Associations of the placental metabolome with immune maturation up to one year of age in the Swedish NICE-cohort.

INTRODUCTION: Allergies and other immune-mediated diseases are thought to result from incomplete maturation of the immune system early in life. We previously showed that infants' metabolites at birth were associated with immune cell subtypes during infancy. The placenta supplies the fetus with nutrients, but may also provide immune maturation signals. OBJECTIVES: To examine the relationship between metabolites in placental villous tissue and immune maturation during the first year of life and infant and maternal characteristics (gestational length, birth weight, sex, parity, maternal age, and BMI). METHODS: Untargeted metabolomics was measured using Liquid Chromatography-Mass Spectrometry. Subpopulations of T and B cells were measured using flow cytometry at birth, 48 h, one, four, and 12 months. Random forest analysis was used to link the metabolomics data with the T and B cell sub populations as well as infant and maternal characteristics. RESULTS: Modest associations (Q2 = 0.2-0.3) were found between the placental metabolome and kappa-deleting recombination excision circles (KREC) at birth and naïve B cells and memory T cells at 12 months. Weak associations were observed between the placental metabolome and sex and parity. Still, most metabolite features of interest were of low intensity compared to associations previously found in cord blood, suggesting that underlying metabolites were not of placental origin. CONCLUSION: Our results indicate that metabolomic measurements of the placenta may not effectively recognize metabolites important for immune maturation.

Frequent Occurrence of Perianal Disease and Granuloma Formation in Patients with Crohn's Disease and Coexistent Orofacial Granulomatosis.

BACKGROUND: Orofacial granulomatosis (OFG) is an inflammatory disorder of the perioral region and oral cavity. Crohn's disease (CD) in conjunction with OFG (CD-OFG), has been suggested to constitute a phenotype of CD with distinct features at diagnosis. AIMS: The aim of this project was to investigate whether the distinct phenotypic features of CD-OFG persist in the years following the initial diagnosis of CD. METHODS: Clinical data were extracted from medical records covering the first 5 years post-diagnosis for a cohort of patients with CD-OFG, and were compared to those of references with CD without OFG. RESULTS: The clinical characteristics of our cohort of patients with CD-OFG (N = 25) were evaluated in comparison to references with CD without OFG (ratio 1:2). Five years post-diagnosis, more patients with CD-OFG had a phenotype with perianal disease (cumulative incidence: 16/25, 64% vs 13/50, 26%, P = 0.002) and intestinal granulomas (cumulative incidence: 22/25, 88% vs 24/50, 48%, P = 0.0009) than patients in the CD reference group. The patients with CD-OFG were also more likely to have undergone perianal surgery (12/25, 48% vs 4/50, 8%, P = 0.0002). At the end of the observation period, more of the patients with CD-OFG were receiving combination therapy, i.e., immunomodulators and tumor necrosis factor antagonists, than those in the CD reference group (9/25, 36% vs 5/50, 10%, P = 0.01). CONCLUSION: The results support the notion that CD in conjunction with OFG represents a specific phenotype of CD that is characterized by frequent perianal disease, pronounced intestinal granuloma formation and a need for extensive therapy.

Chromatin remodeling by the NuRD complex regulates development of follicular helper and regulatory T cells.

Lineage commitment and differentiation into CD4+ T cell subsets reflect an interplay between chromatin regulators and transcription factors (TF). Follicular T cell development is regulated by the Bcl6 TF, which helps determine the phenotype and follicular localization of both CD4+ follicular helper T cells (TFH) and follicular regulatory T cells (TFR). Here we show that Bcl6-dependent control of follicular T cells is mediated by a complex formed between Bcl6 and the Mi-2ß-nucleosome-remodeling deacetylase complex (Mi-2ß-NuRD). Formation of this complex reflects the contribution of the intracellular isoform of osteopontin (OPN-i), which acts as a scaffold to stabilize binding between Bcl6 and the NuRD complex that together regulate the genetic program of both TFH and TFR cells. Defective assembly of the Bcl6-NuRD complex distorts follicular T cell differentiation, resulting in impaired TFR development and skewing of the TFH lineage toward a TH1-like program that includes expression of Blimp1, Tbet, granzyme B, and IFNγ. These findings define a core Bcl6-directed transcriptional complex that enables CD4+ follicular T cells to regulate the germinal center response.

