Canonical Wnt signaling negatively modulates regulatory T cell function.

Foxp3 is crucial for both the development and function of regulatory T (Treg) cells; however, the posttranslational mechanisms regulating Foxp3 transcriptional output remain poorly defined. Here, we demonstrate that T cell factor 1 (TCF1) and Foxp3 associates in Treg cells and that active Wnt signaling disrupts Foxp3 transcriptional activity. A global chromatin immunoprecipitation sequencing comparison in Treg cells revealed considerable overlap between Foxp3 and Wnt target genes. The activation of Wnt signaling reduced Treg-mediated suppression both in vitro and in vivo, whereas disruption of Wnt signaling in Treg cells enhanced their suppressive capacity. The activation of effector T cells increased Wnt3a production, and Wnt3a levels were found to be greatly increased in mononuclear cells isolated from synovial fluid versus peripheral blood of arthritis patients. We propose a model in which Wnt produced under inflammatory conditions represses Treg cell function, allowing a productive immune response, but, if uncontrolled, could lead to the development of autoimmunity.

Nutritional Interventions to Prevent the Development of Atopic Diseases: A Focus on Cow's Milk Allergy.

In the western world the prevalence of atopic diseases such as food allergies is increasing highly significantly. One of the earliest and most prevalent food allergies occurring in the first year of life is cow's milk allergy. No treatment is available and only avoidance of the cow's milk allergens prevents the occurrence of an allergic reaction. Since cow's milk allergic children have an increased risk of developing other allergies later in life, investigating nutritional strategies to prevent the development of cow's milk allergy by developing oral tolerance is of high interest. Nutritional components such as prebiotics, probiotics, synbiotics and long-chain polyunsaturated fatty acids possess potential to support the maturation of the immune system early in life that might prevent the development of cow's milk allergy. The available research, so far, shows promising results particularly on the development of eczema. However, the preventive effects of the nutritional interventions on the development of food allergy are inconclusive. Future research may benefit from the combination of various dietary components. To clarify the preventive effects of the nutritional components in food allergy more randomized clinical trials are needed.

The Quantity of Autocrine IL-2 Governs the Expansion Potential of CD8+ T Cells.

Adequate responsiveness of CD8(+) T cell populations is of utmost importance for the efficacy of many vaccines and immunotherapeutic strategies against intracellular pathogens and cancer. In this study, we show in mouse models that the relative number of IL-2-producing cells within Ag-specific CD8(+) T cell populations predicts the population expansion capacity upon challenge. We further demonstrate that IL-2 producers constitute the best responding subset. Notably, we show that elevated production of IL-2 by CD8(+) T cells results in concomitant improved population expansion capacity and immunity. The amount of IL-2 produced on a per-cell basis essentially connects directly to the superior CD8(+) T cell population expansion. Together, our findings identified that autocrine IL-2 production operates in a dose-dependent fashion to facilitate the expansion potential of Ag-specific CD8(+) T cell populations, which may instigate ways to augment therapies depending on fit CD8(+) T cells.

T Cell factor 1 represses CD8+ effector T cell formation and function.

The Wnt-responsive transcription factor T cell factor 1 (Tcf1) is well known for its role in thymic T cell development and the formation of memory CD8(+) T cells. However, its role in the initial phases of CD8(+) T effector cell formation has remained unexplored. We report that high levels of Wnt signaling and Tcf1 are operational in naive and memory CD8(+) T cells, whereas Wnt signaling and Tcf1 were low in effector CD8(+) T cells. CD8(+) T cells deficient in Tcf1 produce IFN-γ more rapidly, coinciding with increased demethylation of the IFN-γ enhancer and higher expression of the transcription factors Tbet and Blimp1. Moreover, virus-specific Tcf1(-/-) CD8(+) T cells show accelerated expansion in acute infection, which is associated with increased IFN-γ and TNF production and lower viral load. Genetic complementation experiments with various Tcf1 isoforms indicate that Tcf1 dosage and protein stability are critical in suppressing IFN-γ production. Isoforms lacking the ß-catenin binding domain are equally effective in inhibiting CD8(+) effector T cell formation. Thus, Tcf1 functions as a repressor of CD8(+) effector T cell formation in a ß-catenin/Wnt-independent manner.

