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Neonatal and infant immune responses are characterized by a limited capability to generate protective Ab titers and memory B cells as seen in adults. Multiple studies support an immature or even impaired character of umbilical cord blood (UCB) B cells themselves. In this study, we provide a comprehensive molecular and functional comparison of B cell subsets from UCB and adult peripheral blood. Most UCB B cells have a mature, naive B cell phenotype as seen in adults. The UCB Ig repertoire is highly variable but interindividually conserved, as BCR clonotypes are frequently shared between neonates. Furthermore, UCB B cells show a distinct transcriptional program that confers accelerated responsiveness to stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into Ab-secreting cells, presumably limiting memory B cell formation. Humanized mice suggest that the distinctness of UCB versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice, albeit our findings suggest only partial comparability to murine B-1 cells. Our study shows that UCB B cells are not immature or impaired but differ from their adult mature counterpart in a conserved BCR repertoire, efficient IgA class switching, and accelerated, likely transient response dynamics.

Objective Treatment with CTLA-4Ig blocks T-cell activation and is clinically effective in RA. However, it is unknown if specific CD4+ T-cell subsets in blood at baseline predict remission after CTLA-4Ig, or other biological treatments with different modes of action, and how treatment affects CD4+ T cells in patients with untreated early RA (eRA). Methods This study included 60 patients with untreated eRA from a larger randomized trial. They were treated with methotrexate combined with CTLA-4Ig (abatacept, n = 17), anti-IL6 receptor (tocilizumab, n = 21) or anti-TNF (certolizumab-pegol, n = 22). Disease activity was assessed by clinical disease activity index (CDAI), DAS28, swollen joint counts, tender joint counts, CRP and ESR. The primary outcome was CDAI remission (CDAI ≤ 2.8) at week 24. Proportions of 12 CD4+ T-cell subsets were measured by flow cytometry at baseline and after 4, 12 and 24 weeks of treatment. Results In patients treated with CTLA-4Ig, the proportions of PD-1+TFh and CTLA-4+ conventional CD4+ T cells at baseline predicted CDAI remission at week 24. CD4+ T-cell subset proportions could not predict remission after treatment with anti-IL6R or anti-TNF. The percentage of regulatory T cells (Tregs) expressing CTLA-4 decreased in all treatment arms by 24 weeks, but only CTLA-4Ig treatment significantly reduced the proportions of Tregs and PD-1+T follicular helper (TFh) cells. Conclusion These findings indicate that circulating proportions PD-1+TFh and CTLA-4+ conventional CD4+ T cells at baseline may serve as predictive biomarkers for remission in early RA after CTLA-4Ig treatment.

Background Population-based neonatal screening using T-cell receptor excision circles (TRECs) identifies infants with profound T lymphopenia, as seen in cases of severe combined immunodeficiency, and in a subgroup of infants with 22q11 deletion syndrome (22q11DS). Purpose To investigate the long-term prognostic value of low levels of TRECs in newborns with 22q11DS. Methods Subjects with 22q11DS and low TRECs at birth (22q11Low, N=10), matched subjects with 22q11DS and normal TRECs (22q11Normal, N=10), and matched healthy controls (HC, N=10) were identified. At follow-up (median age 16 years), clinical and immunological characterizations, covering lymphocyte subsets, immunoglobulins, TRECs, T-cell receptor repertoires, and relative telomere length (RTL) measurements were performed. Results At follow-up, the 22q11Low group had lower numbers of naïve T-helper cells, naïve T-regulatory cells, naïve cytotoxic T cells, and persistently lower TRECs compared to healthy controls. Receptor repertoires showed skewed V-gene usage for naïve T-helper cells, whereas for naïve cytotoxic T cells, shorter RTL and a trend towards higher clonality were found. Multivariate discriminant analysis revealed a clear distinction between the three groups and a skewing towards Th17 differentiation of T-helper cells, particularly in the 22q11Low individuals. Perturbations of B-cell subsets were found in both the 22q11Low and 22q11Normal group compared to the HC group, with larger proportions of naïve B cells and lower levels of memory B cells, including switched memory B cells. Conclusions This long-term follow-up study shows that 22q11Low individuals have persistent immunologic aberrations and increased risk for immune dysregulation, indicating the necessity of lifelong monitoring. Clinical Implications This study elucidates the natural history of childhood immune function in newborns with 22q11DS and low TRECs, which may facilitate the development of programs for long-term monitoring and therapeutic choices.

