Human-engineered Treg-like cells suppress FOXP3-deficient T cells but preserve adaptive immune responses in vivo

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.

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.

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.

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.