Maaike W. G. Looman

DCIR is endocytosed into human dendritic cells and inhibits TLR8-mediated cytokine production

C‐type lectin receptors (CLRs) expressed on APCs play a pivotal role in the immune system as pattern‐recognition and antigen‐uptake receptors. In addition, they may signal directly, leading to cytokine production and immune modulation. To this end, some CLRs, like dectin‐1 and dendritic cell immunoreceptor (DCIR), contain intracellular ITIMs or ITAMs. In this study, we explored expression and function of the ITIM‐containing CLR DCIR on professional APCs. DCIR is expressed on immature and mature monocyte‐derived DCs (moDC) but also on monocytes, macrophages, B cells, and freshly isolated myeloid and plasmacytoid DCs. We show that endogenous DCIR is internalized efficiently into human moDC after triggering with DCIR‐specific mAb. DCIR internalization is clathrin‐dependent and leads to its localization in the endo‐/lysosomal compartment, including lysosome‐associated membrane protein‐1+ lysosomes. DCIR triggering affected neither TLR4‐ nor TLR8‐mediated CD80 and CD86 up‐regulation. Interestingly, it did inhibit TLR8‐mediated IL‐12 and TNF‐α production significantly, and TLR2‐, TLR3‐, or TLR4‐induced cytokine production was not affected. Collectively, the data presented characterize DCIR as an APC receptor that is endocytosed efficiently in a clathrin‐dependent manner and negatively affects TLR8‐mediated cytokine production. These data provide further support to the concept of CLR/TLR cross‐talk in modulating immune responses.

DC-STAMP, a novel multimembrane-spanning molecule preferentially expressed by dendritic cells

Dendritic cells (DC) are unique in their ability to present antigen to naive T cells, and therefore play a central role in the initiation of immune responses. Characterization of DC‐specific genes may help to unravel the mechanism underlying their potent antigen presenting capacity. Here we describe the identification of a novel transcript, isolated by random sequencing of a cDNA library prepared from monocyte‐derived DC, which we termed DC‐specific transmembrane protein (DC‐STAMP). DC‐STAMP is specifically expressed by DC, and not in a panel of other leukocytes or non‐hematopoietic cells. Interestingly, DC‐STAMP was also detected in activated but not resting blood DC. The DC‐STAMP transcript encodes a 470‐amino acid protein containing seven putative transmembrane domains. Expression of a DC‐STAMP‐GFP fusion protein in 293 cells indicates that DC‐STAMP is expressed at the cell surface, and has an intracellular C terminus. Surprisingly, no sequence homology was found with any other protein or multimembrane‐spanning receptor. Therefore, we propose that DC‐STAMP is a novel DC‐specific multimembrane‐spanning protein, representing a new group of transmembrane proteins.

Intratumoral administration of recombinant human interleukin 12 in head and neck squamous cell carcinoma patients elicits a T-helper 1 profile in the locoregional lymph nodes.

The objective of this Phase II study was to evaluate the pharmacodynamic and immune effects of intratumorally administered recombinant human interleukin-12 (IL-12) on regional lymph nodes, primary tumor, and peripheral blood. Ten previously untreated patients with head and neck squamous cell carcinoma were injected in the primary tumor two to three times, once/week, at two dose levels of 100 or 300 ng/kg, before surgery. We compared these patients with 20 control (non-IL-12-treated) patients. Toxicity was high, with unexpected dose-limiting toxicities at the 300 ng/kg dose level. Dose-dependent plasma IFN-γ and IL-10 increments were detected. These cytokine levels were higher after the first injection than after the subsequent injections. A rapid, transient reduction in lymphocytes, monocytes, and all lymphocyte subsets, especially natural killer cells, was observed, due to a redistribution to the lymph nodes. In the enlarged lymph nodes of the IL-12-treated patients, a higher percentage of natural killer cells and a lower percentage of T-helper cells were found compared with control patients. The same pattern was detected in the infiltrate in the primary tumor. Real-time semiquantitative PCR analysis of peripheral blood mononuclear cells in the peripheral blood showed a transient decrease of T-bet mRNA. Interestingly, the peripheral blood mononuclear cells in the lymph nodes showed a 128-fold (mean) increase of IFN-γ mRNA. A switch from the Th2 to a Th1 profile in the lymph nodes compared with the peripheral blood occurred in the IL-12-treated patients. In conclusion, in previously untreated head and neck squamous cell carcinoma patients, recombinant human IL-12 intratumorally showed dose-limiting toxicities at the dose level of 300 ng/kg and resulted in measurable immunological responses locoregionally at both dose levels.

