Generation of alloantigen-specific Tregs. A) For the generation of monocyte-derived dendritic cells (Mo-DCs), CD14+ monocytes from PBMCs are stimulated with exogenous IL-4 and GM-CSF for 8 days. B) To obtain allospecific-Tregs, FACS-sorted CD4+CD25hiCD127- Tregs (tTregs) are stimulated with allogeneic Mo-DCs in the presence of retinoic acid and IL-2 for 7 days.
What?
Regulatory T cells (Tregs) are a subtype of CD4+ T cells that suppress the immune response and play a key role in preventing the development of autoimmune diseases as well as restraining exacerbated immune responses, thereby maintaining self-tolerance and immune homeostasis. Thymic Tregs (tTregs) and induced Tregs (iTregs) are characterized by the expression of a transcription factor Foxp3, considered as the master regulator of Treg phenotype and function (1). In contrast, Tr1 cells (Foxp3 negative Tregs) exert their suppressive function mainly by the production of IL-10 (2). Due to their antigen specificity Tregs are currently being used in clinical trials both to regulate autoimmune responses and to prevent the development of graft versus host disease (GVHD) and allograft rejection in solid organ transplantation. Our group has obtained three types of allospecific regulatory T cells (allo-Tregs) using either tTregs or naïve T cells that are expanded or de novo-induced (iTregs and Tr1) in vitro, respectively, in the presence of antigen presenting cells from a HLA mismatched donor. These cells have been denominated as allo-tTregs (3), allo-iTregs (4) and exp-allo-Tr1 (5). These allo-Tregs have the advantage over other cellular products to be highly pure and can be expanded to achieve a high number of antigen-specific Tregs that maintain their phenotype and suppressive function even in the presence of proinflammatory cytokines.
Why?
Current therapies to prevent long term allograft rejection are mainly based on the use of immunosuppressive drugs. Nonetheless, several unfavorable outcomes associated with long term immunosuppression have been documented, including the development of neoplasia and infections, as well as drug toxicity leading to renal/hepatic failure or cardiovascular disease, that results in a reduction of survival of transplanted patients (6). In this context, polyclonal Treg-based therapies allow for minimal immunosuppression regimes in kidney transplanted patients (7, 8). Interestingly, animal models have indicated that the use of antigen-specific Tregs may be more effective at inducing long term tolerance (9), providing an advantage over the use of polyclonal T regs in that, lower numbers of allo-Tregs may be needed to achieve tolerance in the infused patients and, additionally, this could result in a significant reduction in the production costs (10, 11). Although the obtention of a pure population of allospecific Treg cells required for application to patients was initially a difficult challenge to overcome, we have demonstrated that it is possible to highly purify and effectively in vitro expand allospecific Treg populations, which are phenotypically and functionally stable, and can be expanded at a large scale with a potential clinical use in kidney transplanted patients (3-5).
Who?
Initial studies performed in a number of research labs investigated the biology of Tregs and their potential role in inducing and/or maintaining tolerance in specific inflammatory conditions (allograft rejection, autoimmunity, GVHD etc). Different Treg-based products have been produced and scaled up in compliance with current Good Manufacturing Practice (GMP), and Phase I and Phase II clinical trials have demonstrated the safety and efficacy of polyclonal Treg infusion both in hematopoietic stem cell transplantation (HSCT) and solid organ transplantation settings (12). However, only a few trials have been conducted using allospecific Tregs (13). The promising results obtained so far have supported the establishment of new companies aiming to produce Treg products for implementing personalized immunotherapy for the treatment of autoimmunity and or for transplantation (12). Even though companies have reaped the benefits of developing these new therapies, research laboratories continue to conduct research to improve or develop more and better protocols for the use of these therapies in various autoimmune diseases and in transplantation (e.g. kidney transplant, which is the most urgent need worldwide) and to bring these new treatment options to developing countries. Our group is setting up the first GMP facility for Treg therapy in Mexico, to produce three Treg subtypes (tTreg, iTreg, and Tr1 cells) and will be used for the first time in Mexican patients undergoing a kidney transplant, providing a new promising antigen specific Treg therapy (3-5).
When?
