Herbert Strobl

Nomenclature of monocytes and dendritic cells in blood

Monocytes and cells of the dendritic cell lineage circulate in blood and eventually migrate into tissue where they further mature and serve various functions, most notably in immune defense. Over recent years these cells have been characterized in detail with the use of cell surface markers and flow cytometry, and subpopulations have been described. The present document proposes a nomenclature for these cells and defines 3 types of monocytes (classical, intermediate, and nonclassical monocytes) and 3 types of dendritic cells (plasmacytoid and 2 types of myeloid dendritic cells) in human and in mouse blood. This classification has been approved by the Nomenclature Committee of the International Union of Immunological Societies, and we are convinced that it will facilitate communication among experts and in the wider scientific community.

Mixed-phenotype acute leukemia: clinical and laboratory features and outcome in 100 patients defined according to the WHO 2008 classification

The features of 100 mixed-phenotype acute leukemias (MPALs), fulfilling WHO 2008 criteria, are documented. Myeloid and T-lineage features were demonstrated by cytoplasmic myeloperoxidase and CD3; B-lineage features were demonstrated by at least 2 B-lymphoid markers. There were 62 men and 38 women; 68% were adults. Morphology was consistent with acute lymphoblastic leukemia (ALL; 43%), acute myeloid leukemia (AML; 42%), or inconclusive (15%). Immunophenotyping disclosed B + myeloid (59%), T + myeloid (35%), B + T (4%), or trilineage (2%) combinations. Cytogenetics evidenced t(9;22)/(Ph(+)) (20%), 11q23/MLL rearrangements (8%), complex (32%), aberrant (27%), or normal (13%) karyotypes. There was no correlation between age, morphology, immunophenotype, or cytogenetics. Response to treatment and outcome were available for 67 and 70 patients, respectively; 27 received ALL, 34 AML, 5 a combination of ALL + AML therapy, and 1 imatinib. ALL treatment induced a response in 85%, AML therapy in 41%; 3 of 5 patients responded to the combination therapy. Forty (58%) patients died, 33 of resistant disease. Overall median survival was 18 months and 37% of patients are alive at 5 years. Age, Ph(+), and AML therapy were predictors for poor outcome (P < .001; P = .002; P = .003). MPAL is confirmed to be a poor-risk disease. Adults and Ph(+) patients should be considered for transplantation in first remission.

miR-146a Is Differentially Expressed by Myeloid Dendritic Cell Subsets and Desensitizes Cells to TLR2-Dependent Activation

Langerhans cells (LCs) in epithelia and interstitial dendritic cells (intDCs) in adjacent connective tissues represent two closely related myeloid-derived DC subsets that exert specialized functions in the immune system and are of clinical relevance for cell therapy. Both subsets arise from monocyte-committed intermediates in response to tissue-associated microenvironmental signals; however, molecular mechanisms underlying myeloid DC subset specification and function remain poorly defined. Using microarray profiling, we identified microRNA (miRNA) miR-146a to be constitutively expressed at higher levels in human LCs compared with intDCs. Moreover, miR-146a levels were low in monocytes and nondetectable in neutrophil granulocytes. Interestingly, constitutive high miR-146a expression in LCs is induced by the transcription factor PU.1 in response to TGF-β1, a key microenvironmental signal for epidermal LC differentiation. We identified miR-146a as a regulator of monocyte and DC activation but not myeloid/DC subset differentiation. Ectopic miR-146a in monocytes and intDCs interfered with TLR2 downstream signaling and cytokine production, without affecting phenotypic DC maturation. Inversely, silencing of miR-146a in LCs enhanced TLR2-dependent NF-κB signaling. We therefore conclude that high constitutive miR-146a levels are induced by microenvironmental signals in the epidermis and might render LCs less susceptible to inappropriate activation by commensal bacterial TLR2 triggers at body surfaces.

