Figure 1. Mucosal antibody secretion and small intestinal IgA⁺ Plasma Cell activity exhibit diurnal rhythmicity.

A) Serial fecal sampling of C57BL/6 mice at five 6 hour intervals over a circadian day (ZT 0, 6, 12, 18, 0), n=10 (pooled from two independent data sets). Data representative of at least 4 independent experiments. B) Exemplar flow plots of small intestinal CD138⁺ IgA⁺ PC, pregated as Live CD45⁺CD3^-CD5^-NK1.1^-MHCII^+/-B220^-IgD^-, at ZT0 and ZT12 and C) Quantification of IgA⁺ plasma cell frequencies at ZT0, 6, 12 and 18. B+C n=5 and representative of three independent experiments. D) Ex vivo secretion of IgA by sort-purified IgA⁺ PC (from ZT0 and ZT12) cultured for 18 hours. Data pooled from two independent experiments, n=8. E) Heatmap of significantly oscillatory genes (JTK Cycle, p<0.01) identified from bulk RNA Sequencing of sort-purified small intestinal IgA⁺ PC taken at ZT0, 6, 12 and 18, z-score of average relative gene expression (fpkm) values of n=5 per timepoint. F) GO-Term pathway enrichment analysis on oscillatory gene signatures. Selected relative expression (fpkm) values for oscillatory gene signatures related to G) Plasma Cell function, survival and identitity, H) Extrinsic survival and antibody secretion signals, and I) Cellular Metabolism, values representative of n=5 per timepoint. P values were determined using JTK Cycle, with the exception of panel D which was determined via a parametric, unpaired t-test. All data shown as +/- SEM, * p< 0.05, ** p< 0.01, *** p< 0.001, **** p< 0.0001.