Forkhead box P3 (FOXP3) is the master transcription factor of regulatory T cells (Tregs) and is essential for maintaining immune tolerance. FOXP3 is stably expressed in Tregs, driving their suppressive function, and it is transiently induced in conventional T cells (Tconv) upon activation, where it fine-tunes immune responses. In humans, but not in mice, FOXP3 is expressed as two major isoforms: the full-length protein (FOXP3FL) and an isoform lacking exon 2 (FOXP3Δ2), with additional variants such as FOXP3Δ7 and FOXP3Δ2Δ7 described. The precise contribution of each isoform to Treg development, stability, and function in health and disease remains incompletely defined, although clinical observations indicate that patients expressing only FOXP3Δ2 develop autoimmunity, highlighting a nonredundant role for FOXP3FL in Treg-mediated tolerance. Multiple studies have further associated increased FOXP3Δ2 expression with autoimmune disease and reduced Treg stability, suggesting that the relative abundance of FOXP3FL and FOXP3Δ2 may critically shape Treg function and the balance between tolerance and pathology. However, the physiological expression levels and ratios of FOXP3FL and FOXP3Δ2 in Tregs and activated Tconvs remain poorly characterized, hindering understanding of the distinct functions of these isoforms, the correlation of specific FOXP3 mutations with isoform expression patterns and disease severity, and the development of Treg-targeted and gene transfer–based therapies. To address this, we quantified isoform-specific expression in Tregs and activated Tconvs from healthy donors and in patients with loss-of-function mutations in FOXP3 that cause immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome, a severe monogenic disorder that typically presents in early infancy, affects male patients, and is almost uniformly fatal without definitive therapy. Our data indicate that FOXP3Δ2 is the dominant isoform in activated Tconvs and Tregs across both cohorts, with a FOXP3FL:FOXP3Δ2 ratio of 0.52 ± 0.08, suggesting that regulated co-expression of FOXP3 isoforms but not exclusive expression of a single isoform underpins effective Treg development and function. Consequently, therapeutic strategies for IPEX and autoimmunity should aim to restore a physiologic FOXP3 isoform balance rather than solely achieving full-length FOXP3 expression.
