Microflora reactive IL-10 producing regulatory T cells are present in the colon of IL-2 deficient mice but lack efficacious inhibition of IFN-γ and TNF-α production

Background: Inflammatory bowel disease in interleukin 2 (IL-2) deficient (1L-2(-/-)) mice is triggered by the intestinal microflora and mediated by CD4(+) T cells. Aims: To determine the characteristics of microflora specific intestinal T cells, including migration and cytokine production. Methods: Intestinal T cell populations and cytokine mRNA expression of specific pathogen free (SPF) and germ free (GF) IL-2(-/-) and IL-2(+/+) mice were compared by flow cytometry and reverse transcription-polymerase chain reaction. Cytokine production of intestinal mononuclear cells on stimulation with microflora antigens was assessed by ELISA. In vivo migration of T cells was assessed by adoptive transfer of Cr-51 labelled CD4(+)CD25(-)alphabeta(+) T cells. The ability of intestinal T cell lines to promote colitis was determined by adoptive transfer experiments. Results: SPF IL-2(-/-) mice produced higher interferon gamma (IFN-gamma) and tumour necrosis factor alpha mRNA levels than GF IL-2(-/-) mice, which was accompanied by an increased number of CD4(+)alphabeta T cells in the colon. Tracking of Cr-51 labelled and adoptively transferred T cells revealed an increased MAdCAM-1 dependent but VCAM-1 independent recruitment of these cells into the colon of SPF IL-2(-/-) mice. Colon lamina propria lymphocytes (LPL) from SPF IL-2(-/-) mice showed increased spontaneous IFN-gamma production in vitro. On stimulation with bacterial microflora antigens, intraepithelial lymphocytes and LPL did not produce IFN-gamma, but high quantities of IL-10, which did not suppress IFN-gamma production. Bacterial antigen specific cell lines established from colon LPL of SPF IL-2 mice with colitis showed a regulatory T cell-like cytokine profile and only marginally modulated the course of colitis and survival of IL-2(-/-) mice. Conclusions: Our results suggest that microflora reactive regulatory T cells are present in the colon of SPF IL-2(-/-) mice. However, IL-10 produced by these cells did not significantly modulate a possible secondary proinflammatory CD4 Th1 cell population to produce IFN-gamma.