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. 2022 Oct 3;13:1015585. doi: 10.3389/fimmu.2022.1015585

Figure 4.

Figure 4

Human AACs show antigen encapsulation and, after uptake, induce MoDC maturation to activate antigen-specific CD8+ T cells in vitro. (A) Annexin V staining and FAM-labeled E7 SLP delivery to human RBCs following squeeze. (B) Left: graph displaying mean of 3 donors (see methods section), anti-human CD235a (blue) and FAM-E7 (green) fluorescence intensity along line-scan drawn across the length of the AAC-HPV. Right: line-scan is shown in representative microscopy images of a single human AAC-HPV squeezed with (top) FAM-E7 or (bottom) unlabeled E7 stained with erythrocyte marker anti-human CD235a. (C) Uptake of PKH26-AAC-HPV by HLA-A*02+ CD11c+ MoDCs at 37°C or 4°C. For display purposes, conditions with unlabeled AAC-HPV were plotted on the x-axis at 0.2, since zero cannot be plotted on a log scale (n = 3 independent experiments with 3 distinct RBC donors). (D) Expression of maturation markers CD86, CD80 and MHC class II on MoDCs following 2-day culture with AAC-HPV. Data is shown as fold change in gMFI in comparison to media control (n = 5 MoDC donors). Each colored dot represents a different donor. (E) Manufacturing scale SQZ-AAC-HPV and HLA-A*02+ MoDCs were cultured overnight with E711-20 -specific CD8+ T cells. Supernatants analyzed for IFNγ release by ELISA (n = 6 different RBC donors). *P < 0.05, **P < 0.01, ****P < 0.0001, unpaired t-test.