Peripheral Expression of FOXP3 in Brazilian Patients with IPEX Syndrome: From FOXP3 Biomarker to Targeted FOXP3 Therapy Within the CNE3I

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a monogenic disorder caused by loss-of-function mutations in the FOXP3 gene, which is critical for the development of functional regulatory T cells (Tregs). In IPEX patients, Tregs are unable to inhibit effector T cell proliferation and cytokine production, leading to a loss of peripheral immune tolerance. The disease presents with heterogeneous clinical manifestations, severe early-onset autoimmunity, the classic triad (enteropathy, eczema, and type 1 diabetes), as well as atypical or late-onset symptoms. A clear genotype–phenotype correlation has not been established for IPEX, and immunological assessments that could contribute to the diagnosis are scarce. Therefore, we aim to characterize the peripheral Treg cells in IPEX patients in Brazil through combined quantitative and qualitative flow cytometry analysis. We enrolled 6 male patients with a confirmed IPEX diagnosis (clinical, genetic, and immunological). All patients exhibited autoimmunity (enteropathy: 5/6; arthritis: 3/6; hemolytic anemia: 2/6; type 1 diabetes: 2/6), and allergic manifestations (rhinitis: 6/6; asthma: 4/6), eczema (4/6), and recurrent sinusitis (4/6) were also observed. Up to the last follow-up, two patients were alive after bone marrow transplantation and one after gene therapy (Figure 1). Peripheral FOXP3 expression was assessed in peripheral blood mononuclear cells (PBMCs) by flow cytometry (CD3, CD4, CD25, CD127, and FOXP3). Quantitative analysis determined the frequency of Tregs (CD4+CD25+CD127-FOXP3+), while qualitative analysis measured FOXP3 protein expression via median fluorescence intensity (MFI). The frequency of circulating Tregs in IPEX patients from our cohort was highly variable (14.6%–81.2%; mean: 58.5%) and overlapped with the control range (59.6%–84.1%; mean: 72.4%). In contrast, FOXP3 MFI was significantly lower in patient Tregs (mean: 986, range: 863–1,276) compared to controls (mean: 1,937, range: 1,089–4,132; p<0.05), another indication of quantitative Treg defects. Different patterns of FOXP3 expression have been reported, depending on the type and location of the mutation. While Treg numbers can be preserved in IPEX syndrome, FOXP3 protein expression per cell is consistently and significantly reduced. This defect in the expression level of FOXP3 likely underlies Treg dysfunction and disease pathogenesis. Assessment of FOXP3 MFI emerges as an additional valuable complementary diagnostic tool, providing functional insight beyond genetic sequencing alone. Figure 1. Clinical, immunologic, genetic, and therapeutic findings for IPEX syndrome within the CNE3i (Centro Nacional de Erros Inatos da Imunidade e Imunodesregulação) in Brazil. (1) Demonstrates the main clinical and geographical findings of the six IPEX patients; (2) general and advanced laboratory findings; and (3) genetics and therapeutics. SP, São Paulo; MG, Minas Gerais; PE, Pernambuco; DF, Distrito Federal; ES, Espírito Santo; IgE, immunoglobulin E; IgG immunoglobulin G; DNT, double negative T cells; IFN-SG, interferon-stimulated genes; MFI, median fluorescence intensity; BMT, bone marrow transplantation; GT, gene therapy; LFW, last follow-up. FINEP funding: 0956/24; FAPESP funding: 2023/09965-0; Instituto de Investigação em Imunologia funding CNPQ/MCTI: 408685/2024-7.