Background Protein profiles that can predict allergy development in children are lacking and the ideal sampling age is unknown. By applying an exploratory proteomics approach in the prospective FARMFLORA birth cohort, we sought to identify previously unknown circulating proteins in early life that associate to protection or risk for development of allergy up to 8 years of age. Methods We analyzed plasma prepared from umbilical cord blood (n = 38) and blood collected at 1 month (n = 42), 4 months (n = 39), 18 months (n = 42), 36 months (n = 42) and 8 years (n = 44) of age. We profiled 230 proteins with a multiplexed assay and evaluated the global structure of the data with principal component analysis (PCA). Protein profiles informative to allergic disease at 18 months, 36 months and/or 8 years were evaluated using Lasso logistic regression and random forest. Results Two clusters emerged in the PCA analysis that separated samples obtained at birth and at 1 month of age from samples obtained later. Differences between the clusters were mostly driven by abundant plasma proteins. For the prediction of allergy, both Lasso logistic regression and random forest were most informative with samples collected at 1 month of age. A Lasso model with 27 proteins together with farm environment differentiated children who remained healthy from those developing allergy. This protein panel was primarily composed of antigen-presenting MHC class I molecules, interleukins and chemokines. Conclusion Sampled at one month of age, circulating proteins that reflect processes of the immune system may predict the development of allergic disease later in childhood.

Additional file 2: Table S1. Diagnostic criteria for eczema, asthma, food allergy and allergic rhinoconjunctivitis.

Additional file 1: Figure S1. Age related differences in plasma proteins. Exploratory analysis with samples marked based on age, visualized with T-distributed Stochastic Neighbour Embedding (tSNE).

Additional file 3: Table S2. Allergy diagnosis in allergic children at 18 months, 36 months and 8 years of age.

Allergy is one of the most common diseases among young children yet all factors that affect development of allergy remain unclear. In a small cohort of 65 children living in the same rural area of south-west Sweden, we have previously found that maternal factors, including prenatal diet, affect childhood allergy risk, suggesting that in utero conditions may be important for allergy development. Here, we studied if metabolites in the umbilical cord blood of newborns may be related to development of childhood allergy, accounting for key perinatal factors such as mode of delivery, birth order and sex. Available umbilical cord blood plasma samples from 44 of the participants were analysed using gas chromatography-mass spectrometry metabolomics; allergy was diagnosed by specialised paediatricians at ages 18 months, 3 years and 8 years and included eczema, asthma, food allergy and allergic rhinoconjunctivitis. Nineteen cord blood metabolites were related to future allergy diagnosis though there was no clear pattern of up- or downregulation of metabolic pathways. In contrast, perinatal factors birth order, sex and mode of delivery affected several energy and biosynthetic pathways, including glutamate and aspartic acid—histidine metabolism (p = 0.004) and the tricarboxylic acid cycle (p = 0.006) for birth order; branched chain amino acid metabolism (p = 0.0009) and vitamin B6 metabolism (p = 0.01) for sex; and glyoxylate and dicarboxylic acid metabolism (p = 0.005) for mode of delivery. Maternal diet was also related to some of the metabolites associated with allergy. In conclusion, the cord blood metabolome includes individual metabolites that reflect lifestyle, microbial and other factors that may be associated with future allergy diagnosis, and also reflects temporally close events/factors. Larger studies are required to confirm these associations, and perinatal factors such as birth order or siblings must be considered in future cord-blood metabolome studies.

Children growing up on farms have low rates of allergy, but the mechanism for this protective effect has not been fully elucidated. Short chain fatty acids (SCFAs) produced by the gut microbiota may play a role in protection from allergy. We measured fecal SCFA levels in samples collected from 28 farming and 37 control children over the first 3 years of life using gas chromatography. Data on diet and other host factors were recorded and allergy was diagnosed at 8 years of age. Among all children, median propionic and butyric acid concentration increased over the first 3 years, and longer SCFAs typically appeared by 1 year of age. Farm children had higher levels of iso-butyric, iso-valeric and valeric acid at 3 years of age than rural controls. In addition, children with elder siblings had higher levels of valeric acid at 3 years of age, and dietary factors also affected SCFA pattern. High levels of valeric acid at 3 years of age were associated with low rate of eczema at 8 years of age. The fecal SCFA pattern in farm children suggests a more rapid maturation of the gut microbiota. Valeric acid or associated microbes may have protective potential against eczema.

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-1β 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-1β 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.

