CBirTox Selectively Induces CD4+Foxp3+ T Cells to Microbiota Flagellin

We have previously defined a protective regulatory T cell (Treg)- Immunoglobulin A (IgA) pathway toward microbiota antigens. Tregs provide critical survival factors to IgA⁺ B cells in the intestine in order to maintain mucosal homeostasis. Cholera toxin B (CTB) has previously been demonstrated to induce Foxp3⁺ Tregs in vitro and in vivo when conjugated to defined antigens. In order to further study the Treg-IgA pathway, we have generated a construct, termed CBirTox, composed of a distal fragment of CBir1 flagellin fused to the A2 subunit of cholera toxin and expressed with CTB as a GM-1 ganglioside-binding pseudotoxin. CD11c⁺ dendritic cells (DCs) pulsed with CBirTox for as little as five minutes are capable of activating B6.CBir1 TCR Tg CD4⁺ T cells in vitro, while CD19⁺ B cells require pulse times of approximately one hour. CBirTox, but not CBir1 peptide, pulsed CD11c⁺ DCs from multiple tissues, including the spleen, MLN, and lamina propria, induced Foxp3 expression in approximately 20-30% of B6.CBir1 TCR Tg CD4⁺CD25^- T cells in vitro. Neither Th1 nor Th17 cells are induced with CBirTox, though they can develop under polarizing conditions, demonstrating a selective induction of Foxp3⁺ Tregs but not inhibition of Th1 or Th17 subsets. While retinoic acid inhibitors had no effect, anti-TGF-β ameliorated Foxp3 expression. CBirTox pulsed B cells downregulate expression of phosphorylated p70S6 kinase, a downstream target of mammalian target of rapamycin (mTOR), suggesting CBirTox acts to induce Foxp3⁺ Tregs by partially inhibiting antigen presenting cell (APC) mTOR signaling. Collectively, our data demonstrates CBirTox may be used as an effective probe of the Treg-IgA pathway.