Objectives: Genetic or acquired defects in FOXP3⁺ regulatory T cells (Tregs) play a key role in many immune‐mediated diseases including immune dysregulation polyendocrinopathy, enteropathy, X‐linked (IPEX) syndrome. Previously, we demonstrated CD4⁺ T cells from healthy donors and IPEX patients can be converted into functional Treg‐like cells by lentiviral transfer of FOXP3 (CD4^LVFOXP3). These CD4^LVFOXP3 cells have potent regulatory function, suggesting their potential as an innovative therapeutic. Here, we present molecular and preclinical in vivo data supporting CD4^LVFOXP3 cell clinical progression.Methods: The molecular characterisation of CD4^LVFOXP3 cells included flow cytometry, qPCR, RNA‐seq and TCR‐seq. The in vivo suppressive function of CD4^LVFOXP3 cells was assessed in xenograft‐versus‐host disease (xeno‐GvHD) and FOXP3‐deficient IPEX‐like humanised mouse models. The safety of CD4^LVFOXP3 cells was evaluated using peripheral blood (PB) humanised (hu)‐ mice testing their impact on immune response against pathogens, and immune surveillance against tumor antigens.Results: We demonstrate that the conversion of CD4⁺ T cells to CD4^LVFOXP3 cells leads to specific transcriptional changes as compared to CD4⁺ T‐cell transduction in the absence of FOXP3, including upregulation of Treg‐related genes. Furthermore, we observe specific preservation of a polyclonal TCR repertoire during in vitro cell production. Both allogeneic and autologous CD4^LVFOXP3 cells protect from xeno‐GvHD after two sequential infusions of effector T cells. CD4^LVFOXP3 cells prevent hyper‐proliferation of CD4⁺ memory T cells in the FOXP3‐deficient IPEX‐like hu‐mice. CD4^LVFOXP3 cells do not impede in vivo expansion of antigen‐primed T cells or tumor clearance in the PB hu‐mice.Conclusion: These data support the clinical readiness of CD4^LVFOXP3 cells to treat IPEX syndrome and other immune‐mediated diseases caused by insufficient or dysfunctional FOXP3⁺ Tregs.

FOXP3 is the master transcription factor in both murine and human FOXP3⁺regulatory T cells (Tregs), a T-cell subset with a central role in controlling immune responses. Loss of the functional Foxp3 protein inscurfymice leads to acute early-onset lethal lymphoproliferation. Similarly, pathogenic FOXP3 mutations in humans lead to immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome, which are characterized by systemic autoimmunity that typically begins in the first year of life. However, although pathogenicFOXP3mutations lead to overlapping phenotypic consequences in both systems, FOXP3 in human Tregs, but not mouse, is expressed as two predominant isoforms, the full length (FOXP3FL) and the alternatively spliced isoform, delta 2 (FOXP3Δ2). Here, using CRISPR/Cas9 to generate FOXP3 knockout CD4⁺T cells (FOXP3KO^GFPCD4+ T cells), we restore the expression of each isoform by lentiviral gene transfer to delineate their functional roles in human Tregs. When compared to FOXP3FL or FOXP3Δ2 alone, or double transduction of the same isoform, co-expression of FOXP3FL and FOXP3Δ2 induced the highest overall FOXP3 protein expression in FOXP3KO^GFPCD4+ T cells. This condition, in turn, led to optimal acquisition of Treg-like cell phenotypes including downregulation of cytokines, such as IL-17, and increased suppressive function. Our data confirm that co-expression of FOXP3FL and FOXP3Δ2 leads to optimal Treg-like cell function and supports the need to maintain the expression of both when engineering therapeutics designed to restore FOXP3 function in otherwise deficient cells.

Regulatory T cells (Tregs) play a central role in modulating adaptive immune responses in humans and mice. The precise biological role of non-canonical nuclear factor ‘κ-light-chain-enhancer’ of activated B cells (NFKB) signaling in human Tregs has yet to be fully elucidated. To gain insight into this process, a Treg-like cell line (MT-2) was genetically modified using CRISPR/Cas9. Interestingly, NFKB2 knockout MT-2 cells exhibited downregulation of FOXP3, while NFKB1 knockout did not. Additionally, mRNA expression of FOXP3-dependent molecules was significantly reduced in NFKB2 knockout MT-2 cells. To better understand the functional role of the NFKB signaling, the NFKB1/NFKB2 loci of human primary Tregs were genetically edited using CRISPR/Cas9. Similar to MT-2 cells, NFKB2 knockout human Tregs displayed significantly reduced FOXP3 expression. Furthermore, NFKB2 knockout human Tregs showed downregulation of FOXP3-dependent molecules and a diminished suppressive function compared to wild-type and NFKB1 knockout Tregs. These findings indicate that non-canonical NFKB signaling maintains a Treg-like phenotype and suppressive function in human Tregs through the FOXP3-dependent regulatory T cell program.

This report describes the pathologic findings for a patient with Hunter syndrome who underwent aortic valve replacement at 10 years of age, 3 years after the initiation of enzyme replacement therapy. Aortic valve pathology showed mild thickening and fibrosis as well as massive glycosaminoglycan accumulation. This suggests that enzyme replacement therapy has limited efficacy for cardiac valve disease both clinically and pathologically.

Forkhead box P3 (FOXP3) is an essential transcription factor for regulatory T cell (Treg) function. Defects in Tregs mediate many immune diseases including the monogenic autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX), which is caused by FOXP3 mutations. Treg cell products are a promising modality to induce allograft tolerance or reduce the use of immunosuppressive drugs to prevent rejection, as well as in the treatment of acquired autoimmune diseases. We have recently opened a phase I clinical trial for IPEX patients using autologous engineered Treg-like cells, CD4^LVFOXP3. To facilitate the pre-clinical studies, a novel humanized-mouse (hu-mouse) model was developed whereby immune-deficient mice were transplanted with human hematopoietic stem progenitor cells (HSPCs) in which the FOXP3 gene was knocked out (FOXP3KO) using CRISPR-Cas9. Mice transplanted with FOXP3KO HSPCs had impaired survival, developed lymphoproliferation 10–12 weeks post-transplant and T cell infiltration of the gut, resembling human IPEX. Strikingly, injection of CD4^LVFOXP3 into the FOXP3KO hu-mice restored in vivo regulatory functions, including control of lymphoproliferation and inhibition of T cell infiltration in the colon. This hu-mouse disease model can be reproducibly established and constitutes an ideal model to assess pre-clinical efficacy of human Treg cell investigational products.