Generation of Functional Regulatory T Cells By FOXP3 Gene Transfer into CD4 T Cells from Scurfy Mice and IPEX Patients
Authors: Delville Marianne , Six Emmanuelle , Bellier Florence , Sigrist Nelly , Zemmour David , Bessard Marie-André , Mazerolles Fabienne , Gross David , Schambach Axel , Rieux-Laucat Frederic , Benoist Christoph , Andre-Schmutz Isabelle , Cavazzana Marina 
Published: October 2019

Abstract
IPEX (Immunodysregulation Polyendocrinopathy Enteropathy X-linked) syndrome is the prototype of primary immunodeficiency with prevailing autoimmunity. The disease is caused by mutations in the gene encoding the transcription factor forkhead box P3 (FOXP3), which leads to the loss of function of thymus-derived CD4+CD25+ regulatory T (tTreg) cells. In IPEX patients, the absence of a functional Treg cell compartment leads to the development of multiple autoimmune manifestations (including severe enteropathy, type 1 diabetes and eczema) usually in the first months or years of life.
The current treatments for IPEX syndrome include immunosuppressive, hormone replacement therapies. Unfortunately, immunosuppressive treatments are usually only partially effective and their dose is often limited because of the occurrence of infectious complications and toxicity. Currently, the only curative treatment for IPEX syndrome is allogeneic hematopoietic stem cell transplantation (HSCT). The absence of an HLA-compatible donor for all patients and their poor clinical condition particularly expose them to a risk of mortality when HLA partially compatible donors are used. For all these reasons, effective alternative therapeutic approaches are urgently needed.
Various preclinical studies have shown that partial donor chimerism is sufficient for complete remission meaning that a small number of functional natural Treg is sufficient to restore immune tolerance. This suggests that a gene therapy approach designed to selectively induce a Treg program in T cells by expressing FOXP3 could be a promising potential cure for IPEX. However, several issues might compromise the success of this strategy: (i) will the introduction of FOXP3 alone be sufficient to induce a stable Treg program or will it require additional transcription factors to lock the Treg function and sustain the stability of transduced cells? (ii) Targeting effector CD4+ T cells might be an issue in terms of T-cell receptor repertoire, since the TCR repertoire of nTregs is different from the one of effector CD4+ T cells, (iii) will FOXP3-transduced T cells be able to migrate to appropriate tissues to control auto-immune reactions?, (iv) infusion of nTreg prevents the appearance of some autoimmune manifestations in murine models, however the infusion was done in prophylaxis before the appearance of the symptoms.
In order to address these questions, we have developed a mouse scurfy model to evaluate the functional and stability of the correction in vivo in parallel to the characterization of gene corrected human CD4 T cells from IPEX patients. Scurfy mice develop a disease very close to human pathology due to a spontaneous mutation of Foxp3 gene. We improved Scurfy mice model to improve animal production and increase the timeline of treatement.
We demonstrated that FOXP3 gene transfer into murine CD4+ T cells enable the generation of potent regulatory T cells. Indeed we showed the functional suppressive properties of the generated CD4-FOXP3 cells in an optimized flow-cytometry-based in vitro suppression assay. The ability of CD4-FOXP3 to prevent Scurfy disease by adoptive transfer in the first days of life is currently under evaluation.
Similarly in humans, we demonstrated that FOXP3 gene transfer into CD4+ T cells from IPEX patients enable the generation of potent regulatory T cells, as shown through the functional in vitro suppressive properties of the generated CD4IPEX-FOXP3. Moreover comparison of the transcriptional profile of these regulatory CD4IPEX-FOXP3 cells to natural Treg by RNA-seq analysis demonstrated a good repression of cytokine transcripts (IL4/5/13/CSF2, CD40L), a strong repression of IL7R, a strong induction of IL1R2, and a moderate activation of typical Treg genes (IL2RA, IKZF2, CTLA4).
Therefore, the introduction of a functional copy of the FOXP3 gene into an IPEX patient's T cells may be enough to restore immune tolerance and thus avoid the complications of allogenic HSCT. We will also discuss the challenge of generating a large, homogenous and stable population of cells in vitro for adoptive transfer and whether it can ensure long-term disease correction without generating a context of generalized immunosuppression.

Disclosures
No relevant conflicts of interest to declare.