AHR Signaling Dampens Inflammatory Signature in Neonatal Skin γδ T Cells.

Background Aryl hydrocarbon receptor (AHR)-deficient mice do not support the expansion of dendritic epidermal T cells (DETC), a resident immune cell population in the murine epidermis, which immigrates from the fetal thymus to the skin around birth. Material and Methods In order to identify the gene expression changes underlying the DETC disappearance in AHR-deficient mice, we analyzed microarray RNA-profiles of DETC, sorted from the skin of two-week-old AHR-deficient mice and their heterozygous littermates. In vitro studies were done for verification, and IL-10, AHR repressor (AHRR), and c-Kit deficient mice analyzed for DETC frequency. Results We identified 434 annotated differentially expressed genes. Gene set enrichment analysis demonstrated that the expression of genes related to proliferation, ion homeostasis and morphology differed between the two mouse genotypes. Importantly, with 1767 pathways the cluster-group "inflammation" contained the majority of AHR-dependently regulated pathways. The most abundant cluster of differentially expressed genes was "inflammation." DETC of AHR-deficient mice were inflammatory active and had altered calcium and F-actin levels. Extending the study to the AHRR, an enigmatic modulator of AHR-activity, we found approximately 50% less DETC in AHRR-deficient mice than in wild-type-littermates. Conclusion AHR-signaling in DETC dampens their inflammatory default potential and supports their homeostasis in the skin.

A Recombinant Bispecific CD20×CD95 Antibody With Superior Activity Against Normal and Malignant B-cells.

Monoclonal antibodies directed to the B-cell-specific CD20-antigen are successfully used for the treatment of lymphomas and autoimmune diseases. Here, we compare the anti-B-cell activity of three different antibodies directed to CD20: (i) a chimeric, monospecific antibody, (ii) an Fc-optimized variant thereof, and (iii) a bispecific CD20×CD95-antibody in a newly developed recombinant format, termed Fabsc. The bispecific antibody specifically triggers the CD95 death receptor on malignant, as well as activated, normal B-cells. We found that the capability of this antibody to suppress the growth of malignant B-cells in vitro and in vivo and to specifically deplete normal, activated B-cells from peripheral blood mononuclear cell (PBMC) cultures was superior to that of the Fc-optimized monospecific antibody. This antibody in turn was more effective than its nonoptimized variant. Moreover, the bispecific antibody was the only reagent capable of significantly suppressing antibody production in vitro. Our findings imply that the bispecific CD20×CD95-antibody might become a new, prototypical reagent for the treatment of B-cell-mediated autoimmune disease.

HOXB4 Increases Runx1 Expression to Promote the de novo Formation of Multipotent Hematopoietic Cells.

BACKGROUND: The de novo generation of patient-specific hematopoietic stem and progenitor cells from induced pluripotent stem cells (iPSCs) has become a promising approach for cell replacement therapies in the future. However, efficient differentiation protocols for producing fully functional human hematopoietic stem cells are still missing. In the mouse model, ectopic expression of the human homeotic selector protein HOXB4 has been shown to enforce the development of hematopoietic stem cells (HSCs) in differentiating pluripotent stem cell cultures. However, the mechanism how HOXB4 mediates the formation of HSCs capable of long-term, multilineage repopulation after transplantation is not well understood yet. METHODS: Using a mouse embryonic stem (ES) cell-based differentiation model, we asked whether retrovirally expressed HOXB4 induces the expression of Runx1/AML1, a gene whose expression is absolutely necessary for the formation of definitive, adult HSCs during embryonic development. RESULTS: During ES cell differentiation, basal expression of Runx1 was observed in all cultures, irrespective of ectopic HOXB4 expression. However, only in those cultures ectopically expressing HOXB4, substantial amounts of hematopoietic progenitors were generated which exclusively displayed increased Runx1 expression. CONCLUSIONS: Our results strongly suggest that HOXB4 does not induce basal Runx1 expression but, instead, mediates an increase of Runx1 expression which appears to be a prerequisite for the formation of hematopoietic stem and progenitor cells.

Generation of an induced pluripotent stem cell line (IUFi001) from a Cockayne syndrome patient carrying a mutation in the ERCC6 gene.

Human fibroblasts from a Cockayne Syndrome (CS) patient carrying the compound heterozygous c.1131 A > T and c.2571C > T within ERCC Excision Repair 6 (ERCC6) were reprogramed to generate integration-free induced pluripotent stem cells (iPSCs). Characterization of IUFi001-iPSCs demonstrated that this iPSC line is free of exogenous reprogrammed genes and maintains the genomic integrity. The pluripotency of IUFi001-iPSCs was confirmed by the expression of the pluripotency-associated markers and by embryoid body-based differentiation into cell types representative of the three germ layers. The generated iPSC line provides a powerful tool to dissect the molecular mechanisms underlying CS caused by mutations within ERCC6.

