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SUMMARY Autotaxin (ATX; ENPP2) produces lysophosphatidic acid (LPA) that regulates multiple biological functions via cognate G protein-coupled receptors LPAR1–6. ATX/LPA promotes tumor cell migration and metastasis via LPAR1 and T cell motility via LPAR2, yet its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes (TILs) and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T cell responses but, importantly, suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from melanoma tumors are consistent with intratumoral ATX acting as a T cell repellent. These findings highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities., In brief Through LPA production, ATX modulates the tumor microenvironment in autocrine-paracrine manners. Matas-Rico et al. show that ATX/LPA is chemorepulsive for T cells with a dominant inhibitory role for Gα12/13-coupled LPAR6. Upon anticancer vaccination, tumor-intrinsic ATX suppresses the infiltration of CD8+ T cells without affecting their cytotoxic quality., Graphical Abstract

The CD4+ regulatory T (Treg) cell lineage, as defined by FOXP3 expression, comprises thymus-derived (t)Treg cells and peripherally induced (p)Treg cells. In human, naïve tTreg cells can be isolated from blood, while effector tTreg and pTreg cells cannot be purified reliably for lack of cell surface markers. As a model for Treg cells, studies often employ TGF-β-induced (i)Treg cells generated from CD4+ conventional T (Tconv) cells in vitro. Here, we describe the relationship of iTreg cells to tTreg and Tconv cells, as optimally purified from human blood. Proteomic analysis revealed that each of these cell populations has a unique protein expression pattern before and after CD3/CD28-mediated activation. iTreg cells had limited overlap in protein expression with tTreg cells and exhibited markedly differential expression of proteins involved in signal transduction and metabolism. Whereas iTreg and Tconv cells responded strongly to CD3/CD28-mediated activation, tTreg cells showed a modest response, which reflects their adaptations in signal transduction pathways. As a benchmark, we used a previously defined proteomic signature that discerns ex vivo naïve and effector Treg cells from Tconv cells and includes conserved Treg cell properties. This Treg cell core signature was largely absent in iTreg cells, as exemplified by STAT4 expression that may contribute to pro-inflammatory functions. In addition, we used a proteomic signature that distinguishes ex vivo effector Treg cells from Tconv cells and naïve Treg cells. This effector Treg cell signature was partially present in iTreg cells, indicating that they do have protein expression features in common with ex vivo effector Treg cells. In conclusion, iTreg cells are distinct from tTreg cells and largely lack the Treg cell core proteomic signature, indicating that caution is warranted when using iTreg cells as a model for Treg cell populations found in vivo.

SummaryAutotaxin (ATX) is secreted by diverse cell types to produce lysophosphatidic acid (LPA) that regulates multiple biological functions via G protein-coupled receptors LPAR1-6. ATX/LPA promotes tumor cell migration and metastasis mainly via LPAR1; however, its actions in the tumor immune microenvironment remain unclear. Here, we show that ATX secreted by melanoma cells is chemorepulsive for tumor-infiltrating lymphocytes and circulating CD8+ T cells ex vivo, with ATX functioning as an LPA-producing chaperone. Mechanistically, T-cell repulsion predominantly involves Gα12/13-coupled LPAR6. Upon anti-cancer vaccination of tumor-bearing mice, ATX does not affect the induction of systemic T-cell responses but suppresses tumor infiltration of cytotoxic CD8+ T cells and thereby impairs tumor regression. Moreover, single-cell data from patient samples are consistent with intra-tumor ATX acting as a T-cell repellent. These studies highlight an unexpected role for the pro-metastatic ATX-LPAR axis in suppressing CD8+ T-cell infiltration to impede anti-tumor immunity, suggesting new therapeutic opportunities.

Following activation, conventional T (T

Contains fulltext : 200117.pdf (Publisher’s version ) (Open Access) 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-alpha and IFN-gamma, while other TIL subsets lacked such cytokine production. Whereas, CD103(+)CD4(+)PD-1(low) 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.

Dietary intervention with short-chain galacto-oligosaccharides (scGOS), long-chain fructo-oligosaccharides (lcFOS) and

We investigated the effects of minocycline, a microglia suppressant, on olfactory bulbectomized (OBX) rats, a model of cognitive and behavioral impairments arising from neurodegenerative processes. Previously, we demonstrated that the major OBX-induced behavioral and cognitive impairments develop between day 3 and 7 following bulbectomy. Here we show that the onset of these cognitive changes parallel in time with signs of microglia activation (increased mRNA levels of IL-1β and CD68) in hippocampus. Next, rats were treated with minocycline (50 mg/kg, i.p.) once daily for 4 weeks. OBX surgery was done at day 3 of drug treatment. Animals were tested in a battery of behavioral assays: open field, passive avoidance (fear learning and memory-acquired prior to OBX) and T-maze (spatial memory, conducted post bulbectomy). Minocycline normalized OBX-induced hyperactivity in the open field. Minocycline failed to prevent fear memory loss, but protected the OBX rats against hippocampal-dependent spatial memory deficit. Our findings suggest that treatment with minocycline may be effective in the early phase of a neurodegenerative disease.