The nuclear effector of Wnt-signaling, Tcf1, functions as a T-cell-specific tumor suppressor for development of lymphomas.

The HMG-box factor Tcf1 is required during T-cell development in the thymus and mediates the nuclear response to Wnt signals. Tcf1(-/-) mice have previously been characterized and show developmental blocks at the CD4-CD8- double negative (DN) to CD4+CD8+ double positive transition. Due to the blocks in T-cell development, Tcf1(-/-) mice normally have a very small thymus. Unexpectedly, a large proportion of Tcf1(-/-) mice spontaneously develop thymic lymphomas with 50% of mice developing a thymic lymphoma/leukemia at the age of 16 wk. These lymphomas are clonal, highly metastatic, and paradoxically show high Wnt signaling when crossed with Wnt reporter mice and have high expression of Wnt target genes Lef1 and Axin2. In wild-type thymocytes, Tcf1 is higher expressed than Lef1, with a predominance of Wnt inhibitory isoforms. Loss of Tcf1 as repressor of Lef1 leads to high Wnt activity and is the initiating event in lymphoma development, which is exacerbated by activating Notch1 mutations. Thus, Notch1 and loss of Tcf1 functionally act as collaborating oncogenic events. Tcf1 deficiency predisposes to the development of thymic lymphomas by ectopic up-regulation of Lef1 due to lack of Tcf1 repressive isoforms and frequently by cooperating activating mutations in Notch1. Tcf1 therefore functions as a T-cell-specific tumor suppressor gene, besides its established role as a Wnt responsive transcription factor. Thus, Tcf1 acts as a molecular switch between proliferative and repressive signals during T-lymphocyte development in the thymus.

TCF-1-mediated Wnt signaling regulates Paneth cell innate immune defense effectors HD-5 and -6: implications for Crohn's disease.

Wnt signaling regulates small intestinal stem cell maintenance and Paneth cell differentiation. In patients with ileal Crohn's disease (CD), a decrease of Paneth cell α-defensins has been observed that is partially caused by impaired TCF-4 and LRP6 function. Here we show reduced expression of the Wnt signaling effector TCF-1 (also known as TCF-7) in patients with ileal CD. Reporter gene assays and in vitro promoter binding analysis revealed that TCF-1 activates α-defensin HD-5 and HD-6 transcription in cooperation with ß-catenin and that activation is mediated by three distinct TCF binding sites. EMSA analysis showed binding of TCF-1 to the respective motifs. In ileal CD patients, TCF-1 mRNA expression levels were significantly reduced. Moreover, we found specifically reduced expression of active TCF-1 mRNA isoforms. Tcf-1 knockout mice exhibited reduced cryptdin expression in the jejunum, which was not consistently seen at other small intestinal locations. Our data provide evidence that TCF-1-mediated Wnt signaling is disturbed in small intestinal CD, which might contribute to the observed barrier dysfunction in the disease.

Multipotent stromal cells induce human regulatory T cells through a novel pathway involving skewing of monocytes toward anti-inflammatory macrophages.

Multipotent stromal cells (MSC) have been shown to possess immunomodulatory capacities and are therefore explored as a novel cellular therapy. One of the mechanisms through which MSC modulate immune responses is by the promotion of regulatory T cell (Treg) formation. In this study, we focused on the cellular interactions and secreted factors that are essential in this process. Using an in vitro culture system, we showed that culture-expanded bone marrow-derived MSC promote the generation of CD4(+) CD25(hi) FoxP3(+) T cells in human PBMC populations and that these populations are functionally suppressive. Similar results were obtained with MSC-conditioned medium, indicating that this process is dependent on soluble factors secreted by the MSC. Antibody neutralization studies showed that TGF-ß1 mediates induction of Tregs. TGF-ß1 is constitutively secreted by MSC, suggesting that the MSC-induced generation of Tregs by TGF-ß1 was independent of the interaction between MSC and PBMC. Monocyte-depletion studies showed that monocytes are indispensable for MSC-induced Treg formation. MSC promote the survival of monocytes and induce differentiation toward macrophage type 2 cells that express CD206 and CD163 and secrete high levels of IL-10 and CCL-18, which is mediated by as yet unidentified MSC-derived soluble factors. CCL18 proved to be responsible for the observed Treg induction. These data indicate that MSC promote the generation of Tregs. Both the direct pathway through the constitutive production of TGF-ß1 and the indirect novel pathway involving the differentiation of monocytes toward CCL18 producing type 2 macrophages are essential for the generation of Tregs induced by MSC.