The human infant B cell system is considered premature or impaired. Here we show that most cord blood B cells are mature and functional as seen in adults, albeit with distinct transcriptional programs providing accelerated responsiveness to T cell-independent and T cell-dependent stimulation and facilitated IgA class switching. Stimulation drives extensive differentiation into antibody-secreting cells, thereby presumably limiting memory B cell formation. The neonatal Ig-repertoire is highly variable, but conserved, showing recurrent B cell receptor (BCR) clonotypes frequently shared between neonates. Our study demonstrates that cord blood B cells are not impaired but differ from their adult counterpart in a conserved BCR repertoire and rapid but transient response dynamics. These properties may account for the sensitivity of neonates to infections and limited effectivity of vaccination strategies. Humanized mice suggest that the distinctness of cord blood versus adult B cells is already reflected by the developmental program of hematopoietic precursors, arguing for a layered B-1/B-2 lineage system as in mice. Still, our findings reveal overall limited comparability of human cord blood B cells and murine B-1 cells.Significance StatementNeonates and infants suffer from enhanced susceptibility to infections. Our study contrasts with the current concept of a premature or impaired B cell system in neonates, by showing that most cord blood B cells are mature and functional. However, their responses are rapid but provide only short-term protection, which may help to improve infant vaccination strategies. We propose an altered perspective on the early human B cell system, which looks similar to but functions differently from the adult counterpart. Finally, our analysis indicates that cord blood- and adult B cell development occur layered as in mice, but certain mouse models still may offer a limited view on human neonatal B cell immunity.

Background Adiponectin is an adipokine circulating in blood in three forms, whereof the high molecular weight (HMW) is supposed to mediate the metabolic effects of adiponectin. Subjects with rheumatoid arthritis (RA) have elevated levels of adiponectin in serum and synovial fluid but it is unclear if circulating adiponectin associates with disease activity markers in subjects with RA. Here we aimed to determine whether total and/or HMW adiponectin levels associate with markers of disease activity and plasma chemokines in a cohort of subjects with untreated newly diagnosed RA.Methods Inflammation and disease activity were assessed in a cohort of 70 subjects with untreated newly diagnosed RA using erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), number of tender or swollen joints, as well as disease activity scores (DAS28 and CDAI) Plasma levels of 15 chemokines were measured using flow cytometry bead-based immunoassay or ELISA. Total and HMW adiponectin plasma levels were determined with ELISA.Results Both total and HMW adiponectin were positively associated with CRP (both P = 0.001) and ESR (P = 0.001 and P P = 0.021), CCL2 (P = 0.012) and CXCL9 (P = 0.044), whereas HMW adiponectin associated with CXCL9 only (P = 0.033).Conclusion Total and HMW adiponectin are associated with markers of inflammation as well as pro-inflammatory chemokines in a cohort of 70 subjects with untreated newly diagnosed RA. Our results support the hypothesis that adiponectin might be a pro-inflammatory factor involved in the pathogenesis of RA.

Boys present with higher proportions of immature/naïve CD5+ B cells than girls up to 3 years of age. Boys also have higher fractions of regulatory T cells (Tregs) in early infancy, but the mechanisms for these sex-related differences are unknown. In the prospective FARMFLORA follow-up study of 23 boys and 25 girls, we investigated if these immunological differences remained at 8 years of age. We also examined if testosterone or dihydrotestosterone (DHT) levels at birth and at 8 years of age were associated with immune maturation. Immunological variables and androgen levels were examined and measured in blood samples obtained at birth, 3–5 days and at 8 years of age. Boys had higher proportions of CD5+ and immature/transitional CD24hiCD38hi B cells, whereas girls had higher fractions of B cells with a memory phenotype at 8 years of age. School-aged boys also presented with higher frequencies of Tregs, and a greater capacity to produce T-cell-associated cytokines. Among boys, higher cord blood DHT levels were associated with higher proportions of CD5+ B cells in early infancy and at 8 years of life. These results suggest that DHT actions in utero might be involved in the mechanism for delayed peripheral B-cell maturation in boys.

Androgen exposure of the fetus during gestation plays an important role in human physiology and pathophysiology, but assessment of androgens, in particular dihydrotestosterone (DHT), in human umbilical cord blood is technically challenging. The aim of this study was to assess umbilical cord androgen levels, including DHT, at birth by a highly sensitive assay, and study their association with sex of the infant, sex-hormone-binding globulin (SHBG) levels, and gestational age at delivery. Swedish infants (27 girls, 26 boys) were recruited at maternity care clinics in Southern Sweden. Umbilical cord blood levels of dehydroepiandrosterone (DHEA), androstenedione, testosterone and DHT at delivery were assessed by a gas chromatography–tandem mass spectrometry assay. Cord blood levels of DHT were 2.4-fold higher in boys (median 27.8 pg/mL) than in girls (11.5 pg/mL), while the sex difference was less pronounced for testosterone (1.3-fold higher in boys) and non-significant for DHEA and androstenedione. Gestational age at delivery associated inversely with DHT levels in boys and with DHEA levels in girls. There was a strong inverse correlation between SHBG and DHEA in both sexes, while there were no associations between SHBG and testosterone or DHT levels. In conclusion, using state of the art technology, we report that there is a pronounced sexual dimorphism in human umbilical cord blood DHT levels. The possibility to assess a complete androgen profile in human cord blood opens up for future increased understanding of the biological impact of the fetal androgen milieu.

