Evidence that a neutrophil-keratinocyte crosstalk is an early target of IL-17A inhibition in psoriasis.

The response of psoriasis to antibodies targeting the interleukin (IL)-23/IL-17A pathway suggests a prominent role of T-helper type-17 (Th17) cells in this disease. We examined the clinical and immunological response patterns of 100 subjects with moderate-to-severe psoriasis receiving 3 different intravenous dosing regimens of the anti-IL-17A antibody secukinumab (1 × 3 mg/kg or 1 × 10 mg/kg on Day 1, or 3 × 10 mg/kg on Days 1, 15 and 29) or placebo in a phase 2 trial. Baseline biopsies revealed typical features of active psoriasis, including epidermal accumulation of neutrophils and formation of microabscesses in >60% of cases. Neutrophils were the numerically largest fraction of infiltrating cells containing IL-17 and may store the cytokine preformed, as IL-17A mRNA was not detectable in neutrophils isolated from active plaques. Significant clinical responses to secukinumab were observed 2 weeks after a single infusion, associated with extensive clearance of cutaneous neutrophils parallel to the normalization of keratinocyte abnormalities and reduction of IL-17-inducible neutrophil chemoattractants (e.g. CXCL1, CXCL8); effects on numbers of T cells and CD11c-positive dendritic cells were more delayed. Histological and immunological improvements were generally dose dependent and not observed in the placebo group. In the lowest-dose group, a recurrence of neutrophils was seen in some subjects at Week 12; these subjects relapsed faster than those without microabscesses. Our findings are indicative of a neutrophil-keratinocyte axis in psoriasis that may involve neutrophil-derived IL-17 and is an early target of IL-17A-directed therapies such as secukinumab.

Notch is active in Langerhans cell histiocytosis and confers pathognomonic features on dendritic cells.

Langerhans cell histiocytosis (LCH) is an enigmatic disease defined by the accumulation of Langerhans cell-like dendritic cells (DCs). In the present study, we demonstrate that LCH cells exhibit a unique transcription profile that separates them not only from plasmacytoid and myeloid DCs, but also from epidermal Langerhans cells, indicating a distinct DC entity. Molecular analysis revealed that isolated and tissue-bound LCH cells selectively express the Notch ligand Jagged 2 (JAG2) and are the only DCs that express both Notch ligand and its receptor. We further show that JAG2 signaling induces key LCH-cell markers in monocyte-derived DCs, suggesting a functional role of Notch signaling in LCH ontogenesis. JAG2 also induced matrix-metalloproteinases 1 and 12, which are highly expressed in LCH and may account for tissue destruction in LCH lesions. This induction was selective for DCs and was not recapitulated in monocytes. The results of the present study suggest that JAG2-mediated Notch activation confers phenotypic and functional aspects of LCH to DCs; therefore, interference with Notch signaling may be an attractive strategy to combat this disease.

T cell stimulator cells, an efficient and versatile cellular system to assess the role of costimulatory ligands in the activation of human T cells.

It is well established that full activation of T cells requires the interaction of the TCR complex with the peptide-MHC complex (Signal 1) and additional signals (Signal 2). These second signals are generated by the interaction of costimulatory ligands expressed on antigen presenting cells with activating receptors on T cells. In addition, T cell responses are negatively regulated by inhibitory costimulatory pathways. Since professional antigen presenting cells (APC) harbour a plethora of stimulating and inhibitory surface molecules, the contribution of individual costimulatory molecules is difficult to assess on these cells. We have developed a system of stimulator cells that can give signal 1 to human T cells via a membrane bound anti-CD3 antibody fragment. By expressing human costimulatory ligands on these cells, their role in T cell activation processes can readily be analyzed. We demonstrate that T cell stimulator cells are excellent tools to study various aspects of human T cell costimulation, including the effects of immunomodulatory drugs or how costimulatory signals contribute to the in vitro expansion of T cells. T cell stimulator cells are especially suited for the functional evaluation of ligands that are implicated in costimulatory processes. In this study we have evaluated the role of the CD2 family member CD150 (SLAM) and the TNF family member TL1A (TNFSF15) in the activation of human T cells. Whereas our results do not point to a significant role of CD150 in T cell activation we found TL1A to potently costimulate human T cells. Taken together our results demonstrate that T cell stimulator cells are excellent tools to study various aspects of costimulatory processes.

