Generation of type 1-like regulatory T cells via retroviral transduction of human CD4+ T cells with membrane-attached IL-10

Introduction
Tr1 cells are a subset of CD4+ regulatory T cells (Tregs) characterized by a CD4+/FoxP3‒/CD49b+/LAG-3+ phenotype that are induced in the periphery following chronic antigen exposure in the presence of IL-10. Activated Tr1 cells secrete exceedingly more IL-10 than FoxP3+ Tregs, which is translated into superior ability to enforce bystander tolerance in a paracrine manner. These attributes make Tr1 cells promising candidates for adoptive cell therapy targeting autoimmune and inflammatory diseases. However, clinical use requires a scalable and reproducible protocol to generate cells with high purity, stability, and antigen specificity. Traditional reliance on soluble IL-10 carries risks of systemic dissemination and diminished tissue specificity, necessitating improved methods.

Materials and Methods
To address these challenges, we engineered two derivatives of membrane-attached IL-10 (memIL-10) to ensure localized IL-10 activity while avoiding systemic spread. Human CD4+ T cells were retrovirally transduced with memIL-10 constructs under optimized conditions. Transduced cells were purified using magnetic beads to isolate memIL-10+ populations. Flow cytometry was used to assess the expression of Tr1 markers, including CD49b, LAG-3 and phosphorylated (p)STAT3. Cytokine profiles were evaluated following polyclonal activation.

Results and Discussion
Transduced cells expressed high levels of memIL-10, which were sustained for at least 28 days with excellent cell viability. Purified memIL-10+ cells included a homogenous CD49b+/LAG-3+ double-positive population, displaying hallmark Tr1-like characteristics. Upon polyclonal activation, sorted double-positive, but not double-negative cells secreted cytokines that are consistent with the Tr1 profile such as IL-10, IL-5, Granzyme B and IFN-γ and stained positive for pSTAT3. The protocol required only a single transduction step and eliminated the need for feeder mixtures, antigen-presenting cells or other specialized reagents.

Conclusions
Our approach offers a robust, efficient, and scalable method for generating Tr1-like cells. By leveraging memIL-10, this protocol enhances antigen-dependent IL-10 activity, minimizes systemic effects, and simplifies purification. These findings provide a strong foundation for the clinical application of Tr1 cells in treating autoimmune and inflammatory diseases, underscoring their therapeutic potential and advancing immune-modulatory strategies.