Background CD4+ regulatory T cells (Tregs) in human newborns express FOXP3 and CTLA-4. Although they are naïve they are functional and essential in the maintenance of self-tolerance and immune homeostasis. Tregs can be subdivided into Helios+ natural Tregs derived from the thymus and Helios– induced Tregs that are generated from conventional CD4 T cells in the periphery after stimulation. The gut microbiota is a stong stimulus for the development of the immune system in neonates and germ-free animal models have demonstrated that intestinal bacterial colonization is important for the induction of Tregs and tolerance.
Objectives We here asked whether specific bacterial strains that colonize the human infant gut are able to expand the proportion of Tregs from newborns in vitro. We also asked whether the expanded cells were still suppressive and if they were dominated by natural or induced Tregs. Finally we asked whether an association could be found in vivo between early gut bacterial colonization and proportion of CTLA-4+ putative Tregs.
Methods Mononuclear cells were isolated from cord blood of healthy newborns and peripheral blood of healthy adults. We defined the regulatory T cells as the CD4+CD25+CD127low/neg T cells (Tregs) both in the subsequent cell sorting, the suppression assays and and when subdividing them into Helios+ natural Tregs and peripherally induced Helios- Tregs. The cell cultures were stimulated with UV-killed bacteria isolated from infants, here Escherichia coli, Staphylococcus aureus or Lactobacilli paracaseii. We also analyzed the association between gut bacterial colonization and regulatory T cells in a prospective newborn-infant cohort of 64 children using multivariate factor analysis.
Results The proportion of Tregs out of CD4+ cells was significantly lower in newborns compared to adults and newborns had a significantly higher fraction of Helios+ natural Tregs compared to adults. Tregs from newborns suppressed autologous naïve CD4+ responder cells equally well as Tregs from adults. Stimulation with E. coli significantly increased the proportion of Tregs of CD4+ T cells in newborns in contrast to adults. When subdivided into natural Helios+ and induced Helios– Tregs we found that the expansion of the Tregs in newborns was more pronounced in the natural than in the induced Treg fraction. The Tregs maintained their suppressive ability in vitro after bacterial stimulation. In the prospective newborn-infant cohort we found a positive association between early gut colonization with E. coli and proportion of CTLA-4+ cells out of CD4+ T cells at 1 month of age.
Conclusions E. coli expands the proportion of preferentially Helios+ natural CD25+CD127low/neg regulatory T cells from newborn children in vitro and these cells maintain their suppressive capacity. These results were supported by the finding that early colonization with E. coli and no other bacteria was associated with higher proportions of CTLA-4+ cells of CD4+ T cells in 1-month-old infants in the prospective newborn-infant cohort. Thus, early intestinal colonization with E. coli may play an important role in increasing the proportion of natural Tregs in infants, which may lead to development of stronger self-tolerance and immune homeostasis in children.
Disclosure of Interest None Declared
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