Induction of targeted cell migration by cutaneous administration of a DNA vector encoding a biologically active chemokine CCL21.

Skin inflammation can induce local expression of CCL21, which is subsequently drained to lymph nodes (LNs) influencing their cellular composition. To determine whether the same can be achieved by dermal administration of a plasmid DNA (pDNA) encoding CCL21, we generated a pDNA-based gene construct allowing high-level expression of CCL21. Expression and secretion of biologically active CCL21 were confirmed in vitro by immunohistochemistry, western blot analysis, ELISA, and transwell chemotactic assays. In vivo experiments showed cellular expression of transgenic CCL21 after particle-mediated gene gun delivery of pDNA into skin. CCL21 was expressed in the epidermis, consequently secreted into the upper dermis, and transported into the draining LNs, which resulted in increased CCL21 concentration, total cell number, and frequencies of CD11c(+) DCs and CD4(+)/CD62L(+) naïve, CD4(+)/CD62L(-), and CD8(+)/CD62L(-) effector memory T-cells (expressing CCL21 receptors CCR7 or CXCR3), as well as retention of adoptively transferred T-lymphocytes, in the draining LNs of plt/plt mice (lacking endogenous expression of CCL21). Our studies show that biologically active CCL21 can be overexpressed by genetic means in vitro and in vivo. This strategy allows reconstitution of a genetic defect and colocalization of different cell types in the secondary lymphoid organs, an important prerequisite for targeted cell migration.

Pemphigus vegetans--immunopathological findings in a rare variant of pemphigus vulgaris.

A patient with painful erosions of the oral cavity and the labia minora developed multifocal blisters in inter-triginous areas. These blisters eroded and evolved into papillomatous erosive vegetations. Histopathology and immunopathological investigations confirmed the diagnosis of pemphigus vegetans, mediated by IgG autoantibodies. The circulating IgG1 and IgG4 autoantibodies were exclusively directed against desmoglein 3, as shown by ELISA and indirect immunofluorescence studies. These IgG1 and IgG4 isotypes were also in vivo bound, as demonstrated with immunoperoxidase staining of perilesional skin. Our clinical, biochemical and immunopathological observations confirm the hypothesis that pemphigus vegetans is a variant of pemphigus vulgaris.

Serum IgE autoantibodies target keratinocytes in patients with atopic dermatitis.

Previous studies have shown that sera of patients with severe atopic dermatitis (AD) contain IgE specific for self-proteins, supporting the hypothesis of autoreactivity as a pathogenic factor in AD. In this study, we screened a large panel of AD patients (n=192) by western blotting (WB) for IgE reactivity not only against the human epithelial cell line A431 but also against primary keratinocytes (KCs). To investigate autoantigenic cell structures in detail, normal human skin and primary KCs were incubated with sera from both WB-reactive patients and, for control purposes, healthy individuals, and analyzed by immunohistology, confocal laser microscopy, and flow cytometry. Our analysis revealed that 28% of AD patients, but not healthy individuals, display serum IgE autoreactivity by WB analysis. The individual IgE reaction patterns of the sera pointed to the existence of unique as well as common specificities against epidermal or A431-derived proteins. Immunostainings identified cytoplasmic and, occasionally, also cell membrane-associated moieties as targets for autoreactive IgE antibodies. Interestingly, in certain autoreactive patients, the surface-staining pattern was accentuated at cellular contact sites. We conclude that IgE autoreactivity is common, particularly among severe AD patients, and that non-transformed primary cells are needed for characterization of the entire spectrum of IgE-defined autoantigens.

Down-modulation of CXCR3 surface expression and function in CD8+ T cells from cutaneous T cell lymphoma patients.