Wnt signaling in leukemias and myeloma: T-cell factors are in control.

Aberrant activation of the Wnt pathway has been implicated in the pathogenesis of many malignancies, especially solid tumors. During the past decade it also became clear that in hematological malignancies abnormal regulation of the Wnt pathway can either be causative or enhance disease progression, which will be discussed in detail in this review.

FAS/FAS-L dependent killing of activated human monocytes and macrophages by CD4+CD25- responder T cells, but not CD4+CD25+ regulatory T cells.

Conclusive resolution of an immune response is critical for the prevention of autoimmunity and chronic inflammation. We report that following co-culture with autologous CD4+CD25- responder T cells, human CD14+ monocytes and monocyte-derived macrophages become activated but also significantly more prone to apoptosis than monocytes/macrophages cultured alone. In contrast, in the presence of CD4+CD25+ regulatory T cells (Tregs), monocytes and macrophages survive whilst adopting an anti-inflammatory phenotype. The induction of monocyte death requires responder T cell activation and cell-contact between responder T cells and monocytes. We demonstrate a critical role for FAS/FAS-L ligation in responder T cell-induced monocyte killing since responder T cells, but not Tregs, upregulate FAS-ligand (FAS-L) mRNA, and induce FAS expression on monocytes. Furthermore, responder T cell-induced monocyte apoptosis is blocked by neutralising FAS/FAS-L interaction, and is not observed when monocytes from an autoimmune lymphoproliferative syndrome (ALPS) patient with complete FAS-deficiency are used as target cells. Finally, we show that responder T cell-induced killing of monocytes is impaired in patients with active rheumatoid arthritis (RA). Our data suggest that resolution of inflammation in the course of a healthy immune response is aided by the unperturbed killing of monocytes with inflammatory potential by responder T cells and the induction of longer-lived, Treg-induced, anti-inflammatory monocytes.

Protection against Marburg Virus and Sudan Virus in NHP by an Adenovector-Based Trivalent Vaccine Regimen Is Correlated to Humoral Immune Response Levels.

The Marburg virus (MARV) and Sudan virus (SUDV) belong to the filovirus family. The sporadic human outbreaks occur mostly in Africa and are characterized by an aggressive disease course with high mortality. The first case of Marburg virus disease in Guinea in 2021, together with the increased frequency of outbreaks of Ebola virus (EBOV), which is also a filovirus, accelerated the interest in potential prophylactic vaccine solutions against multiple filoviruses. We previously tested a two-dose heterologous vaccine regimen (Ad26.Filo, MVA-BN-Filo) in non-human primates (NHP) and showed a fully protective immune response against both SUDV and MARV in addition to the already-reported protective effect against EBOV. The vaccine-induced glycoprotein (GP)-binding antibody levels appear to be good predictors of the NHP challenge outcome as indicated by the correlation between antibody levels and survival outcome as well as the high discriminatory capacity of the logistic model. Moreover, the elicited GP-specific binding antibody response against EBOV, SUDV, and MARV remains stable for more than 1 year. Overall, the NHP data indicate that the Ad26.Filo, MVA-BN-Filo regimen may be a good candidate for a prophylactic vaccination strategy in regions at high risk of filovirus outbreaks.

The poly(I:C)-induced maternal immune activation model; a systematic review and meta-analysis of cytokine levels in the offspring.