Staphylococcus aureus convert neonatal conventional CD4(+) T cells into FOXP3(+) CD25(+) CD127(low) T cells via the PD-1/PD-L1 axis.

The gut microbiota provides an important stimulus for the induction of regulatory T (Treg) cells in mice, whether this applies to newborn children is unknown. In Swedish children, Staphylococcus aureus has become a common early colonizer of the gut. Here, we sought to study the effects of bacterial stimulation on neonatal CD4(+) T cells for the induction of CD25(+) CD127(low) Treg cells in vitro. The proportion of circulating CD25(+) CD127(low) Treg cells and their expression of FOXP3, Helios and CTLA-4 was examined in newborns and adults. To evaluate if commensal gut bacteria could induce Treg cells, CellTrace violet-stained non-Treg cells from cord or peripheral blood from adults were co-cultured with autologous CD25(+) CD127(low) Treg cells and remaining mononuclear cells and stimulated with S. aureus. Newborns had a significantly lower proportion of CD25(+) CD127(low) Treg cells than adults, but these cells were Helios(+) and CTLA-4(+) to a higher extent than in adults. FOXP3(+) CD25(+) CD127(low) T cells were induced mainly in neonatal CellTrace-stained non-Treg cells after stimulation with S. aureus. In cell cultures from adults, S. aureus induced CD25(+) CD127(low) T cells only if sorted naive CD45RA(+) non-Treg cells were used, but these cells expressed less FOXP3 than those induced from newborns. Sorted neonatal CD25(+) CD127(low) T cells from S. aureus-stimulated cultures were still suppressive. Finally, blocking PD-L1 during stimulation reduced the induction of FOXP3(+) CD25(+) CD127(low) T cells. These results suggest that newborns have a higher proportion of circulating thymically derived Helios(+) Treg cells than adults and that S. aureus possess an ability to convert neonatal conventional CD4(+) T cells into FOXP3(+) CD25(+) CD127(low) Treg cells via the PD-1/PD-L1 axis.

In vitro stimulation with nontuberculous mycobacteria induced a stronger cytokine response in leukocytes isolated from individuals with latent tuberculosis compared to those isolated from active tuberculosis or cystic fibrosis patients.

Mycobacterium tuberculosis and opportunistic environmental non-tuberculous mycobacteria (NTM) can cause severe infection. Why latent tuberculosis infection advances to active disease, and why some individuals with cystic fibrosis (CF) develop pulmonary infections with NTM is still poorly understood. The aim of this study was to investigate the effector function of peripheral blood mononuclear cells (PBMC) from individuals with active or latent tuberculosis, individuals with CF with or without pulmonary NTM-infection and healthy controls, by measuring cytokine response to in vitro stimulation with different species of NTMs. The cytokine concentrations of IL-17A, IL-22, IL-23, IL-10, IL12p70 and IFN-γ were measured in PBMC-culture supernatants after stimulation with NTMs. PBMCs from individuals with latent tuberculosis infection showed strong IL-17A, IL-22, and IFN-γ responses compared to individuals with active tuberculosis or CF. IL-10 production was low in both tuberculosis groups compared to the CF groups and controls. This study suggests that IL-17A and IL-22 might be important to keep tuberculosis in a latent phase and that individuals with CF with an ongoing NTM infection seem to have a low cytokine response.

Short-chain fatty acids (SCFA) in infants' plasma and corresponding mother's milk and plasma in relation to subsequent sensitisation and atopic disease.

