Introduction to Regulatory T Cells
The human adaptive immune system can respond to an infinite array of foreign signals while remaining tolerant to self-cells and tissues. This ability of the immune system to tolerate “self” antigens (and not attack the body’s own tissues), is supported by a complex system of regulatory mechanisms, involving different types of immune cells and signaling pathways, which work together to maintain a balanced immune response and prevent autoimmune reaction.
T regulatory cells (Tregs) are a specialized subset of immune cells that actively suppress the activation and subsequent function of other immune cells (1). First identified in the 1990’s, Tregs have gained the interest of immunologists and translational scientists alike given their potential therapeutic application in an array of inflammatory diseases (1-3).
Dr. Megan Levings, a professor in the Department of Surgery and School of Biomedical Engineering at the University of British Columbia and BC Children’s Hospital Research Institute, is an accomplished investigator in Treg biology. Dr. Levings is focused on the elucidation of critical biological mechanisms of Tregs as well as the application of these cells in clinical indications. We sat down with Dr. Levings to discuss her past experiences in the Treg space, to understand the current clinical status of Tregs in the treatment of autoimmunity, and to note the current challenges and future trends in the space.
Journey as a Scientist and Immunologist and Current Research Focus
Dr. Levings did her early training in basic biology and then moved onto a Ph.D. at UBC in genetics. Although she enjoyed her work, she felt a calling to a more clinical application and found the emerging and, at that time, hotly contested field of regulatory T cell biology. Dr. Levings went on to study Tregs at the San Raffaele Scientific Institute in Milan, Italy. After returning to Canada, Dr. Levings started a unique, human immunology-focused research program at UBC. After many years of “at the bench” science, one aspect of her program is now focused on the clinical translation of Treg therapy to clinical testing. The furthest advanced approach involves isolation of Tregs from human thymus tissue, which is often removed and discarded generated during routine, pediatric cardiac surgery. Given the benefits observed in preclinical models, this novel source of allogeneic Tregs is moving forward to clinical evaluation as a therapy to prevent graft vs host disease (GvHD) (4,5). Further, Dr. Levings noted that these thymic Tregs can also be used in the autologous format in the context of heart transplantation (6).
Tregs in Autoimmune Diseases
Dr. Levings noted that the basic function of Tregs, often defective in autoimmune conditions, is what makes them an attractive cell type for further clinical development. “They are built to suppress autoimmune response. In common forms of autoimmunity, it is logical to seek ways to restore the defective function of this cell type” Dr. Levings noted. She went on to state that other potential applications, such as chronic inflammatory diseases including atherosclerosis, stroke, and Parkinson’s disease are emerging as potential targets for Treg therapy.
Dr. Levings went on to discuss the underlying multimodal mechanisms involved in Treg-mediated immune tolerance in the context of autoimmunity. These include
• The trans endocytosis (trogocytosis) of key molecules on effector immune cells, such as CD80 and CD86 on antigen presenting cells through high levels of CTLA4, resulting in blunting of the overall immune response.
• The expression of T cell inhibitory cytokines, such as TGF-beta, is another key mechanism of Tregs. TGF-beta can induce de novo FOXP3 expression, which in turn limits T cell proliferation and proinflammatory function.
• The inability of Tregs to produce the T cell survival factor IL-2 and then act as an IL-2 “sink” to deprive other immune cells of the critical cytokine.
The Thymus as an Alternative Source of Tregs
Traditionally, Tregs have been isolated from peripheral blood. However, due to their low occurrence, isolating homogeneous population of these cells in sufficient numbers to generate a therapeutic dose has proven difficult. Dr. Levings highlighted some of the key advantages of utilizing Tregs from pediatric thymus tissue, obtained through the removal of part of the thymus during routine surgery. These include:
• The stable expression of FOXP3 and long telomeres.
• Functional capability to suppress proliferation and cytokine production of activated allogeneic T cells in vitro
• More potent suppression in xenogenic GvHD in vivo models than their peripherally isolated counterparts (7,8).

Figure: Optimized protocol to expand and cryopreserve allogeneic thymic Tregs (adapted from Mcdonald et al, Cytotherapy, 2019.)
Dr. Levings also noted Thymic Tregs are not limited in terms of cell number, as is the case with peripheral Tregs. However, thymic Treg isolation has been a challenge for multiple reasons. Dr. Levings stated that the initial amount of surgical specimen obtained can be quite variable, affecting the overall yield of Tregs from individual surgical specimen. Further, the actual isolation process has been a particular issue due to the incompatibility of standard reagents with thymic tissues. “Commercial T cell selection reagents are not designed for thymuses so we had to develop a custom process” said Dr. Levings. Finally, the thymus contains an additional population of progenitor Tregs, identified by their CD25+FOXP3- status. Once stimulated, Dr. Levings stated that these cells will differentiate into fully mature Tregs (9).
