CD4⁺ and CD4⁻ cDC are equivalent in their ability to activate CD8⁺ T lymphocytes.

<p>(A, B) CD4⁺ and CD4⁻ cDC subsets were sensitized with graded doses of OVA peptide (A) or whole OVA (B) and co-cultured for 3 and 5 days with naive CD8⁺ T cells purified from OT-I mice. The production of IFN-γ was quantified by ELISA. Results represent the mean ± SD of a representative experiment out of two.</p

CD4⁺ and CD4⁻ cDC are equivalent in their ability to activate CD4⁺ T lymphocytes.

<p>(A) Splenic CD4⁺ and CD4⁻ cDC were sorted on the basis of CD11c, CD11b, CD8 and CD4 expression. The presence of contaminating plasmacytoid DC and CD8α⁺ cDC precursors in the CD4⁻ cDC fraction was evaluated by using anti-Siglec-H and Sirp-α mAbs, respectively. (B, C) Both cDC subsets were sensitized with graded doses of OVA peptide (B) or whole OVA (C) and co-cultured for 3 and 5 days with naive CD4⁺ T cells purified from OT-II mice. The production of IFN-γ and IL-13 was quantified by ELISA. Results represent the mean ± SD of a representative experiment out of two.</p

CD4⁺ and CD4⁻ cDC differ <i>in vitro</i> in their capacity to activate iNKT cells.

<p>(A, C) Sorted CD4⁺ and CD4⁻ cDC were exposed to graded doses of α-GalCer and then co-cultured for 48 h with sorted iNKT cells (A) or with the iNKT cell hybridoma DN32.D3 (C). Cytokine production was quantified by ELISA. Results represent the mean ± SD of 3 (A) or 2 (C) independent experiments. (B) CD1d expression on CD4⁺ and CD4⁻ cDC was assessed by flow cytometry. Of note, the staining with the isotype control was identical on both cDC subsets. For clarity, the isotype control on the CD4⁺, but not CD4⁻, cDC subset is shown. Shown is a representative experiment out of three. (D) Sorted CD4⁺ and CD4⁻ cDC were exposed, or not (medium), to α-GalCer (100 ng/ml) and then co-cultured for 6 h with sorted iNKT cells. RNAs were prepared and IL-12p35 (<i>Il12p35</i>) mRNA copy numbers were measured by quantitative RT-PCR. Data are normalized to expression of <i>Gapdh</i> and are expressed as fold increase over average gene expression in vehicle-treated cDC. Of note, the basal level of IL-12p40 transcript in <i>ex vivo</i> sorted cDC is relatively elevated (Ct: 25–26) (Ct of <i>gapdh</i>: 20, Ct of <i>il12p35</i>: 31–32). Data represent the mean ± SD (triplicates) of an experiment out of two performed. (E) α-GalCer-loaded cDC subsets were co-cultured for 48 h with sorted iNKT cells in the presence of a neutralizing IL-12 Ab or an isotype control Ab. Shown is a representative experiment (mean ± SD) out of three performed. * p<0.05; ** p<0.01; *** p<0.001.</p

Biomimetic Cryptic Site Surfaces for Reversible Chemo- and Cyto-Mechanoresponsive Substrates

Chemo-mechanotransduction, the way by which mechanical forces are transformed into chemical signals, plays a fundamental role in many biological processes. The first step of mechanotransduction often relies on exposure, under stretching, of cryptic sites buried in adhesion proteins. Likewise, here we report the first example of synthetic surfaces allowing for specific and fully reversible adhesion of proteins or cells promoted by mechanical action. Silicone sheets are first plasma treated and then functionalized by grafting sequentially under stretching poly(ethylene glycol) (PEG) chains and biotin or arginine-glycine-aspartic acid (RGD) peptides. At unstretched position, these ligands are not accessible for their receptors. Under a mechanical deformation, the surface becomes specifically interactive to streptavidin, biotin antibodies, or adherent for cells, the interactions both for proteins and cells being fully reversible by stretching/unstretching, revealing a reversible exposure process of the ligands. By varying the degree of stretching, the amount of interacting proteins can be varied continuously

Chemically Detachable Polyelectrolyte Multilayer Platform for Cell Sheet Engineering

Human gingival fibroblasts (HGFs) cell sheets have a potential use for in vivo wound healing due to the ability of HGFs to adopt a contractile phenotype which is typically expressed during extracellular matrix tissue remodeling. For this purpose, we developed a chemically detachable platform based on poly­(allylamine hydrochloride)/poly­(styrene sulfonate) multilayer film built on a sacrificial precursor film which served as a substrate for HGF cell layer formation. The sacrificial precursor film, based on disulfide-containing polycation and polyanion, is degradable under mild conditions compatible for cell sheet detachment. Cellular viability and cell phenotype analysis of HGF show that the designed platform promotes cell phenotype switch into contractile phenotype, maintained after cell sheet lift-off. This contractile phenotype is acquired by fibroblasts during in vivo wound healing and tissue remodeling. HGFs cell sheet fragments, obtained by this detachment process, could be cultured later on showing a good retention of the typical spindle-shape of differentiated cells after 10 days of culture. HGFs cell sheets have great potential applications as autologous substrates for tissue repair and cellular synthetic platforms for research on connective tissue diseases or evaluation of novel therapeutic agents