Functional Relevance of CTLA4 Variants: an Upgraded Approach to Assess CTLA4-Dependent Transendocytosis by Flow Cytometry.

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.

A Toolkit for Monitoring Immunoglobulin G Levels from Dried Blood Spots of Patients with Primary Immunodeficiencies.

PURPOSE: This study assessed whether measuring immunoglobulin G (IgG) from dried blood spots (DBSs) using nephelometry is a suitable remote monitoring method for patients with primary immunodeficiencies (PID). METHODS: Patients receiving immunoglobulin replacement therapy for PID were included in this non-interventional single-arm study (DRKS-ID: DRKS00020522) conducted in Germany from December 4, 2019, to December 22, 2020. Three blood samples, two capillary DBSs (one mail-transferred and the other direct-transferred to the laboratory), and one intravenous were collected from each patient. IgG levels were determined using nephelometry. IgG levels were summarized descriptively, and significant differences were assessed using Wilcoxon matched-pairs signed-rank tests. Correlation and agreement between IgG levels were assessed using Spearman correlation and Bland-Altman analyses, respectively. RESULTS: Among 135 included patients, IgG levels measured from DBS samples were lower than those measured in serum (p < 0.0001). There was no significant difference between IgG levels in direct- and mail-transferred DBS samples. There was a high degree of correlation between IgG levels in serum samples and DBS samples (r = 0.94-0.95). Although there was a bias for higher levels of IgG in serum than in DBS samples, most samples were within the 95% interval of agreement. There was a high degree of correlation between IgG levels measured in direct- and mail-transferred DBS samples (r = 0.96) with no bias based on the shipment process and most samples within the 95% interval of agreement. CONCLUSION: Monitoring IgG levels from DBS samples is a suitable alternative to the standard method, and results are not substantially affected by mailing DBS cards.

Fecal Immunoglobulin Levels as a Modifier of the Gut Microbiome in Patients with Common Variable Immunodeficiency.

OBJECTIVE: Common variable immunodeficiency (CVID) is the most common clinically relevant entity of inborn errors of immunity. In these patients, an altered gut microbiome composition with reduced diversity has been described. We sought to investigate the fecal immunoglobulin levels and their impact on the gut microflora in patients with CVID. METHODS: We analyzed the gut microbiome of 28 CVID patients and 42 healthy donors (HDs), including 21 healthy household controls, by sequencing the V3 and V4 regions of the bacterial 16S rRNA gene extracted from stool samples. The fecal levels of immunoglobulin A, M, and G of 27 CVID patients and 41 HDs were measured in the supernatant by ELISA and normalized for protein concentration. RESULTS: We measured decreased IgA and increased IgG in stool samples from CVID patients compared to HDs. Decreased levels of fecal IgA and IgM were associated with reduced microbial diversity and increased dysbiosis. We identified a large number of significantly differentially abundant taxa, especially in patients with decreased IgA levels, but also in patients with decreased IgM levels compared to their counterparts. CONCLUSIONS: CVID patients have an altered gut microbiota composition, which is most prevalent in patients with decreased fecal IgA and IgM levels. In this study, we identify fecal immunoglobulins as a potential modifier of the gut microbiome in CVID patients.

Establishing the Molecular Diagnoses in a Cohort of 291 Patients With Predominantly Antibody Deficiency by Targeted Next-Generation Sequencing: Experience From a Monocentric Study.

Predominantly antibody deficiencies (PAD) are a heterogeneous group of disorders characterized by dysfunctional antibody production, low immunoglobulin levels in serum and impaired vaccine responses. The clinical picture is variable, ranging from mild symptoms to severe complications, which may include autoimmunity, gastrointestinal disease, allergy, and malignancies. If left untreated, PAD patients are at risk of enduring disease progression, irreversible organ damage, and reduced life expectancy. A timely diagnosis has been shown to significantly improve disease prognosis. Here, we report on our experience using targeted gene panel sequencing by employing Agilent's HaloPlex or SureSelect and Illumina's MiSeq technologies in a cohort of 291 individuals who presented with low or absent immunoglobulin levels in combination with or without other clinical features. In total, we have detected over 57 novel or previously reported relevant mutations in ADA, ADA2, BTK, CTLA4, LRBA, NFKB1, NFKB2, PIK3CD, STAT3, and TNFRSF13B. Overall, a genetic diagnosis could be made in 24.7% of the investigated patients. The percentage of coverage for the targeted regions ranged from 90% to 98% in this study. Moreover, functional assays were performed on a defined group of the patients carrying candidate variants in CTLA4, LRBA, NFKB1 and BTK, which confirmed their deleterious effect on protein expression and/or function. This study reiterates that the immunological heterogeneity of predominantly antibody deficiencies may have a diverse genetic origin, although certain clinical features may hint towards a specific group of defects. Employing targeted sequencing panels proves to be a very time- and cost-efficient, yet reliable, method for the establishment of a genetic diagnosis in individuals with PAD. However, in case of negative panel results, or if functional testing reveals inconspicuous observations in patients with a clear indication for genetic testing, further work-up including whole exome or whole genome sequencing should be considered.