Scaling Treg Therapies: Workflow Steps, Challenges, and Solutions
Given the therapeutic potential of Tregs across a multitude of different inflammatory and autoimmune disease conditions, determining how to scale the manufacturing of these cells is crucial. Ideally, true Tregs must exhibit high levels of the transcription factors FOXP3 and HELIOS, lack IL-2 production, as well as exhibit a highly unmethylated status of the TSDR locus. However, Dr. Levings noted that culture conditions used during Treg expansion can potentially change these cells and reduce their function once delivered in vivo. “One of the challenges of Tregs is you don’t want them to expand uncontrollably. Methods that drive rapid cell proliferation, tend to result in a downregulation of FOXP3” stated Dr. Levings.
Thus, cell activation and cell growth must be gentle as well as consistent. To this end, CTS™ Dynabeads™ Treg Xpander beads are ideal candidates for Treg manufacturing. These superparamagnetic beads are coated with an optimized combination of anti-human CD3 and anti-human CD28 antibodies and are intended for ex vivo activation and expansion/proliferation of human Tregs. “The reason we chose these beads (Dynabeads™) for our process was the consistency in our process…in FOXP3 expression” said Dr. Levings. Likewise, previous studies have demonstrated that the use of CTS™ Dynabeads™ Treg Xpander gave the best combination of Treg fold expansion, viability, and FOXP3 expression (9).
Advancing Tregs Beyond the Bench
Specific approaches can be utilized to increase the potency as well as reduce the demand for cells in a therapeutic context. One key tactic is to engineer chimeric antigen receptors (CARs) into Tregs. Although the approach seems straight forward, signficant technical hurdles have been encountered. Dr. Levings stated that key learnings obtained in engineering T cells for oncology applications rarely hold true for Tregs. Thus, a key question that has emerged from the field is centered around optimized constructs to provide CARs with optimal function in the context of Tregs. Further, Dr. Levings raised the question of how these engineered Tregs would function in different patient populations with variable immunosuppressant regimens used as a standard of care. She noted that this question of optimal construct design and Treg interface with different immunosuppressants was a core focus of her lab currently.
However, even with the construct complexities and variable patient backgrounds that are the new norm, Dr. Levings noted two independent clinical studies utilizing CAR Treg. The first, a Phase 1/2 led by Sangamo Therapeutics, is focused on utilizing genetically modified Treg in the context of kidney transplantation to significantly reduce the levels of immunosuppressant drugs that have detrimental long term side effects. The second trial, sponsored by Quell Therapeutics, is an HLA-A2 targeted CAR Treg that aims to eliminate the need for lifelong immunosuppression after liver transplant.
Final Thoughts
Discovered three decades ago, investigators like Dr. Levings are still unraveling the complex and intricate network of Treg-mediated immune system regulation. In the process, Dr. Levings is helping to develop Tregs into a therapeutic for a vast array of inflammatory and autoimmune disorders. As an industry, a healthy preclinical pipeline of novel Treg-based therapies will soon make the transition into the clinic. Together, these Treg-based therapies will give hope to the patients who need them most.
For more information on how Thermo Fisher can support your cell therapy manufacturing journey, please visit our website here.
For a deeper dive into the expansion methods for Tregs, read this article.
References
1. Ferreira, L. M. R., Muller, Y. D., Bluestone, J. A. & Tang, Q. Next-generation regulatory T cell therapy. Nature Reviews Drug Discovery vol. 18 749–769 Preprint at https://doi.org/10.1038/s41573-019-0041-4 (2019).
2. Esensten, J. H., Muller, Y. D., Bluestone, J. A. & Tang, Q. Regulatory T-cell therapy for autoimmune and autoinflammatory diseases: The next frontier. Journal of Allergy and Clinical Immunology 142, 1710–1718 (2018).
3. Tang, Q. & Bluestone, J. A. Regulatory T-cell therapy in transplantation: Moving to the clinic. Cold Spring Harb Perspect Med 3, (2013).
4. Steiner, D. et al. Overcoming T cell-mediated rejection of bone marrow allografts by T-regulatory cells: Synergism with veto cells and rapamycin. Exp Hematol 34, 802–808 (2006).
5. Pierini, A. et al. Donor Requirements for Regulatory T Cell Suppression of Murine Graft-versus-Host Disease. The Journal of Immunology 195, 347–355 (2015).
6. Bernaldo-de-quirós, E. et al. First-in-human therapy with Treg produced from thymic tissue (thyTreg) in a heart transplant infant. Journal of Experimental Medicine 220, (2023).
7. Hoeppli, R. E. et al. Tailoring the homing capacity of human Tregs for directed migration to sites of Th1-inflammation or intestinal regions. American Journal of Transplantation 19, 62–76 (2019).
8. Dijke, I. E. et al. Discarded human thymus is a novel source of stable and long-lived therapeutic regulatory T cells. American Journal of Transplantation 16, 58–71 (2016).
9. MacDonald, K. N. et al. Cryopreservation timing is a critical process parameter in a thymic regulatory T-cell therapy manufacturing protocol. Cytotherapy 21, 1216–1233 (2019).
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