Vitamin D controls murine and human plasmacytoid dendritic cell function

Topical application of the vitamin D (VitD) analog calcipotriol is a highly effective standard treatment modality of psoriatic skin lesions. However, the immune modulatory effects of the treatment are incompletely understood. VitD is well known to induce tolerogenic responses in conventional dendritic cells (cDCs). Plasmacytoid DCs (pDCs) comprise a specialized, naturally occurring DC subset known to be important in autoimmune diseases including psoriasis. pDCs from the blood rapidly infiltrate psoriatic skin and are key to the initiation of the immune-mediated pathogenesis of the disease. We now demonstrate that pDCs express various proteins of the VitD receptor (VDR) pathway, including the VitD-metabolizing enzymes Cyp27B1 and Cyp24A1, and that VDR is transcriptionally active in pDCs. Moreover, VitD impairs the capacity of murine and human pDCs to induce T-cell proliferation and secretion of the T-helper 1 cytokine IFNγ. The inhibitory effect of VitD is dependent on the expression of the VDR in the DCs. This study demonstrates that VitD signaling can act as a natural inhibitory mechanism on both cDCs and pDCs, which may instigate the development of VitD-based therapeutic applications for psoriasis and other inflammatory skin diseases.

The dendritic cell-derived protein DC-STAMP is highly conserved and localizes to the endoplasmic reticulum.

Recently, we described the molecular identification of dendritic cell‐specific TrAnsMembrane protein (DC‐STAMP), a multimembrane‐spanning protein preferentially expressed by human DC (hDC). In this report, we describe the identification and expression profile of the murine homologue of DC‐STAMP (mDC‐STAMP) as well as the characterization of the DC‐STAMP protein. The results demonstrate that mDC‐STAMP is over 90% homologous to hDC‐STAMP and is also preferentially expressed by DC in vitro and ex vivo. mDC‐STAMP expression is enhanced by interleukin‐4 and down‐regulated upon DC maturation. Analysis of differently tagged DC‐STAMP proteins further demonstrates that hDC‐STAMP and mDC‐STAMP are glycosylated and primarily localize to an intracellular compartment. Applying confocal microscopy and electron microscopy, we demonstrate that hDC‐STAMP localizes to the endoplasmic reticulum (ER) in human embryonic kidney 293 cells as well as hDC transduced with an adenovirus encoding hDC‐STAMP‐green fluorescent protein fusion protein. These data imply that DC‐STAMP may exert its effect in the ER.

Identification and characterization of DC-SCRIPT, a novel dendritic cell-expressed member of the zinc finger family of transcriptional regulators

Dendritic cells (DC) compose a heterogeneous population of cells that hold a leading role in initiating and directing immune responses. Although their function in recognizing, capturing, and presenting Ags is well defined, the molecular mechanisms that control their differentiation and immune functions are still largely unknown. In this study, we report the isolation and characterization of DC-SCRIPT, a novel protein encoded by an 8-kb mRNA that is preferentially expressed in DC. DC-SCRIPT is expressed in multiple DC subsets in vivo, including myeloid DC, plasmacytoid DC, and Langerhans cells. At the protein level, DC-SCRIPT consists of a proline-rich region, 11 C2H2-type zinc fingers, and an acidic region. Localization studies reveal that DC-SCRIPT resides in the nucleus and that nuclear localization is critically dependent on the zinc fingers. The protein displays no transcriptional activation properties according to assorted transactivation assays, but interacts with the corepressor C-terminal binding protein 1. Taken together, our results show that we have isolated a novel DC marker that could be involved in transcriptional repression. In contrast to other DC molecules, DC-SCRIPT identifies all DC subsets tested to date.