The maternal polyinosinic:polycytidylic acid (poly(I:C)) animal model is frequently used to study how maternal immune activation may impact neuro development in the offspring. Here, we present the first systematic review and meta-analysis on the effects of maternal poly(I:C) injection on immune mediators in the offspring and provide an openly accessible systematic map of the data including methodological characteristics. Pubmed and EMBASE were searched for relevant publications, yielding 45 unique papers that met inclusion criteria. We extracted data on immune outcomes and methodological characteristics, and assessed the risk of bias. The descriptive summary showed that most studies reported an absence of effect, with an equal number of studies reporting an increase or decrease in the immune mediator being studied. Meta-analysis showed increased IL-6 concentrations in the offspring of poly(I:C) exposed mothers. This effect appeared larger prenatally than post-weaning. Furthermore, poly(I:C) administration during mid-gestation was associated with higher IL-6 concentrations in the offspring. Maternal poly(I:C) induced changes in IL-1ß, Il-10 and TNF-α concentrations were small and could not be associated with age of offspring, gestational period or sampling location. Finally, quality of reporting of potential measures to minimize bias was low, which stresses the importance of adherence to publication guidelines. Since neurodevelopmental disorders in humans tend to be associated with lifelong changes in cytokine concentrations, the absence of these effects as identified in this systematic review may suggest that combining the model with other etiological factors in future studies may provide further insight in the mechanisms through which maternal immune activation affects neurodevelopment.

CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human monocytes/macrophages.

CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) are potent suppressors of the adaptive immune system, but their effects on innate immune cells are less well known. Here we demonstrate a previously uncharacterized function of Tregs, namely their ability to steer monocyte differentiation toward alternatively activated macrophages (AAM). AAM are cells with strong antiinflammatory potential involved in immune regulation, tissue remodeling, parasite killing, and tumor promotion. We show that, after coculture with Tregs, monocytes/macrophages display typical features of AAM, including up-regulated expression of CD206 (macrophage mannose receptor) and CD163 (hemoglobin scavenger receptor), an increased production of CCL18, and an enhanced phagocytic capacity. In addition, the monocytes/macrophages have reduced expression of HLA-DR and a strongly reduced capacity to respond to LPS in terms of proinflammatory mediator production (IL-1beta, IL-6, IL-8, MIP-1alpha, TNF-alpha), NFkappaB activation, and tyrosine phosphorylation. Mechanistic studies reveal that CD4(+)CD25(+)CD127(low)Foxp3(+) Tregs produce IL-10, IL-4, and IL-13 and that these cytokines are the critical factors involved in the suppression of the proinflammatory cytokine response. In contrast, the Treg-mediated induction of CD206 is entirely cytokine-independent, whereas the up-regulation of CD163, CCL18, and phagocytosis are (partly) dependent on IL-10 but not on IL-4/IL-13. Together these data demonstrate a previously unrecognized function of CD4(+)CD25(+)Foxp3(+) Tregs, namely their ability to induce alternative activation of monocytes/macrophages. Moreover, the data suggest that the Treg-mediated induction of AAM partly involves a novel, cytokine-independent pathway.

Functional definition of a transcription factor hierarchy regulating T cell lineage commitment.

T cell factor 1 (Tcf1) is the first T cell-specific protein induced by Notch signaling in the thymus, leading to the activation of two major target genes, Gata3 and Bcl11b. Tcf1 deficiency results in partial arrests in T cell development, high apoptosis, and increased development of B and myeloid cells. Phenotypically, seemingly fully T cell-committed thymocytes with Tcf1 deficiency have promiscuous gene expression and an altered epigenetic profile and can dedifferentiate into more immature thymocytes and non-T cells. Restoring Bcl11b expression in Tcf1-deficient cells rescues T cell development but does not strongly suppress the development of non-T cells; in contrast, expressing Gata3 suppresses their development but does not rescue T cell development. Thus, T cell development is controlled by a minimal transcription factor network involving Notch signaling, Tcf1, and the subsequent division of labor between Bcl11b and Gata3, thereby ensuring a properly regulated T cell gene expression program.

Decreased Histone Acetylation Levels at Th1 and Regulatory Loci after Induction of Food Allergy.

