Poster #013, Stanford University
Optimizing FOXP3 Isoform Ratios to Induce Regulatory T Cell Transcriptional Signatures in Human CD4+ T Cells
Mentors: Hope Lancero, PhD and Rosa Bacchetta, MD
IPEX (immune dysregulation, polyendocrinopathy, enteropathy, X-linked) syndrome is a rare, life-threatening autoimmune disorder resulting from mutations in FOXP3, a master transcription factor of regulatory T cells (Tregs). Human FOXP3 is expressed as two predominant splice isoforms: full-length (FOXP3FL) and a variant lacking exon 2 (FOXP3Δ2), whose balance is thought to shape Treg identity.
We hypothesized that forced co-expression of FOXP3FL and FOXP3Δ2 at varying ratios would induce a stronger regulatory transcriptional profile in human CD4+ T cells than either isoform alone. To map these ratio-dependent effects, healthy primary CD4+ T cells—along with FOXP3-negative Jurkat cells serving as a supplementary model—were transduced with varying multiplicities of infection (MOIs) of lentiviral constructs driving FOXP3FL and FOXP3Δ2.
Successful lentiviral generation and MOI determination were established via HEK293T cell titration, with titratable FOXP3 expression confirmed by Western blot and flow cytometry of the HEK293T cells. Primary CD4+ T cells and Jurkat cells were subsequently transduced with NGFR-FL and EGFR-Δ2 lentiviruses. Flow cytometric analysis of the CD4+ T cells (utilizing anti-FOXP3 clones 150D and 259D) confirmed successful, dose-dependent co-expression of both FOXP3 isoforms. To evaluate transcriptional shifts, cells were cultured with and without PMA/ionomycin activation, followed by RT-qPCR to quantify the expression of Treg signature genes, such as LAP (TGFB1) and GARP (LRRC32). Ongoing analysis of these primary T cell gene expression profiles will establish how tuning the FOXP3Δ2:FL ratio directly modulates Treg-like transcriptional programming. Together, these data support isoform balance as a novel lever for dissecting Treg biology and engineering regulatory T cell therapies.