(A, B) HLA-DR (A) or CD83 (B) expression on the surface of DCs stimulated for 24 hr on PSLB prepared as indicated to present SE. Each symbol represents the mean fluorescent intensity of a cell (n ≥ 20) from five independent donors, which are represented by the different donors. Lines and errors represent means ± SD. ns, not significant; *, p≤0.05; ***, p<0.001; Kruskal-Wallis with Tukey`s post-hoc test. (C) After 24 hr of culture, the supernatant of DCs stimulated as in A) and B) were analyzed for the presence of different secreted factors. Results from seven independent donors. The fold-change was normalized to the results from the supernatant of DCs incubated with PSLB containing ICAM-1 alone. (D) For assessing the biological significance of presenting CD40L in vesicular structures compared to the proposed biologically active form of soluble trimeric CD40L [i, green], we developed 86 nm diameter synthetic unilamellar vesicles (SUV; Supplementary file 2D) using phospholipids either without (ii, gray) or with His-tag, and hence CD40L, binding activity (iii, blue). An equal total mass (Mass eq.) of N-terminal His-tagged recombinant human CD40L was either incubated with DOPC liposomes (mock-SUV [ii, gray]) or used to load 12.5% DOGS-NTA and make vesicular CD40L (SUV:CD40L [iii, green]). As control, a Mass eq. of CD40L was used as soluble, free protein in culture. (E) FCM analyses of moDCs stimulated for 24 hr with either SUVs containing increasing amounts of CD40L monomers (blue; approximately 0, 40, 80, 120, 190 and 230 CD40L molec./SUV respectively) or an equivalent concentration of soluble CD40L (green). Data represent fold change in the GMFIs for ICAM-1, CD80 and CD86 of treated moDCs over unpulsed controls. (F) As in B, moDCs were stimulated using SUV:CD40L at a final load of approximately 230 molec./SUV. An equivalent concentration of soluble CD40L either delivered alone or in combination with SUVs lacking NTA lipids (soluble CD40L + SUV) were used as controls. After 24 hr of stimulation moDCs were collected, FcR blocked, stained and analysed by multicolor FCM for the expression of maturation markers. Given the high variability of arbitrary fluorescence values, the response was normalized as a fold change compared to unpulsed, immature DC controls. Data represent mean + /- SEM collected from five different donors and five independent experiments. One-way ANOVA and Dunn´s multiple comparisons test to the mock treated control was performed. P values < 0.05 (*);<0.002 (**);<0.0002 (***);<0.0001 (****) were considered significant.