The secretory SEA component IPSE/alpha-1 induces Breg cell development.

<p><b>(A)</b> Splenic B cells were incubated with PF-647-labeled SEA (20 μg/ml), natural IPSE/alpha-1 (nIPSE, 1–5–10 μg/ml) or left untreated and the frequency of SEA- and IPSE-positive cells measured after 60 minutes by flow cytometry. Representative FACS plots and summary of 3 experiments is shown (N = 3). <b>(B)</b> Splenic B cells from naïve mice were cultured for 3 days with 10 μg/ml nIPSE, 20 μg/ml SEA, 20 μg/ml SEAΔIPSE or medium as control, with or without addition of anti-CD40 mAb (2 μg/ml). Cytokine concentration in supernatants after 3 days as determined by ELISA. Summary of 4 experiments. <b>(C)</b> Splenic B cells from naïve mice were cultured for 3 days with 10 μg/ml recombinant, plant-derived IPSE (pIPSE), 20 μg/ml SEA or medium as control. Cytokine concentration in supernatants after 3 days as determined by ELISA. Summary of 4 experiments. <b>(D)</b> Splenic B cells were cultured for 3 days with nIPSE (10 μg/ml), SEA (20 μg/ml) or medium alone as control, and subsequently co-cultured with CD4⁺CD25^- sorted splenic T cells from C57BL/6 or DEREG mice. After 4 days, the frequency of CD25⁺Foxp3⁺ Treg cells within the CD4 T cell population was determined by flow cytometry. Summary of 3 experiments. Significant differences to medium control are indicated with * p < 0.05, as tested by Mann-Whitney test. ^# p < 0.05 indicates significant difference by one-sample t-test of log-transformed data.