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Blood. 2017 Mar 16;129(11):1458-1468. doi: 10.1182/blood-2016-10-745174. Epub 2017 Feb 3.

Identifying functional defects in patients with immune dysregulation due to LRBA and CTLA-4 mutations.

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Institute of Immunity and Transplantation, Division of Infection & Immunity, School of Life and Medical Sciences, University College London, Royal Free Hospital, London, United Kingdom.
Immunology Department, Great Ormond Street Hospital for Children National Health Service (NHS) Foundation Trust, London, United Kingdom.
Papworth and Addenbrooke's Hospital NHS Foundation Trusts, Cambridge, United Kingdom.
Clinical Immunology Department, Royal Free Hospital, London, United Kingdom.
Sección de Infectología e Inmunopatología, Unidad de Pediatría, Hospital Virgen del Rocío/Instituto de Biomedicina de Sevilla (IBiS), Sevilla, Spain.
Primary Immunodeficiency Group, Institute of Cellular Medicine, Medical School, Newcastle University, Newcastle upon Tyne, United Kingdom; and.
Department of Immunology, University of Manchester, Royal Manchester Children's Hospital, Manchester, United Kingdom.


Heterozygous CTLA-4 deficiency has been reported as a monogenic cause of common variable immune deficiency with features of immune dysregulation. Direct mutation in CTLA-4 leads to defective regulatory T-cell (Treg) function associated with impaired ability to control levels of the CTLA-4 ligands, CD80 and CD86. However, additional mutations affecting the CTLA-4 pathway, such as those recently reported for LRBA, indirectly affect CTLA-4 expression, resulting in clinically similar disorders. Robust phenotyping approaches sensitive to defects in the CTLA-4 pathway are therefore required to inform understanding of such immune dysregulation syndromes. Here, we describe assays capable of distinguishing a variety of defects in the CTLA-4 pathway. Assessing total CTLA-4 expression levels was found to be optimal when restricting analysis to the CD45RA-Foxp3+ fraction. CTLA-4 induction following stimulation, and the use of lysosomal-blocking compounds, distinguished CTLA-4 from LRBA mutations. Short-term T-cell stimulation improved the capacity for discriminating the Foxp3+ Treg compartment, clearly revealing Treg expansions in these disorders. Finally, we developed a functionally orientated assay to measure ligand uptake by CTLA-4, which is sensitive to ligand-binding or -trafficking mutations, that would otherwise be difficult to detect and that is appropriate for testing novel mutations in CTLA-4 pathway genes. These approaches are likely to be of value in interpreting the functional significance of mutations in the CTLA-4 pathway identified by gene-sequencing approaches.

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