In the 1990s, the role of Tregs in suppressing inflammatory responses was described. Since then, different preclinical models have shown that Tregs are essential to induce long-term tolerance to allografts. Since 2009, the first therapy with Tregs was performed in two patients with GVHD (14) and several phase I or I/IIa clinical trials have been initiated or completed to evaluate the feasibility and efficacy of infusion of Tregs to prevent allograft rejection. In early trials, transfer of polyclonal Tregs in the perioperative period was reported to reduce rates of acute and chronic GvHD, opportunistic infections or other adverse effects (15-19), even in the absence of immunosuppression (17, 18), supporting the therapeutic potential of Tregs to prevent the development of GvHD. Moreover, for solid organ transplantation, trials using autologous polyclonal Tregs in kidney (7, 8, 20-22) or liver (23) transplanted patients showed no serious adverse events attributable to Treg infusion and no increased rejection episodes. Interestingly, three studies (7, 8, 22) reported a lower incidence of opportunistic infections in patients treated with polyclonal Tregs, and more importantly, 39% to 73% of those, were maintained with immunosuppressive monotherapy. Overall, these trials demonstrated that adoptive polyclonal Treg cell therapy is feasible, safe, and potentially effective in the transplant setting. However, no clinical trials administering purified alloantigen-specific Tregs in organ transplanted patients have yet been published.
Where?
Currently, countries in the EU and the USA have carried out various studies based on the transfer of Tregs. In the context of HSCT, studies have been carried out in Poland (14), USA (15, 16, 19) and Italy (17, 18). It is important to highlight the ONE Study, a pioneering international multicenter phase I/IIa study that included eight hospitals in France, Germany, Italy, UK and the USA, to test the safety and feasibility of cellular therapies in kidney transplantation (22). The information collected in these clinical trials has been essential for the design of new therapeutic strategies with Tregs in humans (12).
How?
Regulatory T cells can be obtained from peripheral blood by isolating CD4+ T cells that are highly positive for CD25 and have low or null expression for CD127 (CD4+CD25++CD127 lo/-) (called tTregs). Alternatively, Tregs can be generated from naïve T cells by stimulating them in vitro through the TCR in the presence of IL-2 plus TGF-β (called iTregs) or IL-10 (called TR1). To obtain a large number of regulatory T cells, they need to be expanded through several cycles of stimulation and resting. This can be achieved by using anti-CD3/CD28 beads to obtain polyclonal regulatory T cells or by using antigen-presenting cells such as monocyte-derived Dendritic Cells to obtain antigen-specific Tregs that are then FACS-purified and expanded. Specific conditions are required for cellular expansion to maintain their phenotype and regulatory function. These conditions include the addition of IL-2 and rapamycin (for tTregs), as well as TGFβ (for iTregs) or IL-10 (for TR1). To use Tregs in clinical therapy, their cellular production must meet GMP requirements and fulfill the established product release criteria (24). Currently, one of the main challenges currently is to obtain highly pure and stable Treg cells after long-term expansion, as both Foxp3 expression downregulation and epigenetic modifications may result in impaired Treg function. Several strategies have been reported to ensure long-term stability, such as the use of other surface markers (CD27+, CD45RA+) for peripheral blood isolation of tTregs (25) or the addition of epigenetic modifiers such as vitamin C during the generation of iTregs (26). Moreover, tonic stimulation of Tregs can result in Treg exhaustion, which has been associated to the expression of a specific gene-profile , which may interfere with the in vivo long-term Treg suppressive function (27). However, this could be resolved by limiting the number of expansion cycles or by using a population of Tregs that exhibit more effective suppressive functions, such as with the use of antigen-specific Tregs (9). On the other hand, in contrast to commonly used methods for Treg purification based on magnetic separation (CliniMacs Miltenyi), which have the advantage of providing a fully-closed system, highly purified antigen-specific allo-Tregs can be obtained after aseptic FACS (Fluorescence Activated Cell Sorting) (i.e. FACS Aria Fusion from Becton & Dickinson) with the additional challenge to fulfill the sterility requirements of a GMP grade product. Additionally, gene editing systems such as CRISPR/Cas are promising innovations to obtain a highly stable population for the use of Tregs in immunotherapy (28).
Did you know that… the importance of regulatory T cells is reflected in the number of clinical assays approved by the FDA, where Tregs are used for the treatment of autoimmune, inflammatory diseases, graft-versus-host disease, and organ/tissue allograft rejection. In addition to their role in regulating the immune system, Tregs can exert various other functions in non-lymphoid tissues, including tissue repair, regulation of angiogenesis, maintenance of stem cells, and modulation of metabolism, among others (29). Finally, the diverse activities of Tregs suggest that they can be used for treating a wide range of other disorders, such as myocardial infarction, ischemic brain injury or even exercise-induced muscle inflammation, where Tregs confer protection to damaged tissue and promote its reparation (30-32).
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