Fig. 1: The scFv CD103 fusion protein specifically binds CD103+ DCs populations.
Schematic representation and in silico model of the adjuvant active fusion proteins; CTA1-II-DD, CTA1-I/II-DD, CTA1-II-CD103, and CTA1-I/II-CD103 molecules, carrying the MHC I-restricted SIINFEKL-peptide and/or class II-restricted p323-peptide from ovalbumin (a). Western blot analysis of the CTA1-II-DD (1), CTA1-I/II-DD (2), CTA1-II-CD103 (3), and CTA1-I/II-CD103 (4) molecules (b). The ADP-ribosylating activity of CT holotoxin and the CTA1-enzyme or mutant CTA1R7K in the respective fusion proteins as assessed by the agmatine in vitro test. (c). Binding-specificity of the fusion proteins was assessed in vitro by gating on migratory DCs (MHC IIhigh CD11chigh) from mesenteric LN (MLN) from WT or CD103−/− C57Bl/6 mice and flow cytometry with labeled DC-relevant Mabs (CD11c, MHC-II, CD103, CD11b) and labeled anti-Flag Mab to detect the fusion proteins used at 1 μM conc (d). Similar to D, but this in vitro analysis used WT migratory DCs or B cells (CD19+) from mediastinal LN (mLN) to assess the ability of the fusion proteins (MFI of labeled anti-Flag Mabs) at 1 and 0.1 μM concentrations to bind the different DC-subsets; CD103+CD11b−, CD103+CD11b+, CD103−CD11b+ or B cells using flow cytometry (e). Results are representative of three independent experiments with mean values±SD for means of triplicate cultures (d, e). Statistical significance was calculated using student t test with p value * p < 0.05, **p < 0.01, ***p < 0.001.
