First Comprehensive Analysis of Full-Length and Δ2 Foxp3 Isoforms Distribution in PBMCs from Healthy Volunteers

FOXP3 is the master transcriptional regulator of regulatory T cells (Tregs) and is expressed in humans as two main alternatively spliced isoforms: full-length FOXP3 (FOXP3-FL) and the exon 2-deficient variant (FOXP3-Δ2). While the role of these isoforms has been mainly studied in CD4+ T cells, their distribution across peripheral blood leukocyte populations and their relationship with immune checkpoint expression remain incompletely defined. In this study, we used a multiparametric flow cytometry panel allowing isoform-specific detection of FOXP3-FL and FOXP3-Δ2, together with PD-1 and CTLA-4, to analyze peripheral blood samples from six healthy donors under basal conditions. Major leukocyte populations, including CD4+CD25+ and CD4+CD25− T cells, CD8+ T cells, monocytes, and neutrophils, were evaluated. FOXP3-FL predominated in CD4+CD25+ T cells, whereas FOXP3-Δ2 was more frequently detected in CD8+ T cells, monocytes, and neutrophils. However, the absolute frequencies of these FOXP3-Δ2-positive populations were low, consistent with the overall low levels of FOXP3 expression observed in these cell types. In CD4+ T-cell subsets, PD-1 expression was generally higher than CTLA-4, regardless of FOXP3 isoform, and FOXP3-Δ2+ cells showed relatively higher PD-1 expression compared to FOXP3-FL+ cells. In contrast, checkpoint expression in non-CD4+ populations was limited. The observed FOXP3-FL+/FOXP3-Δ2+ ratios across immune cell populations were consistent with a predominant role of FOXP3-FL in maintaining immune tolerance under basal conditions; whether these patterns are preserved or altered in pathological settings warrants further investigation. These results provide a descriptive overview of FOXP3 isoform distribution and checkpoint expression across peripheral blood immune cell subsets in healthy individuals, which may serve as a reference for future studies in immune-mediated diseases.