During pregnancy, the maternal immune system must tolerate the developing foetus, and yet retain a potent antimicrobial response to prevent infections. Mucosal associated invariant T (MAIT) cells recognize microbial-derived vitamin B metabolites presented on the MR1 molecule, but their presence and function at the foetal-maternal interface is not known. We here isolated mononuclear cells from paired samples of peripheral blood (PB), intervillous blood (IVB), and decidua parietalis (DP) following uncomplicated term pregnancies. Interestingly, MAIT cells were highly enriched in IVB compared to PB and DP. The activation status of IVB MAIT cells was similar to that of PB MAIT cells, except for a lower expression of PD-1. Both IVB MAIT cells and conventional T cells were more dominated by an effector memory phenotype compared to PB MAIT cells and T cells. IVB MAIT cells also responded more vigorously with expression of IFN-γ, granzyme B, and perforin in response to Escherichia coli stimulation compared to PB. MR1 was not expressed in syncytiotrophoblasts, but in placental villous and decidual macrophages. These data indicate that maternal MAIT cells accumulate in the intervillous space of the placenta and that they are highly armed to quickly respond if bacteria are encountered at the foetal-maternal interface.

Background: The pathogenesis of rheumatoid arthritis (RA) involves the action of immune cells and fibroblast-like synoviocytes (FLS). Adiponectin, an adipokine produced mainly by adipocytes, is elevated in serum of RA patients compared to healthy controls; moreover, synovial fluid from RA subjects has higher levels of adiponectin compared to controls with osteoarthritis1,2. Adiponectin induces the production of pro-inflammatory cytokines, such as interleukin 6 (IL-6) and IL-8, by FLS from RA patients 3,4. However, it remains unclear whether the response to adiponectin in terms of cytokine production is different in immune blood cells from healthy donors compared to RA patients; moreover, it is not known if FLS from healthy subjects can also produce pro-inflammatory cytokines upon adiponectin stimulation. Objectives: The study aims to analyse if adiponectin induces pro-inflammatory cytokine production by peripheral blood mononuclear cells (PBMCs) from both early RA patients and healthy controls. We also aim to study whether adiponectin induces pro-inflammatory cytokine production by FLS from healthy subjects. Methods: PBMCs were isolated from whole blood obtained from 5 healthy donors and 3 early untreated newly diagnosed RA patients using Ficoll-Paque PLUS. FLS were isolated from synovial tissue obtained from 3 healthy donors after knee surgery due to injury. Healthy FLS were cultured in Dulbecco’s Modified Eagle Medium (DMEM GlutaMAX) supplemented with 10% FBS, 1% Penicillin-streptomycin and 0.5% Gentamycin. Harvested PBMCs and FLS were resuspended in X-VIVO 15 serum-free hematopoietic cell medium and stimulated with different doses of human recombinant adiponectin (1, 5 and 10 μg/ml), and the culture media were collected at different time points. Concentrations of IL-6, IL-8 and TNF-α were measured with ELISA. Results: Unstimulated PBMCs from early RA patients produced higher levels of IL-6 compared to healthy subjects (P Adiponectin was able to induce the production of IL-6 and IL-8 by FLS isolated from heathy donors (n=3, P=0.03 for IL-6 and P=0.02 for IL-8), but not TNF-α. Conclusion: Our results show that adiponectin induces the production of IL-6 and TNF-α from PBMCs from both healthy subjects and patients with RA and that adiponectin is able to stimulate the production of IL-6 and IL-8 by FLS isolated from healthy subjects. Those results suggest that adiponectin may play a role in the pathogenesis of RA. References [1] Laurberg TB, et al. Plasma adiponectin in patients with active, early, and chronic rheumatoid arthritis who are steroid- and disease-modifying antirheumatic drug-naive compared with patients with osteoarthritis and controls. J Rheumatol2009 [2] Tan W, et al. High adiponectin and adiponectin receptor 1 expression in synovial fluids and synovial tissues of patients with rheumatoid arthritis. Semin Arthritis Rheum2009 [3] Tang CH, et al. Adiponectin enhances IL-6 production in human synovial fibroblast via an AdipoR1 receptor, AMPK, p38, and NF-kappa B pathway. J Immunol2007 [4] Kitahara K, et al. Adiponectin stimulates IL-8 production by rheumatoid synovial fibroblasts. Biochem Biophys Res Commun2009 Disclosure of Interests: Yuan Zhang: None declared, Jonathan Aldridge: None declared, Anna-Carin Lundell: None declared, Anna Rudin Grant/research support from: I have received research grants fran AstraZeneca (2017-2018), Consultant for: I was paid consultant for AstraZeneca (2015-2018), Cristina Maglio: None declared