Aryl Hydrocarbon Receptor Activation Inhibits In Vitro Differentiation of Human Monocytes and Langerhans Dendritic Cells

The transcription factor aryl hydrocarbon receptor (AhR) represents a promising therapeutic target in allergy and autoimmunity. AhR signaling induced by the newly described ligand VAF347 inhibits allergic lung inflammation as well as suppresses pancreatic islet allograft rejection. These effects are likely mediated via alterations in dendritic cell (DC) function. Moreover, VAF347 induces tolerogenic DCs. Langerhans cells (LCs) are immediate targets of exogenous AhR ligands at epithelial surfaces; how they respond to AhR ligands remained undefined. We studied AhR expression and function in human LCs and myelopoietic cell subsets using a lineage differentiation and gene transduction model of human CD34+ hematopoietic progenitors. We found that AhR is highly regulated during myeloid subset differentiation. LCs expressed highest AhR levels followed by monocytes. Conversely, neutrophil granulocytes lacked AhR expression. AhR ligands including VAF347 arrested the differentiation of monocytes and LCs at an early precursor cell stage, whereas progenitor cell expansion or granulopoiesis remained unimpaired. AhR expression was coregulated with the transcription factor PU.1 during myeloid subset differentiation. VAF347 inhibited PU.1 induction during initial monocytic differentiation, and ectopic PU.1 restored monocyte and LC generation in the presence of this compound. AhR ligands failed to interfere with cytokine receptor signaling during LC differentiation and failed to impair LC activation/maturation. VAF347-mediated antiproliferative effect on precursors undergoing LC lineage differentiation occurred in a clinically applicable serum-free culture model and was not accompanied by apoptosis induction. In conclusion, AhR agonist signaling interferes with transcriptional processes leading to monocyte/DC lineage commitment of human myeloid progenitor cells.

Identification of bone morphogenetic protein 7 (BMP7) as an instructive factor for human epidermal Langerhans cell differentiation

Bone morphogenetic protein 7 (BMP7) promotes the differentiation of Langerhans cells in the epidermis during prenatal development.

Identification of Axl as a downstream effector of TGF-β1 during Langerhans cell differentiation and epidermal homeostasis

Axl expression is induced by TGF-β1 during Langerhans cell differentiation and enhances apoptotic cell uptake and blocks proinflammatory cytokine production.

ETV6-NCOA2: A Novel Fusion Gene in Acute Leukemia Associated with Coexpression of T-Lymphoid and Myeloid Markers and Frequent NOTCH1 Mutations

Purpose: The ETV6 gene has been reported to be fused to a multitude of partner genes in various hematologic malignancies with 12p13 aberrations. Cytogenetic analysis of six cases of childhood acute lymphoblastic leukemia revealed a novel recurrent t(8;12)(q13;p13), suggesting involvement of ETV6. Experimental Design: Fluorescence in situ hybridization was used to confirm the involvement of ETV6 in the t(8;12)(q13;p13) and reverse transcription-PCR was used to identify the ETV6 partner gene. Detailed immunologic characterization was done, and owing to their lineage promiscuity, the leukemic blast cells were analyzed for NOTCH1 mutations. Results: We have identified a novel recurrent t(8;12)(q13;p13), which results in a fusion between the transcriptional repressor ETV6 (TEL) and the transcriptional coactivator NCOA2 (TIF2) in six cases of childhood leukemia expressing both T-lymphoid and myeloid antigens. The ETV6-NCOA2 transcript encodes a chimeric protein that consists of the pointed protein interaction motif of ETV6 that is fused to the COOH terminus of NCOA2, including the cyclic AMP–responsive element binding protein–binding protein (CBP) interaction and the AD2 activation domains. The absence of the reciprocal NCOA2-ETV6 transcript in one of the cases suggests that the ETV6-NCOA2 chimeric protein and not the reciprocal NCOA2-ETV6 is responsible for leukemogenesis. In addition, ETV6-NCOA2 leukemia shows a high frequency of heterozygous activating NOTCH1 mutations, which disrupt the heterodimerization or the PEST domains. Conclusions: The ETV6-NCOA2 fusion may define a novel subgroup of acute leukemia with T-lymphoid and myeloid features, which is associated with a high prevalence of NOTCH1 mutations.

Lack of DNA synthesis among CD34+ cells in cord blood and in cytokine-mobilized blood.

