Tolerance to food antigen manifests in the absence and/or suppression of antigen-specific immune responses locally in the gut but also systemically, a phenomenon known as oral tolerance. Oral tolerance is thought to originate in the gut-draining lymph nodes, which support the generation of FoxP3(+) regulatory T (Treg) cells. Here we use several mouse models to show that Treg cells, after their generation in lymph nodes, need to home to the gut to undergo local expansion to install oral tolerance. Proliferation of Treg cells in the intestine and production of interleukin-10 by gut-resident macrophages was blunted in mice deficient in the chemokine (C-X3-C motif) receptor 1 (CX3CR1). We propose a model of stepwise oral tolerance induction comprising the generation of Treg cells in the gut-draining lymph nodes, followed by migration into the gut and subsequent expansion of Treg cells driven by intestinal macrophages.

Seminal observations demonstrated that lymphocytes isolated from gut-associated lymphoid tissues (GALT) preferentially home to mucosal tissues, whereas lymphocytes isolated from skin-draining LN are biased to enter cutaneous sites ( 1, 2 ). Since then the molecular mechanisms underlying these divergent homing properties have been studied in exquisite detail (for a recent review see reference 3 ). The migration of antigen-experienced T cells into the intestine requires ␣ 4 ␤ 7-integrin and the chemokine receptor CCR9. ␣ 4 ␤ 7-Integrin interacts with mucosal vascular addressin cell adhesion molecule 1, which is uniformly expressed by intestinal venules, and ␤ 7-integrin -defi cient cells are impaired in entering the intestine ( 4, 5 ). Expression of the CCR9 ligand CCL25 builds up a gradient with highest expression in the proximal small intestine, low expression in the ileum, and no detectable expression in the colon ( 6 ). Consistently, homing of T cells into the proximal small intestine more stringently depends on CCL25 -CCR9 interaction compared with homing into the ileum ( 7 ). In contrast, the migration of eff ector T cells into the skin requires expression of E-and P-selectin ligand and the chemokine receptor CCR4, which endow T cells to interact with the respective ligands expressed in the skin ( 8 ). Thus the anatomical site of T cell activation and expansion determines the array of homing factors, including chemokine receptors and integrins, expressed by antigen-experienced T cells. The particular combination of these homing factors specifi cally targets antigen-experienced T cells to diff erent lymphoid and extralymphoid tissues expressing the respective ligands.Numerous studies indicated that DC play a decisive role in the imprinting of tissue tropism. In vitro stimulation by GALT-derived DC is suffi cient to induce expression of ␣ 4 ␤ 7-integrin and CCR9 on T cells ( 9, 10 ), whereas DC isolated from skin-draining LN confer expression

High-throughput sequencing reveals stability of the intestinal IgA repertoire after plasma cell depletion and changes in repertoire diversity with age and microbial colonization.

Secretory immunoglobulin A (SIgA) shields the gut epithelium from luminal antigens and contributes to host-microbe symbiosis. However, how antibody responses are regulated to achieve sustained host-microbe interactions is unknown. We found that mice and humans exhibited longitudinal persistence of clonally related B cells in the IgA repertoire despite major changes in the microbiota during antibiotic treatment or infection. Memory B cells recirculated between inductive compartments and were clonally related to plasma cells in gut and mammary glands. Our findings suggest that continuous diversification of memory B cells constitutes a central process for establishing symbiotic host-microbe interactions and offer an explanation of how maternal antibodies are optimized throughout life to protect the newborn.

Eosinophils are potent effector cells that are recruited to sites of inflammation. However, in some tissues, in particular in the gastrointestinal tract, eosinophils constitute an abundant leukocyte population also under homeostatic conditions. The lack of suitable isolation protocols restricted the analysis of these cells to histological assessment of cell numbers while important aspects of their phenotype, turnover, and functions remain unresolved. In this study, we report a protocol that allows the quantitative isolation of intestinal eosinophils. We characterized small intestinal eosinophils by flow cytometry as SSChighCD11b+CD11c+CCR3+Siglec-F+ cells. Intestinal eosinophils resembled eosinophils isolated from thymus and uterus but differed from eosinophils isolated from lung or blood. Eosinophils in intestine, thymus, and uterus showed in vivo a markedly higher life time compared with eosinophils present in lung and blood measured by incorporation of BrdU. This indicates that under steady-state conditions homeostasis of eosinophils is controlled by regulation of cell survival. Intestinal eosinophils are severely reduced in the intestines of Rag-2/common γ-chain double-deficient mice but not Rag-2-deficient mice, correlating with differential expression of GM-CSF and CCL11 in both mouse strains. Moreover, under steady-state conditions, intestinal eosinophils constitutively express high levels of the common γ-chain transcripts compared with lung eosinophils as well as eosinophils present under inflammatory conditions. These observations reveal a hitherto unrecognized diversity in phenotypic and functional properties of eosinophils and suggest that tissue-specific common γ-chain-dependent signals might profoundly affect eosinophil function and homeostasis.

