Multi-parametric flow cytometric and genetic investigation of the peripheral B cell compartment in human type 1 diabetes.

The appearance of circulating islet-specific autoantibodies before disease diagnosis is a hallmark of human type 1 diabetes (T1D), and suggests a role for B cells in the pathogenesis of the disease. Alterations in the peripheral B cell compartment have been reported in T1D patients; however, to date, such studies have produced conflicting results and have been limited by sample size. In this study, we have performed a detailed characterization of the B cell compartment in T1D patients (n = 45) and healthy controls (n = 46), and assessed the secretion of the anti-inflammatory cytokine interleukin (IL)-10 in purified B cells from the same donors. Overall, we found no evidence for a profound alteration of the B cell compartment or in the production of IL-10 in peripheral blood of T1D patients. We also investigated age-related changes in peripheral B cell subsets and confirmed the sharp decrease with age of transitional CD19(+) CD27(-) CD24(hi) CD38(hi) B cells, a subset that has recently been ascribed a putative regulatory function. Genetic analysis of the B cell compartment revealed evidence for association of the IL2-IL21 T1D locus with IL-10 production by both memory B cells (P = 6·4 × 10(-4) ) and islet-specific CD4(+) T cells (P = 2·9 × 10(-3) ). In contrast to previous reports, we found no evidence for an alteration of the B cell compartment in healthy individuals homozygous for the non-synonymous PTPN22 Trp(620) T1D risk allele (rs2476601; Arg(620) Trp). The IL2-IL21 association we have identified, if confirmed, suggests a novel role for B cells in T1D pathogenesis through the production of IL-10, and reinforces the importance of IL-10 production by autoreactive CD4(+) T cells

Anti-SARS-CoV2 antibody responses in serum and cerebrospinal fluid of COVID-19 patients with neurological symptoms

Antibody responses to SARS-CoV-2 in serum and CSF from 16 COVID-19 patients with neurological symptoms were assessed using two independent methods. IgG specific for the virus spike protein was found in 81% of cases in serum and in 56% in CSF. SARS-CoV-2 IgG in CSF was observed in two cases with negative serology. Levels of IgG in both serum and CSF were associated with disease severity (p<0.05). All patients with elevated markers of CNS damage in CSF also had CSF antibodies (p=0.002), and CSF antibodies had the highest predictive value for neuronal damage markers of all tested clinical variables

Cells with Treg-specific FOXP3 demethylation but low CD25 are prevalent in autoimmunity

Identification of alterations in the cellular composition of the human immune system is key to understanding the autoimmune process. Recently, a subset of FOXP3+ cells with low CD25 expression was found to be increased in peripheral blood from systemic lupus erythematosus (SLE) patients, although its functional significance remains controversial. Here we find in comparisons with healthy donors that the frequency of FOXP3+ cells within CD127lowCD25low CD4+ T cells (here defined as CD25lowFOXP3+ T cells) is increased in patients affected by autoimmune disease of varying severity, from combined immunodeficiency with active autoimmunity, SLE to type 1 diabetes. We show that CD25lowFOXP3+ T cells share phenotypic features resembling conventional CD127lowCD25highFOXP3+ Tregs, including demethylation of the Treg-specific epigenetic control region in FOXP3, HELIOS expression, and lack of IL-2 production. As compared to conventional Tregs, more CD25lowFOXP3+HELIOS+ T cells are in cell cycle (33.0% vs 20.7% Ki-67+; P = 1.3 × 10−9) and express the late-stage inhibitory receptor PD-1 (67.2% vs 35.5%; P = 4.0 × 10−18), while having reduced expression of the early-stage inhibitory receptor CTLA-4, as well as other Treg markers, such as FOXP3 and CD15s. The number of CD25lowFOXP3+ T cells is correlated (P = 3.1 × 10−7) with the proportion of CD25highFOXP3+ T cells in cell cycle (Ki-67+). These findings suggest that CD25lowFOXP3+ T cells represent a subset of Tregs that are derived from CD25highFOXP3+ T cells, and are a peripheral marker of recent Treg expansion in response to an autoimmune reaction in tissues.This work was supported by the JDRF UK Centre for Diabetes - Genes, Autoimmunity and Prevention (D-GAP; 4-2007-1003) in collaboration with M. Peakman and T. Tree at Kings College London, a strategic award to the Diabetes and Inflammation Laboratory from the JDRF (9-2011-253) and the Wellcome Trust (WT; WT061858/091157), and the National Institute for Health Research Cambridge Biomedical Research Centre. RCF is funded by an advanced JDRF post-doctoral fellowship (2-APF-2017-420-A-N). CW is funded by the Wellcome Trust (088998)