Flow cytometric DNA analysis was performed in combination with three‐colour immunological staining of cell surface antigens on density‐separated mononuclear cells (MNC) obtained from peripheral blood (PB) before, during and after cytokine stimulation of healthy adults. The aim of the study was to determine the cell‐cycling status of haemopoietic progenitor cells mobilized into the blood of healthy volunteers during a 5 d treatment period with 5 μg per kg body weight of either granulocyte colony‐stimulating factor (G‐CSF) or granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). Despite considerably increasing numbers of CD34+ PB MNC, the latter were not found to be in S/G2M phase, whereas, among the CD34− MNC, the proportion of cells in S/G2M phase increased from <0.1% to 0.75 ± 0.4% (GM‐CSF) and to 1.34 ± 0.75% (G‐CSF) and dropped again after discontinuation of the cytokine stimulation. These cells expressed CD33 but were negative for CD45RA, CD3, CD19 and CD14 and were thus considered granulopoietic cells. Analogous results were obtained from analyses of cord blood (CB). In contrast, CD34+ cells from bone marrow (BM) were partially (between 9% and 15%) found to be in S/G2M phase. The non‐cycling status of PB and CB progenitor cells was confirmed by the analysis of CD34+ cells enriched from the two cell sources. However, in vitro stimulation of these progenitor cells using IL3, GM‐CSF, erythropoietin and steel factor (SF) revealed that, after 48 h in suspension culture, up to 30% of the CD34+ cells were in S/G2M phase. The fact that cycling CD34+ cells are only detectable in BM but not in PB or CB may suggest different adhesive properties of migrating/mobilized ‘stem cells’ which may require the BM micro‐environment for adequate proliferation in vivo

Identification of TROP2 (TACSTD2), an EpCAM-Like Molecule, as a Specific Marker for TGF-β1-Dependent Human Epidermal Langerhans Cells

Langerin (CD207) expression is a hallmark of epidermal Langerhans cells (LCs); however, CD207(+) cells comprise several functional subsets. Murine studies showed that epidermal, but not dermal, CD207(+) cells require transforming growth factor-β 1 (TGF-β1) for development, whereas human data are lacking. Using gene profiling, we found that the surface molecule TROP2 (TACSTD2) is strongly and rapidly induced during TGF-β1-dependent LC commitment of human CD34(+) hematopoietic progenitor cells or monocytes. TROP2 is conserved between mouse and human, and shares substantial amino-acid identity with EpCAM, a marker for murine epidermal LCs. To our knowledge, neither TROP2 nor EpCAM expression has been analyzed in human dendritic cell (DC) subsets. We found that (i) all human epidermal LCs are TROP2(+)EpCAM(+); (ii) human dermis lacks CD207(+)EpCAM(-) or CD207(+)TROP2(-) DCs, i.e., equivalents of murine dermal CD207(+) DCs; and (iii) pulmonary CD207(+) cells are TROP2(-)EpCAM(-). Moreover, although EpCAM was broadly expressed by pulmonary and intestinal epithelial cells, as well as by bone marrow erythroid progenitor cells, these cells lacked TROP2. However, although TROP2 is expressed by human LCs as well as by human and murine keratinocytes, most murine LCs, except of a small subset, lacked TROP2. Therefore, TROP2 is a marker for human TGF-β1-dependent epidermal LCs.

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) and CD99 are critical in lymphatic transmigration of human dendritic cells.

The reverse transmigration (RT) of tissue-resident dendritic cells (DCs) across lymphatic endothelia is prerequisite for the initiation of adaptive immune responses and might be regulated in a manner similar to diapedesis. Specifically, CD31 and CD99, which act as gatekeepers during diapedesis, might have a role in RT of DCs. We found that human lymphatic endothelial cells (LECs) and DCs in vitro and in human skin explants express CD31 and CD99. In human skin, CD31 was enriched along intercellular surfaces of LECs, whereas CD99 was preferentially confined to luminal surfaces as evidenced by immunoelectron microscopy. Confocal microscopy analysis revealed that tumor necrosis factor-alpha (TNF-α) and CXCL12 acted as inducers of RT in vitro, but only CXCL12 stimulation resulted in a significant increase in migration rate of DCs. Upon TNF-α stimulation, CXCL12 mRNA levels transiently increased in human fibroblasts and LECs, whereas CXCL12 protein expression levels did not significantly change. Blocking mAbs to CD31 and CD99 significantly reduced RT of DCs across cultured human LEC monolayers and blocked CXCL12-induced migration of DCs in whole-skin explants. In sum, this study shows that CD31 and CD99 are involved in the RT of DCs across LECs and that similar mechanisms promote both diapedesis and RT.