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Variants of uncertain significance (VUS) in CTLA4 are frequently identified in patients with antibody deficiency or immune dysregulation syndromes including, but not limited to, patients with multi-organ autoimmunity and autoinflammation. However, to ascertain the diagnosis of CTLA4 insufficiency, the functional relevance of each variant needs to be determined. Currently, various assays have been proposed to assess the functionality of CTLA4 VUS, including the analysis of transendocytosis, the biological function of CTLA4 to capture CD80 molecules from antigen presenting cells. Challenges of this assay include weak fluorescence intensity of the internalized ligand, poor reproducibility, and poor performance upon analyzing thawed cells. In addition, the distinction of pathogenic from non-pathogenic variants and from wild-type CTLA4, and the classification of the different VUS according to its level of CTLA4 dysfunction, would be desirable. We developed a novel CD80-expressing cell line for the evaluation of CD80-transendocytosis and compared it to the published transendocytosis assay. Our approach showed lower inter-assay variability and better robustness regardless the type of starting material (fresh or thawed peripheral mononuclear cells). In addition, receiver operating characteristic analysis showed 100% specificity, avoiding false positive results and allowing for a clear distinction between pathogenic and non-pathogenic variants in CTLA4-variant carriers. With our transendocytosis assay, we assessed the pathogenicity of 24 distinct CTLA4 variants from patients submitted to our diagnostic unit. Significantly impaired transendocytosis was demonstrated for 17 CTLA4 variants, whereas seven variants tested normal. In conclusion, our upgraded transendocytosis assay allows a reliable assessment of newly identified variants in CTLA4.
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Células Apresentadoras de Antígenos , Autoimunidade , Humanos , Antígeno CTLA-4/genética , Citometria de Fluxo , Reprodutibilidade dos TestesRESUMO
The gut microbiome is a dense and diverse community of different microorganisms that deeply influence human physiology and that have important interactions with pathogens. For the correct antibiotic treatment of infections, with its twin goals of effective inhibition of the pathogen and limitation of collateral damage to the microbiome, the identification of infectious organisms is key. Microbiological culturing is still the mainstay of pathogen identification, and anaerobic species are among the most demanding bacterial communities to culture. This study aimed to evaluate the impact of growth media on the culture of an-aerobic bacteria from human stool samples. Stool samples from eight human subjects were cultured each on a yeast extract cysteine blood agar (HCB) and a modified peptone-yeast extract-glucose (MPYG) plate and subjected to Illumina NGS analysis after DNA extraction and amplification. The results showed tight clustering of sequencing samples belonging to the same human subject. Various differences in bacterial richness and evenness could be observed between the two media, with HCB plates supporting the growth of a more diverse microbial community, and MPYG plates improving the growth rates of certain taxa. No statistical significance was observed between the groups. This study highlights the importance of choosing the appropriate growth media for anaerobic bacterial culture and adjusting culture conditions to target specific pathological conditions. HCB plates are suitable for standard microbiological diagnostics, while MPYG plates may be more appropriate for targeting specific conditions. This work emphasizes the role of next-generation sequencing in supporting future research in clinical microbiology.
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[This corrects the article DOI: 10.3389/fimmu.2022.1011646.].
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Background: Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) insufficiency and lipopolysaccharide-responsive and beige-like anchor protein (LRBA) deficiency are both complex immune dysregulation syndromes with an underlying regulatory T cell dysfunction due to the lack of CTLA-4 protein. As anticipated, the clinical phenotypes of CTLA-4 insufficiency and LRBA deficiency are similar. Main manifestations include hypogammaglobulinemia, lymphoproliferation, autoimmune cytopenia, immune-mediated respiratory, gastrointestinal, neurological, and skin involvement, which can be severe and disabling. The rationale of this clinical trial is to improve clinical outcomes of affected patients by substituting the deficient CTLA-4 by administration of CTLA4-Ig (abatacept) as a causative personalized treatment. Objectives: Our objective is to assess the safety and efficacy of abatacept for patients with CTLA-4 insufficiency or LRBA deficiency. The study will also investigate how treatment with abatacept affects the patients' quality of life. Methods: /Design: ABACHAI is a phase IIa prospective, non-randomized, open-label, single arm multi-center trial. Altogether 20 adult patients will be treated with abatacept 125 mg s.c. on a weekly basis for 12 months, including (1) patients already pretreated with abatacept, and (2) patients not pretreated, starting with abatacept therapy at the baseline study visit. For the evaluation of drug safety infection control during the trial, for efficacy, the CHAI-Morbidity Score will be used. Trial registration: The trial is registered in the German Clinical Trials Register (Deutsches Register Klinischer Studien, DRKS) with the identity number DRKS00017736, registered: 6 July 2020, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00017736.