Immunoglobulin E (IgE)-mediated allergy against cow's milk protein fractions such as whey is one of the most common food-related allergic disorders of early childhood. Histone acetylation is an important epigenetic mechanism, shown to be involved in the pathogenesis of allergies. However, its role in food allergy remains unknown. IgE-mediated cow's milk allergy was successfully induced in a mouse model, as demonstrated by acute allergic symptoms, whey-specific IgE in serum, and the activation of mast cells upon a challenge with whey protein. The elicited allergic response coincided with reduced percentages of regulatory T (Treg) and T helper 17 (Th17) cells, matching decreased levels of H3 and/or H4 histone acetylation at pivotal Treg and Th17 loci, an epigenetic status favoring lower gene expression. In addition, histone acetylation levels at the crucial T helper 1 (Th1) loci were decreased, most probably preceding the expected reduction in Th1 cells after inducing an allergic response. No changes were observed for T helper 2 cells. However, increased histone acetylation levels, promoting gene expression, were observed at the signal transducer and activator of transcription 6 (Stat6) gene, a proallergic B cell locus, which was in line with the presence of whey-specific IgE. In conclusion, the observed histone acetylation changes are pathobiologically in line with the successful induction of cow's milk allergy, to which they might have also contributed mechanistically.

Management of Cow's Milk Allergy from an Immunological Perspective: What Are the Options?

The immunological mechanism underlying Immunoglobuline E (IgE)-mediated cow's milk allergy has been subject to investigations for many years. Identification of the key immune cells (mast cells, B cells) and molecules (IgE) in the allergic process has led to the understanding that avoidance of IgE-crosslinking epitopes is effective in the reduction of allergic symptoms but it cannot be envisioned as a treatment. For the treatment and prevention of IgE-mediated cow's milk allergy, it is thought that the induction of a sustained state of immunological tolerance is needed. In this review, we will discuss various approaches aimed at achieving immunological tolerance and their success. Furthermore, we will speculate on the involved immunological mechanism.

An inactivated poliovirus vaccine using Sabin strains produced on the serum-free PER.C6® cell culture platform is immunogenic and safe in a non-human primate model.

BACKGROUND: The World Health Organization recommends the development of affordable next-generation inactivated poliovirus vaccines (IPV) using attenuated poliovirus Sabin strains. Previously, we introduced a novel PER.C6® cell culture platform, which allows for high yield production of an affordable trivalent Sabin IPV vaccine. METHODS: Immunogenicity and safety of this novel PER.C6®-based Sabin-IPV (sIPV) was assessed in rats and non-human primates (NHPs). NHPs received one of four different dose dilutions vaccine according to current human schedule (three prime-immunizations and one boost immunization). For comparison, NHPs received commercially available reference Salk IPV or sIPV. RESULTS: Dose-dependent immunogenicity and good tolerability was observed for the PER.C6®-based sIPV formulations in rats and NHPs. In NHPs, the lowest tested dose that induced anti-Sabin virus-neutralizing antibody titers that were non-inferior to commercial sIPV after three immunizations was 5-7.5-25 D-antigen units for type 1, 2 and 3 respectively. DISCUSSION: PER.C6®-based sIPV induced comparable immunogenicity to commercial Salk IPV and sIPV vaccines in NHPs. Together with the absence of any preclinical safety signals, these data warrant further testing in clinical trials. sIPV produced on the PER.C6® cell platform could be one solution to the need for an affordable and immunogenic IPV to achieve and maintain global polio eradication.

Lack of suppressive CD4+CD25+FOXP3+ T cells in advanced stages of primary cutaneous T-cell lymphoma.

Mycosis fungoides and its leukemic variant, Sezary syndrome, are the most common primary cutaneous T-cell lymphomas (CTCLs). In an ex vivo study, we investigated the percentage, phenotype, and suppressive function of CD4+CD25+ regulatory T cells (Tregs) from peripheral blood of CTCL patients. The percentage of Tregs did not differ significantly between patients and controls. Functional assays demonstrated a dichotomy in Treg function: in four out of 10 patients CD4+CD25+ T cells were incapable of suppressing autologous CD4+CD25- T-cell proliferation, whereas suppressive function was intact in the other six patients. Suppressive activity of Tregs inversely correlated with the peripheral blood tumor burden. T-plastin gene expression, used as a Sezary cell marker, confirmed that Sezary cells were heterogeneous for CD25 expression. Mixed lymphocyte reactions demonstrated that CD4+CD25- T cells from patients who lacked functional Tregs were susceptible to suppression by Tregs from healthy controls, and had not become suppressive themselves. Furthermore, we found reduced expression of Foxp3 in the CD4+CD25+ Tregs of these patients relative to the other six CTCL patients and controls. Our findings thus indicate a dysfunction of peripheral Tregs in certain CTCL patients, which correlates with tumor burden.