To cite this article: van Esch BCAM, Schouten B, de Kivit S, Hofman GA, Knippels LMJ, Willemsen LEM, Garssen J. Oral tolerance induction by partially hydrolyzed whey protein in mice is associated with enhanced numbers of Foxp3+ regulatory T-cells in the mesenteric lymph nodes. Pediatr Allergy Immunol 2011: 22: 820–826. Abstract Background: Hypoallergenic formulas are considered a good option for infants at risk for cow’s milk allergy. The aim of this animal study was to investigate whether whey hydrolyzates (WH) have the capacity to induce oral tolerance to whey. Methods: Whey, partial or extensive WH was given via gavages to naive mice prior to oral whey sensitization using cholera toxin as an adjuvant. The acute allergic skin response, mouse mast cell protease-1 (mMCP-1), whey-specific IgE, IgG1 and effector Th2-cells, Th1-cells, and Foxp3+ regulatory T-cells were determined in the mesenteric lymph nodes (MLN). MLN cells from tolerized mice were adoptively transferred to naive recipient mice prior to whey sensitization. Results: In contrast to the extensive WH, pre-treatment of naive mice with whey or partial WH reduced the acute allergic skin response and mast cell degranulation after whey challenge. However, only treatment with whey prevented the generation of serum-specific IgE/IgG1. In partial WH tolerized mice, Foxp3+ regulatory T-cell numbers in the MLN were increased compared to whey-sensitized mice. Both whey and partial WH treatment showed a tendency toward a decreased number of effector Th2-cells. Transfer of MLN cells from tolerized mice protected recipient mice from developing an acute allergic skin response. Conclusion: 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.

Hypoallergenic infant formulas (HA) are considered a good alternative for infants at high risk for developing allergy if breastfeeding is not possible. Dietary intervention studies with HA combined with a specific mixture of non-digestible oligosaccharides, have been shown to reduce allergic symptoms in these children. However, the mechanisms by which these oligosaccharides exert their effect are yet to be explored. In this study, the contribution of this specific oligosaccharides mixture on the tolerizing capacity of a partial whey hydrolysate (WH) was investigated in a mice model of cow's milk allergy. Mice were sensitized orally with whey using cholera toxin as adjuvant. Prior to sensitization mice were pre-treated orally with partial WH, PBS, with or without supplementation with the specific oligosaccharide mixture containing short chain-galacto-, long chain-fructo- and acidic-oligosaccharides (9:1:1). After challenge, the acute allergic skin response, the mast cell mediator mMCP-1 and whey-specific antibodies were measured. The presence of Foxp3+regulatory T-cells and CD103+DC were determined in mesenteric lymph nodes. Oral pre-treatment of mice fed the partial WH induced tolerance as refl ected by a reduced acute allergic skin response and a suppressed mMCP-1 release without affecting whey-specific IgE levels. This effect coincided with increased CD103+DC and Foxp3+regulatory T-cell numbers. Interestingly, a combination of the partial WH and oligosaccharide diet completely abolished the acute allergic skin response and mMCP-1 release. In addition, a tendency towards decreased IgE levels and a further increase in intestinal CD103+DC numbers was observed. A specific mixture of non-digestible oligosaccharides enhanced the capacity of a partial WH to induce oral tolerance. This effect was associated with increased numbers of CD103+DC in the mesenteric lymph nodes, suggesting a role of these cells in the observed tolerance inducing capacity of this specific oligosaccharide mixture combined with partial WH.

Dietary non-digestible carbohydrates reduce the development of cows' milk allergy in mice. In the present study, the contribution of CD25+ regulatory T-cells (Treg) was investigated using in vivo Treg depletion and adoptive transfer studies. Mice were orally sensitised with casein and fed a diet containing 2 % short-chain galacto-, long-chain fructo- and acidic oligosaccharides (GFA) or a control diet. Donor splenocytes of mice sensitised with casein and fed the GFA or control diet were adoptively transferred to naive recipient mice, which were casein- or sham-sensitised and fed the control diet. In addition, in vivo or ex vivo CD25+ Treg depletion was performed using anti-CD25 (PC61). The acute allergic skin response upon intradermal casein challenge and casein-specific Ig were determined. Furthermore, T-helper (TH) 1 and TH2 cell numbers were analysed in the mesenteric lymph nodes. The oligosaccharide diet strongly reduced the development of the acute allergic skin response, which was abrogated by the in vivo anti-CD25 treatment. The diet enhanced the percentage of TH1 cells and tended to reduce the percentage of TH2 cells in casein-sensitised mice. Recipient mice were protected against the development of an acute allergic skin response when transferred with splenocytes from casein-sensitised GFA-fed donor mice before sensitisation. Ex vivo depletion of CD25+ Treg abrogated this transfer of tolerance. Splenocytes from sham-sensitised GFA-fed donor mice did not suppress the allergic response in recipient mice. In conclusion, CD25+ Treg contribute to the suppression of the allergic effector response in casein-sensitised mice induced by dietary intervention with non-digestible carbohydrates.