BACKGROUND: Short-chain fatty acids (SCFAs) in intestinal contents may influence immune function, while less is known about SCFAs in blood plasma. The aims were to investigate the relation between infants' and maternal plasma SCFAs, as well as SCFAs in mother's milk, and relate SCFA concentrations in infant plasma to subsequent sensitisation and atopic disease. METHODS: Infant plasma (N = 148) and corresponding mother's milk and plasma were collected four months postpartum. Nine SCFA (formic, acetic, propionic, isobutyric, butyric, succinic, valeric, isovaleric, and caproic acid) were analysed by UPLC-MS. At 12 months of age, atopic disease was diagnosed by a pediatric allergologist, and sensitisation was measured by skin prick test. All families participated in the Swedish birth cohort NICE (Nutritional impact on Immunological maturation during Childhood in relation to the Environment). FINDINGS: Infants with sensitisation, atopic eczema, or food allergy had significantly lower concentrations of five, three, and two SCFAs, respectively, in plasma at four months. Logistic regressions models showed significant negative associations between formic, succinic, and caproic acid and sensitisation [ORadj (95% CI) per SD: 0.41 (0.19-0.91); 0.19 (0.05-0.75); 0.25 (0.09-0.66)], and between acetic acid and atopic eczema [0.42 (0.18-0.95)], after adjusting for maternal allergy. Infants' and maternal plasma SCFA concentrations correlated strongly, while milk SCFA concentrations were unrelated to both. Butyric and caproic acid concentrations were enriched around 100-fold, and iso-butyric and valeric acid around 3-5-fold in mother's milk, while other SCFAs were less prevalent in milk than in plasma. INTERPRETATION: Butyric and caproic acid might be actively transported into breast milk to meet the needs of the infant, although mechanistic studies are needed to confirm this. The negative associations between certain SCFAs on sensitisation and atopic disease adds to prior evidence regarding their immunoregulatory potential. FUNDING: Swedish Research Council (Nr. 2013-3145, 2019-0137 and 2023-02217 to A-S.S.), Swedish Research Council for Health, Working Life and Welfare FORTE, Nr 2018-00485 to A.W.), The Swedish Asthma and Allergy Association's Research Fund (2020-0020 to A.S.).

Transplantation of a bioengineered tissue patch promotes uterine repair in the sheep.

Innovative bioengineering strategies utilizing extracellular matrix (ECM) based scaffolds derived from decellularized tissue offer new prospects for restoring damaged uterine tissue. Despite successful fertility restoration in small animal models, the translation to larger and more clinically relevant models have not yet been assessed. Thus, our study investigated the feasibility to use a 6 cm2 graft constructed from decellularized sheep uterine tissue, mimicking a future application to repair a uterine defect in women. Some grafts were also recellularized with fetal sheep bone marrow-derived mesenchymal stem cells (SF-MSCs). The animals were followed for six weeks post-surgery during which blood samples were collected to assess the systemic immune cell activation by fluorescence-activated cell sorting (FACS) analysis. Tissue regeneration was assessed by histology, immunohistochemistry, and gene expression analyses. There was a large intra-group variance which prompted us to implement a novel scoring system to comprehensively evaluate the regenerative outcomes. Based on the regenerative score each graft received, we focused our analysis to map potential differences that may have played a role in the success or failure of tissue repair following the transplantation therapy. Notably, three out of 15 grafts exhibited major regeneration that resembled native uterine tissue, and an additional three grafts showed substantial regenerative outcomes. For the better regenerated grafts, it was observed that the systemic T-cell subgroups were significantly different compared with the failing grafts. Hence, our data suggest that the T-cell response play an important role for determining the uterus tissue regeneration outcomes. The remarkable regeneration seen in the best-performing grafts after just six weeks following transplantation provides compelling evidence that decellularized tissue for uterine bioengineering holds great promise for clinically relevant applications.

Investigating immune profile by CyTOF in individuals with long-standing type 1 diabetes.

