Figure 1. Expression of FOXP3 ΔE2 isoform in IPEX patients with deletion mutations within exon 2 of FOXP3 gene.

(A) Flow cytometric analysis of FOXP3 ΔE2 isoform expression in IPEX patient #3. PBMCs from the patient and a healthy donor were stained with two anti-FOXP3 antibodies. Clone 150D is exon 2-specific while clone 259D recognizes an epitope after exon 2 common for both isoforms. Cells were analyzed either ex vivo (Rest) or stimulated with anti-CD3/anti-CD28 coated beads for 24 hours (Activated). Gated on CD4⁺ T cells. (B) Flow cytometric analysis of CD25 expression on CD4⁺ T cells before and after activation. (C) Expression of FOXP3 and CD25 by Tregs (gated on CD4⁺259D⁺) from healthy control (green line) and patient #3 (blue line) before or after activation. Gray lines were 259D⁻ cells. Numbers in the parenthesis represent mean fluorescence intensity (MFI). Similar results as in A-C were obtained with PBMCs from patient #2. (D) Deletion mutations in FOXP3 exon 2 identified in IPEX patients #1, #2 and #4 lead to expression of only the FOXP3 ΔE2 isoform. Genomic DNA fragment containing exons 1–7 of the FOXP3 gene from a healthy control and patients #1, #2, and #4 were cloned into the pcDNA3-NV5 vector in-frame with an N-terminal V5 epitope tag (upper panel). Jurkat T cells transfected with the expression constructs were examined by Western blot with an anti-V5 antibody.