BackgroundDespite a myriad of attempts in the last three decades to diagnose ovarian cancer (OC) earlier, this clinical aim still remains a significant challenge. Aberrant methylation patterns of linked CpGs analyzed in DNA fragments shed by cancers into the bloodstream (i.e. cell-free DNA) can provide highly specific signals indicating cancer presence.MethodsWe analyzed 699 cancerous and non-cancerous tissues using a methylation array or reduced representation bisulfite sequencing to discover the most specific OC methylation patterns. A three-DNA-methylation-serum-marker panel was developed using targeted ultra-high coverage bisulfite sequencing in 151 women and validated in 250 women with various conditions, particularly in those associated with high CA125 levels (endometriosis and other benign pelvic masses), serial samples from 25 patients undergoing neoadjuvant chemotherapy, and a nested case control study of 172 UKCTOCS control arm participants which included serum samples up to two years before OC diagnosis.ResultsThe cell-free DNA amount and average fragment size in the serum samples was up to ten times higher than average published values (based on samples that were immediately processed) due to leakage of DNA from white blood cells owing to delayed time to serum separation. Despite this, the marker panel discriminated high grade serous OC patients from healthy women or patients with a benign pelvic mass with specificity/sensitivity of 90.7% (95% confidence interval [CI] = 84.3–94.8%) and 41.4% (95% CI = 24.1–60.9%), respectively. Levels of all three markers plummeted after exposure to chemotherapy and correctly identified 78% and 86% responders and non-responders (Fisher’s exact test, p = 0.04), respectively, which was superior to a CA125 cut-off of 35 IU/mL (20% and 75%). 57.9% (95% CI 34.0–78.9%) of women who developed OC within two years of sample collection were identified with a specificity of 88.1% (95% CI = 77.3–94.3%). Sensitivity and specificity improved further when specifically analyzing CA125 negative samples only (63.6% and 87.5%, respectively).ConclusionsOur data suggest that DNA methylation patterns in cell-free DNA have the potential to detect a proportion of OCs up to two years in advance of diagnosis and may potentially guide personalized treatment. The prospective use of novel collection vials, which stabilize blood cells and reduce background DNA contamination in serum/plasma samples, will facilitate clinical implementation of liquid biopsy analyses.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-017-0500-7) contains supplementary material, which is available to authorized users.

De novo induction of Foxp3⁺ regulatory T cells (Tregs) is particularly efficient in gut-draining mesenteric and celiac lymph nodes (mLN and celLN). Here we used LN transplantations to dissect the contribution of stromal cells and environmental factors to the high Treg-inducing capacity of these LN. After transplantation into the popliteal fossa, mLN and celLN retained their high Treg-inducing capacity, whereas transplantation of skin-draining LN into the gut mesenteries did not enable efficient Treg induction. However, de novo Treg induction was abolished in the absence of dendritic cells (DC), indicating that this process depends on synergistic contributions of stromal and DC. Stromal cells themselves were influenced by environmental signals as mLN grafts taken from germ-free donors and celLN grafts taken from vitamin A-deficient donors did not show any superior Treg-inducing capacity. Collectively, our observations reveal a hitherto unrecognized role of LN stromal cells for the de novo induction of Foxp3⁺ Tregs.

Dominant tolerance to self-antigen requires the presence of sufficient numbers of CD4 1 Foxp31 Treg cells with matching antigen specificity. However, the size and role of TCR repertoire diversity for antigen-specific immuno-regulation through Treg cells is not clear. Here, we developed and applied a novel high-throughput (HT) TCR sequencing approach to analyze the TCR repertoire of Treg cells and revealed the importance of high diversity for Treg-cell homeostasis and function. We found that highly polyclonal Treg cells from WT mice vigorously expanded after adoptive transfer into non-lymphopenic TCR-transgenic recipients with low Treg-cell diversity. In that system, we identified specific Treg-cell TCR preferences in distinct anatomic locations such as the mesenteric LN indicating that Treg cells continuously compete for MHC class-II-presented self-, food-, or flora-antigen. Functionally, we showed that high TCR diversity was required for optimal suppressive function of Treg cells in experimental acute graft versus host disease (GvHD). In conclusion, we suggest that efficient immuno-regulation by Treg cells requires high TCR diversity. Thereby, continuous competition of peripheral Treg cells for limited self-antigen shapes an organ-optimized, yet highly diverse, local TCR repertoire.