Co-cultures were incubated overnight in a CO2 incubator at 37 C

Co-cultures were incubated overnight in a CO2 incubator at 37 C. for positive or negative terminal charges, respectively, while oligoethylene glycol (OEG) or propyl groups adjusted the relative hydrophilicity. We then created intermixed nanofibers containing both epitope-bearing peptides (OVAQ11, containing the OVA323C339 mixed B cell/T cell epitope peptide) and the variously functionalized peptides. We have previously demonstrated that several Q11 based peptides carrying different terminal functional sequences co-assemble into the same nanofiber, and we have developed preparation procedures for maximizing intermixing.23 In addition, two different peptides with N, N-dimethylated glutamine residues in the Fumalic acid (Ferulic acid) Q11 domain were also studied, where the Fumalic acid (Ferulic acid) dimethylated Gln residues were expected to disrupt -sheet formation by disrupting side chain hydrogen bonding.40 Table 1 Summary of peptides investigated imaging. Draining lymph nodes (brachial, axillary, and inguinal) were collected and imaged using an IVIS Fumalic acid (Ferulic acid) 200 imager 24 h after subcutaneous immunization. This time point was chosen to emphasize draining of the assemblies rather than slower trafficking of the materials by antigen-presenting cells. By this measure, all peptide nanofibers were drained to the lymph nodes (Figure 6). Interestingly, Q11-6 drained most efficiently, with considerably Fumalic acid (Ferulic acid) more accumulation in all lymph nodes tested than Q11. It is not clear why this was the case, but it is known that size is an important determinant of whether a particle can enter the lymphatic system and drain efficiently to lymph nodes, and that particles smaller than 200 nm drain with particular efficiency.44 It is not clear to what extent such metrics would apply to the high aspect ratio nanofibers studied here, but it is possible that Q11-6s increased lymph node drainage could have arisen from smaller size (widths of about ? that of other fibers, Table 2) and potential degradation into smaller particles. KQ11 and EEEQ11 trafficked similarly to each other (Figure 6), at a level slightly greater than Q11 (Figure 6). These findings indicated that surface charge did not significantly affect gross lymphatic draining, and that the diminished responses seen with the negatively charged assemblies could not be attributed to reduced entry into lymph nodes. Open in a separate window Figure 6 Surface potentials did not compromise lymphatic drainingDraining lymph nodes were collected at 24 h after immunization, imaged (a), and quantified (b). Mice were Rabbit Polyclonal to PRKAG1/2/3 immunized subcutaneously with 100 uL TAMRA-labeled nanofibers (1.8 mM peptide + 0.2 mM OVAQ11 + 0.02 mM TAMRA-OVAQ11). Images shown were from one representative experiment (n=2) and data plotted were combined from Fumalic acid (Ferulic acid) two experiments (n=4) and analyzed by two-way ANOVA and Dunnetts multiple comparison test (compared to Q11 group). *:p 0.05, ***: p 0.001. Differential uptake and presentation efficiency by antigen presenting cells For the activation of antigen-specific T helper cells, antigens need to be internalized by antigen-presenting cells (APCs), proteolyzed (processed) into short T cell epitopes, loaded into class-II major histocompatibility molecules (MHC class II), and presented to T cells on the APC surface. Antigen-specific T cells, once activated, then go on to help B cells produce antibodies. Owing to the fact that both T cell responses and B cell responses were eliminated by negatively charged EEEQ11, we hypothesized that significant charge on the nanofibers may interfere with the process of antigen uptake, processing, and presentation. To test this hypothesis, we studied nanofiber internalization using fluorescent nanofibers and flow.