Abstract: 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

Abstract: 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