Viruses can escape destruction by the immune system by exploitation of the chemokine-chemokine receptor system. It is less established whether human cancers can adopt similar strategies to evade immunologic control. In this study, we show that advanced cutaneous T cell lymphoma (CTCL) is associated with selective and efficient inactivation of CXCR3-dependent T cell migration. Our studies demonstrate that this alteration is at least in part due to CXCR3 down-regulation in vivo by elevated serum levels of CXCR3 ligands. The T cell population most affected by this down-regulatory mechanism are CD8+ cytotoxic effector T cells. In CTCL patients, cytotoxic effector T cells have strongly reduced surface CXCR3 expression, accumulate in peripheral blood, but are virtually absent from CTCL tumor lesions, indicating an inability to extravasate into lymphoma tissue. CTCL-associated inactivation of effector cell recruitment may be a paradigmatic example of a new type of immune escape mechanisms shielding the neoplasm from a tumoricidal attack.

Blood and lymphatic endothelial cell-specific differentiation programs are stringently controlled by the tissue environment.

The discovery of marker proteins of human blood (BECs) and lymphatic endothelial cells (LECs) has allowed researchers to isolate these cells. So far, efforts to unravel their transcriptional and functional programs made use of cultured cells only. Hence, it is unknown to which extent previously identified LEC- and BEC-specific programs are representative of the in vivo situation. Here, we define the human BEC- and LEC-specific in vivo transcriptomes by comparative genomewide expression profiling of freshly isolated cutaneous EC subsets and of non-EC skin cells (fibroblasts, mast cells, dendritic cells, epithelial cells). Interestingly, the expression of most of the newly identified EC subset-discriminating genes depends strictly on the in vivo tissue environment as revealed by comparative analyses of freshly isolated and cultured EC subsets. The identified environment-dependent, EC subset-restricted gene expression regulates lineage fidelity, fluid exchange, and MHC class II-dependent antigen presentation. As an example for a BEC-restricted in vivo function, we show that non-activated BECs in situ, but not in vitro, assemble and display MHC class II protein complexes loaded with self-peptides. Thus, our data demonstrate the key importance of using precisely defined native ECs for the global identification of in vivo relevant cell functions.

Liver X receptors regulate dendritic cell phenotype and function through blocked induction of the actin-bundling protein fascin.

Liver X receptors (LXRs) are nuclear receptors regulating lipid and cholesterol metabolism. Recent data revealed a cross talk between LXR and Toll-like receptor signaling in macrophages, indicating a role in immunity. Here, we show that LXRalpha is expressed in human myeloid dendritic cells (DCs) and induced during differentiation of monocyte-derived DCs, whereas LXRbeta is expressed constitutively at a very low level. LXR activation by 2 different LXR agonists strongly interfered with lipopolysaccharide (LPS)-induced but not with CD40L-induced DC maturation by altering DC morphology and suppressing interleukin-12-but enhancing interleukin-10-secretion. LXR activation in DCs largely blocked their T-cell stimulatory ability despite essentially unaltered expression of various antigen-presenting and costimulatory molecules. Immunologic synapse formation was significantly inhibited by LXR activation along with a complete block in LPS- but not CD40L-induced expression of the actin-bundling protein fascin. Notably, overexpression of fascin in LXR agonist-treated DCs restored immunologic synapse formation and restored their ability to activate T cells. In conclusion, our data reveal LXR as a potent modulator of DC maturation and function mediated in part by blocking the expression of fascin. Due to the central position of DCs in immunity, LXRalpha could be a potential novel target for immunomodulation.

Balance between NF-kappaB and JNK/AP-1 activity controls dendritic cell life and death.