Problem Circulating B-cell numbers are lower during pregnancy compared with non-pregnant women, but the underlying reasons for this are unknown. Pregnancy-related hormones could influence B-cell lymphopoiesis in the bone marrow, but B cells may also be recruited to the placenta. To investigate the latter, we examined whether the proportions of total B cells and B cells at different maturational stages in placental intervillous blood (IVB) differ compared with peripheral blood (PB). Method of study From 23 paired samples of PB and IVB following full-term healthy pregnancies, total B cells and immature/transitional, mature/naive, and memory B cells were identified by flow cytometry. Chemokine levels in blood were analyzed using a Luminex assay. Placental explant-derived supernatant was assayed for B-cell chemotactic activity. Results The proportions of total B cells and mature/naive B cells were significantly higher in IVB relative to PB, while the fractions of immature/transitional cells and memory B cells were higher in PB. Multivariate factor analysis demonstrated that a specific chemokine profile in IVB, including CCL20, positively associated with higher proportions of mature/naive B cells in the intervillous space. All B cells expressed CCR6, the corresponding receptor for CCL20, but the intensity of CCR6 expression was significantly higher in mature/naive B cells relative to immature/transitional B cells. Migration assays showed that placental explant-derived supernatants attract B cells. Conclusion These results indicate that B cells, and mature/naive B cells in particular, are retained in the intervillous blood in response to certain chemokines produced by the placenta during late healthy pregnancy.

Objective Women with SLE face an increased risk of adverse pregnancy outcomes compared with healthy women, but the underlying immunological mechanisms are unknown. Given the recognised association of neutrophil activation with SLE pathogenesis, we examined whether there is increased neutrophil activation and inflammation in blood and placenta in SLE relative to healthy pregnancy. Methods At delivery, peripheral blood, maternal-derived intervillous blood and placentas were collected from 12 SLE and 10 healthy control pregnancies. The proportion of low-density granulocytes (LDGs) and the activation status of LDG and normal-density granulocytes were examined with flow cytometry. The chemokines CXCL8 and CXCL1 were quantified with a cytometric bead-based assay and interferon alpha (IFN alpha) protein levels with a Simoa method. IFN alpha-stimulated maternal-derived decidual stromal cells were examined for CXCL8 gene expression with qPCR. A pathologist, blinded to the patient background, examined all placentas. Results Women with SLE had significantly higher proportions of LDG in peripheral blood compared with controls (p=0.02), and LDG in both peripheral and intervillous blood were more activated in SLE relative to healthy pregnancies (peripheral blood: p=0.002 and intervillous blood: p=0.05). There were higher levels of CXCL8 and CXCL1 in intervillous compared with peripheral blood in women with SLE (p=0.004 and p=Funding Agencies|Swedish government; county councils; ALF agreement; Swedish Rheumatism Association; Gothenburg Society of Medicine; Ingegerd Johanssons Donation; foundations of King Gustaf Vs 80th Anniversary; Queen Victorias Freemasons; Swedish Research CouncilSwedish Research CouncilEuropean Commission

Adiponectin, leptin, and resistin are adipocytokines whose levels are elevated in blood and synovial fluid from patients with rheumatoid arthritis (RA). However, their role in RA pathogenesis is unclear. Here, we examined whether adipocytokines are associated with circulating chemokines, markers of inflammation and RA disease activity in patients with untreated newly diagnosed RA. Plasma levels of 15 chemokines, adiponectin, leptin, and resistin were measured using flow cytometry bead-based immunoassay or enzyme-linked immunosorbent assay (ELISA) in a cohort of 70 patients with untreated newly diagnosed RA. Markers of inflammation and disease activity were also assessed in all patients. Positive association was found between total adiponectin and CXCL10 (β = 0.344, p = 0.021), CCL2 (β = 0.342, p = 0.012), and CXCL9 (β = 0.308, p = 0.044), whereas high-molecular weight (HMW) adiponectin associated only with CXCL9 (β = 0.308, p = 0.033). Furthermore, both total and HMW adiponectin were associated with C-reactive protein (β = 0.485, p = 0.001, β = 0.463, p = 0.001) and erythrocyte sedimentation rate (β = 0.442, p = 0.001, β = 0.507, p &lt, 0.001). Leptin and resistin were not associated with plasma chemokines, markers of inflammation, or disease activity scores. Our study shows an association between circulating adiponectin and pro-inflammatory chemokines involved in RA pathogenesis as well as markers of inflammation in a well-characterized cohort of patients with untreated newly diagnosed RA.