Impaired plasma cell differentiation associates with increased oxidative metabolism in IkappaBNS-deficient B cells

Mutations causing loss of the NF-kappaB regulator IkappaBNS, result in impaired development of innate-like B cells and defective plasma cell (PC) differentiation. Since productive PC differentiation requires B cell metabolic reprogramming, we sought to investigate processes important for this transition using the bumble mouse strain, deficient for IkappaBNS. We report that LPS-activated bumble B cells exhibited elevated mTOR activation levels, mitochondrial accumulation, increased OXPHOS and mROS production, along with a reduced capacity for autophagy, compared to wildtype B cells. Overall, our results demonstrate that PC differentiation in the absence of IkappaBNS is characterized by excessive activation during early rounds of B cell division, increased mitochondrial metabolism and decreased autophagic capacity, thus improving our understanding of the role of IkappaBNS in PC differentiation

Seropositivity in blood donors and pregnant women during the first year of SARS-CoV-2 transmission in Stockholm, Sweden.

BACKGROUND: In Sweden, social restrictions to contain SARS-CoV-2 have primarily relied upon voluntary adherence to a set of recommendations. Strict lockdowns have not been enforced, potentially affecting viral dissemination. To understand the levels of past SARS-CoV-2 infection in the Stockholm population before the start of mass vaccinations, healthy blood donors and pregnant women (n = 5,100) were sampled at random between 14 March 2020 and 28 February 2021. METHODS: In this cross-sectional prospective study, otherwise-healthy blood donors (n = 2,600) and pregnant women (n = 2,500) were sampled for consecutive weeks (at four intervals) throughout the study period. Sera from all participants and a cohort of historical (negative) controls (n = 595) were screened for IgG responses against stabilized trimers of the SARS-CoV-2 spike (S) glycoprotein and the smaller receptor-binding domain (RBD). As a complement to standard analytical approaches, a probabilistic (cut-off independent) Bayesian framework that assigns likelihood of past infection was used to analyse data over time. SETTING: Healthy participant samples were randomly selected from their respective pools through Karolinska University Hospital. The study was carried out in accordance with Swedish Ethical Review Authority: registration number 2020-01807. PARTICIPANTS: No participants were symptomatic at sampling, and blood donors were all over the age of 18. No additional metadata were available from the participants. RESULTS: Blood donors and pregnant women showed a similar seroprevalence. After a steep rise at the start of the pandemic, the seroprevalence trajectory increased steadily in approach to the winter second wave of infections, approaching 15% of all individuals surveyed by 13 December 2020. By the end of February 2021, 19% of the population tested seropositive. Notably, 96% of seropositive healthy donors screened (n = 56) developed neutralizing antibody responses at titres comparable to or higher than those observed in clinical trials of SARS-CoV-2 spike mRNA vaccination, supporting that mild infection engenders a competent B-cell response. CONCLUSIONS: These data indicate that in the first year since the start of community transmission, seropositivity levels in metropolitan in Stockholm had reached approximately one in five persons, providing important baseline seroprevalence information prior to the start of vaccination.Swedish Research Council (agreement 2017-00968) National Institutes of Health (agreement 400 SUM1A44462-02) Wellcome Trust (WT107881) Medical Research Council (MC_UP_1302/5) European Union-funded CoroNAb project (coordination number 101003653

Cells with Treg-specific FOXP3 demethylation but low CD25 are prevalent in autoimmunity

Identification of alterations in the cellular composition of the human immune system is key to understanding the autoimmune process. Recently, a subset of FOXP3+ cells with low CD25 expression was found to be increased in peripheral blood from systemic lupus erythematosus (SLE) patients, although its functional significance remains controversial. Here we find in comparisons with healthy donors that the frequency of FOXP3+ cells within CD127lowCD25low CD4+ T cells (here defined as CD25lowFOXP3+ T cells) is increased in patients affected by autoimmune disease of varying severity, from combined immunodeficiency with active autoimmunity, SLE to type 1 diabetes. We show that CD25lowFOXP3+ T cells share phenotypic features resembling conventional CD127lowCD25highFOXP3+ Tregs, including demethylation of the Treg-specific epigenetic control region in FOXP3, HELIOS expression, and lack of IL-2 production. As compared to conventional Tregs, more CD25lowFOXP3+ HELIOS+ T cells are in cell cycle (33.0% vs 20.7% Ki-67+ ; P = 1.3 x 10-9 ) and express the late-stage inhibitory receptor PD-1 (67.2% vs 35.5%; P = 4.0 x 10-18 ), while having reduced expression of the early-stage inhibitory receptor CTLA-4, as well as other Treg markers, such as FOXP3 and CD15s. The number of CD25lowFOXP3+ T cells is highly correlated (P = 3.1 x 10−7 ) with the proportion of CD25highFOXP3+ T cells in cell cycle (Ki-67+ ). These findings suggest that CD25lowFOXP3+ T cells represent a subset of Tregs that are derived from CD25highFOXP3+ T cells, and are a peripheral marker of recent Treg expansion in response to an autoimmune reaction in tissues

