Concentrations of S100B and neurofilament light chain in blood as biomarkers for checkpoint inhibitor-induced CNS inflammation.

BACKGROUND: Cancer treatment with immune checkpoint inhibition (ICI) can cause immune-related adverse events in the central nervous system (CNS irAE). There are no blood biomarkers to detect CNS irAE. We investigated if concentrations of S100-calcium-binding protein B (S100B) and neurofilament light chain (NfL) in blood can be used as biomarkers for CNS irAE and assessed the incidence of CNS irAE in a cohort of ICI-treated patients. METHODS: In this single-centre, retrospective cohort study, we examined medical records and laboratory data of 197 consecutive patients treated with combined CTLA-4 and PD-1 inhibition (ipilimumab; ipi + nivolumab; nivo) for metastatic melanoma or renal cell carcinoma. CNS irAE was diagnosed using established criteria. Concentrations of S100B and NfL in blood were measured in patients with CNS irAE and in 84 patients without CNS irAE. FINDINGS: Nine of 197 patients (4.6%) fulfilled criteria for CNS irAE. S100B and NfL in blood increased during CNS inflammation and normalized during immunosuppression. CNS irAE was detected with a sensitivity of 100% (S100B) and 79% (NfL) and a specificity of 89% (S100B) and 74% (NfL). Patients with CNS irAE had simultaneous increased concentration of C-reactive protein (CRP) (9/9) and alanine aminotransferase (ALT) and/or aspartate aminotransferase (AST) in blood (8/9). INTERPRETATION: Analysis of S100B, NfL and CRP in blood facilitates the diagnosis of CNS irAE. CNS irAE may be more common than previously reported. There may be shared immune mechanisms between CNS and hepatitis irAE. FUNDING: Supported by funding from the Swedish Cancer Foundation, the ALF-agreement, and Jubileumsklinikens Cancerfond.

Blood chemokine levels are markers of disease activity but not predictors of remission in early rheumatoid arthritis.

OBJECTIVES: In early rheumatoid arthritis (eRA) plasma levels of specific chemokines have been shown to correlate with disease activity. However, it is unclear whether pre-treatment chemokine levels can predict disease remission at week 24, and it is not known how biological treatments with different modes of action affect plasma chemokine levels in patients with untreated eRA. METHODS: This study included 347 Swedish patients with untreated eRA from the larger NORD-STAR randomised treatment trial. Here, eRA patients were treated with methotrexate combined with either prednisolone, anti-TNF (certolizumab-pegol), CTLA-4Ig (abatacept) or anti-IL6 receptor (tocilizumab). The primary clinical outcome was remission by clinical disease activity index (CDAI) defined as CDAI ≤ 2.8. Disease activity was assessed by CDAI, DAS28-ESR, DAS28-CRP, swollen joint counts, tender joint counts, ESR and CRP. The plasma concentrations of 14 chemokines were measured at baseline and after 24 weeks of treatment by bead-based immunoassay or ELISA. RESULTS: Baseline plasma concentrations of CXCL10, CXCL8, CXCL9, CXCL11, CXCL5 and CCL2 correlated with baseline disease activity measures. After 24 weeks of treatment, plasma levels of CXCL10, CXCL8, CXCL9, CXCL11 and CXCL13 decreased in all treatment groups except in patients treated with anti-IL6 receptor. In multivariate factor analysis, plasma chemokine levels at baseline could not differentiate patients who attained remission by week 24 from those who did not in any of the treatment groups. CONCLUSIONS: In patients with untreated eRA, plasma levels of several chemokines correlate with disease activity at baseline but cannot predict remission after 24 weeks of treatment with methotrexate combined with prednisolone, anti­TNF, CTLA­4Ig or anti­IL6R.

Human Cord Blood B Cells Differ from the Adult Counterpart by Conserved Ig Repertoires and Accelerated Response Dynamics.

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.

Adipocytokines in Untreated Newly Diagnosed Rheumatoid Arthritis: Association with Circulating Chemokines and Markers of Inflammation.

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 (ß = 0.344, p = 0.021), CCL2 (ß = 0.342, p = 0.012), and CXCL9 (ß = 0.308, p = 0.044), whereas high-molecular weight (HMW) adiponectin associated only with CXCL9 (ß = 0.308, p = 0.033). Furthermore, both total and HMW adiponectin were associated with C-reactive protein (ß = 0.485, p = 0.001; ß = 0.463, p = 0.001) and erythrocyte sedimentation rate (ß = 0.442, p = 0.001; ß = 0.507, p < 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.

Recombinant Adiponectin Induces the Production of Pro-Inflammatory Chemokines and Cytokines in Circulating Mononuclear Cells and Fibroblast-Like Synoviocytes From Non-Inflamed Subjects.

