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. 2023 Jul 21;11(7):e007198. doi: 10.1136/jitc-2023-007198

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© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See http://creativecommons.org/licenses/by-nc/4.0/.

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Pathogen vaccines can function as naPRRa to activate both murine and human DCs. Patient PBMCs or murine splenocytes were activated with naPRRa and analyzed by spectral flow cytometry. (A) Immune cell populations in PBMCs from two patients visualized by viSNE (left). Representative viSNE plots show indicated activation marker expression (middle), and bar graph (right) quantifies mean fluorescence intensity (MFI) fold changes of indicated activation marker expression in the aggregate cDC cluster (DN DC, DC1, DC2). Statistical significance was calculated against No PRRa condition by two-way ANOVA with Dunnett’s multiple comparison test. (B) Representative viSNE plots showing murine splenocyte subsets (left) indicated activation marker expression (middle) and bar graph (right) of mean MFI fold changes of activation marker expression in the aggregate cDC cluster (DN DC, DC1, DC2). Statistical significance was calculated comparing against No PRRa by two-way ANOVA with Dunnett’s multiple comparison test; data from three independent experiments. (C) Representative cytomountain plots comparing cDC and B cell costimulatory marker expression in response to naPRRa. (D) Representative cytomountain plots depicting activation of in vivo Flt3L-generated DCs by the naPRRa BCG. (E) Heatmap summarizing CD11c⁺ DC cytokine production and type I interferon response after naPRRa stimulation, as measured by multiplex bead assay and qPCR, respectively. DC cytokines that did not change from baseline on stimulation are not shown. ANOVA, analysis of variance; DC, dendritic cells; DN, double-negative; PRRa, pattern recognition receptor agonists.