The life cycle of dendritic cells (DCs) must be precisely regulated for proper functioning of adaptive immunity. However, signaling pathways actively mediating DC death remain enigmatic. Here we describe a novel mechanism of hierarchical transcriptional control of DC life and death. Ligation of tumor necrosis factor receptor superfamily (TNFR-SF) members on DCs and cognate contact with T cells resulted in quantitatively balanced nuclear factor-kappaB (NF-kappaB) and c-Jun N-terminal kinase (JNK)-mediated activator protein-1 (AP-1) induction and strongly enhanced DC longevity. Specific blockade of NF-kappaB in DCs induced strongly augmented JNK/AP-1 activity because of elevated levels of reactive oxygen species. In this scenario, DC activation by TNFR-SF members or T cells induced DC apoptosis. Specific inhibition of JNK/AP-1 rescued DCs from this activation-induced cell death program and restored TNFR-SF member- and T-cell-mediated survival. We conclude that JNK/AP-1 activity is under negative feedback control of NF-kappaB and can execute apoptosis in DCs. Thus, feedback-controlled signaling amplitudes of 2 transcriptional pathways decide the fate of a DC.

Plasmacytoid dendritic cell recruitment by immobilized CXCR3 ligands.

Plasmacytoid dendritic cells (pDCs) recognize microbes, viruses in particular, and provide unique means of innate defense against them. The mechanism of pDC tissue recruitment remained enigmatic because the ligands of CXCR3, the cardinal chemokine receptor on pDCs, have failed to induce in vitro chemotaxis of pDCs in the absence of additional chemokines. In this study, we demonstrate that CXCR3 is sufficient to induce pDC migration, however, by a migratory mechanism that amalgamates the features of haptotaxis and chemorepulsion. To mediate "haptorepulsion" of pDCs, CXCR3 requires the encounter of its cognate ligands immobilized, optimally by heparan sulfate, in a form of a negative gradient. This is the first report of the absolute requirement of chemokine immobilization and presentation for its in vitro promigratory activity. The paradigmatic example of pDC haptorepulsion described here may represent a new pathophysiologically relevant migratory mechanism potentially used by other cells in response to other chemokines.

Lipid raft-associated GTPase signaling controls morphology and CD8+ T cell stimulatory capacity of human dendritic cells.

Their eponymous morphology and unique ability to activate naive T cells are hallmark features of dendritic cells (DCs). Specific properties of the actin cytoskeleton may define both characteristics. In search for regulators that coordinate DC phenotype and function, we observed strongly increased expression of the actin-remodeling GTPases Cdc42 and Rac1 during DC development from human stem cells. Cdc42 and Rac1 are constitutively active in immature DCs, and their activity is further up-regulated by maturational stimuli such as LPS or CD40L. Activation of Rac1 is associated with its rapid recruitment into lipid rafts. Cdc42 is not recruited into rafts, but readily activated by raft-associated moieties. The functional interplay of rafts, GTPases, and cortical actin is further shown by GTPase activation and actin remodeling after pharmacological disruption of lipid rafts and by the loss of the actin-based DC morphology by transfection of dominant-negative Cdc42 and Rac1. Both Cdc42 and Rac1 also control the transport of essential immunostimulatory molecules to the DC surface. Transfection with dominant-negative GTPases led to reduced surface expression of MHC class I and CD86. Consecutively, DCs display a reduced stimulatory capacity for CD8(+) T cells, whereas MHC class II-dependent stimulation of CD4(+) T cells remains unperturbed. We conclude that Cdc42 and Rac1 signaling controls DC morphology and conditions DCs for efficient CD8(+) T cell stimulation.

Different heparan sulfate proteoglycans serve as cellular receptors for human papillomaviruses.

Papillomaviruses replicate in stratified epithelia of skin and mucosa. Infection with certain human papillomavirus (HPV) types is the main cause of anogenital neoplasia, in particular cervical cancer. Early events of papillomavirus infectivity are poorly understood. While heparan sulfate proteoglycans (HSPGs) mediate initial binding to the cell surface, the class of proteins carrying heparan sulfates has not been defined. Here we examined two processes of papillomavirus infection, attachment of virus-like particles (VLP) to cells and infection with authentic HPV type 11 (HPV11) virions. Of the HSPGs, syndecan-1 is the major epithelial form and is strongly upregulated in wound edge keratinocytes. We employed K562 cells, which lack HSPGs except minor amounts of endogenous betaglycan, and stable clones that express cDNAs of syndecan-1, syndecan-4, or glypican-1. Binding of VLP correlated with levels of heparan sulfate on the cell surface. Parental K562 bound HPV16 VLP weakly, whereas all three K562 transfectants demonstrated enhanced binding, with the highest binding capacity observed for syndecan-1-transfected cells, which also expressed the most HSPG. For HPV11 infectivity assays, a high virion inoculum was required to infect K562 cells, whereas ectopic expression of syndecan-1 increased permissiveness eightfold and expression of syndecan-4 or glypican-1 fourfold. Infection of keratinocytes was eliminated by treatment with heparitinase, but not phospholipase C, further implicating the syndecan family of integral membrane proteins as receptor proteins. Human keratinocytes with a homozygous deletion of alpha6 integrin are permissive for HPV11 infection. These results indicate that several HSPGs can serve as HPV receptors and support a putative role for syndecan-1, rather than alpha6 integrin, as a primary receptor protein in natural HPV infection of keratinocytes.