Intestinal epithelial cells (IEC) separate the mucosal immune system from the external milieu. Under inflammatory conditions, Toll-like receptor (TLR) expression by IEC is increased. In a transwell co-culture model immune modulation by IEC upon TLR ligation was studied. Human IEC (HT-29 and T84) grown on filters were apically or basolaterally exposed to TLR4 or TLR9 ligands and co-cultured with CD3/CD28-activated healthy donor PBMC in the basolateral compartment. TLR4 ligation of IEC (HT-29) enhanced the production of TNF-α and IEC-derived MDC and decreased numbers of Foxp3(+) regulatory T cells. Neutralization of TSLP abrogated TLR4-induced TNF-α secretion. In contrast, apical TLR9 ligation of IEC (HT-29 and T84) enhanced IFN-γ and IL-10 secretion and increased the number of activated T(h)1 cells. The increase in IFN-γ secretion depended on the presence of IEC. Furthermore, CD14 expression on monocytes was reduced coinciding with enhanced intracellular IL-10 and decreased TNF-α production. However, basolateral TLR9 ligand exposure of HT-29 cells resulted in enhanced IFN-γ, IL-6 and TNF-α, while IL-10 secretion remained unaltered. TLR4 and TLR9 ligands reduced IL-13 secretion in presence and absence of apically exposed IEC and enhanced IL-12 secretion in presence of IEC. These data suggest that TLR4 ligation of IEC drives an inflammatory, while apical TLR9 ligation drives a regulatory T(h)1 effector immune response in vitro in a polarized manner. IEC may be important modulators of the mucosal effector immune response.

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 FOXP3+CD4+ 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. Using high-resolution mass spectrometry and transcriptomics, Cuadrado et al. provide a molecular characterization of regulatory and conventional CD4+ T cell subsets, yielding markers to distinguish cells with different properties and insights into mechanisms that prevent regulatory T cells from exhibiting undesirable functional activities of the related but functionally antithetical conventional T cells.

Background: Intestinal bacteria and intestinal epithelial cells (IEC) may modulate the mucosal immune response. In this study, immune modulation by Lactobacillus GG (LGG) and Bifidobacterium breve (Bb1, Bb2) in the presence or absence of IEC was addressed in an in vitro transwell co-culture model. Methods: UV-killed LGG,Bb1, Bb2 or Toll-like receptor (TLR) 2 or nucleotide oligomerization domain (NOD) 2 ligands were added directly to unstimulated or anti-CD3/CD28-stimulated PBMC, or applied apically to human IEC (HT-29) co-cultured with PBMC. A mixture of live bacteria was used as reference. The effect on T helper 1 (IFN-γ, IL-12), T helper 2 (IL-13), inflammatory (TNF-α) and regulatory (IL-10) cytokine secretion was determined. Results: Both UV-killed LGG and Bb enhanced IL-12, IFN-γ, TNF-α and IL-10, and reduced IL-13 secretion when added directly to stimulated PBMC, similar to live bacteria. IEC reduced IL-13, IFN-γ and IL-10 secretion by stimulated PBMC. Apically added LGG, TLR2 and NOD2 ligands,but not Bb, enhanced IFN-γ, IL-12 and/or TNF-α secretion. Bacteria did not induce cytokine secretion when added to HT-29/unstimulated PBMC co-cultures, whereas direct incubations with PBMC did. Conclusion: UV-killed LGG as well as Bb supported a T helper 1 and/or regulatory phenotype when added directly to activated PBMC, similar to live bacteria. In contrast, LGG, TLR2 or NOD2 ligands – but not Bb – enhanced T helper 1 type cytokine secretion when added to IEC, while IL-10 secretion remained suppressed. Co-cultures combining IEC and PBMC may reveal differences between bacterial strains relevant for the in vivo situation.