Chromosome contacts in activated T cells identify autoimmune disease candidate genes

Autoimmune disease-associated variants are preferentially found in regulatory regions in immune cells, particularly CD4+ T cells. Linking such regulatory regions to gene promoters in disease-relevant cell contexts facilitates identification of candidate disease genes.Within 4 h, activation of CD4+ T cells invokes changes in histone modifications and enhancer RNA transcription that correspond to altered expression of the interacting genes identified by promoter capture Hi-C. By integrating promoter capture Hi-C data with genetic associations for five autoimmune diseases, we prioritised 245 candidate genes with a median distance from peak signal to prioritised gene of 153 kb. Just under half (108/245) prioritised genes related to activation-sensitive interactions. This included IL2RA, where allele-specific expression analyses were consistent with its interaction-mediated regulation, illustrating the utility of the approach.Our systematic experimental framework offers an alternative approach to candidate causal gene identification for variants with cell state-specific functional effects, with achievable sample sizes

IL-21 production by CD4+ effector T cells and frequency of circulating follicular helper T cells are increased in type 1 diabetes patients

This is the final published version. It first appeared at http://link.springer.com/article/10.1007%2Fs00125-015-3509-8.Aims/hypothesis\ud \ud Type 1 diabetes results from the autoimmune destruction of insulin-secreting pancreatic beta cells by T cells. Despite the established role of T cells in the pathogenesis of the disease, to date, with the exception of the identification of islet-specific T effector (Teff) cells, studies have mostly failed to identify reproducible alterations in the frequency or function of T cell subsets in peripheral blood from patients with type 1 diabetes.\ud Methods\ud \ud We assessed the production of the proinflammatory cytokines IL-21, IFN-? and IL-17 in peripheral blood mononuclear cells from 69 patients with type 1 diabetes and 61 healthy donors. In an additional cohort of 30 patients with type 1 diabetes and 32 healthy donors, we assessed the frequency of circulating T follicular helper (Tfh) cells in whole blood. IL-21 and IL-17 production was also measured in peripheral blood mononuclear cells (PBMCs) from a subset of 46 of the 62 donors immunophenotyped for Tfh.\ud Results\ud \ud We found a 21.9% (95% CI 5.8, 40.2; p?=?3.9???10?3) higher frequency of IL-21+ CD45RA? memory CD4+ Teffs in patients with type 1 diabetes (geometric mean 5.92% [95% CI 5.44, 6.44]) compared with healthy donors (geometric mean 4.88% [95% CI 4.33, 5.50]). Consistent with this finding, we found a 14.9% increase in circulating Tfh cells in the patients (95% CI 2.9, 26.9; p?=?0.016).\ud Conclusions/interpretation\ud \ud These results indicate that increased IL-21 production is likely to be an aetiological factor in the pathogenesis of type 1 diabetes that could be considered as a potential therapeutic target.This work was supported by the JDRF UK Centre for\ud Diabetes - Genes, Autoimmunity and Prevention (D-GAP; 4-2007-1003) in collaboration with M. Peakman and T. Tree at King?s College\ud London, the JDRF, the Wellcome Trust (WT; WT061858/091157 and\ud 083650/Z/07/Z) and the National Institute for Health Research\ud Cambridge Biomedical Research Centre (CBRC). The Cambridge\ud Institute for Medical Research (CIMR) is in receipt of a Wellcome Trust\ud Strategic Award (100140). RCF is funded by a JDRF post-doctoral fellowship\ud (3-2011-374). CW is funded by the Wellcome Trust (088998).\ud The funding organisations had no involvement with the design and\ud conduct of the study; collection,management, analysis, and interpretation\ud of the data; and preparation, review, or approval of the manuscript