Identification and characterization of intestinal antigen-presenting cells involved in uptake and processing of a nontoxic recombinant chimeric mucosal immunogen based on cholera toxin using imaging flow cytometry

Abstract

Intragastric immunization with recombinant chimeric immunogen, SBR-CTA2/B, constructed from the saliva-binding region (SBR) of Streptococcus mutans antigen AgI/II and the A2/B subunits of cholera toxin (CT) induces salivary and circulating antibodies against S. mutans that protect against dental caries. We previously found that SBR-CTA2/B activated dendritic cells (DC) in the Peyer's patches (PP) and mesenteric lymph nodes (MLN). To identify the cells involved in the intestinal uptake of SBR-CTA2/B and the initiation of immune responses, mice were immunized intragastrically with fluorescein-labeled SBR-CTA2/B or SBR, and intestinal cells were examined by imaging flow cytometry after fluorescent staining for cell surface markers. SBR-CTA2/B was preferentially taken up by CD103+ DC in the PP and by both CD103+ and CD11c + DC in intestinal lamina propria (LP), whereas SBR was taken up to a lesser extent by PP CD11c+ DC, within 2 to 16 h. By 16 h, CD103 + and CD11c+ DC containing fluorescein-labeled SBR-CTA2/B were found in MLN and showed upregulation of the chemokine receptor CCR7. Large numbers of SBR-CTA2/B-containing DC were found interacting with CD4+ (T helper) cells, which costained for nuclear transcription factors T-bet or RORγt, identifying them as Th1 or Th17 cells. In contrast, SBR-containing CD11c"1" DC interacted preferentially with GATA3 + (Th2) cells. No SBR- or SBR-CTA2/B-containing DC were found interacting with Foxp3+ (T regulatory) cells. We conclude that the coupling of SBR to CTA2/B enhances its immunogenicity by promoting uptake by DC in both PP and LP and that these antigen-containing DC migrated to MLN and interacted preferentially with Th1 and Th17 cells to induce active immune responses. Copyright © 2014, American Society for Microbiology. All Rights Reserved.