic cell" may help researchers to understand migration of other types of dendritic cells.

Interleukin-16 supports the migration of Langerhans cells, partly in a CD4-independent way.

Migration of cutaneous dendritic cells is essential for the induction of primary immune responses. Chemotaxis plays an important part in guiding migrating cells through the skin. Therefore, we investigated the influence of interleukin-16, a potent chemoattractant, on the migratory properties of cutaneous dendritic cells. Interleukin-16 added to murine and human skin explant cultures, enhanced emigration of Langerhans cells as well as dermal dendritic cells out of the skin. In contrast to tumor necrosis factor-alpha, intradermally injected interleukin-16 did not reduce the density of Langerhans cells suggesting a chemotactic rather than a mechanistic migration-inducing effect of interleukin-16. In support of these findings, the known migration-promoting effect of tumor necrosis factor-alpha in skin explant cultures could be neutralized by anti-interleukin-16 antibody and vice versa, indicating different but cooperative ways of action for both cytokines. In whole skin explant cultures blocking of the interleukin-16 effect was also achieved with a monoclonal antibody against CD4, the receptor for interleukin-16. In contrast, in cultures of murine epidermis alone no blocking by anti-CD4 became obvious and in CD4-deficient mice Langerhans cell migration in response to interleukin-16 was maintained. This suggests that another receptor for interleukin-16 might be operative for Langerhans cells in the mouse epidermis. Finally, we detected interleukin-16-positive cells in the dermis of skin explants, tumor necrosis factor-alpha-treated and contact allergen-treated skin. Taken together, it seems likely that locally secreted interleukin-16 might serve to enhance the migration of cutaneous dendritic cells and optimize the response to foreign antigen encountering the skin.

Generation of large numbers of human dendritic cells from whole blood passaged through leukocyte removal filters: an alternative to standard buffy coats.

Many blood banks now use whole blood inline filtration to produce leukocyte-depleted blood products. As a result, a common source of large numbers of human dendritic cells (DC) for research purposes, namely standard buffy coats, has been lost. Therefore, we have adapted our conventional method for growing DC from CD14(+) precursors in order to make use of these filter units. A dextran solution containing human serum albumin was used to flush back the filters. After pelleting, mononuclear cells were obtained by standard density gradient centrifugation (Lymphoprep). To eliminate T cells, we used rosetting with sheep red blood cells. In addition to the classical PBMC, the cell population obtained after Lymphoprep centrifugation was found to contain high numbers of CD14(+) granulocytes which could be depleted by separation on an additional Percoll gradient. At this stage, FACS analysis revealed a cell population that resembled the CD14(+) monocyte-enriched population, obtained from traditional buffy coat preparations after Lymphoprep centrifugation and T cell elimination. Culture of the cells and the induction of maturation was identical to the previously described procedures, except that the culture time was reduced from 7 to 5 days and the maturation time from 3 to 2 days. Analyses of the major molecules indicative of DC maturation (CD83, CD86, CD208/DC-LAMP) and functional analyses of the T cell-stimulatory capacity of the DC population (using the MLR assay with normal peripheral T cells and naive T cells) revealed no major differences from buffy coat-derived DC preparations.

Network of vascular-associated dendritic cells in intima of healthy young individuals.

In earlier studies, our group has established a new "immunological" hypothesis for atherogenesis supported by experimental and clinical studies showing that inflammatory immunological reactions against heat shock protein 60 initiate the development of atherosclerosis. In the present study, we describe the discovery of a so-far-unknown network of dendritic cells in the innermost layer of arteries, the intima, but not veins of healthy humans and rabbits. The number of these dendritic cells is comparable to that of Langerhans cells in the skin, and dendritic cells show a similar phenotype (CD1a(+) S-100(+) lag(+) CD31(-) CD83(-) CD86(-) and no staining for von Willebrand factor or smooth muscle cell myosin). These vascular-associated dendritic cells accumulate most densely in those arterial regions that are subjected to major hemodynamic stress by turbulent flow conditions and are known to be predisposed for the later development of atherosclerosis. These results open new perspectives for the activation of the immune system within the arterial wall.

Production of IL-12 by human monocyte-derived dendritic cells is optimal when the stimulus is given at the onset of maturation, and is further enhanced by IL-4.

Dendritic cells produce IL-12 both in response to microbial stimuli and to T cells, and can thus skew T cell reactivity toward a Th1 pattern. We investigated the capacity of dendritic cells to elaborate IL-12 with special regard to their state of maturation, different maturation stimuli, and its regulation by Th1/Th2-influencing cytokines. Monocyte-derived dendritic cells were generated with GM-CSF and IL-4 for 7 days, followed by another 3 days +/- monocyte-conditioned media, yielding mature (CD83(+)/dendritic cell-lysosome-associated membrane glycoprotein(+)) and immature (CD83(-)/dendritic cell-lysosome-associated membrane glycoprotein(-)) dendritic cells. These dendritic cells were stimulated for another 48 h, and IL-12 p70 was measured by ELISA. We found the following: 1) Immature dendritic cells stimulated with CD154/CD40 ligand or bacteria (both of which concurrently also induced maturation) secreted always more IL-12 than already mature dendritic cells. Mature CD154-stimulated dendritic cells still made significant levels (up to 4 ng/ml). 2) Terminally mature skin-derived dendritic cells did not make any IL-12 in response to these stimuli. 3) Appropriate maturation stimuli are required for IL-12 production: CD40 ligation and bacteria are sufficient; monocyte-conditioned media are not. 4) Unexpectedly, IL-4 markedly increased the amount of IL-12 produced by both immature and mature dendritic cells, when present during stimulation. 5) IL-10 inhibited the production of IL-12. Our results, employing a cell culture system that is now being widely used in immunotherapy, extend prior data that IL-12 is produced most abundantly by dendritic cells that are beginning to respond to maturation stimuli. Surprisingly, IL-12 is only elicited by select maturation stimuli, but can be markedly enhanced by the addition of the Th2 cytokine, IL-4.