ABSTRACTProbiotics play an important role in the maintenance of the gastrointestinal barrier. In addition to direct effects on mucosal integrity, the interaction with the intestinal mucosa may have an active immunoregulatory effect. In the present work, we exposed HT29 intestinal epithelial cells to twoBifidobacteriumspecies to determine their effect on gene expression profile, enterocyte monolayer integrity, and T-cell response.Bifidobacterium breveIPLA 20004 triggered a more pronounced increase in the transepithelial resistance of the enterocyte monolayer thanBifidobacterium bifidumLMG13195. The transcriptome profile of HT29 cells cultured in the presence ofB. bifidumLMG13195 showed an increased expression of immune mediators and, interestingly, chemotactic molecules (CXCL10, CCL20, CXCL11 and CCL22) able to recruit lymphocytes. Since regulatory T cells (Treg cells) may express receptors for specific chemokines, we cultured peripheral blood mononuclear cells with supernatants of HT29 cells previously treated withBifidobacteriumstrains and analyzed FOXP3 and CD25 Treg markers and CCR6, CXCR3, CCR4, and CCR3 expression on CD4+lymphocytes. The proportion of CD25highFOXP3+cells was significantly increased after culture withB. bifidumLMG13195-conditioned HT29 supernatant. Moreover, this treatment led to the largest amount of CCR6+CXCR3−CCR4+CCR3+CD4+cells expressing high levels of CD25, corresponding to the Treg population. These results suggest that soluble factors secreted afterB. bifidumLMG13195 contact with intestinal epithelial cells favored the generation of CD4+CD25highlymphocytes expressing chemokine receptor Treg markers, thus making possible their recruitment to the intestinal mucosa.