To obtain a molecular definition of regulatory T (Treg) cell identity, we performed proteomics and transcriptomics on various populations of human regulatory and conventional CD4 T (Tconv) cells. A protein expression signature was identified that defines all Treg cells, and another signature that defines effector Treg cells. These signatures could not be extrapolated from transcriptome data. Unique cell-biological and metabolic features in Treg cells were defined, as well as specific adaptations in cytokine, TCR, and costimulatory receptor signaling pathways. One such adaptation-selective STAT4 deficiency-prevented destabilization of Treg cell identity and function by inflammatory cytokines, while these signals could still induce critical transcription factors and homing receptors via other pathways. Furthermore, our study revealed surface markers that identify FOXP3CD4 T cells with distinct functional properties. Our findings suggest that adaptation in signaling pathways protect Treg cell identity and present a resource for further research into Treg cell biology.

Dietary supplementation with GF/Bb enhances serum galectin-9 levels, which associates with the prevention of allergic symptoms.

The intestinal mucosa is constantly facing a high load of antigens including bacterial antigens derived from the microbiota and food. Despite this, the immune cells present in the gastrointestinal tract do not initiate a pro-inflammatory immune response. Toll-like receptors (TLRs) are pattern recognition receptors expressed by various cells in the gastrointestinal tract, including intestinal epithelial cells (IEC) and resident immune cells in the lamina propria. Many diseases, including chronic intestinal inflammation (e.g., inflammatory bowel disease), irritable bowel syndrome (IBS), allergic gastroenteritis (e.g., eosinophilic gastroenteritis and allergic IBS), and infections are nowadays associated with a deregulated microbiota. The microbiota may directly interact with TLR. In addition, differences in intestinal TLR expression in health and disease may suggest that TLRs play an essential role in disease pathogenesis and may be novel targets for therapy. TLR signaling in the gut is involved in either maintaining intestinal homeostasis or the induction of an inflammatory response. This mini review provides an overview of the current knowledge regarding the contribution of intestinal epithelial TLR signaling in both tolerance induction or promoting intestinal inflammation, with a focus on food allergy. We will also highlight a potential role of the microbiota in regulating gut immune responses, especially through TLR activation.

Dietary intervention using nondigestible oligosaccharides, short-chain galacto-oligosaccharides (scGOS)/long-chain fructo-oligosaccharides (lcFOS), in combination with Bifidobacterium breve M-16V prevents allergic disease involving galectin-9. In addition, apical TLR9 signaling contributes to intestinal homeostasis. We studied the contribution of galectin-9 secreted by intestinal epithelial cells (IEC; HT-29 and T84) in T_h1 and regulatory T-cell (T_reg) polarization in vitro. IEC were grown in transwell filters, cocultured with CD3/CD28-activated human peripheral blood mononuclear cells (PBMC) and apically exposed to genomic DNA derived from B. breve M-16V or synthetic TLR9 ligand in the absence or presence of scGOS/lcFOS. Cytokine production and T-cell phenotype were determined and galectin expression by IEC was assessed. Galectin-9 was neutralized using lactose or a TIM-3-Fc fusion protein. IEC exposed to DNA from B. breve M-16V or TLR9 ligand in the presence of scGOS/lcFOS enhanced IFN-γ secretion by PBMC and increased the percentage of T_h1 and T_reg cells. Expression and secretion of galectin-9 by IEC was increased and neutralization of galectin-9 prevented the induction of IFN-γ secretion and also suppressed the production of IL-10 by PBMC. Furthermore, we show that galectin-9 induces T_reg and T_h1 polarization through interaction with antigen-presenting cells. Our findings show that galectin-9 secreted by IEC apically exposed to TLR9 ligand in the presence of scGOS/lcFOS is involved in T_h1 and T_reg polarization and may be a promising target to prevent or treat allergic disease.

