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Purpose Increased circulating concentrations of short-chain fatty acids (SCFA) achieved by ingestion of high-fibre diets is associated with anti-inflammatory effects through promotion of FoxP3+ regulatory T(reg) cells in mouse models. This study aimed to determine whether similar increments in blood SCFA levels can be achieved in humans and whether these are associated with similar immune modulatory effects. Methods In a pilot single-blinded, randomised, controlled cross-over study in ten healthy subjects, the effects were determined of high- (39 g/day) and low-fibre (18 g/day) intake (all food provided) on SCFA (gas chromatography), proportions of Treg cells (flow cytometry) and a panel of cytokines (multiplex methodology) measured in peripheral blood at day 5 of each diet. Results Actual fibre intake differed between the diets by 19 [16–21] g/day ( P < 0.001). Median [range] total plasma SCFA levels with high-fibre intake were 174.5 [104.8–249.5] µmol/L, which were greater than those associated with low-fibre intake at 59.0 [26.5–79.9] ( P  < 0.001). Differences were significantly different for both acetate and propionate. The frequencies of total CD4 T cells and T-regulatory cells, and concentrations of inflammatory and anti-inflammatory cytokines were not significantly different between the dietary interventions. Conclusions Plasma SCFA levels can be modulated by altering dietary fibre consumption in healthy individuals with increments similar to those achieved in murine studies. Five days of diet intervention did not result in changes in regulatory T-cell proportions and cytokine concentrations in peripheral blood, and may require longer duration of dietary change.

Predominantly antibody deficiency (PAD) is the most prevalent form of primary immunodeficiency, and is characterized by broad clinical, immunological and genetic heterogeneity. Utilizing the current gold standard of whole exome sequencing for diagnosis, pathogenic gene variants are only identified in less than 20% of patients. While elucidation of the causal genes underlying PAD has provided many insights into the cellular and molecular mechanisms underpinning disease pathogenesis, many other genes may remain as yet undefined to enable definitive diagnosis, prognostic monitoring and targeted therapy of patients. Considering that many patients display a relatively late onset of disease presentation in their 2nd or 3rd decade of life, it is questionable whether a single genetic lesion underlies disease in all patients. Potentially, combined effects of other gene variants and/or non-genetic factors, including specific infections can drive disease presentation. In this review, we define (1) the clinical and immunological variability of PAD, (2) consider how genetic defects identified in PAD have given insight into B-cell immunobiology, (3) address recent technological advances in genomics and the challenges associated with identifying causal variants, and (4) discuss how functional validation of variants of unknown significance could potentially be translated into increased diagnostic rates, improved prognostic monitoring and personalized medicine for PAD patients. A multidisciplinary approach will be the key to curtailing the early mortality and high morbidity rates in this immune disorder.

Measles causes a transient immune suppression, leading to increased susceptibility to opportunistic infections. In experimentally infected non-human primates (NHPs) measles virus (MV) infects and depletes pre-existing memory lymphocytes, causing immune amnesia. A measles outbreak in the Dutch Orthodox Protestant community provided a unique opportunity to study the pathogenesis of measles immune suppression in unvaccinated children. In peripheral blood mononuclear cells (PBMC) of prodromal measles patients, we detected MV-infected memory CD4 + and CD8 + T cells and naive and memory B cells at similar levels as those observed in NHPs. In paired PBMC collected before and after measles we found reduced frequencies of circulating memory B cells and increased frequencies of regulatory T cells and transitional B cells after measles. These data support our immune amnesia hypothesis and offer an explanation for the previously observed long-term effects of measles on host resistance. This study emphasises the importance of maintaining high measles vaccination coverage. The mechanisms by which measles virus infection induces transient immune suppression in humans are poorly understood. Here, Laksono and colleagues characterise the pathogenesis of measles-associated immune suppression in unvaccinated children, and shed new light on the long-term effects of measles on the host.