Academic application of Good Cell Culture Practice for induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSC) are a promising tool for replacing animal-based experiments. To warrant data reproducibility, quality-controlled research material is recommended. While the need for global harmonization of quality standards for stem cell banking centers, commercial providers, pre-clinical and clinical use of cells is well doc­umented, there are no recommendations available for quality control of hiPSC in an academic research environment to date. To fill this gap, we here give an example of a quality-controlled, two-tiered banking process producing a fully characterized master cell bank (MCB) and partially characterized respective working cell banks (WCB). Characteri­zation includes the study of morphology, mycoplasma contamination, cell line identity, karyotype stability, cell antigen expression and viability, gene expression, pluripotency, and post-thaw recovery. Costs of these procedures are cal­culated. We present the results of the proposed testing panel of two hiPSC MCBs and show that both fulfil the defined specifications regarding the above-mentioned characterization assays during and upon banking. In conclusion, we propose a panel of eight assays, which are practical and useful for an academic research laboratory working with hiPSCs. Meeting these proposed specifications ensures the quality of pluripotent stem cells throughout diverse experi­ments at moderate costs.

The small chain fatty acid butyrate antagonizes the TCR-stimulation-induced metabolic shift in murine epidermal gamma delta T cells.

The metabolic requirements change during cell proliferation and differentiation. Upon antigen-stimulation, effector T cells switch from adenosine-triphospate (ATP)-production by oxidative phosphorylation in the mitochondria to glycolysis. In the gut it was shown that short chain fatty acids (SCFA), fermentation products of the microbiota in colon, ameliorate inflammatory reactions by supporting the differentiation of regulatory T cells. SCFA are a major energy source, but they are also anabolic metabolites, histone-deacetylase-inhibitors and activators of G protein receptors. Recently, it was reported that a topical application of the SCFA butyrate promotes regulatory T cells in the skin. Here we ask if the SCFA butyrate, propionate and acetate affect the energy metabolism and inflammatory potential of dendritic epidermal T cells (DETC), the innate resident skin γδ T cell population. Using the Seahorse™ technology, we measured glycolysis and oxidative phosphorylation (OXPHOS) in a murine DETC cell line, 7-17, upon TCR-stimulation by CD3/CD28 crosslinking, with or without SCFA addition. TCR engagement resulted in a change of the ratio glycolysis/OXPHOS. A similar metabolic shift has been described for activated CD4 T cells. Addition of 5 mM SCFA, in particular butyrate, antagonized the effect. Stimulated DETC secrete cytokines, e.g. the pro-inflammatory cytokine interferon-gamma (IFNγ), and thereby regulate skin homeostasis. Addition of butyrate and propionate to the cultures at non-toxic concentrations decreased secretion of IFNγ by DETC and increased the expression of the immunoregulatory surface receptor CD69. We hypothesize that SCFA can dampen the inflammatory activity of DETC.

HOXB4 Promotes Hemogenic Endothelium Formation without Perturbing Endothelial Cell Development.

Generation of hematopoietic stem cells (HSCs) from pluripotent stem cells, in vitro, holds great promise for regenerative therapies. Primarily, this has been achieved in mouse cells by overexpression of the homeotic selector protein HOXB4. The exact cellular stage at which HOXB4 promotes hematopoietic development, in vitro, is not yet known. However, its identification is a prerequisite to unambiguously identify the molecular circuits controlling hematopoiesis, since the activity of HOX proteins is highly cell and context dependent. To identify that stage, we retrovirally expressed HOXB4 in differentiating mouse embryonic stem cells (ESCs). Through the use of Runx1(-/-) ESCs containing a doxycycline-inducible Runx1 coding sequence, we uncovered that HOXB4 promoted the formation of hemogenic endothelium cells without altering endothelial cell development. Whole-transcriptome analysis revealed that its expression mediated the upregulation of transcription of core transcription factors necessary for hematopoiesis, culminating in the formation of blood progenitors upon initiation of Runx1 expression.

In Vitro Generation of Vascular Wall-Resident Multipotent Stem Cells of Mesenchymal Nature from Murine Induced Pluripotent Stem Cells.

The vascular wall (VW) serves as a niche for mesenchymal stem cells (MSCs). In general, tissue-specific stem cells differentiate mainly to the tissue type from which they derive, indicating that there is a certain code or priming within the cells as determined by the tissue of origin. Here we report the in vitro generation of VW-typical MSCs from induced pluripotent stem cells (iPSCs), based on a VW-MSC-specific gene code. Using a lentiviral vector expressing the so-called Yamanaka factors, we reprogrammed tail dermal fibroblasts from transgenic mice containing the GFP gene integrated into the Nestin-locus (NEST-iPSCs) to facilitate lineage tracing after subsequent MSC differentiation. A lentiviral vector expressing a small set of recently identified human VW-MSC-specific HOX genes then induced MSC differentiation. This direct programming approach successfully mediated the generation of VW-typical MSCs with classical MSC characteristics, both in vitro and in vivo.