Unsupervised Flow Cytometry Reveals a Constant Shift Towards Activated <scp>CD4</scp> ⁺ T Cell Subsets in <scp>APECED</scp>

ABSTRACT Autoimmune polyendocrinopathy‐candidiasis‐ectodermal dystrophy (APECED) is a rare monogenic autoimmune disease caused by loss‐of‐function mutations in the Autoimmune regulator gene and the subsequent impairment of negative selection in the thymus. Previous studies have identified many T cell abnormalities in APECED patients, with increased signs of activation and failure of regulation. These findings, however, have been demonstrated using traditional flow cytometry studies, prone to human misinterpretation of multidimensional data. Here we present an unsupervised flow cytometry analysis of CD4 + T cells in 20 APECED patients. Our workflow consists of an optimized sequence of well documented unsupervised analysis algorithms for data quality control, batch correction and clustering with multiple options evaluated. We show the overall increase in T cell activation to extend from the previously reported CD8 + compartment to both the T helper and regulatory T cells. We demonstrate the regulatory T cells in APECED to adopt a population distribution dominated by cells of low suppressive potential and present a novel CD45RA neg , CCR7 neg , CD31 high , CD127 mid population unique to APECED. In general, however, we find the population structure of the APECED CD4 + T cell compartment largely similar to that of the controls as supported by both our clustering and dimensionality reduction analyses. Our analysis replicates previous findings and provides novel information on APECED while also presenting an optimized unsupervised workflow for flow cytometry analysis in general. Our approach offers multiple advantages over manual flow cytometry analysis with its rigorous data quality control, well‐documented batch correction and optimized automated clustering.