Modulation of monocyte/macrophage function by human CD4+CD25+ regulatory T cells.

The suppressive effects of CD4+CD25+ regulatory T cells (Tregs) on T cells have been well documented. Here we investigated whether human CD4+CD25+ Tregs can inhibit the proinflammatory properties of monocytes/macrophages. Monocytes and T cells were isolated from peripheral blood of healthy volunteers by magnetic cell separation and cocultured for 40 h. Monocytes were analyzed directly for cytokine production and phenotypic changes or repurified and used in T-cell stimulation and lipopolysaccharide challenge assays. Coculture with CD4+CD25+ Tregs induced minimal cytokine production in monocytes, whereas coculture with CD4+CD25- T cells resulted in large amounts of proinflammatory (tumor necrosis factor-alpha, interferon-gamma, interleukin-6) and regulatory (interleukin-10) cytokines. Importantly, when these CD4+CD25+ Treg-treated monocytes were repurified after coculture and challenged with lipopolysaccharide, they were severely inhibited in their capacity to produce tumor necrosis factor-alpha and interleukin-6 compared with control-treated monocytes. In addition, monocytes that were precultured with CD4+CD25+ Tregs displayed limited upregulation of human leukocyte antigen class II, CD40 and CD80, and downregulation of CD86 compared with control-treated monocytes. This altered phenotype had functional consequences, as shown by the reduction in T cell-stimulatory capacity of Treg-treated monocytes. Together, these data demonstrate that CD4+CD25+ Tregs can exert direct suppressive effects on monocytes/macrophages, thereby affecting subsequent innate and adaptive immune responses.

WNT signalling in the immune system: WNT is spreading its wings.

WNT proteins are secreted morphogens that are required for basic developmental processes, such as cell-fate specification, progenitor-cell proliferation and the control of asymmetric cell division, in many different species and organs. In blood and immune cells, WNT signalling controls the proliferation of progenitor cells and might also affect the cell-fate decisions of stem cells. Recent studies indicate that WNT proteins also regulate effector T-cell development, regulatory T-cell activation and dendritic-cell maturation. WNT signalling seems to function as a universal mechanism in leukocytes to establish a pool of undifferentiated cells for further selection, effector-cell maturation and terminal differentiation. WNT signalling is therefore subject to strict molecular control, and dysregulated WNT signalling is implicated in the development of haematological malignancies.

Cow's milk-specific T-cell reactivity of children with and without persistent cow's milk allergy: key role for IL-10.

BACKGROUND: The role of antigen-specific T cells in the mechanism of food allergy or maintenance of tolerance toward an innocuous antigen, such as cow's milk, is not yet fully understood. OBJECTIVE: The cow's milk-specific T-cell response of donors with various allergic backgrounds was investigated. METHODS: Cow's milk-specific T-cell clones (TCCs) were generated from the blood of children with persistent cow's milk allergy (CMA) and the blood of cow's milk-tolerant allergic and nonallergic control subjects. The TCCs were characterized by their antigen-specific proliferation, cytokine production, and activation status. RESULTS: Cow's milk-specific TCCs of children with persistent CMA were T(H)2 skewed, and the production of IL-4 and IL-13 was significantly correlated with the expression of the activation marker CD25. TCCs of the allergic control subjects were characterized by a high production of IL-10, which was positively correlated with the production of IL-4 and IFN-gamma and with the expression of CD25. TCCs derived from nonallergic control subjects had an attenuated response toward cow's milk in that they did not produce high levels of cytokines nor did they express high levels of surface markers. As in the allergic control subjects, in the nonallergic control subjects IL-10 production was positively correlated with the expression of CD25. CONCLUSION: The activation status of T cells derived from persistent donors with CMA was associated with the production of IL-4 and IL-13, whereas activated TCCs of cow's milk-tolerant control subjects were characterized by the production of IL-10 and, to a lesser extent, IFN-gamma. These findings suggest that activated CD4(+) T cells (characterized by a high CD25 expression) might contribute to the tolerogenic immune response toward an antigen, such as cow's milk, through the production of IL-10.