The pathogenic role and frequency of T cell subtypes in early rheumatoid arthritis are still unclear. We therefore performed a comprehensive analysis of the circulating T cell subtype pattern in patients with untreated early rheumatoid arthritis compared to healthy control subjects. Peripheral blood mononuclear cells were obtained from 26 patients with untreated early rheumatoid arthritis and from with 18 age- and sex-matched healthy control subjects. T helper cell types Th0, Th1, Th2, Th17, and Th1/17 and nonclassic T helper subsets were defined by flow cytometry based on the expression of chemokine receptors CCR4, CCR6, and CXCR3. Regulatory T cells were defined by expression of CD25+ CD127low and also FOXP3. CXCR5+ cells among regulatory and nonregulatory T cells were defined as T follicular regulatory and T follicular helper cells, respectively. The phenotype of T cell subsets was confirmed by transcription factor and cytokine secretion analyses. Multivariate discriminant analysis showed that patients with untreated early rheumatoid arthritis were segregated from healthy control subjects based on the circulating T cell subset profile. Among the discriminator subsets, CCR4+CXCR3− (Th2 and Th17), CTLA4+ and FOXP3+ subsets were present in significantly higher frequencies, whereas CCR4− (Th1/Th17, CCR6+CCR4−CXCR3−, and Th1) subsets were present in lower frequencies in patients with untreated early rheumatoid arthritis compared with healthy control subjects. The proportions of Th2 and Th17 subsets associated positively with each other and negatively with the CXCR3+/interferon γ-secreting subsets (Th1 and Th1/Th17) in patients with untreated rheumatoid arthritis. The proportions of Th2 cells increased with age in patients with untreated early rheumatoid arthritis and healthy control subjects. The dominance of circulating CCR4+CXCR3– T helper subsets (Th2 and Th17) in untreated early rheumatoid arthritis point toward a pathogenic role of these cells in early stages of the disease.

Figure S1. Differences in the proportions of T cell subsets between untreated early RA and healthy controls. OPLS-DA scatter and loading plots resulting from multivariate factor analysis of ueRA patients (male + female) vs HC (male + female). Table S1. The conjugate, clone, reference number and company of respective antibodies used for flow cytometry analysis. List of antibodies used in flow cytometry analysis and the respective conjugate, clone, reference number and company for each of these. (PPTX 99 kb)

Background/purpose Pathogenic mechanisms in early and preclinical stages of rheumatoid arthritis are unclear. We have recently analysed the profile of T cell subtypes based on chemokine receptor expression in blood from untreated early rheumatoid arthritis (ueRA) patients compared to healthy controls (HC). Here, we compared the levels of the respective chemokines in blood plasma of ueRA patients with those of HC. We also studied the association of chemokine levels with the proportions of circulating T cell subsets and the clinical disease activity. Methods Peripheral blood was obtained from 43 patients with ueRA satisfying the ACR 2010 criteria and who had not received any DMARD or prednisolone, and from 14 sex- and age-matched HC. Proportions of T helper cells in blood, including Th0, Th1, Th2, Th17, Th1Th17, TFh, and regulatory T cells were defined by flow cytometry. Fifteen chemokines, including several CXCL and CCL chemokines related to the T cell subtypes as well as to other major immune cells were measured in blood plasma using flow cytometry bead-based immunoassay or ELISA. Clinical disease activity in patients was evaluated by assessing the following parameters: DAS28, Clinical Disease Activity Index (CDAI), Swollen Joint Counts (SJC), Tender Joint Counts (TJC), CRP and ESR. The data was analysed using multivariate factor analyses followed by univariate analyses. Results Multivariate discriminant analysis showed that patients with ueRA separate from HC based on the blood plasma chemokine profile. The best discriminators were CXCL9, CXCL10, CXCL13, CCL4 and CCL22, which were significantly higher in ueRA compared to HC in univariate analyses. Among the chemokines analysed, only CXCL10 correlated with multiple disease activity measures, including DAS28-CRP, DAS28-ESR, CDAI, SJC in 66 joints, CRP and ESR. Conclusions High circulating levels of CXCL10 in plasma of ueRA and the association with the clinical disease activity suggests that CXCL10 may serve as a disease activity marker in early RA.