BackgroundDespite the success of combined cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and programmed cell death protein-1 (PD-1) immune checkpoint blockade (cICB), the majority of patients with melanoma fail to respond or experience severe treatment-related toxicity. Currently, there are no reliable biomarkers available to predict these events and guide treatment choices. We here evaluated the peripheral immune compartment to identify features associated with cICB outcome and toxicity.MethodsBlood samples were collected from 51 patients with advanced melanoma prior to commencing and after one cycle of cICB. Patients were classified as responders or non-responders based on radiographic best overall response to treatment, and grouped by the occurrence of severe toxicity. Absolute immune cell counts were obtained and peripheral blood mononuclear cells were cryopreserved prior to spectral flow-cytometric T-cell immunophenotyping.Results20 patients (39%) failed to respond to treatment, and 29 (57%) experienced severe toxicity. Pre-treatment, patients had fewer T cells than age-matched healthy controls (median 892 vs 1297 cells/µL, p=0.0004), mostly due to reduced naive CD4+ (p=0.0038) and CD8+ (p=0.0031) T cells. One cycle of cICB restored patient T cells to levels equivalent to healthy controls through expansion and activation of CD4+ and CD8+ memory and regulatory, but not naive subsets, and skewed the T-cell compartment towards an activated phenotype. This T-cell expansion correlated strongly with pre-treatment PD-1 (r=0.88, p=0.0003) but not CTLA-4 (r=0.32, p=0.34) expression levels, and was accompanied by upregulation of molecules including Ki67, inducible co-stimulator of T cells (ICOS), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3), and T cell immunoreceptor with Ig and ITIM domains (TIGIT) on effector CD4+ and CD8+ T cells. Greater upregulation of Ki67 in CD4+ central memory cells significantly differentiated responders and non-responders after one cycle of treatment (p=0.0086, area under the curve (AUC)=0.74, 95% CI 0.59 to 0.88), while higher on-treatment TIM-3 frequency within CD8+ T cells differentiated patients who experienced severe toxicity (p=0.0086, AUC=0.74, 95% CI 0.59 to 0.88).ConclusionsWe here show that response and toxicity to cICB in advanced melanoma are driven by distinct immune features evident after only one cycle of treatment. These could serve as prognostic biomarkers upon validation in larger cohorts.

The mechanisms involved in sequential immunoglobulin G (IgG) class switching are still largely unknown. Sequential IG class switching is linked to higher levels of somatic hypermutation (SHM) in vivo, but it remains unclear if these are generated temporally during an immune response or upon activation in a secondary response. We here aimed to uncouple these processes and to distinguish memory B cells from primary and secondary immune responses. SHM levels and IgG subclasses were studied with 454 pyrosequencing on blood mononuclear cells from young children and adults as models for primary and secondary immunological memory. Additional sequencing and detailed immunophenotyping with IgG subclass‐specific antibodies was performed on purified IgG+ memory B‐cell subsets. In both children and adults, SHM levels were higher in transcripts involving more downstream‐located IGHG genes (esp. IGHG2 and IGHG4). In adults, SHM levels were significantly higher than in children, and downstream IGHG genes were more frequently utilized. This was associated with increased frequencies of CD27+IgG+ memory B cells, which contained higher levels of SHM, more IGHG2 usage, and higher expression levels of activation markers than CD27−IgG+ memory B cells. We conclude that secondary immunological memory accumulates with age and these memory B cells express CD27, high levels of activation markers, and carry high SHM levels and frequent usage of IGHG2. These new insights contribute to our understanding of sequential IgG subclass switching and show a potential relevance of using serum IgG2 levels or numbers of IgG2‐expressing B cells as markers for efficient generation of memory responses.

CD molecules are surface molecules expressed on cells of the immune system that play key roles in immune cell-cell communication and sensing the microenvironment. These molecules are essential markers for the identification and isolation of leukocytes and lymphocyte subsets. Here, we present the results of the first phase of the CD Maps study, mapping the expression of CD1-CD100 ( = 110) on 47 immune cell subsets from blood, thymus, and tonsil using an eight-color standardized EuroFlow approach and quantification of expression. The resulting dataset included median antibody binding capacities (ABCs) and percentage of positivity for all markers on all subsets and was developed into an interactive CD Maps web resource. Using the resource, we examined differentially expressed proteins between granulocyte, monocyte, and dendritic cell subsets, and profiled dynamic expression of markers during thymocyte differentiation, T-cell maturation, and between functionally distinct B-cell subset clusters. The CD Maps resource will serve as a benchmark of antibody reactivities ensuring improved reproducibility of flow cytometry-based research. Moreover, it will provide a full picture of the surfaceome of human immune cells and serves as a useful platform to increase our understanding of leukocyte biology, as well as to facilitate the identification of new biomarkers and therapeutic targets of immunological and hematological diseases.

