Figure 2. Fibrin matrices functionalized with FNIII 9-10 + FNIII 11-EDA stimulate an antigen-specific CD8⁺ T cell response.

(a) Immunization schedule. C57BL/6J mice were immunized with 50 μg OVA and 20 μg LPS on day 0. The ability of FNIII EDA-containing fibrin-binding FN fragments to boost a CD8⁺ T cell response was tested on day 14 by implanting s.c. fibrin matrices functionalized with 5 nmol of TG-OVA_250–264 (comprising the MHC-I binding immunodominant peptide of ovalbumin, OVA_257–264) + 5 nmol of FNIII 11-EDA or FNIII 9-10-EDA (each with an N-terminal TG domain); alternatively mice were injected i.d. with 5 nmol of soluble OVA_257–264 + 50 μg of LPS or with PBS only. On day 19, mice were sacrificed and the spleen was harvested. (b) Fibrin gels functionalized with fibrin-binding FNIII 9-10 + FNIII 11-EDA (each with an N-terminal TG domain) + TG-OVA_250–264 induced levels of OVA_257–264-MHC-I-specific CD8⁺ T cells in the spleen comparable to that induced by i.d. injections of LPS + OVA_257–264. (c, d) IFN-γ production from splenocytes restimulated for 6 h ex vivo with OVA_257–264 were evaluated by flow cytometry. The proportion of IFN-γ producing CD8⁺ T cell was similar among the different groups. However, the production of IFN-γ by CD8⁺ T cells was greater, as shown by the mean fluorescence intensity (MFI) of IFN-γ intracellular staining, in mice that were boosted with fibrin matrices functionalized with FNIII 9-10 + FNIII 11-EDA (each with an N-terminal TG domain) + TG-OVA_250–264 or with LPS and OVA_257–264; the group boosted with FNIII 11-EDA yielded statistically similar responses as the group boosted with LPS. Box plots represent median ± 95% confidence interval (n = 5). *P < 0.05; ns, not significant.