Hematopoietic stem cell (HSC)-derived hematopoietic progenitors migrate into the thymus and develop into different types of T cells. The transcription factors Aire (largely expressed in medullary thymic epithelial cells - mTECs) and FoxP3 have key functions in clonal deletion and T_reg selection (3). There are links between Aire expression, FoxP3 up-regulation and T_reg selection; Aire deficiency affects the negative selection of self-reactive T cells, and FoxP3 controls the development and function of the naturally occurring T_regs (nT_regs) (4). Our laboratory has shown the development of stable T_regs from CD4⁺ T cells by over-expressing FoxP3 and bcl-xL (5).

Recent advances in the use of large-scale in vitro expansion of T_regs followed by in vivo re-infusion of these cells raises the possibility that this strategy may be successfully utilized for the treatment of T1D. Although polyclonally expanded populations of T_regs exhibit suppressive activity, Ag-specific T_regs are more efficient at suppressing local autoimmune disorders such as RA, type-1 diabetes (T1D), inflammatory bowel diseases (IBD), allergic reactions and graft-versus-host disease (GVHD) (67891011). In addition, tissue/organ-associated T_reg targeting stabilizes FoxP3 expression and avoids induction of a potentially detrimental systemic immunosuppression (1213). For T_reg-based immunotherapy, in vitro generation of tissue/organ (e.g., islets)-associated and non-terminally differentiated effector T_regs for in vivo re-infusion is an optimal approach. However, current methodologies are limited in terms of the capacity to generate, isolate, and expand a sufficient quantity of such T_regs from patients for therapeutic interventions.

There are a number of challenges in T_reg-based immunotherapy. First: Only low numbers of T_regs can be harvested from the peripheral blood mononuclear cells (PBMCs). CD4 and CD25 have been used to isolate T_regs for ex vivo expansion. CD4⁺CD25⁺ T cells are not homogenous and contain both T_regs and conventional effector T cells (T_effs). Current expansion protocols activate both T_regs and T_effs, and because it takes a longer time for T_regs to enter the S phase of cell cycle, T_effs outgrow T_regs (14). In addition, T_regs can lose suppressive activity after repetitive stimulation with α-CD3 plus α-CD28 Abs with or without rIL-2 in vitro. Second: No approach to date has demonstrated the capacity to isolate the entire Treg population with 100% specificity from patients (the current clinical approach). Even FoxP3 or more recently Eos, a transcriptional factor that is considered the gold standard for identification of T_regs, is expressed transiently in some activated non-regulatory human T cells (15), highlighting the difficulty in both identifying and isolating a pure T_reg population. Adoptive transfer of non-regulatory T_effs with T_regs has a potential to worsen autoimmune diseases. Third: Gene transduction of CD4⁺ T cells from PBMCs with Ag-specific TCR (16) or chimeric Ag receptor (CAR) (17) and/or TCR with FoxP3 elicits the generation of suppressive T cell populations (7) and overcomes the hurdle of the limited numbers of Ag-specific T cells. However, the engineered T_regs express endogenous and exogenous polyclonal TCRs, which reduce their therapeutic potential (the current experimental approach). Also, TCR mispairing is a concern with regards to the safety of TCR gene-transferred T_regs for clinical use, because the formation of new heterodimers of TCR can induce immunopathology (18). Therefore, there is a need to improve this strategy and generate monoclonal T_regs. Fourth: The differentiation state of T_regs is inversely related to their capacity to proliferate and persist. The "right" T_regs resist terminal differentiation, maintain high replicative potential (e.g., expression of common γc, CD132), are less prone to apoptosis (e.g., low expression of PD-1), and have the ability to respond to homeostatic cytokines (19), which facilitates their survival. In addition, the "right" T_regs express high levels of molecules that facilitate their homing to lymph nodes (LNs), such as CD62L and CC-chemokine receptors (e.g., CCR4, CCR7), and maintain stability or plasticity under certain inflammatory conditions. Furthermore, after an effective immune response, the "right" T_regs persist and provide protective immunity. Fifth: Because there are too few cells, harvesting sufficient numbers of tissue-associated T_regs from PBMCs for TCR gene transduction can be problematic.

Taken together, strong arguments support the development of T_reg-based therapies in autoimmune diabetes using engineered T_regs. While clinical trials show safety, feasibility, and potential therapeutic activity of T_reg-based therapies using this approach, concerns about autoimmunity due to cross-reactivity with healthy tissues remains a major safety issue (2021). In addition, genetically modified T_regs using current approaches are usually intermediate or later effector T_regs (22), which only have short-term persistence in vivo.