Resident memory T cells (TRM) inhabit peripheral tissues and are critical for protection against localized infections. Recently, it has become evident that CD103+ TRM are not only important in combating secondary infections, but also for the elimination of tumor cells. In several solid cancers, intratumoral CD103+CD8+ tumor infiltrating lymphocytes (TILs), with TRM properties, are a positive prognostic marker. To better understand the role of TRM in tumors, we performed a detailed characterization of CD8+ and CD4+ TIL phenotype and functional properties in non-small cell lung cancer (NSCLC). Frequencies of CD8+ and CD4+ T cell infiltrates in tumors were comparable, but we observed a sharp contrast in TRM ratios compared to surrounding lung tissue. The majority of both CD4+ and CD8+ TILs expressed CD69 and a subset also expressed CD103, both hallmarks of TRM. While CD103+CD8+ T cells were enriched in tumors, CD103+CD4+ T cell frequencies were decreased compared to surrounding lung tissue. Furthermore, CD103+CD4+ and CD103+CD8+ TILs showed multiple characteristics of TRM, such as elevated expression of CXCR6 and CD49a, and decreased expression of T-bet and Eomes. In line with the immunomodulatory role of the tumor microenvironment, CD8+ and CD4+ TILs expressed high levels of inhibitory receptors 2B4, CTLA-4, and PD-1, with the highest levels found on CD103+ TILs. Strikingly, CD103+CD4+ TILs were the most potent producers of TNF-α and IFN-γ, while other TIL subsets lacked such cytokine production. Whereas, CD103+CD4+PD-1low TILs produced the most effector cytokines, CD103+CD4+PD-1++ and CD69+CD4+PD-1++ TILs produced CXCL13. Furthermore, a large proportion of TILs expressed co-stimulatory receptors CD27 and CD28, unlike lung TRM, suggesting a less differentiated phenotype. Agonistic triggering of these receptors improved cytokine production of CD103+CD4+ and CD69+CD8+ TILs. Our findings thus provide a rationale to target CD103+CD4+ TILs and add co-stimulation to current therapies to improve the efficacy of immunotherapies and cancer vaccines.

No abstract

These results show that partial WH with limited sensitizing properties reduced the effector response upon whey challenge. This effect is transferable using MLN cells and was associated with enhanced Foxp3(+) regulatory T-cell numbers in the MLN. Partial WH retained the capacity to induce active immune suppression in mice which may be relevant for allergy prevention.

Upon activation, conventional T (Tconv) cells undergo an mTOR-driven glycolytic switch. Regulatory T (Treg) cells reportedly repress the mTOR pathway and avoid glycolysis. However, here we demonstrate that human thymusderived (t)Treg cells can become glycolytic in response to tumor necrosis factor receptor 2 (TNFR2) costimulation. This costimulus increases proliferation and induces a glycolytic switch in CD3-activated tTreg cells, but not in Tconv cells. Glycolysis in CD3/TNFR2-activated tTreg cells is driven by PI3-kinase/mTOR signaling and supports tTreg cell identity and suppressive function. Contrary to glycolytic Tconv cells, glycolytic tTreg cells do not show net lactate secretion and shuttle glucose-derived carbon into the tricarboxylic acid cycle. Ex vivo characterization of blood-derived TNFR2 high CD4 + CD25 high CD127 low effector T cells, which were FOXP3 + IKZF2 + , revealed an increase in glucose consumption and intracellular lactate levels, identifying them as glycolytic tTreg cells. Our study links TNFR2 costimulation in human tTreg cells to metabolic remodeling, providing an additional avenue for drug targeting.receptor 2 (TNFR2, TNFRSF1B, CD120b) costimulation in tTreg and Tconv cells. TNFR2 was previously shown to be important for Treg cell responses and protection against autoimmunity in human and mouse 30,31 and is considered a clinical target for selective Treg expansion or inhibition in transplant rejection, autoimmunity, or cancer 9, 10 . We here report that CD3-activated tTreg cells selectively respond to TNFR2 costimulation by proliferation and a PI3K/mTOR-driven glycolytic switch that is important for tTreg cell identity and function. We also identify unique elements of the glycolytic program in tTreg cells and validate our findings in tTreg cells directly isolated from human blood. RESULTS A novel strategy allows for stable human Treg cell expansion in the absence of rapamycinHuman Treg cells occur in low frequency in the blood and therefore, expansion protocols are used for clinical application 32 . In such protocols, Treg cells are flow cytometrically sorted and expanded in presence of the mTOR inhibitor rapamycin that selectively inhibits proliferation of contaminating Tconv cells 12 . However, since rapamycin affects many aspects of metabolism, these expansion protocols are not suitable to generate Treg cells for metabolic studies. Also, such cultures may still be contaminated with pTreg cells that can convert back to Tconv cells and confound data interpretation. We therefore employed a novel method to purify stable human tTreg cells, based on the marker glycoprotein (GP)A33 33 . Among CD4 + T cells, naïve Tconv cells were purified by flow cytometry on the basis of a CD25 low CD127 high CD45RA + GPA33 int phenotype and naïve tTreg cells on the basis of a CD25 high CD127 low CD45RA + GPA33 high phenotype (Extended Data Figure 1a). Phenotypic analysis of these populations indicated that the naïve tTreg cells could be discriminated from Tconv cells as previously defined 34 by expressi...