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Common variable immunodeficiency (CVID) is the most prevalent form of symptomatic primary immunodeficiency in humans. The genetic cause of CVID is still unknown in about 70% of cases. Ten percent of CVID patients carry heterozygous mutations in the tumor necrosis factor receptor superfamily member 13B gene (TNFRSF13B), encoding TACI. Mutations in TNFRSF13B alone may not be sufficient for the development of CVID, as 1% of the healthy population carry these mutations. The common hypothesis is that TACI mutations are not fully penetrant and additional factors contribute to the development of CVID. To determine these additional factors, we investigated the perturbations of transcription factor (TF) binding and the transcriptome profiles in unstimulated and CD40L/IL21-stimulated naïve B cells from CVID patients harboring the C104R mutation in TNFRSF13B and compared them to their healthy relatives with the same mutation. In addition, the proteome of stimulated naïve B cells was investigated. For functional validation, intracellular protein concentrations were measured by flow cytometry. Our analysis revealed 8% less accessible chromatin in unstimulated naïve B cells and 25% less accessible chromatin in class-switched memory B cells from affected and unaffected TACI mutation carriers compared to healthy donors. The most enriched TF binding motifs in TACI mutation carriers involved members from the ETS, IRF, and NF-κB TF families. Validation experiments supported dysregulation of the NF-κB and MAPK pathways. In steady state, naïve B cells had increased cell death pathways and reduced cell metabolism pathways, while after stimulation, enhanced immune responses and decreased cell survival were detected. Using a multi-omics approach, our findings provide valuable insights into the impaired biology of naïve B cells from TACI mutation carriers.
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Imunodeficiência de Variável Comum , NF-kappa B , Linfócitos B , Cromatina/metabolismo , Humanos , Mutação , NF-kappa B/metabolismoRESUMO
Purpose: Heterozygous mutations in CTLA4 lead to an inborn error of immunity characterized by immune dysregulation and immunodeficiency, known as CTLA-4 insufficiency. Cohort studies on CTLA4 mutation carriers showed a reduced penetrance (around 70%) and variable disease expressivity, suggesting the presence of modifying factors. It is well studied that infections can trigger autoimmunity in humans, especially in combination with a genetic predisposition. Methods: To investigate whether specific infections or the presence of specific persisting pathogens are associated with disease onset or severity in CTLA-4 insufficiency, we have examined the humoral immune response in 13 CTLA4 mutation carriers, seven without clinical manifestation and six with autoimmune manifestations, but without immunoglobulin replacement therapy against cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes simplex virus 1/2 (HSV 1/2), parvovirus B19 and Toxoplasma gondii. Additionally, we have measured FcγRIII/CD16A activation by EBV-specific IgG antibodies to examine the functional capabilities of immunoglobulins produced by CTLA4 mutation carriers. Results: The seroprevalence between affected and unaffected CTLA4 mutation carriers did not differ significantly for the examined pathogens. Additionally, we show here that CTLA4 mutation carriers produce EBV-specific IgG, which are unimpaired in activating FcγRIII/CD16A. Conclusions: Our results show that the investigated pathogens are very unlikely to trigger the disease onset in CTLA-4-insufficient individuals, and their prevalence is not correlated with disease severity or expressivity.
