Thus, transcriptome profiles, TCR repertoire analysis, as well as analysis of neuropilin-1 expression, indicate that Treg cells in the gut are quite different compared with Treg cells at other sites, and, in particular, the gut Treg-cell population is comprised of substantial numbers of iTreg cells besides nTreg cells. It is tempting to speculate that a higher prevalence of iTreg cells in the gut might be due

to the particular intense contact with foreign antigen in that location and, in fact, Treg cells in the LP have been noted to encode TCRs directed against the intestinal microbiota [16]; however, this seemingly straightforward correlation between antigen load and iTreg-cell numbers needs to be tempered by considering the total number of Treg cells in the gut. Although Foxp3+ cells are abundant

in Ixazomib order the gut LP, they are still less frequent as compared with macrophages, plasma cells, and some other T-cell subsets. By carefully counting the number of Treg cells in longitudinal 7 μm ileum cryosections for mice we observed, on average, 0.35 cells per villus (O. Pabst, unpublished observation). We expect this number might vary depending on the housing conditions and intestinal microbiota composition, as both are GSI-IX known to skew the Treg-cell pool in the gut [17, 18]. In any case, the actual number of Treg cells per villus seems too limited, rendering it unlikely that the Treg-cell pool with its TCR specificities might fully cover the complexity of the total antigen load. It is therefore possible that the antigen-driven generation of iTreg cells

does not account for immunoregulation covering the full antigen load but might rather constitute a sophisticated pathway to deal with particularly “problematic” antigens. In vitro, TGF-β and IL-2 are sufficient to induce expression of Foxp3 in a substantial eltoprazine fraction of activated CD4+ T cells [19] and this fraction can be further increased by the addition of retinoic acid (RA) [20]. TGF-β and RA have also been suggested to enable iTreg-cell generation following antigen administration through the oral route [21, 22]. One commonly used experimental setup to quantify Treg-cell conversion in the intestinal immune system involves the adoptive transfer of TCR-transgenic Foxp3− T cells to recipient mice. Subsequent antigen feeding results in T-cell activation and proliferation, and the formation of a sizable number of Foxp3+ T cells (Fig. 1) [3, 21, 23]. In the gut-draining mesenteric lymph nodes (mLNs), this frequency is considerably higher as compared with that of other lymphoid compartments. Such a high capacity to generate iTreg cells could be recapitulated in vitro by stimulating Foxp3− cells via “intestinal” DCs, that is, DCs isolated from mLNs or intestinal LP, but not those from pLNs or splenic DCs [21, 24].