Prevention of CD40-triggered dendritic cell maturation and induction of T-cell hyporeactivity by targeting of Janus kinase 3.

Pharmacological targeting of Janus kinase 3 (JAK3) has been employed successfully to control allograft rejection and graft-vs.-host disease (GVHD). Recent evidence suggests that in addition to its involvement in common-gamma chain (cgamma) signaling of cytokine receptors, JAK3 is also engaged in the CD40 signaling pathway of peripheral blood monocytes. In this study, we assessed the consequences of JAK3 inhibition during CD40-induced maturation of myeloid dendritic cells (DCs), and tested the impact thereof on the induction of T-cell alloreactivity. Dendritic cells triggering through CD40 induced JAK3 activity, the expression of costimulatory molecules, production of IL-12, and potent allogeneic stimulatory capacity. In contrast, JAK3 inhibition with the rationally designed JAK3 inhibitor WHI-P-154 prevented these effects arresting the DCs at an immature level. Interestingly, DCs exposed to the JAK3-inhibitor during CD40-ligation induced a state of hyporeactivity in alloreactive T cells that was reversible upon exogenous IL-2 supplementation to secondary cultures. These results suggest that immunosuppressive therapies targeting the tyrosine kinase JAK3 may also affect the function of myeloid cells. This property of JAK3 inhibitors therefore represents a further level of interference, which together with the well-established suppression of cgamma signaling could be responsible for their clinical efficacy.

Tumor-associated macrophages express lymphatic endothelial growth factors and are related to peritumoral lymphangiogenesis.

Formation of lymphatic metastasis is the initial step of generalized spreading of tumor cells and predicts poor clinical prognosis. Lymphatic vessels generally arise within the peritumoral stroma, although the lymphangiopoietic vascular endothelial growth factors (VEGF)-C and -D are produced by tumor cells. In a carefully selected collection of human cervical cancers (stage pT1b1) we demonstrate by quantitative immunohistochemistry and in situ hybridization that density of lymphatic microvessels is significantly increased in peritumoral stroma, and that a subset of stromal cells express large amounts of VEGF-C and VEGF-D. The density of cells producing these vascular growth factors correlates with peritumoral inflammatory stroma reaction, lymphatic microvessel density, and indirectly with peritumoral carcinomatous lymphangiosis and frequency of lymph node metastasis. The VEGF-C- and VEGF-D-producing stroma cells were identified in situ as a subset of activated tumor-associated macrophages (TAMs) by expression of a panel of macrophage-specific markers, including CD68, CD23, and CD14. These TAMs also expressed the VEGF-C- and VEGF-D-specific tyrosine kinase receptor VEGFR-3. As TAMs are derived from monocytes in the circulation, a search in peripheral blood for candidate precursors of VEGFR-3-expressing TAMs revealed a subfraction of CD14-positive, VEGFR-3-expressing monocytes, that, however, failed to express VEGF-C and VEGF-D. Only after in vitro incubation with tumor necrosis factor-alpha, lipopolysaccharide, or VEGF-D did these monocytes start to synthesize VEGF-C de novo. In conclusion VEGF-C-expressing TAMs play a novel role in peritumoral lymphangiogenesis and subsequent dissemination in human cancer.