Generation of CD4⁺ Regulatory T Cells by Retroviral Transduction of CD4⁺CD25⁻ T Cells Either with the Full-Length or with a Common Exon-2 Negative Variant of Human Foxp3.

The forkhead/winged helix transcription factor, Foxp3 is a key element for the differentiation of CD4⁺CD25⁺ regulatory T (Treg) cells. While murine Foxp3 gene is expressed as a single full-length transcript, we observed that transcription of human Foxp3 gene usually reveals two different mRNA products with ~ 100 base pair difference. Sequencing of these products revealed that one transcript represents the full-length Foxp3, while the other transcript appears to be an alternative splicing product that lacks the exon-2. Using a specific primer set that amplifies the region between exon-1 and exon-3 we found that both exon-2^pos and exon-2^neg variants are preferentially expressed in CD4⁺CD25⁺ cells with high levels of expression in CD4⁺CD25^hi cells. Analysis of individual Treg clones generated by limiting dilution of CD4⁺CD25^hi cells revealed that both Foxp3 variants are simultaneously expressed in Treg clones. However, quantitative-real time PCR analyses, performed using a primer set that amplifies only the full-length Foxp3 and another set that amplifies both exon-2^neg and exon-2^pos variants, indicated that expression levels of the full-length Foxp3 gene do not correlate with expression levels of total Foxp3 in individual Treg clones, in PBMC of healthy donors and in leukemia patients after donor lymphocyte infusions. Remarkably, some donors expressed little or no full-length Foxp3 while expressing considerable amounts of total Foxp3, suggesting a predominant expression of the exon-2^neg variant in CD4⁺CD25⁺ T cells of these donors. To determine the role of individual Foxp3 isoforms in Treg cell-differentiation, the full-length and the exon-2^neg variants of the Foxp3 gene were cloned into retroviral vectors and transduced separately into highly purified CD4⁺CD25⁻ cells. After a brief in vitro culture, T cells transduced with the Foxp3 isoforms were sorted to high purity by flow cytometry using the GFP marker gene and expanded to high quantities by nonspecific stimulation with allogeneic feeder cells, PHA and IL-2. Phenotypical and functional analyses of expanded cells revealed that T cells transduced with both Foxp3 variants expressed high levels of CD25, intacellular CTLA-4 and CD62L; were anergic to stimulation via CD3 and suppressed the CD3/CD28 triggered proliferation of autologous and allogeneic CD4⁺CD25⁻ cells. These results reveal that both isoforms of human Foxp3 are functionally intact and can convert CD4⁺CD25⁻ cells into Treg cells. Current efforts are focused on testing the in vivo capacity of in vitro expanded, Foxp3 transduced T cells to prevent Graft versus Host Disease (GvHD) in a xenogeneic model where GvHD is induced by administration of human T cells in Rag2, gamma chain double knockout mice.