Induction of FoxP3 Pre-mRNA Alternative Splicing to Enhance the Suppressive Activity of Regulatory T Cells from Amyotrophic Lateral Sclerosis Patients

Abstract

Forkhead box protein 3 (FoxP3) is a key transcription factor responsible for the development, maturation, and function of regulatory T cells (Tregs). The FoxP3 pre-mRNA is subject to alternative splicing, resulting in the translation of multiple splice variants. We have shown that Tregs from patients with amyotrophic lateral sclerosis (ALS) have reduced expression of full-length (FL) FoxP3, while other truncated splice variants are expressed predominantly. A correlation was observed between the reduced number of Tregs in the peripheral blood of ALS patients, reduced total FoxP3 mRNA, and reduced mRNA of its FL splice variant. Induction of FL FoxP3 was achieved using splice-switching oligonucleotides capable of base pairing with FoxP3 pre-mRNA and selectively modulating the inclusion of exons 2 and 7 in the mature mRNA. Selective expression of FL FoxP3 resulted in the induction of CD127low, CD152, and Helios-positive cells, while the cell markers CD4 and CD25 were not altered. Such Tregs had an increased proliferative activity and a higher frequency of cell divisions per day. The increased suppressive activity of Tregs with the induced FL FoxP3 splice variant was associated with the increased synthesis of the pro-apoptotic granzymes A and B, and perforin, IL-10, and IL-35, which are responsible for contact-independent suppression, and with the increased ability to suppress telomerase in target cells. The upregulation of Treg suppressive and proliferative activity using splice-switching oligonucleotides to induce the predominant expression of the FoxP3 FL variant is a promising approach for regenerative cell therapy in Treg-associated diseases.

Many Faces of Regulatory T Cells: Heterogeneity or Plasticity?

“…Impaired FoxP3 isoform ratios are observed in several diseases, affecting the Treg immunophenotype. In a recent paper, we demonstrated that patients suffering from amyotrophic lateral sclerosis have reduced expression of FL FoxP3, and the expression of other truncated variants predominates [ 29 ]. The induction of selective FL FoxP3 expression resulted in the appearance of a more suppressive and proliferative phenotype [ 29 ].…”

“…In a recent paper, we demonstrated that patients suffering from amyotrophic lateral sclerosis have reduced expression of FL FoxP3, and the expression of other truncated variants predominates [ 29 ]. The induction of selective FL FoxP3 expression resulted in the appearance of a more suppressive and proliferative phenotype [ 29 ]. Therefore, FoxP3-detecting antibodies must be used with caution when identifying Tregs.…”