B cell activating factor (BAFF) is a critical cytokine for maturation of immature B cells. In murine lymph nodes, BAFF is mainly produced by podoplanin-expressing stromal cells. We have previously shown that circulating BAFF levels are maximal at birth, and that farmers’ children exhibit higher BAFF levels in cord blood than non-farmers’ children. Here, we sought to investigate whether maternal-derived decidual stromal cells from placenta secrete BAFF and examine what factors could stimulate this production. We found that podoplanin is expressed in decidua basalis and in the underlying villous tissue as well as on isolated maternal-derived decidual stromal cells. Decidual stromal cells produced BAFF when stimulated with IFN-γ and IFN-α, and NK cells and NK-T-like cells competent of IFN-γ production were isolated from the decidua. Finally, B cells at different maturational stages are present in decidua and all expressed BAFF-R, while stromal cells did not. These findings suggest that decidual stromal cells are a cellular source of BAFF for B cells present in decidua during pregnancy.

Figure S1. Gating strategy describing T-cell subsets. Figure S2. OPLS-DA score scatter plot showing differences in the profile of T-cell subset proportions between untreated early RA and healthy controls. Figure S3. OPLS-DA column loading plots and univariate analyses showing sex-based differences in the levels of blood chemokines in healthy controls and untreated early RA patients. Figure S4. OPLS plots showing association of eotaxin with clinical disease activity in untreated early RA and sex-based differences. Figure S5. OPLS plots showing association of CCL20 with clinical disease activity in untreated early RA and sex-based differences. Figure S6. OPLS-DA column loading plots showing comparison in the levels of blood chemokines between untreated early RA subgroups based on ACPA or RF positivity. (PPTX 655 kb)

SummaryBackground We previously reported that exposure to a farming environment is allergy-protective, while high proportions of neonatal immature/naive CD5+ B cells and putative regulatory T cells (Tregs) are risk factors for development of allergic disease and sensitization up to 3 years of age. Objective To examine if B and T cell maturation are associated with allergic disease and farming environment over the first 8 years in life. Methods In the prospective FARMFLORA study, including both farming and non-farming families, 48 of 65 children took part in the 8-year follow-up study. Various B and T cell maturation variables were examined in blood samples obtained at several occasions from birth to 8 years of age and related to doctors' diagnosed allergic disease and sensitization, and to farming environment. Results We found that the incidence of allergic disease was lower among farmers' compared to non-farmers' children during the 8-year follow-up period, and that farmers' children had higher proportions of memory B cells at 8 years of age. Moreover, a high proportion of neonatal CD5+ B cells was a risk factor for and may predict development of allergic disease at 8 years of age. A high proportion of Tregs was not protective against development of these conditions. Conclusion and Clinical Relevance High proportions of neonatal naive B cells remained as a risk factor for allergic disease in school-aged children. Thus, the accelerated B cell maturation observed among farmers' children may be crucial for the allergy-protective effect of a farming environment.

Germ-free animal models have demonstrated that commensal bacterial colonization of the intestine induces B cell differentiation and activation. Whether colonization with particular bacterial species or groups is associated with B cell development during early childhood is not known. In a prospective newborn/infant cohort including 65 Swedish children, we examined the numbers and proportions of CD20+, CD5+, and CD27+ B cells in blood samples obtained at several time points during the first 3 y of life using flow cytometry. Fecal samples were collected and cultured quantitatively for major facultative and anaerobic bacteria at 1, 2, 4, and 8 wk of life. We found that the numbers of CD20+ B cells and CD5+CD20+ B cells reached their highest levels at 4 mo, whereas CD20+ B cells expressing the memory marker CD27 were most numerous at 18 and 36 mo of age. Using multivariate analysis, we show that early colonization with Escherichia coli and bifidobacteria were associated with higher numbers of CD20+ B cells that expressed the memory marker CD27 at 4 and 18 mo of age. In contrast, we were unable to demonstrate any relation between bacterial colonization pattern and numbers of CD20+ or CD5+CD20+ B cells. These results suggest that the intestinal bacterial colonization pattern may affect the B cell maturation also in humans, and that an early gut microbiota including E. coli and bifidobacteria might promote this maturation.