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Infecções por Vírus Epstein-Barr , Herpesvirus Humano 4 , Humanos , Antígeno CTLA-4/genética , Herpesvirus Humano 4/fisiologia , Infecções por Vírus Epstein-Barr/complicações , Infecções por Vírus Epstein-Barr/epidemiologia , Estudos Soroepidemiológicos , Anticorpos Antivirais , Imunoglobulina GRESUMO
Most of the currently known heterozygous pathogenic NFKB1 (Nuclear factor kappa B subunit 1) variants comprise deleterious defects such as severe truncations, internal deletions, and frameshift variants. Collectively, these represent the most frequent monogenic cause of common variable immunodeficiency (CVID) identified so far. NFKB1 encodes the transcription factor precursor p105 which undergoes limited proteasomal processing of its C-terminal half to generate the mature NF-κB subunit p50. Whereas p105/p50 haploinsufficiency due to devastating genetic damages and protein loss is a well-known disease mechanism, the pathogenic significance of numerous NFKB1 missense variants still remains uncertain and/or unexplored, due to the unavailability of accurate test procedures to confirm causality. In this study we functionally characterized 47 distinct missense variants residing within the N-terminal domains, thus affecting both proteins, the p105 precursor and the processed p50. Following transient overexpression of EGFP-fused mutant p105 and p50 in HEK293T cells, we used fluorescence microscopy, Western blotting, electrophoretic mobility shift assays (EMSA), and reporter assays to analyze their effects on subcellular localization, protein stability and precursor processing, DNA binding, and on the RelA-dependent target promoter activation, respectively. We found nine missense variants to cause harmful damage with intensified protein decay, while two variants left protein stability unaffected but caused a loss of the DNA-binding activity. Seven of the analyzed single amino acid changes caused ambiguous protein defects and four variants were associated with only minor adverse effects. For 25 variants, test results were indistinguishable from those of the wildtype controls, hence, their pathogenic impact remained elusive. In summary, we show that pathogenic missense variants affecting the Rel-homology domain may cause protein-decaying defects, thus resembling the disease-mechanisms of p105/p50 haploinsufficiency or may cause DNA-binding deficiency. However, rare variants (with a population frequency of less than 0.01%) with minor abnormalities or with neutral tests should still be considered as potentially pathogenic, until suitable tests have approved them being benign.
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Mutação de Sentido Incorreto , NF-kappa B , DNA , Células HEK293 , Humanos , NF-kappa B/metabolismo , Subunidade p50 de NF-kappa B/genética , Subunidade p50 de NF-kappa B/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-rel/metabolismoRESUMO
'There is no gene for fate' (citation from the movie 'GATTACA') - and there is no gene for CVID. Common Variable ImmunoDeficiency (CVID) is the most prevalent primary immunodeficiency in humans. CVID is characterized by an increased susceptibility to infections, hypogammaglobulinemia, reduced switched memory B cell numbers in peripheral blood and a defective response to vaccination, often complicated by autoimmune and autoinflammatory conditions. However, as soon as a genetic diagnosis has been made in a patient with CVID, the diagnosis must be changed to the respective genetic cause (www.esid.org). Therefore, there are genetic causes for primary antibody deficiencies, but not for CVID. Primary antibody deficiencies (PADs) are a heterogeneous group of disorders. Several attempts have been made to gain further insights into the pathogenesis of PAD, using unbiased approaches such as whole exome or genome sequencing. Today, in just about 35% of cases with PAD, monogenic mutations (including those in the gene TNFRSF13B) can be identified in a set of 68 genes [1â¢]. These mutations occur either sporadically or are inherited and do explain an often complex phenotype. In our review, we not only discuss gene defects identified in PAD patients previously diagnosed with CVID and/or CVID-like disorders such as IKZF1, CTNNBL1, TNFSF13 and BACH2, but also genetic defects which were initially described in non-CVID patients but have later also been observed in patients with PAD such as PLCG2, PIK3CG, PMS2, RNF31, KMT2D, STAT3. We also included interesting genetic defects in which the pathophysiology suggests a close relation to other known defects of the adaptive immune response, such as DEF6, SAMD9 and SAMD9L, and hence a CVID-like phenotype may be observed in the future. However, alternative mechanisms most likely add to the development of an antibody-deficient phenotype, such as polygenic origins, epigenetic changes, and/or environmental factors.