Antibody deficiencies constitute the largest group of symptomatic primary immunodeficiency diseases. In several patients, mutations in CD19 have been found to underlie disease, demonstrating the critical role for the protein encoded by this gene in antibody responses; CD19 functions in a complex with CD21, CD81, and CD225 to signal with the B cell receptor upon antigen recognition. We report here a patient with severe nephropathy and profound hypogammaglobulinemia. The immunodeficiency was characterized by decreased memory B cell numbers, impaired specific antibody responses, and an absence of CD19 expression on B cells. The patient had normal CD19 alleles but carried a homozygous CD81 mutation resulting in a complete lack of CD81 expression on blood leukocytes. Retroviral transduction and glycosylation experiments on EBV-transformed B cells from the patient revealed that CD19 membrane expression critically depended on CD81. Similar to CD19-deficient patients, CD81-deficient patients had B cells that showed impaired activation upon stimulation via the B cell antigen receptor but no overt T cell subset or function defects. In this study, we present what we believe to be the first antibody deficiency syndrome caused by a mutation in the CD81 gene and consequent disruption of the CD19 complex on B cells. These findings may contribute to unraveling the genetic basis of antibody deficiency syndromes and the nonredundant functions of CD81 in humans.

Background Umbilical cord blood (UCB) is commonly used in epigenome-wide association studies of prenatal exposures. Accounting for cell type composition is critical in such studies as it reduces confounding due to the cell specificity of DNA methylation (DNAm). In the absence of cell sorting information, statistical methods can be applied to deconvolve heterogeneous cell mixtures. Among these methods, reference-based approaches leverage age-appropriate cell-specific DNAm profiles to estimate cellular composition. In UCB, four reference datasets comprising DNAm signatures profiled in purified cell populations have been published using the Illumina 450 K and EPIC arrays. These datasets are biologically and technically different, and currently, there is no consensus on how to best apply them. Here, we systematically evaluate and compare these datasets and provide recommendations for reference-based UCB deconvolution. Results We first evaluated the four reference datasets to ascertain both the purity of the samples and the potential cell cross-contamination. We filtered samples and combined datasets to obtain a joint UCB reference. We selected deconvolution libraries using two different approaches: automatic selection using the top differentially methylated probes from the function pickCompProbes in minfi and a standardized library selected using the IDOL (Identifying Optimal Libraries) iterative algorithm. We compared the performance of each reference separately and in combination, using the two approaches for reference library selection, and validated the results in an independent cohort (Generation R Study, n = 191) with matched Fluorescence-Activated Cell Sorting measured cell counts. Strict filtering and combination of the references significantly improved the accuracy and efficiency of cell type estimates. Ultimately, the IDOL library outperformed the library from the automatic selection method implemented in pickCompProbes . Conclusion These results have important implications for epigenetic studies in UCB as implementing this method will optimally reduce confounding due to cellular heterogeneity. This work provides guidelines for future reference-based UCB deconvolution and establishes a framework for combining reference datasets in other tissues.

In the rapidly evolving field of primary immunodeficiencies (PID), the EuroFlow consortium decided to develop a PID orientation and screening tube that facilitates fast, standardized, and validated immunophenotypic diagnosis of lymphoid PID, and allows full exchange of data between centers. Our aim was to develop a tool that would be universal for all lymphoid PIDs and offer high sensitivity to identify a lymphoid PID (without a need for specificity to diagnose particular PID) and to guide and prioritize further diagnostic modalities and clinical management. The tube composition has been defined in a stepwise manner through several cycles of design-testing-evaluation-redesign in a multicenter setting. Equally important appeared to be the standardized pre-analytical procedures (sample preparation and instrument setup), analytical procedures (immunostaining and data acquisition), the software analysis (a multidimensional view based on a reference database in Infinicyt software), and data interpretation. This standardized EuroFlow concept has been tested on 250 healthy controls and 99 PID patients with defined genetic defects. In addition, an application of new EuroFlow software tools with multidimensional pattern recognition was designed with inclusion of maturation pathways in multidimensional patterns (APS plots). The major advantage of the EuroFlow approach is that data can be fully exchanged between different laboratories in any country of the world, which is especially of interest for the PID field, with generally low numbers of cases per center.