data_sheet_1_Phenotype, Function, and Mobilization of 6-Sulfo LacNAc-Expressing Monocytes in Atopic Dermatitis.docx

<p>Mononuclear phagocytes (MPs) are important immune regulatory cells in atopic dermatitis (AD). We previously identified 6-sulfo LacNAc-expressing monocytes (slanMo) as TNF-α- and IL-23-producing cells in psoriatic skin lesions and as inducers of IFN-γ-, IL-17-, and IL-22-producing T cells. These cytokines are also upregulated in AD and normalize with treatment, as recently shown for dupilumab-treated patients. We here asked for the role of slanMo in AD. Increased numbers of slanMo were found in AD skin lesions. In difference to other MPs in AD, slanMo lacked expression of FcɛRI, CD1a, CD14, and CD163. slanMo from blood of patients with AD expressed increased levels of CD86 and produced IL-12 and TNF-α at higher amounts than CD14⁺ monocytes and myeloid dendritic cells. While CD14⁺ monocytes from patients with AD revealed a reduced IL-12 production, we observed no difference in the cytokine production comparing slanMo in AD and healthy controls. Interestingly, experimentally induced mental stress, a common trigger of flares in patients with AD, rapidly mobilized slanMo which retained their high TNF-α-producing capacity. This study identifies slanMo as a distinct population of inflammatory cells in skin lesions and as proinflammatory blood cells in patients with AD. slanMo may, therefore, represent a potent future target for treatment of AD.</p

Highly Versatile Catalytic Hydrogenation of Carboxylic and Carbonic Acid Derivatives using a Ru-Triphos Complex: Molecular Control over Selectivity and Substrate Scope

The complex [Ru­(Triphos)­(TMM)] (Triphos = 1,1,1-tris­(diphenylphosphinomethyl)­ethane, TMM = trimethylene methane) provides an efficient catalytic system for the hydrogenation of a broad range of challenging functionalities encompassing carboxylic esters, amides, carboxylic acids, carbonates, and urea derivatives. The key control factor for this unique substrate scope results from selective activation to generate either the neutral species [Ru­(Triphos)­(Solvent)­H₂] or the cationic intermediate [Ru­(Triphos)­(Solvent)­(H)­(H₂)]⁺ in the presence of an acid additive. Multinuclear NMR spectroscopic studies demonstrated together with DFT investigations that the neutral species generally provides lower energy pathways for the multistep reduction cascades comprising hydrogen transfer to CO groups and C–O bond cleavage. Carboxylic esters, lactones, anhydrides, secondary amides, and carboxylic acids were hydrogenated in good to excellent yields under these conditions. The formation of the catalytically inactive complexes [Ru­(Triphos)­(CO)­H₂] and [Ru­(Triphos)­(μ-H)]₂ was identified as major deactivation pathways. The former complex results from substrate-dependent decarbonylation and constitutes a major limitation for the substrate scope under the neutral conditions. The deactivation via the carbonyl complex can be suppressed by addition of catalytic amounts of acids comprising non-coordinating anions such as HNTf₂ (bis­(trifluoromethane)­sulfonimide). Although the corresponding cationic cycle shows higher overall barriers of activation, it provides a powerful hydrogenation pathway at elevated temperatures, enabling the selective reduction of primary amides, carbonates, and ureas in high yields. Thus, the complex [Ru­(Triphos)­(TMM)] provides a unique platform for the rational selection of reaction conditions for the selective hydrogenation of challenging functional groups and opens novel synthetic pathways for the utilization of renewable carbon sources

Analysis for proliferation of CD4+ T cells after delivery of the TT-derived T-cell epitope to slanDCs via the novel immuno targeting system.

<p>(A) CFSE labeled PBMCs were prepared and incubated at 37°C for eight days with either full length TT (b) or the linker module containing (d) or lacking (c) the TT-derived T-cell epitope TT_p and analysed by FACS. The data indicate that PBMCs of the selected donor contain anti-TT memory T cells that can be recalled by full length TT and to a less extent by the TT_p peptide linker but not by the linker peptide lacking the TT_p epitope. Untreated PBMCs (a). (B) CFSE labelled PBMCs were incubated with either the antigen-containing scaffold (c) or the single components (a,b) at 4°C. Unbound material was removed by washing.</p

Immuno targeting of slan(+) Jurkat cells with a multivalent anti-slan scaffold containing a TT-derived T-cell epitope.

<p>(a, black graph) Binding of the anti-slan mab DD2 to slan(+) Jurkat cells. (c,d, red graph) Binding of anti-slan scaffolds consisting of the SL scBsDb and trivalent linker peptides either containing (c) or lacking (d) theTT-derived peptide epitope TT_p. (b,c,d, black graph) Control binding of the single components of the anti-slan scaffolds including the SL scBsDb (b), the trivalent linker l-l-GFP-l (c), and the trivalent linker l- TT_p -l-GFP-l (d).</p

Characterisation of purified linker peptides.