Type 1 diabetes (T1D) is an autoimmune disease caused by T-cell mediated destruction of pancreatic beta cells. Eosinophils are found in pancreatic tissue from individuals with T1D. Eosinophilic suppression of T cells is dependent of the protein galectin-10. Little is known when it comes to the role of eosinophil granulocytes in type 1 diabetes. Here we show that individuals with long-standing T1D had lower levels of galectin-10hi eosinophils and a subgroup of galectin-10hi eosinophils were entirely absent in all T1D patients. In addition, 7% immature eosinophils were present in the circulation of T1D patients whereas 0.8% in healthy individuals. Furthermore, higher levels of CD4+CD8+ T cells and Th17 cells were observed in patients with T1D. Blood samples from 12 adult individuals with long-standing T1D and 12 healthy individuals were compared using cytometry by time-of-flight. Lower levels of galectin-10hi eosinophils, which are potent T cell suppressors, in individuals with T1D could indicate that activated T cells are enabled to unrestrictedly kill the insulin producing beta cells. This is the first study showing absence of galectin-10hi eosinophilic subgroup in individuals with T1D compared with healthy controls. This study is a first important step toward unraveling the role of the eosinophils in patients with T1D.

Development of gut-homing receptors on circulating B cells during infancy.

B cell gut-homing is mainly mediated by α4ß7, CCR9 and CCR10. We here studied the expression of these receptors on B cells from cord blood and from peripheral blood at 1, 4, 18 and 36 months of age in a prospective cohort of Swedish infants. The proportion of all B cells expressing α4ß7 as well as the fraction of CCR10+ B cells expressing α4ß7 was highest in early infancy. Nearly all naïve B cells in all age groups expressed α4ß7, whereas the expression on class-switched B cells decreased with age. Moreover, the proportion of both IgA+ and IgG+ B cells expressing α4ß7, CCR9 and CCR10 were higher during the first months when compared to adults. In conclusion, the high fraction of circulating IgA+ and IgG+ B cells expressing CCR9 and CCR10 in the first months of life indicates activation of naïve B cells in the gut, coinciding with bacterial colonization.

Associations of maternal and infant metabolomes with immune maturation and allergy development at 12 months in the Swedish NICE-cohort.

Allergic diseases are the most common chronic diseases in childrenin the Western world, but little is know about what factors influence immune maturation and allergy development. We therefore aimed to associate infant and maternal metabolomes to T- and B-cell subpopulations and allergy diagnosis. We performed liquid chromatography-mass spectrometry based untargeted metabolomics on blood plasma from mothers (third trimester, n = 605; delivery, n = 558) and from the umbilical cord (n = 366). The measured metabolomes were associated to T- and B-cell subpopulations up to 4 months after delivery and to doctor´s diagnosed eczema, food allergy and asthma at one year of age using random forest analysis. Maternal and cord plasma at delivery could predict the number of CD24+CD38low memory B-cells (p = 0.033, n = 26 and p = 0.009, n = 22), but future allergy status could not be distinguished from any of the three measured metabolomes. Replication of previous literature findings showed hypoxanthine to be upregulated in the umbilical cord of children with subsequent asthma. This exploratory study suggests foetal immune programming occuring during pregnancy as the metabolomic profiles of mothers and infants at delivery related to infants' B-cell maturation.

Cord Blood Levels of EPA, a Marker of Fish Intake, Correlate with Infants' T- and B-Lymphocyte Phenotypes and Risk for Allergic Disease.

Maternal fish intake during pregnancy has been associated with reduced allergy development in the offspring and here, we hypothesized that components of fish stimulate fetal immune maturation. The aim of this study was to investigate how maternal fish intake during pregnancy and levels of n-3 long-chain polyunsaturated fatty acids (LCPUFAs) in the infant's cord serum correlated with different subsets of B- and T-cells in cord blood and B-cell activating factor (BAFF) in cord plasma, and with doctor-diagnosed allergy at 3 and 8 years of age in the FARMFLORA birth-cohort consisting of 65 families. Principal component analysis showed that infant allergies at 3 or 8 years of age were negatively associated with the proportions of n-3 LCPUFAs (eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid) in infant cord serum, which, in turn correlated positively with maternal fish intake during pregnancy. Both maternal fish intake and cord serum n-3 LCPUFAs correlated negatively to CD5+ B cells and the FOXP3+CD25high of the CD4+ T cell subsets in cord blood, but not to BAFF in cord plasma. Our observational study suggests that fish might contain components that promote maturation of the infant's immune system in a manner that protects against allergy development.

Th17 associated cytokines in sputum samples from patients with cystic fibrosis.

