Monitoring and Modulation of Inducible Foxp3+ Regulatory T-Cell Differentiation in the Lymph Nodes Draining the Small Intestine and Colon.

The mucosa-draining lymphoid tissue favors differentiation of inducible Foxp3+ regulatory T cells. Adoptive transfer of T-cell receptor (TCR) transgenic (Tg) T cells is a powerful tool to study antigen-specific regulatory T-cell differentiation in lymphoid tissues in vivo. The kinetics and nature of the T-cell response largely depend on the route of antigen administration and degree of clonal competition. Here, we describe that adoptive transfer of CD4+ DO11.10 TCR Tg T cells can be used for monitoring Foxp3+ regulatory T-cell differentiation in the gut-draining lymph nodes. We describe two routes of mucosal antigen administration, e.g., the oral and intracolonic route known to induce T-cell responses in the small intestine-draining mesenteric lymph nodes (MLN) and distal colon-draining caudal and iliac lymph nodes (ILN), respectively. In particular, we discuss differences in frequency of inducible Foxp3+ regulatory T cells after adoptive transfer of variable numbers of Tg T cells and various amounts of orally gavaged ovalbumin (OVA), and explain how Foxp3+ regulatory T-cell differentiation can be modulated by coadministration of the adjuvant cholera toxin (CT) with OVA using this adoptive transfer system.

Macrophage-mediated gliadin degradation and concomitant IL-27 production drive IL-10- and IFN-γ-secreting Tr1-like-cell differentiation in a murine model for gluten tolerance.

Celiac disease is caused by inflammatory T-cell responses against the insoluble dietary protein gliadin. We have shown that, in humanized mice, oral tolerance to deamidated chymotrypsin-digested gliadin (CT-TG2-gliadin) is driven by tolerogenic interferon (IFN)-γ- and interleukin (IL)-10-secreting type 1 regulatory T-like cells (Tr1-like cells) generated in the spleen but not in the mesenteric lymph nodes. We aimed to uncover the mechanisms underlying gliadin-specific Tr1-like-cell differentiation and hypothesized that proteolytic gliadin degradation by splenic macrophages is a decisive step in this process. In vivo depletion of macrophages caused reduced differentiation of splenic IFN-γ- and IL-10-producing Tr1-like cells after CT-TG2-gliadin but not gliadin peptide feed. Splenic macrophages, rather than dendritic cells, constitutively expressed increased mRNA levels of the endopeptidase Cathepsin D; macrophage depletion significantly reduced splenic Cathepsin D expression in vivo and Cathepsin D efficiently degraded recombinant γ-gliadin in vitro. In response to CT-TG2-gliadin uptake, macrophages enhanced the expression of Il27p28, a cytokine that favored differentiation of gliadin-specific Tr1-like cells in vitro, and was previously reported to increase Cathepsin D activity. Conversely, IL-27 neutralization in vivo inhibited splenic IFN-γ- and IL-10-secreting Tr1-like-cell differentiation after CT-TG2-gliadin feed. Our data infer that endopeptidase mediated gliadin degradation by macrophages and concomitant IL-27 production drive differentiation of splenic gliadin-specific Tr1-like cells.

Colonic tolerance develops in the iliac lymph nodes and can be established independent of CD103(+) dendritic cells.

Tolerance to harmless exogenous antigens is the default immune response in the gastrointestinal tract. Although extensive studies have demonstrated the importance of the mesenteric lymph nodes (MLNs) and intestinal CD103(+) dendritic cells (DCs) in driving small intestinal tolerance to protein antigen, the structural and immunological basis of colonic tolerance remain poorly understood. We show here that the caudal and iliac lymph nodes (ILNs) are inductive sites for distal colonic immune responses and that colonic T cell-mediated tolerance induction to protein antigen is initiated in these draining lymph nodes and not in MLNs. In agreement, colonic tolerance induction was not altered by mesenteric lymphadenectomy. Despite tolerance development, CD103(+)CD11b(+) DCs, which are the major migratory DC population in the MLNs, and the tolerance-related retinoic acid-generating enzyme RALDH2 were virtually absent from the ILNs. Administration of ovalbumin (OVA) to the distal colon did increase the number of CD11c(+)MHCII(hi) migratory CD103(-)CD11b(+) and CD103(+)CD11b(-) DCs in the ILNs. Strikingly, colonic tolerance was intact in Batf3-deficient mice specifically lacking CD103(+)CD11b(-) DCs, suggesting that CD103(-) DCs in the ILNs are sufficient to drive tolerance induction after protein antigen encounter in the distal colon. Altogether, we identify different inductive sites for small intestinal and colonic T-cell responses and reveal that distinct cellular mechanisms are operative to maintain tolerance at these sites.

Adaptive T-cell responses regulating oral tolerance to protein antigen.

The term oral (or mucosal) tolerance has been classically defined as the suppression of T- and B-cell responses to an antigen by prior administration of the antigen by the oral route. In recent years, it has become clear that both innate and acquired regulatory immune responses are essential for the development of oral tolerance. As such, mucosal microenvironmental factors such as transforming growth factor- ß, prostaglandins but also dietary vitamin A create conditioning of an adaptive regulatory T-cell response that suppresses subsequent antigen-specific responses. Particular resident subsets of antigen presenting dendritic cells are pivotal to convey conditioning signals next to the presentation of antigen. This review discusses the primary mechanisms of adaptive regulatory T-cell induction to ingested soluble protein antigen. However, we also discuss the limitations of our knowledge with respect to understanding the very common food hypersensitivity Celiac disease caused by an aberrant adaptive immune response to the food protein gluten.

Cyclooxygenase-2 in mucosal DC mediates induction of regulatory T cells in the intestine through suppression of IL-4.

Oral intake of protein leads to tolerance through the induction of regulatory T cells (Tr cells) in mesenteric lymph nodes (MLNs). Here we show that the inhibition of cyclooxygenase-2 (COX-2) in vivo suppressed oral tolerance and was associated with enhanced differentiation of interleukin (IL)-4-producing T cells and reduced Foxp3(+) Tr-cell differentiation in MLN. As a result, the functional suppressive capacity of these differentiated mucosal T cells was lost. IL-4 was causally related to loss of tolerance as treatment of mice with anti-IL-4 antibodies during COX-2 inhibition restored tolerance. Dendritic cells (DCs) in the MLN differentially expressed COX-2 and reductionist experiments revealed that selective inhibition of the enzyme in these cells inhibited Foxp3(+) Tr-cell differentiation in vitro. Importantly, the inhibition of COX-2 in MLN-DC caused increased GATA-3 expression and enhanced IL-4 release by T cells, which was directly related to impaired Tr-cell differentiation. These data provide crucial insights into the mechanisms driving de novo Tr-cell induction and tolerance in the intestine.