Vaccines have a critical role for the protection against infectious diseases by inducing specific neutralizing IgG antibodies and immunological memory. In Sweden, measles–mumps–rubella (MMR) vaccination is routinely initiated at 18 months with a booster dose at 6–8 years of age, and 95% of all Swedish 2-year-olds have received the vaccine. There is however a notable inter-individual variation in magnitudes of vaccine-specific antibody titers in vaccinated children. Whether these variations are related to the maturation of the adaptive immune system before and close to vaccination is unknown. The adaptive immunity of newborns is essentially naive, given limited exposure to exogenous antigens in utero. Indeed, the majority of CD4+ T cells and B cells in cord blood are naive, that is, express the surface markers CD45RA and CD5, respectively.1, 2, 3, 4, 5 Along with an age-dependent decrease in naive lymphocytes there is an increase in proportions of memory T and B cells that express CD45RO and CD27, respectively.1, 3, 4, 5 This immune maturation progress is paralleled by an enhanced capacity to produce cytokines and with increased total IgG levels in blood.4, 6, 7, 8, 9 Regulatory T cells (Tregs), which express FOXP3 and/or CTLA-4, impede proliferation and cytokine production of other T cells,10, 11, 12, 13 but also have suppressive effects on dendritic cells as well as on B cells and immunoglobulin responses,13, 14, 15 and reviewed in Shevach.16 Whether higher proportions of Tregs in blood hamper the subsequent MMR vaccine-induced antibody titers in children is not known. Early-life environmental factors have been shown to influence adaptive immune maturation. Growing up on a farm is associated with higher proportions of memory T cells and with an enhanced capacity to produce proinflammatory and T helper-associated cytokines.17, 18 Contrary, children born by cesarean section (CS) have lower proportions of memory T cells in childhood.19 Additionally, we have shown that boys have lower B-cell activating factor levels in cord blood than girls, as well as higher proportions of naive B cells at birth and later in childhood.20 Sex-related differences in vaccine-specific antibody levels have also been reported, demonstrating that girls present with higher mumps and rubella antibody titers.21, 22 Given that certain early-life environmental factors appear to have profound effects on immune maturation, they may also impact inter-individual variations in vaccine-induced antibody responses observed in children. By studying the FARMFLORA birth cohort, we sought to further explore how environmental factors and postnatal adaptive immune maturation relate to the MMR-specific humoral immune response.

Background: The upper airway mucosa of healthy humans contains a dense network of cells with dendritic morphology of which the majority express a macrophage-like phenotype (CD14+CD64+CD68+), whereas the smaller population are immature dendritic cells (DC; CD11c+CD14−). Our aim was to study the proinflammatory response of human monocytes and in vitro-generated macrophages and DC after contact with cat allergens. Methods: Monocyte-derived DC and monocyte-derived macrophages were exposed to cat allergen extract or Escherichia coli. Purified monocytes were stimulated with allergen extracts from cat or house dust mite (HDM) or the major allergenic protein Fel d 1 and induction of proinflammatory cytokines by monocytes was analyzed before and after blocking CD14. Results: We show that cat allergen extract induced tumor necrosis factor (TNF) and interleukin (IL)-6 production by CD14-positive macrophages but not by CD14-negative DC. Moreover, monocytes produced significantly higher levels of TNF in response to cat allergens than in response to HDM allergens. We observed no differences in levels of TNF and IL-6 from either macrophages or monocytes after exposure to cat allergen when comparing healthy and cat-allergic individuals. Finally, the proinflammatory cytokine production from monocytes in response to cat allergen extract but not to HDM allergen was significantly reduced by blocking CD14. Conclusion: These results indicate that closely related innate immune cells from the myeloid lineage respond differentially to cat allergen extract and that the pattern-recognition receptor CD14 might be one of the mediators involved in the inflammatory responses to inhalant allergens.

Delayed maturation of the immune system has been proposed to be a risk factor for development of allergy, but B cell maturation in relation to allergic disease has not been examined. B cells lose CD5 and acquire CD27 during maturation from immature via mature/naive to Ig-secreting cells and memory cells. We sought to investigate B cell maturation in relation to development of allergic disease and sensitization in the FARMFLORA birth cohort including 65 Swedish children. Total B cell numbers, proportions of CD5+ and CD27+ B cells, and levels of IgM, IgG, IgA, and IgE were measured in blood on repeated occasions from birth to 36 mo of age, and related to allergic disease and sensitization at 18 and 36 mo of age with multivariate discriminant analysis. We also compared the expression of CD24 and CD38 within CD5+ and CD5neg B cells in children and in adults. We found that infants with a high proportion of CD5+ B cells at birth and at 1 mo of age had an increased risk for having allergic disease at 18 and 36 mo of life. Further, the proportions of CD5+ B cells at 1 mo of age were inversely correlated with total IgG levels at 18 and 36 mo of age. The majority of the CD5+ B cells were of a CD24hi/+CD38hi/+ immature/naive phenotype at birth (97%), 7 y of age (95%), and in adults (86%). These results suggest that development of allergic disease is preceded by an immaturity in neonatal B cell phenotype.