Novel monoclonal antibodies detect elevated levels of the chemokine CCL18/DC-CK1 in serum and body fluids in pathological conditions

CC chemokine ligand 18/dendritic cell‐chemokine 1 (CCL18/DC‐CK1) is a CC chemokine, preferentially expressed by DC, which acts as a chemoattractant for naive T cells and mantle zone B cells. Applying a newly developed CCL18/DC‐CK1 sandwich enzyme‐linked immunosorbent assay, we demonstrate that DC secrete high amounts of CCL18/DC‐CK1 and that this expression can be increased by interleukin‐10. High levels of CCL18/DC‐CK1 were also detected in human serum (average of 88 ng/ml). Moreover, elevated CCL18/DC‐CK1 levels were detected in synovial fluid from rheumatoid arthritis patients and in drain fluid (average of 254 ng/ml and 122 ng/ml, respectively). Immunoprecipitation experiment using anti‐CCL18/DC‐CK1 monoclonal antibodies revealed a protein of 6–7 kDa in serum and drain fluid that was indistinguishable from recombinant CCL18/DC‐CK1 on Western blot and in re‐aggregation assays. The concentration of CCL18/DC‐CK1 found in human serum is in the same order of magnitude as was previously reported to completely inhibit CCL11/eotaxin‐induced CC chemokine receptor 3 (CCR3) activation and consequent migration of eosinophils. CCL18/DC‐CK1 may therefore function as an agonist (for naive T and B cells) and as an antagonist for CCR3‐expressing leukocytes such as eosinophils.

Dendritic Cell-Specific Transcript: Dendritic Cell Marker and Regulator of TLR-Induced Cytokine Production

Dendritic cells (DCs) are the professional APCs of the immune system that dictate the type and course of an immune response. Molecular understanding of DC biology is important for the design of DC-based immunotherapies and optimal clinical applications in vaccination settings. Previously, we isolated and characterized the cDNA-encoding dendritic cell-specific transcript (DC-SCRIPT; also known as ZNF366). DC-SCRIPT mRNA expression in the immune system was confined to DCs and was reported to be an early hallmark of DC differentiation. In this study, we demonstrate IL-4 to be the dominant factor for DC-SCRIPT expression in human monocyte-derived DCs. In addition, to our knowledge, we show for the first time endogenous DC-SCRIPT protein expression in human DCs both in vitro and in situ. DC-SCRIPT protein is detected early upon differentiation of monocytes into DCs and is also present in multiple freshly isolated DC subsets. Maturation of DCs with TLR ligands further increased DC-SCRIPT mRNA expression, suggesting a role in DC maturation. Indeed, small interfering RNA-mediated knockdown of DC-SCRIPT affected the cytokine response upon TLR stimulation. These DCs displayed enhanced IL-10 and decreased IL-12 production, compared with wild-type DCs. Silencing of IL-10 in DC-SCRIPT knockdown DCs rescued IL-12 expression, suggesting a primary role for DC-SCRIPT in the regulation of IL-10 production.

Molecular characterization of the murine homologue of the DC-derived protein DC-SCRIPT

Dendritic cell‐specific transcript (DC‐SCRIPT) is a putative DC zinc (Zn) finger‐type transcription factor described recently in humans. Here, we illustrate that DC‐SCRIPT is highly conserved in evolution and report the initial characterization of the murine ortholog of DC‐SCRIPT, which is also preferentially expressed in DC as shown by real‐time quantitative polymerase chain reaction, and its distribution resembles that of its human counterpart. Studies undertaken in human embryonic kidney 293 cells depict its nuclear localization and reveal that the Zn finger domain of the protein is mainly responsible for nuclear import. The human and the mouse genes are located in syntenic chromosomal regions and exhibit a similar genomic organization with numerous common transcription factor‐binding sites in their promoter region, including sites for many factors implicated in haematopoiesis and DC biology, such as Gfi, GATA‐1, Spi‐B, and c‐Rel. Taken together, these data show that DC‐SCRIPT is well‐conserved in evolution and that the mouse homologue is more than 80% homologous to the human protein. Therefore, mouse models can be used to elucidate the function of this novel DC marker.

The DC-derived protein DC-STAMP influences differentiation of myeloid cells

differences in the clinical outcome based on the MPLW515 mutational status. Only molecular screening of a larger number of cases of CMPD also mimicking features of RARS-T for MPLW515 mutations will reveal the frequency in these borderline cases. It further remains to be clarified whether JAK2 and MPL mutations can occur in coincidence in RARS-T as it was occasionally reported in CIMF. The different phenotypes of MPLW515-associated disordersFET, CIMF and RARS-TFemphasize that these disorders are probably the result of a more complex genetic network that remains to be clarified by further analysis of positive and negative regulators of the JAK–STAT pathway.