Cystic fibrosis (CF) is a genetic disease leading to chronic bacterial airway infection and inflammation. T helper 17 (Th17) cells are identified by their production of interleukin (IL)-17A, which recruit neutrophils to the site of airway infection. IL-23 is an important inducer of IL-17 and IL-22 production. The aim of this study was to study the role of Th17 cells in CF airway infection by measuring the levels of Th17 associated cytokines in sputum from CF patients with or without airway infection and by comparison with non-CF-controls. In a cross-sectional screening study, cytokine levels were measured with a Th17 multiplex cytokine ELISA. Significantly lower levels of IL-17A and IL-23 were found in sputa from infected CF patients. The lowest levels of IL-17A were found in patients chronically infected with P. aeruginosa, which also had the lowest IL-17/IL-22 ratio, while children had a higher ratio. Children also had higher IL-23 levels than adults. IL-1ß and IL-10 were significantly lower in CF sputum compared to controls. Thus, in our study CF patients with chronic infections had a lower production of Th17 associated cytokines in sputum compared with non-infected CF patients and infected patient without CF.

Inflammatory Mediator Profiles in Secretory Otitis Media in Relationship to Viable Bacterial Pathogens and Bacterial and Viral Nucleic Acids.

Secretory otitis media (SOM) is characterized by persistence of fluid in the middle ear, often following an episode of acute otitis media. Our hypothesis is that failure to eliminate bacterial or viral pathogens may result in persistent low-grade inflammation. In this study, we analyzed inflammatory mediators in middle ear fluids from 67 children with SOM. This was combined with determinations of viable bacteria by culture along with detection of bacterial and viral genetic material by real-time polymerase chain reaction (PCR). The inflammatory mediators found at the highest concentrations (>30 ng/mL) were stem cell growth factor-ß (median 110 ng/mL), CXCL1, IL-16, IL-8, migration inhibitory factor, CXCL10, and CXCL9. Among bacterial pathogens, Moraxella catarrhalis and Haemophilus influenzae dominated, regardless of detection methods, while rhinovirus dominated among viral pathogens. Middle ear fluid levels of interleukin (IL)-1α, IL-17, IL-1ß, fibroblast growth factor basic, and tumor necrosis factor correlated strongly with presence of bacteria detected either by culture or PCR, while IL-1RA, IL-3, IL-6, IL-8, CCL3, CCL4, and granulocyte-colony stimulating factor correlated significantly with real-time PCR values. CXCL10, CXCL9, CCL2, and TRAIL correlated significantly with viral nucleic acid levels. To conclude, persistence of viral and bacterial pathogens may fuel persistent inflammation in SOM. Bacteria caused a broad inflammatory response, while viruses chiefly elicited the interferon-induced chemokines CXCL9 and CXCL10.

Neonatal gut colonization by Bifidobacterium is associated with higher childhood cytokine responses.

The gut microbiota is a major stimulus for the immune system, and late acquisition of bacteria and/or reduced complexity of the gut flora may delay adaptive immune maturation. However, it is unknown how the gut bacterial colonization pattern in human infants is related to T cell activation during early childhood. We followed 65 Swedish children in the FARMFLORA cohort, from birth up to 3 years of age. In fecal samples collected at several time points during the first year of life, the gut colonization pattern was investigated with the use of both 16S rRNA next generation sequencing (NGS) and culture-based techniques. This was related to production of IL-13, IL-5, IL-6, TNF, IL-1ß and IFN-γ by PHA-stimulated fresh mononuclear cells and to proportions of CD4+ T cells that expressed CD45RO at 36 months of age. Both NGS and culture-based techniques showed that colonization by Bifidobacterium at 1 week of age associated with higher production of IL-5, IL-6, IL-13, TNF and IL-1ß at 36 months of age. By contrast, gut colonization by Enterococcus, Staphylococcus aureus or Clostridium in early infancy related inversely to induced IL-13, IL-5 and TNF at 3 years of age. Infants with elder siblings produced more cytokines and had a larger fraction of CD45RO+ T cells compared to single children. However, controlling for these factors did not abolish the effect of colonization by Bifidobacterium on immune maturation. Thus, gut colonization in early infancy affects T cell maturation and Bifidobacterium may be especially prone to induce infantile immune maturation.

