A combination of cyclophosphamide and interleukin-2 allows CD4+ T cells converted to Tregs to control scurfy syndrome.

Immunodysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is caused by mutations in forkhead box P3 (FOXP3), which lead to the loss of function of regulatory T cells (Tregs) and the development of autoimmune manifestations early in life. The selective induction of a Treg program in autologous CD4+ T cells by FOXP3 gene transfer is a promising approach for curing IPEX. We have established a novel in vivo assay of Treg functionality, based on adoptive transfer of these cells into scurfy mice (an animal model of IPEX) and a combination of cyclophosphamide (Cy) conditioning and interleukin-2 (IL-2) treatment. This model highlighted the possibility of rescuing scurfy disease after the latter's onset. By using this in vivo model and an optimized lentiviral vector expressing human Foxp3 and, as a reporter, a truncated form of the low-affinity nerve growth factor receptor (ΔLNGFR), we demonstrated that the adoptive transfer of FOXP3-transduced scurfy CD4+ T cells enabled the long-term rescue of scurfy autoimmune disease. The efficiency was similar to that seen with wild-type Tregs. After in vivo expansion, the converted CD4FOXP3 cells recapitulated the transcriptomic core signature for Tregs. These findings demonstrate that FOXP3 expression converts CD4+ T cells into functional Tregs capable of controlling severe autoimmune disease.

A Nontoxic Transduction Enhancer Enables Highly Efficient Lentiviral Transduction of Primary Murine T Cells and Hematopoietic Stem Cells.

Lentiviral vectors have emerged as an efficient, safe therapeutic tool for gene therapy based on hematopoietic stem cells (HSCs) or T cells. However, the monitoring of transduced cells in preclinical models remains challenging because of the inefficient transduction of murine primary T cells with lentiviral vectors, in contrast to gammaretroviral vectors. The use of this later in preclinical proof of concept is not considered as relevant when a lentiviral vector will be used in a clinical trial. Hence, there is an urgent need to develop an efficient transduction protocol for murine cells with lentiviral vectors. Here, we describe an optimized protocol in which a nontoxic transduction enhancer (Lentiboost) enables the efficient transduction of primary murine T cells with lentiviral vectors. The optimized protocol combines low toxicity and high transduction efficiency. We achieved a high-level transduction of murine CD4+ and CD8+ T cells with a VSV-G-pseudotyped lentiviral vector with no changes in the phenotypes of transduced T cells, which were stable and long-lived in culture. This enhancer also increased the transduction of murine HSCs. Hence, use of this new transduction enhancer overcomes the limitations of lentiviral vectors in preclinical experiments and should facilitate the translation of strategies based on lentiviral vectors from the bench to the clinic.

Direct and indirect effects of alloantibodies link neointimal and medial remodeling in graft arteriosclerosis.

OBJECTIVE: Chronic vascular rejection, the main cause of allograft failure, is characterized by the destruction of smooth muscle cells (SMCs) in the media concomitantly with the proliferation of SMCs in the adjacent neointima. We hypothesized that alloantibodies might be responsible for these 2 opposite but coordinated events. METHODS AND RESULTS: We used the rat aortic interposition model of chronic vascular rejection. During the rejection process, a neointima composed of proliferating SMCs from the recipient developed, whereas the SMCs in the media, all of donor origin, underwent apoptosis. Alloantibody deposition was detected only in the media. Using in vitro cultures experiments, we observed that alloantibody binding to donor SMCs exerts (1) a rapid upregulation of the transcription of growth factors genes, followed by (2) the induction of apoptosis after 24 hours. The transient production of growth factors by donor SMCs in response to the binding of alloantibodies induced the proliferation of recipient SMCs in culture supernatant transfer experiments. Additional data suggest that among the repertoire of alloantibodies, those directed against major histocompatibility complex I might carry the remodeling effect. CONCLUSIONS: Our data suggest that during chronic vascular rejection, alloantibody binding to donor medial SMCs is a crucial event that links neointimal and medial remodeling.