<p>For the formation of bi- or trivalent anti-slan scaffolds peptide linker molecules were constructed containing the l-Tag twice (l-GFP-l) or three times (l-l-GFP-l; l-TT_p-l-GFP-l). The purified peptide linkers were analysed by SDS-PAGE/immunoblotting using the anti-La mab 7B6 directed to the l-Tag. (M) Marker proteins.</p

Binding of the SL scBsDb/linker complex to slan(+) Jurkat cells.

<p>(A) Anti-slan scaffolds were obtained by incubation of the SL scBsDb (SL) with the trivalent linker peptide l-l-GFP-l or the bivalent linker peptide (l-GFP-l, data not shown) at various ratios as indicated and analyzed for binding to slan(+) Jurkat cells. Binding of the maternal IgM anti-slan antibody DD2 was set as 100%. (B) After estimation of the optimal conditions, the binding of the bivalent (SL + l-GFP-l) and trivalent anti-slan scaffolds (SL+l-l-GFP-l) were compared to the binding of the maternal anti-slan IgM antibody DD2 (DD2 IgM). Moreover, the binding of the scaffolds in the presence of the maternal anti-slan IgM antibody DD2 was determined (DD2 IgM+SL+l-l-GFP-l).</p

Stability of immune complexes formed via the slan epitope on isolated slanDCs.

<p>(A), (a, black graph) Staining of slanDCs freshly isolated by magnetic anti-IgM/MDC8 anti-slan beads with a secondary FITC-conjugated anti-mouse IgM antibody. (a, red graph) Freshly isolated slanDCs that are stained in addition with the anti-slan IgM mab DD2 and the secondary FITC-conjugated anti-mouse IgM antibody. (b, black graph) Staining of slanDCs with a secondary FITC-conjugated anti-mouse IgM antibody four hrs after isolation and an incubation at 37°C. (b, red graph) Staining of slanDCs with the anti-slan IgM mab DD2 and the secondary FITC-conjugated anti-mouse IgM antibody four hrs after isolation and an incubation at 37°C. (B), (a, black graph) Binding of the multivalent anti-slan scaffold is blocked to freshly isolated slan DCs. (b, black graph) Binding of the multivalent anti-slan scaffold consisting of the SL scBsDb (SL) and the l-TT_p-l-GFP-l linker module regains four hrs after isolation and an incubation at 37°C. (c, black graph) Staining of slan DCs after an additional incubation of 2.5 h at 37°C. The scBsDb/peptide linker scaffold can no more be detected on the surface of slanDCs.</p

Analysis of binding of the SL ScBsDb/linker complex to DD2 positive Jurkat cells.

<p>(A), (a, black graph) IgM isotype negative control. (a, red graph) Binding of the anti-slan IgM mab DD2 to slan(+) positive Jurkat cells. (b, black graph) Anti-penta-HIS, isotype negative control. (b, red graph) Lack of binding of the monovalent SL scBsDb (SL) in the absence of a linker peptide. (c,d black graph) Isotype negative control. (c,d, red graph) Bivalent or trivalent anti-slan scaffolds consisting of the SL scBsDb and the linker peptide l-GFP-l (c, red graph) or l-l-GFP-l (d, red graph). (B), Detection of anti-slan scaffolds on the surface of Jurkat cells by epifluorescence microscopy. GFP (a), phase contrast (b), bar = 10 µm.</p

Binding of the SL scBsDb/linker complex to native slanDCs in PBMCs.

<p>(A) (a) Estimation of the amount of slan-DCs in a PBMC sample using the anti-slan IgM mab DD2. (d) Estimation of the amount of slanDCs in the same PBMC sample using the anti-slan scaffold consisting of the SL scBsDb and the l-l-GFP-l linker peptide. Control staining of the single components of the anti-slan scaffold including the SL scBsDb (b, SL) or the peptide linker module (c, l-l-GFP-l). (B) GFP-labelled anti-slan scaffolds identified by epifluorescence microscopy. GFP (a), phase contrast (b), bar = 10 µm.</p

Recombinant antibodies derived from IgM antibodies: Restoring avidity by oligomerisation on a modular peptide scaffold.

<p>(A) The IgM anti-slan mab DD2 is a pentameric molecule. Preparation of a scFv (B) resulted in a monovalent molecule which failed to bind to slanDCs (C, data not shown). For recovery of binding avidity a scBsDb (D, SL scBsDb) and suitable linking peptide molecules (F,G) were constructed (H,I). In the scBsDb the variable heavy and light chain domains of the two mabs (A, DD2, S; E, 7B6, L) are recombinantly fused via glycine serine linkers (D; see also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016315#pone-0016315-g003" target="_blank">Fig. 3</a>). This SL scBsDb is on the one hand directed to the slan epitope and on the other hand to a continuous peptide epitope (l-Tag). The linker peptide modules contain the l-Tag either two times (l-GFP-l, F) or three times (l-l-GFP-l, G(i); l-TT_p-l-GFP-l, G(ii)). Binding of the SL scBsDb to the respective linker peptides results in the formation of a divalent (H) or trivalent (I) anti-slan scaffold with increased avidity.</p