Mid-trimester amniotic fluid proteome's association with spontaneous preterm delivery and gestational duration.

BACKGROUND: Amniotic fluid is clinically accessible via amniocentesis and its protein composition may correspond to birth timing. Early changes in the amniotic fluid proteome could therefore be associated with the subsequent development of spontaneous preterm delivery. OBJECTIVE: The main objective of this study was to perform unbiased proteomics analysis of the association between mid-trimester amniotic fluid proteome and spontaneous preterm delivery and gestational duration, respectively. A secondary objective was to validate and replicate the findings by enzyme-linked immunosorbent assay using a second independent cohort. METHODS: Women undergoing a mid-trimester genetic amniocentesis at Sahlgrenska University Hospital/Östra between September 2008 and September 2011 were enrolled in this study, designed in three analytical stages; 1) an unbiased proteomic discovery phase using LC-MS analysis of 22 women with subsequent spontaneous preterm delivery (cases) and 37 women who delivered at term (controls), 2) a validation phase of proteins of interest identified in stage 1, and 3) a replication phase of the proteins that passed validation using a second independent cohort consisting of 20 cases and 40 matched controls. RESULTS: Nine proteins were nominally significantly associated with both spontaneous preterm delivery and gestational duration, after adjustment for gestational age at sampling, but none of the proteins were significant after correction for multiple testing. Several of these proteins have previously been described as being associated with spontaneous PTD etiology and six of them were thus validated using enzyme linked immunosorbent assay. Two of the proteins passed validation; Neutrophil gelatinase-associated lipocalin and plasminogen activator inhibitor 1, but the results could not be replicated in a second cohort. CONCLUSIONS: Neutrophil gelatinase-associated lipocalin and Plasminogen activator inhibitor 1 are potential biomarkers of spontaneous preterm delivery and gestational duration but the findings could not be replicated. The negative findings are supported by the fact that none of the nine proteins from the exploratory phase were significant after correction for multiple testing.

High Frequency of Concomitant Food Allergy Development and Autoantibody Formation in Children Who Have Undergone Liver Transplantation.

BACKGROUND: Allergy and other immune-mediated diseases are more frequently reported in children who have undergone liver transplantation. Furthermore, autoantibodies are also prevalent, suggesting a state of immune dysregulation in these patients. Whether or not these processes occur simultaneously in the same individual has not been studied previously. METHODS: A cohort of 43 children who had undergone liver transplantation for nonautoimmune liver disease at median age of 1.3 years was investigated for allergy and autoimmune disease. Sensitization to food and inhalant allergens was assessed, and autoantibodies were measured. RESULTS: The prevalence of food allergy was 26% and that of respiratory allergy was 23%, whereas 33% and 26% of the subjects were sensitized to food and inhalant allergens, respectively. Autoimmune disease (ie, autoimmune hepatitis) occurred in a single individual (2%), whereas autoantibodies were present in 44% of the children. Food allergy and autoantibodies occurred concomitantly in 19% of the children, which was almost twice the frequency expected by chance (11%, P = 0.04). Respiratory allergy and the presence of autoantibodies were unrelated (12% concurrence versus the expected 10%, P = 0.73). In the logistic regression analysis, autoantibody formation was associated with discontinued immunosuppression and food allergy, with odds ratios of 13 (P = 0.01) and 7.1 (P = 0.03), respectively. CONCLUSIONS: In contrast to respiratory allergy, food allergy and autoantibody formation occurred together in the same children who underwent liver transplantation at a frequency higher than would be expected by chance. This may reflect an underlying immune dysregulation that impairs immune tolerance to both food allergens and autoantigens.

Control of Germinal Center Localization and Lineage Stability of Follicular Regulatory T Cells by the Blimp1 Transcription Factor.