Stem cells have the ability to differentiate into Ag-specific T_regs which can be used for cell-based therapies. To date, pluripotent stem cells (PSCs) are the only source available to generate a high number of the "right" T_regs (1123). Human iPSCs can be easily generated from patients' somatic cells by transduction of various transcription factors and exhibit characteristics identical to those of embryonic stem cells (ESCs) (24). Many genetic methods as well as protein-based approaches have been developed to produce iPSCs with potentially reduced risks, including that of immunogenicity and tumorigenicity (25). Because of the plasticity and the potential for an unlimited capacity for self-renewal, iPSCs have high potential for advancing the field of cell-based therapies.

Our laboratory was the first to show that the development of Ag-specific iPSC-CTLs or iPSC-T_regs can be used for cell-based therapies of cancers and autoimmune disorders (11232627); other groups reported similar results (282930). We demonstrated that genetically modified iPSCs with Ag-specific T cell receptor (TCR) and the transcriptional factor FoxP3, followed by differentiation driven by Notch signaling can enable iPSCs to pass hematopoietic and T lineage differentiation checkpoints, resulting in the development of Ag-specific CD4⁺ T_regs. We have developed a novel system to generate stable auto Ag-specific iPSC-T_regs. Our ongoing studies will validate this system and provide new insights into the methodologies and mechanistic requirements for efficient development of inflamed tissue-associated iPSC-T_regs. Once such strategies become available, there is potential to facilitate the generation of tolerance for autoimmune diabetes. Thus, important advances towards T_reg-based immunotherapy in autoimmune disorders are anticipated from the proposed studies.

Signaling mechanisms that direct differentiation of Ag-specific PSC-T_regs remain to be determined. PSCs are exposed to a number of signals responsible for their progression. Although the exact signals are not fully understood, part of the mechanism known to be critical for directing T-cell fate occurs via Notch signaling, an evolutionarily conserved signaling pathway that regulates cell fate decisions in a number of cell and tissue types. Ligand binding by members of the Jagged or Delta-like (DL) families results in the proteolytic cleavage and release of the intracellular fragment of the Notch heterodimer. Translocation to the nucleus then allows for its regulation of gene expression. Notch-1, specifically, is critical for the establishment of T-cell fate. The loss of function results in the blockade of T cell development and enhanced B cell production, while over-expression results in the blockade of B cell lymphopoiesis and leads to the generation of T cells (31). However, the intracellular signaling pathways by which Notch signaling regulates the differentiation of Ag-specific PSC-T_regs remain unknown. PSCs co-cultured on a monolayer of the bone marrow (BM) stromal cell line OP9 cells transfected with the Notch ligand DL1 or 4 exhibit the ability to differentiate into most hematopoietic lineages and T cells (28). Our current studies will determine critical regulations of Hes1 (32), Runx1 (33), survivin (34) and elongation factor-2 (eEF-2) kinase (35) by Notch signaling during the development of auto Ag-specific PSC-T_regs.

While auto Ag-specific iPSC-T_regs have likely therapeutic effects in T_reg-based immunotherapy against autoimmunity, the effectiveness is restricted by the demand to develop a great number of cells by complicated and exclusive in vitro differentiation. Furthermore, the extensive period for performing the generation of iPSCs can constraint the practice in personalized treatments. As an alternative, we will implement T_reg-based immunotherapy by using the TCR/FoxP3 gene-transduced iPSCs, which have the ability to develop auto Ag-specific iPSC-T_regs in vivo and subsequently control autoimmune diabetes. We will conduct diabetes induction before or after adoptive transfer of the gene-transduced iPSCs. We will then administrate Notch agonists or recombinant cytokines (e.g., rIL-7, rFlt3L) to improve in vivo development of auto Ag-specific iPSC-T_regs. To avoid the potential tumorigenicity of the gene-transduced cells, we will incorporate a suicide gene, the inducible caspase 9 (36), into the vector as this allows the removal of the transduced T_regs by the injection of a bioinert small-molecule dimerizing agent (AP1903) to "shut off" the system.

In summary, a current roadblock to progress in the field is the lack of an efficient system to generate the "right" auto Ag-specific T_regs that could be used for cell-based therapies in autoimmune diabetes (Fig. 1). We are using PSC-T_regs to address this limitation, allowing derivation of a large number of stable auto Ag-specific PSC-T_regs for cell-based therapies. Development of such an approach provides an important step toward personalized therapies for T1D.