Natural occurring regulatory T cells: role of transcription factor FOXP3 and new approaches improving Treg-cell based therapy

The immune system requires a network of regulatory mechanisms that enable the host to maintain immune regulation, homeostasis and tolerance. A functionally committed CD4+CD25+FOXP3+ T cells subset (Treg cells) have a key role in determining the outcomes of protective immunity to a spectrum of foreign antigens while maintaining tolerance to self-antigens and suppressing excessive inflammation that can cause pathology. The transcription factor forkhead P3 (FOXP3) is highly expressed in Treg cells and it is critical for their suppressive function. The importance of FOXP3 is demonstrated in humans with a severe autoimmunity disease called immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) caused by mutations in FOXP3 gene. Therefore, there is an increasing interest in manipulating FOXP3 function and/or using CD4+CD25+FOXP3+ Treg cells as cell therapy to modify immune responses in cancer, autoimmunity and transplantation. The transcription factor FOXP3 has been shown to regulate negatively some genes such as Il2 and positively others, such as Cd25 and Ctla4. To better understand the function of FOXP3 as transcriptional activator, the regulation of CD25, the IL-2Rα chain, by FOXP3 was investigated (Part I). Analyzing a regulatory region of Cd25 promoter, it has found the presence of a κB site and two tandem copies of a non-consensus FOXP3 binding site separated at 5’ ends by 19 nucleotides that allow FOXP3 and NF-κB/RelA subunit binding to DNA. The occupancy of the two FOXP3 binding sites in conjunction with NF-κB/RelA binding site occupancy allows FOXP3 to function as a positive activator of Cd25 gene. Indeed mutations of both FOXP3 binding sites such as mutation of κB site on Cd25 promoter abolished FOXP3 activatory functions. Moreover, FOXP3 mutation ΔE251, that compromises FOXP3 homotypic interactions, failed to trans-activate Cd25 promoter, suggesting that both FOXP3 DNA binding and dimerization are required to trans-activate Cd25 promoter. So, these findings identify a novel mechanism by which RelA and FOXP3 cooperate to mediate transcriptional regulation of target genes and characterize a region on Cd25 promoter where FOXP3 dimer could bridge intra-molecularly two DNA sites and trans- activate Cd25 gene. Then the possibility of using ex vivo expanded CD4+CD25+FOXP3+ regulatory T cells as cell therapy was investigated (Part II). The ability to isolate and expand human Treg cells is crucial for their use in the clinic and many questions regarding the stability of phenotype and function of the transferred Tregs during inflammation remained unresolved. In this study it has been developed a protocol for the expansion of clinically useful numbers of functionally suppressive and stable human Treg cells. CD4+CD25+FOXP3+ Treg cells were expanded in vitro with rapamycin (RAPA) and/or all-trans-retinoic acid (ATRA) and then characterized under inflammatory conditions in vitro. The addition of RAPA to Treg cultures confirmed the generation of high numbers of suppressive and stable Tregs in vitro. In contrast, ATRA-treatment generated Treg cells which retained the capacity to secrete pro-inflammatory cytokine IL-17. However, combined use of RAPA and ATRA abolishes IL-17 production and confered a specific chemokine receptor homing profile upon Treg cell preparations. Moreover, the use of purified Treg subpopulations provided direct evidence that RAPA could confer an early selective advantage to CD45RA+ Treg subset, while ATRA favored CD45RA- Treg subset expansion. So, the expansion of Treg cells using RAPA and ATRA drug combinations provided a new approach for large-scale generation of functionally potent and phenotypically stable human Treg cells, rendering them safe for clinical use. All together, the results reported in this thesis sheds light on the ability of FOXP3 in regulating the expression of CD25 molecule and helps pave the way for use Treg cell therapy in clinic.