Phenotypical and Functional Characteristics of Human Regulatory T Cells during Ex Vivo Maturation from CD4+ T Lymphocytes

Abstract

Regulatory T cells (Tregs) participate in the negative regulation of inflammatory reactions by suppressing effector cells. In a number of autoimmune disorders, the suppressive function and/or the number of Tregs is compromised. The lack of active functioning Tregs can be restored with adoptive transfer of expanded ex vivo autologous Tregs. In our study, we traced the differentiation and maturation of Tregs CD4+CD25+FoxP3+CD127low over 7 days of cultivation from initial CD4+ T cells under ex vivo conditions. The resulting ex vivo expanded cell population (eTregs) demonstrated the immune profile of Tregs with an increased capacity to suppress the proliferation of target effector cells. The expression of the FoxP3 gene was upregulated within the time of expansion and was associated with gradual demethylation in the promotor region of the T cell-specific demethylation region. Real-time RT-PCR analysis revealed changes in the expression profile of genes involved in cell cycle regulation. In addition to FOXP3, the cells displayed elevated mRNA levels of Ikaros zinc finger transcription factors and the main telomerase catalytic subunit hTERT. Alternative splicing of FoxP3, hTERT and IKZF family members was demonstrated to be involved in eTreg maturation. Our data indicate that expanded ex vivo eTregs develop a Treg-specific phenotype and functional suppressive activity. We suggest that eTregs are not just expanded but transformed cells with enhanced capacities of immune suppression. Our findings may influence further development of cell immunosuppressive therapy based on regulatory T cells.

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

“…Transfected cells were cultured ex vivo within 96 h in Treg culture media (RPMI-1640 (Paneco, Moscow, Russia), 10% fetal bovine serum (Capricorn Scientific, Ebsdorfergrund, Germany), 5 µg/mL anti-CD3 monoclonal antibodies (MedBioSpectr, Moscow, Russia), 2 µg/mL anti-CD28 antibodies (eBiosciences, San Diego, CA, USA), and 100 U/mL of rHu IL-2 (R&D Systems, Minneapolis, MN, USA), as we have previously described [25,35].…”

“…Two variants are truncated and encode the proteins with the deletions of either of each exon 2 (∆2 splice variant) or exon 7 (∆7 splice variant) coding regions. The shortest variant encodes the smallest protein with the deletions of both exons 2 and 7 (∆2∆7 splice variant) [24,25].…”

Many Faces of Regulatory T Cells: Heterogeneity or Plasticity?

“…Alternative splicing of FoxP3 results in the existence of four isoforms of the FoxP3 protein: a full-length variant (FL), variants with deletions of either exon 2 (∆2 variant) or 7 (∆7 variant) and a deletion of both exons (∆2∆7 variant) [ 27 ]. It is noteworthy that there are differences in the numbering of these exons in the literature, and exons 2 and 7 (protein coding) are referred to as 3 and 8, respectively, in some sources, considering the non-coding exon as the first [ 28 ]. Importantly, Tregs expressing the FL FoxP3 variant show higher suppressive activity and proliferation rates compared to those expressing truncated FoxP3 variants [ 27 ].…”

“…Given the central role of Tregs in maintaining immune homeostasis, there is a growing interest in developing therapeutic approaches that harness the potential of Tregs. In the context of transplantation and various autoimmune diseases, there is a focus on testing different approaches, including IL-2-based, polyclonal, antigen-specific and cell-engineered (CAR and TCR-transduced) approaches [ 28 , 187 , 188 ]. However, there are two major obstacles to overcome.…”

Modulation of Suppressive Activity and Proliferation of Human Regulatory T Cells by Splice-Switching Oligonucleotides Targeting FoxP3 Pre-mRNA

“…According to GenBank, four isoforms of the FoxP3 protein resulting from alternative splicing of its pre-mRNA have been described: a full-length variant (FL), variants with deletions of each of exons 2 (∆2 variant) or 7 (∆7 variant), and a deletion of both exons (∆2∆7 variant). It should be clarified that there are differences in the numbering of these exons in the literature, and exons 2 and 7 (considering only protein-coding exons) in some sources are designated 3 and 8, respectively, considering the noncoding exon as the first [ 10 , 11 ].…”