Forced Fox-P3 expression can improve the safety and antigen-specific function of engineered regulatory T cells

Regulatory T cells (Tregs) are a unique subset of T cells that play a critical role in maintaining immune homeostasis and preventing autoimmunity. These cells are characterized by their ability to suppress the activation and proliferation of other immune cells, including effector T cells, B cells, and antigen-presenting cells. In the context of immunity, Tregs are essential for preventing excessive and harmful immune responses to self-antigens, as well as to innocuous environmental stimuli. Without the regulatory function of Tregs, the immune system would be more prone to causing tissue damage and inducing autoimmune diseases. Tregs achieve their suppressive effects through multiple mechanisms, including the secretion of inhibitory cytokines such as IL-10 and TGF-β, direct cell-to-cell contact, and metabolic regulation. In autoimmune diseases, Tregs have been shown to be functionally impaired or reduced in number, leading to dysregulated immune responses and tissue damage. Strategies to enhance Treg function, increase their numbers and employ Treg specificity have emerged as potential treatment options for autoimmune diseases. Since the activation of Treg function requires TCR stimulation, engineered Tregs expressing antigen-specific TCRs can mediate more efficient immune suppression compared with polyclonal Tregs of unknown specificity.

The transcription factor Forkhead box protein P3 (FoxP3) is a crucial regulator of the development and function of regulatory T cells (Tregs). FoxP3 expression is necessary for Tregs to exert their suppressive activity, and it is considered a hallmark of Treg identity. Functioning as intracellular transcription factor, FoxP3 regulates the levels of expression of a wide range of genes involved in immune suppression. Loss of function mutations in the FoxP3 gene cause severe autoimmune diseases in humans and mice, highlighting the importance of this protein in maintaining immune homeostasis. Although high endogenous FoxP3 expression in human Treg has been linked with substantial suppressive activity in vivo and in vitro, it has remained unclear whether forced FoxP3 expression above the levels seen in ‘natural’ Tregs would be advantageous or deleterious to the suppressive function.

In a new study published in the Journal of Autoimmunity, University College London researchers: Dr. Jenny McGovern, Dr. Angelika Holler, Dr. Sharyn Thomas, and led by Professor Hans Stauss investigated whether increased expression of FoxP3 under the command of a constitutively active promoter resistant to physiological gene regulation was harmful to Treg function in vitro. The authors assessed whether forced FoxP3 expression in TCR engineered Tergs prevented the accumulation of FoxP3-negative, TCR-positive effector T cells in vivo after adoptive transfer into HLA transgenic mice.

The research team demonstrated that forced FoxP3 expression increased CD25 and CTLA4 expression in transduced Tregs, and that this elevated FoxP3, CD25, and CTLA4 expression was linked to an improved in vitro suppression activity. The supraphysiological FoxP3 expression did not impair engraftment and persistence of engineered Tregs following adoptive transfer into HLA transgenic mice. The forced FoxP3 expression prevented the in vivo accumulation effector T cells that produced IL-2 and IFNγ in response to cognate peptide stimulation. The use of a retroviral vector containing FoxP3 plus TCR transformed the cells into functionally stable Tregs that did not generate effector cytokines when stimulated with peptide antigen. Two processes are probably in action to guarantee a “locked” Treg function. First off, forced FoxP3 expression compensate got loss of endogenous FoxP3 expression, which would result in FoxP3-negative effector T cells. Second, the purified Treg populations employed for genetic engineering may contain contaminating conventional CD4 T cells that are converted into Treg-like cells by forced FoxP3 expression. Together, the FoxP3+TCR vector platform offers a double safety mechanism for the production of therapeutic Treg by transforming contaminating conventional CD4 T cells into Treg-like cells and by preventing the effects of endogenous FoxP3 downmodulation, which can result in the accumulation of effector CD4 T cells. These observations demonstrate that the two methods work well together to avoid the in vivo buildup of sizable populations of antigen-specific effector CD4 T cells that is seen when FoxP3 is missing in the gene transfer vector, even if the relative importance of each pathway is uncertain.

In conclusion, understanding the mechanisms behind the FoxP3 expression enhancing activity of Tregs is crucial for developing therapeutic strategies for immune-related disorders that involve Treg dysfunction. Professor Hans Stauss and colleagues findings suggest that engineering Treg to express high levels of exogenous FoxP3 offers a twofold safety characteristic of adoptive Treg treatment while also enhancing the suppressive effect of the modified cell product. According to the authors, engineered Treg engraftment and persistence are not harmed by forced expression of FoxP3, but prevents instead the buildup of antigen-specific effector T cells.