Adiponectin is an adipokine with a modulatory role in metabolism and exerting both anti- and pro-inflammatory effects. Levels of adiponectin are increased in serum and synovial fluid from patients with rheumatoid arthritis (RA). Adiponectin is able to stimulate the production of different pro-inflammatory factors from peripheral blood mononuclear cells (PBMCs) and fibroblast-like synoviocytes (FLS) from subjects with established RA. As increased circulating adiponectin levels are a risk factor for future development of RA in subjects with obesity, we hypothesize that adiponectin is implicated in the development of RA at an early stage by initiating the pro-inflammatory processes associated with the disease pathogenesis. Therefore, we aimed to determine if adiponectin is able to induce pro-inflammatory responses in cells involved in the pathogenesis of RA, but collected from subjects without any known inflammatory disease. PBMCs and FLS were obtained from non-inflamed subjects and stimulated with 5 µg/ml human recombinant adiponectin. Supernatants collected after 48 h were analyzed for the production of 13 chemokines and 12 cytokines using multiplex assay and ELISA. Adiponectin significantly stimulated the production of CXCL1, CXCL5, and interleukin (IL)-6 in both PBMCs and FLS, whereas it induced CCL20, CCL4, CCL3, CCL17, tumor necrosis factor (TNF), granulocyte-macrophage colony-stimulating factor and IL-10 only in PBMCs, and CXCL8, CXCL10, CCL5, CCL11, and CCL2 only in FLS. Pre-stimulation with TNF of FLS from non-inflamed subjects did not significantly enhance the release of most pro-inflammatory factors compared to adiponectin alone. Our findings indicate that PBMCs and FLS from non-inflamed subjects react to adiponectin stimulation with the secretion of several pro-inflammatory chemokines and cytokines. These results suggest that adiponectin is able to initiate pro-inflammatory responses in cells from non-inflamed subjects and support the hypothesis that adiponectin is implicated in the early phases of RA pathogenesis.

Mature naïve B cells are retained in the placental intervillous blood and positively associate with specific chemokines in full-term healthy pregnancy.

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/naïve, 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/naïve 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/naïve 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/naïve 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/naïve B cells in particular, are retained in the intervillous blood in response to certain chemokines produced by the placenta during late healthy pregnancy.

Dihydrotestosterone levels at birth associate positively with higher proportions of circulating immature/naïve CD5+ B cells in boys.

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.

MAIT cells accumulate in placental intervillous space and display a highly cytotoxic phenotype upon bacterial stimulation.

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.

Umbilical cord blood androgen levels in girls and boys assessed by gas chromatography-tandem mass spectrometry.

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 27.8pg/mL) than in girls (11.5pg/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.

IFN type I and II induce BAFF secretion from human decidual stromal cells.

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.

Higher B-cell activating factor levels at birth are positively associated with maternal dairy farm exposure and negatively related to allergy development.

BACKGROUND: A high proportion of circulating immature/naive CD5(+) B cells during early infancy is a risk factor for allergy development. B-cell activating factor (BAFF) is an important cytokine for B-cell maturation. OBJECTIVE: We sought to investigate whether BAFF levels are related to environmental exposures during pregnancy and early childhood and whether BAFF levels are associated with postnatal B-cell maturation and allergic disease. METHODS: In the FARMFLORA study, including both farming and nonfarming families, we measured BAFF levels in plasma from mothers and their children at birth and at 1, 4, 18, and 36 months of age. Infants' blood samples were also analyzed for B-cell numbers and proportions of CD5(+) and CD27(+) B cells. Allergic disease was clinically evaluated at 18 and 36 months of age. RESULTS: Circulating BAFF levels were maximal at birth, and farmers' children had higher BAFF levels than nonfarmers' children. Higher BAFF levels at birth were positively associated with proportions of CD27(+) memory B cells among farmers' children and inversely related to proportions of CD5(+) immature/naive B cells among nonfarmers' children. Children with allergic disease at 18 months of age had lower cord blood BAFF levels than nonallergic children. At birth, girls had higher BAFF levels and lower proportions of CD5(+) B cells than boys. CONCLUSIONS: Farm exposure during pregnancy appears to induce BAFF production in the newborn child, and high neonatal BAFF levels were associated with more accelerated postnatal B-cell maturation, which lend further strength to the role of B cells in the hygiene hypothesis.

High proportion of CD5+ B cells in infants predicts development of allergic disease.

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 CD5(neg) 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 CD24(hi/+)CD38(hi/+) 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.

Infant B cell memory and gut bacterial colonization.

Under normal conditions, the gut microbiota confers health benefits for the host. The microbiota aids in the nutrient processing and contributes to the construction of the intestinal epithelial barrier. Furthermore, animal models demonstrate the importance of stimulation from gut bacteria for a proper maturation of the immune system. In this addendum, we summarize our recent study in which we demonstrate that colonization with Escherichia coli and bifidobacteria in the first 2 months of life was related to higher numbers of CD27-positive memory B cells later in infancy. The numbers of total B cells or CD5(+) CD20(+) B cells, on the other hand, were not related to the bacterial colonization pattern. Thus, the gut microbiota might affect the B cell maturation also in humans, and our study indicates that an early colonization pattern that includes E. coli and bifidobacteria might promote this maturation early in life.

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.