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

TY - JOUR

T1 - Functional Relevance of CTLA4 Variants: an Upgraded Approach to Assess CTLA4-Dependent Transendocytosis by Flow Cytometry

AU - Rojas-Restrepo, Jessica

AU - Sindram, Elena

AU - Zenke, Simon

AU - Haberstroh, Hanna

AU - Mitsuiki, Noriko

AU - Gabrysch, Annemarie

AU - Huebscher, Katrin

AU - Posadas-Cantera, Sara

AU - Krausz, Máté

AU - Kobbe, Robin

AU - Rohr, Jan C

AU - Grimbacher, Bodo

AU - Gámez-Díaz, Laura

N1 - © 2023. The Author(s).

PY - 2023/11

Y1 - 2023/11

N2 - 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.

AB - 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.

KW - Humans

KW - CTLA-4 Antigen/genetics

KW - Flow Cytometry

KW - Reproducibility of Results

KW - Antigen-Presenting Cells

KW - Autoimmunity

U2 - 10.1007/s10875-023-01582-9

DO - 10.1007/s10875-023-01582-9

M3 - SCORING: Journal article

C2 - 37740092

VL - 43

SP - 2076

EP - 2089

JO - J CLIN IMMUNOL

JF - J CLIN IMMUNOL

SN - 0271-9142

IS - 8

ER -