Highly active antiretroviral therapy (HAART) for the treatment of HIV infection sustains viral suppression and increases CD4(+) T cells in HIV patients. However, in 10-25% of subjects, known as immunological non-responders (INRs), HAART does not increase CD4 count. We investigated a potential role for galectin-9 and TIM-3 in INRs as galectin-9 and TIM-3 have been described to modulate NK and T cell function. PBMCs were isolated from healthy controls, HIV immunological responders (IRs, >350CD4(+) cells/mm(3)) and HIV INRs (

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 Th1 and regulatory T-cell (Treg) 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 Th1 and Treg 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 Treg and Th1 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 Th1 and Treg polarization and may be a promising target to prevent or treat allergic disease.

Hypoallergenic formulas are considered a good option for infants at risk for cow's milk allergy. The aim of this animal study was to investigate whether whey hydrolyzates (WH) have the capacity to induce oral tolerance to whey.Whey, partial or extensive WH was given via gavages to naïve mice prior to oral whey sensitization using cholera toxin as an adjuvant. The acute allergic skin response, mouse mast cell protease-1 (mMCP-1), whey-specific IgE, IgG(1) and effector Th2-cells, Th1-cells, and Foxp3(+) regulatory T-cells were determined in the mesenteric lymph nodes (MLN). MLN cells from tolerized mice were adoptively transferred to naïve recipient mice prior to whey sensitization.In contrast to the extensive WH, pre-treatment of naïve mice with whey or partial WH reduced the acute allergic skin response and mast cell degranulation after whey challenge. However, only treatment with whey prevented the generation of serum-specific IgE/IgG(1) . In partial WH tolerized mice, Foxp3(+) regulatory T-cell numbers in the MLN were increased compared to whey-sensitized mice. Both whey and partial WH treatment showed a tendency toward a decreased number of effector Th2-cells. Transfer of MLN cells from tolerized mice protected recipient mice from developing an acute allergic skin response.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.

The intestinal mucosa is constantly exposed to the luminal content, which includes micro-organisms and dietary components. Prebiotic non-digestible oligosaccharides may be supplemented to the diet to exert modulation of immune responses in the intestine. Short chain galacto- and long chain fructo-oligosaccharides (scGOS/lcFOS), functionally mimicking oligosaccharides present in human milk, have been reported to reduce the development of allergy through modulation of the intestinal microbiota and immune system. Nonetheless, the underlying working mechanisms of scGOS/lcFOS are unclear. Intestinal epithelial cells lining the mucosa are known to express carbohydrate (glycan)-binding receptors that may be involved in modulation of the mucosal immune response. This review aims to provide an overview of glycan-binding receptors, in particular galectins, which are expressed by intestinal epithelial cells and immune cells. In addition, their involvement in health and disease will be addressed, especially in food allergy and inflammatory bowel disease, diseases originating from the gastro-intestinal tract. Insight in the recognition of glycans in the intestinal tract may open new avenues for the treatment of intestinal inflammatory diseases by either nutritional concepts or pharmacological intervention.

Intestinal bacteria and intestinal epithelial cells (IEC) may modulate the mucosal immune response. In this study, immune modulation by Lactobacillus GG (LGG) and Bifidobacterium breve (Bb1, Bb2) in the presence or absence of IEC was addressed in an in vitro transwell co-culture model.UV-killed LGG,Bb1, Bb2 or Toll-like receptor (TLR) 2 or nucleotide oligomerization domain (NOD) 2 ligands were added directly to unstimulated or anti-CD3/CD28-stimulated PBMC, or applied apically to human IEC (HT-29) co-cultured with PBMC. A mixture of live bacteria was used as reference. The effect on T helper 1 (IFN-gamma, IL-12), T helper 2 (IL-13), inflammatory (TNF-alpha) and regulatory (IL-10) cytokine secretion was determined.Both UV-killed LGG and Bb enhanced IL-12, IFN-gamma, TNF-alpha and IL-10, and reduced IL-13 secretion when added directly to stimulated PBMC, similar to live bacteria. IEC reduced IL-13, IFN-gamma and IL-10 secretion by stimulated PBMC. Apically added LGG, TLR2 and NOD2 ligands,but not Bb, enhanced IFN-gamma, IL-12 and/or TNF-alpha secretion. Bacteria did not induce cytokine secretion when added to HT-29/unstimulated PBMC co-cultures, whereas direct incubations with PBMC did.UV-killed LGG as well as Bb supported a T helper 1 and/or regulatory phenotype when added directly to activated PBMC, similar to live bacteria. In contrast, LGG, TLR2 or NOD2 ligands - but not Bb - enhanced T helper 1 type cytokine secretion when added to IEC, while IL-10 secretion remained suppressed. Co-cultures combining IEC and PBMC may reveal differences between bacterial strains relevant for the in vivo situation.