Effect of intestinal microbiota on the induction of regulatory CD25⁺ CD4⁺ T cells

When oral tolerance was induced in either specific pathogen-free (SPF) or germ-free (GF) mice, ovalbumin (OVA) feeding before immunization induced oral tolerance successfully in SPF mice. On the other hand, OVA-specific immunoglobulin G1 (IgG1) and IgE titres in OVA-fed GF mice were comparable to those in phosphate-buffered saline-fed GF mice, thus demonstrating that oral tolerance could not be induced in GF mice. The frequencies of CD25⁺ CD4⁺/CD4⁺ cells in the mesenteric lymph node (MLN) and the absolute number of CD25⁺ CD4⁺ cells in the Peyer's patches and MLN of naive GF mice were significantly lower than those in naive SPF mice. In an in vitro assay, the CD25⁺ CD4⁺ cells from the naive SPF mice suppressed more effectively the proliferation of responder cells in a dose-dependent manner than those from the GF mice. In addition, the CD25⁺ CD4⁺ regulatory T (Treg) cells from the naive SPF mice produced higher amounts of interleukin (IL)-10 and transforming growth factor (TGF)-β than those from the GF mice. When anti-TGF-β neutralizing antibody, but not anti-IL-10 neutralizing antibody, was added to the in vitro proliferation assay, the suppressive effect of the CD25⁺ CD4⁺ Treg cells from the SPF mice was attenuated to the same level as that of the CD25⁺ CD4⁺ cells from the GF mice. In conclusion, the TGF-β-producing CD25⁺ CD4⁺ Treg cells from the MLN of SPF mice played a major role in oral tolerance induction. In addition, as the regulatory function of the CD25⁺ CD4⁺ cells from the naive GF mice was much lower than that of the CD25⁺ CD4⁺ Treg cells from the SPF mice, indigenous microbiota are thus considered to contribute to the induction and maintenance of CD25⁺ CD4⁺ Treg cells.