Follicular regulatory T (TFR) cells are a specialized suppressive subset that controls the germinal center (GC) response and maintains humoral self-tolerance. The mechanisms that maintain TFR lineage identity and suppressive activity remain largely unknown. Here, we show that expression of Blimp1 by FoxP3+ TFR cells is essential for TFR lineage stability, entry into the GC, and expression of regulatory activity. Deletion of Blimp1 in TFR cells reduced FoxP3 and CTLA-4 expression and increased pro-inflammatory cytokines and spontaneous production of autoantibodies, including elevated IgE. Maintenance of TFR stability reflected Blimp1-dependent repression of the IL-23R-STAT3 axis and activation of the CD25-STAT5 pathway, while silenced IL-23R-STAT3 or increased STAT5 activation rescued the Blimp1-deficient TFR phenotype. Blimp1-dependent control of CXCR5/CCR7 expression also regulated TFR homing into the GC. These findings uncover a Blimp1-dependent TFR checkpoint that enforces suppressive activity and acts as a gatekeeper of GC entry.

Nutritional impact on Immunological maturation during Childhood in relation to the Environment (NICE): a prospective birth cohort in northern Sweden.

INTRODUCTION: Prenatal and neonatal environmental factors, such as nutrition, microbes and toxicants, may affect health throughout life. Many diseases, such as allergy and impaired child development, may be programmed already in utero or during early infancy. Birth cohorts are important tools to study associations between early life exposure and disease risk. Here, we describe the study protocol of the prospective birth cohort, 'Nutritional impact on Immunological maturation during Childhood in relation to the Environment' (NICE). The primary aim of the NICE cohort is to clarify the effect of key environmental exposures-diet, microbes and environmental toxicants-during pregnancy and early childhood, on the maturation of the infant's immune system, including initiation of sensitisation and allergy as well as some secondary outcomes: infant growth, obesity, neurological development and oral health. METHODS AND ANALYSIS: The NICE cohort will recruit about 650 families during mid-pregnancy. The principal inclusion criterion will be planned birth at the Sunderby Hospital in the north of Sweden, during 2015-2018. Questionnaires data and biological samples will be collected at 10 time-points, from pregnancy until the children reach 4 years of age. Samples will be collected primarily from mothers and children, and from fathers. Biological samples include blood, urine, placenta, breast milk, meconium, faeces, saliva and hair. Information regarding allergic heredity, diet, socioeconomic status, lifestyle including smoking, siblings, pet ownership, etc will be collected using questionnaires. Sensitisation to common allergens will be assessed by skin prick testing and allergic disease will be diagnosed by a paediatrician at 1 and 4 years of age. At 4 years of age, the children will also be examined regarding growth, neurobehavioural and neurophysiological status and oral health. ETHICS AND DISSEMINATION: The NICE cohort has been approved by the Regional Ethical Review Board in Umeå, Sweden (2013/18-31M). Results will be disseminated through peer-reviewed journals and communicated on scientific conferences.

Higher proportions of circulating FOXP3+ and CTLA-4+ regulatory T cells are associated with lower fractions of memory CD4+ T cells in infants.

In adults, a majority of FOXP3(+) T(regs) expresses CTLA-4, and this costimulatory molecule is essential to control the expansion of other T cells. However, it remains to be investigated whether FOXP3(+) and/or CTLA-4(+) T(regs) are associated with the expression of memory markers and homing receptors on CD4(+) T cells. Thus, in a prospective newborn-infant cohort study, we examined the proportions of FOXP3(+) and CTLA-4(+) T(regs) within the CD4(+)CD25(+) T cell population and the fractions of CD4(+) T cells that expressed CD45RA, CD45RO, HLA-DR, α(4)ß(7), CD62L, and CCR4 at several time-points during the first 3 years of life using flow cytometry. With the use of multivariate factor analysis, we found that a high proportion of FOXP3(+) or CTLA-4(+) T(regs) during the first 18 months of life was associated positively with the fraction of T cells that expressed a naïve phenotype (CD45RA and α(4)ß(7)) and inversely related to the fraction of T cells that expressed a memory phenotype (CD45RO and CCR4) later in childhood. In conclusion, FOXP3(+) or CTLA-4(+) T(regs) may modulate CD4(+) T cell activation and homing receptor expression in children.