The landscape of regulatory immune cells has expanded dramatically beyond traditional CD4+CD25+FOXP3+ T regulatory cells (Tregs), revealing a complex network of immunosuppressive cell populations that collectively maintain immune homeostasis and prevent autoimmune diseases. Recent advances in understanding these diverse regulatory mechanisms are opening new therapeutic avenues for treating immune-mediated disorders and improving transplantation outcomes.
Expanding Universe of Regulatory Immune Cells
Research has identified multiple distinct regulatory cell populations, each employing unique suppressive mechanisms. CD8+ Tregs represent a specialized subset that suppresses immune responses through targeted cytotoxicity against activated immune cells and secretion of anti-inflammatory cytokines like IL-10 and TGF-β. These cells uniquely regulate CD8+ T cell-driven immunity while maintaining peripheral tolerance, with elevated levels observed in various cancers where they contribute to immunosuppressive microenvironments.
Regulatory B cells (Bregs) have emerged as crucial modulators of immune responses, characterized by their ability to produce anti-inflammatory cytokines and induce T cell tolerance. Despite the absence of specific markers analogous to the Treg marker FoxP3, researchers have identified common surface markers including CD19, CD1d, CD5, CD24, CD38, and CD27. Bregs employ multiple suppressive mechanisms, including cytokine production, antigen presentation that promotes tolerance, and direct cell-cell contact interactions.
Myeloid-derived suppressor cells (MDSCs) represent another critical regulatory population, arising from disrupted myeloid progenitor differentiation under pathological conditions. These cells suppress immune responses through multiple mechanisms, including secretion of factors like IL-10, IL-6, and TGF-β, triggering T cell apoptosis through reactive oxygen species production, and depleting essential nutrients such as arginine and tryptophan.
Thymus-Derived Tregs: A Superior Cell Source
While peripheral blood and umbilical cord blood have been traditional sources for Treg therapy, thymus-isolated regulatory T cells (thyTregs) have emerged as a superior alternative. Pediatric thymic tissue, routinely removed during corrective heart surgeries, provides an abundant source of naive Tregs with several advantages over blood-derived cells.
ThyTregs demonstrate longer telomeres than adult blood Tregs, with telomere length remaining stable after ex vivo expansion. This suggests enhanced capacity for cell division cycles and improved in vivo survival and functional capability. The cells also exhibit stable FOXP3 expression levels and maintain suppressive features under pro-inflammatory conditions, contrasting with the variable responses typically seen in adult blood Tregs.
Isolation procedures for thyTregs have been optimized across multiple research groups. Mechanical dissociation using the GentleMACS Dissociator produces high yields of viable thymocytes and has proven applicable to GMP-compliant production. CD25+ enrichment represents the most commonly employed strategy, with some groups including additional CD8+ depletion steps to obtain more defined CD4+ SP thyTreg populations.
Clinical Translation and Manufacturing Advances
Several expansion protocols have been developed to achieve therapeutic cell numbers. The Levings group reported expansion methods using irradiated artificial antigen-presenting cells, achieving 300-fold expansion by day 12 using ImmunoCult-XF medium. Alternative cell-free activation approaches using reagents like Dynabeads Treg Xpander and T Cell TransAct have shown comparable results while reducing manufacturing complexity.
The Correa-Rocha group developed a streamlined 7-day expansion protocol using TexMACS GMP medium supplemented with IL-2 and T cell TransAct, achieving 6.9-fold expansion with high viability (92.41%) and purity (95.2% CD25+FOXP3+ cells). This protocol has been successfully adapted to GMP conditions, producing comparable results to laboratory-scale procedures.
Clinical Applications and Early Results
The first clinical applications of thyTregs have demonstrated promising safety and efficacy profiles. A phase 1/2 clinical trial (NCT04924491) evaluating autologous thyTregs (THYTECH1) for preventing rejection in pediatric heart transplant patients reported no adverse effects attributable to thyTreg administration. The treated patient maintained higher Treg frequencies within CD4+ T cells compared to control patients throughout a 2-year follow-up period.
Building on these results, allogeneic applications are being explored. A phase 1/2 clinical trial (NCT06052436) is evaluating allogeneic thyTregs (THYTECH2) for controlling immune dysregulation associated with SARS-CoV-2 infection and acute respiratory distress syndrome. The reduced immunogenicity of thyTregs, evidenced by lower HLA-ABC and HLA-DR expression levels, supports their potential for "off-the-shelf" allogeneic applications.
Therapeutic Mechanisms and Advantages
ThyTregs offer several advantages over conventional blood-derived Tregs. Single-step CD25+ magnetic enrichment can isolate relatively pure thyTreg populations, reducing manufacturing complexity compared to the multi-step CliniMACS CD8-CD25+ isolation typically required for peripheral blood Tregs. The abundance of cells obtainable from a single infant thymus (200-300 million cells) exceeds the Treg numbers present in adult peripheral blood, potentially reducing expansion requirements.
The undifferentiated character of thyTregs may confer hypoimmunogenic properties, making them less likely to be rejected in allogeneic settings. This characteristic, combined with their stability and potency, positions thyTregs as attractive candidates for developing universal donor cell therapy approaches.
Future Directions and Challenges
Despite promising early results, several challenges remain in optimizing thyTreg therapy. The cells isolated using current methods contain thyTregs at different developmental stages, and their differentiation patterns after in vitro expansion require further investigation. Development of specific markers to isolate more homogeneous mature thyTreg populations would advance targeted therapeutic approaches, particularly for applications requiring genetic engineering such as CAR-Treg development.
Optimization of expansion conditions to achieve high purity, viability, and functionality remains critical for clinical applications. Additionally, the potential for combining thyTreg therapy with in vivo expansion strategies using low-dose IL-2 or engineered IL-2 molecules could eliminate the need for extensive ex vivo culture while enhancing therapeutic efficacy.
The expanding understanding of regulatory immune cell networks and the development of thymus-derived Treg therapies represent significant advances in immunotherapy. As clinical trials progress and manufacturing processes mature, these approaches hold promise for revolutionizing treatment of autoimmune diseases, transplant rejection, and immune-mediated disorders.
