Crohn’s disease (CD) and ulcerative colitis (UC) are the major types of inflammatory bowel disease (IBD). The prevalence and incidence of these inflammatory disorders have risen over the past decade (1, 2). Currently, a stable, yet high incidence level was observed in countries such as North America and Europe, while the incidence increased significantly in newly industrialized countries, including Africa, Asia, and South American countries, such as Brazil (1).

CD is an inflammatory disease that involves all layers of the intestinal wall and can affect any part of the gastrointestinal tract. It is characterized by episodes of remission and exacerbation (3). CD can have extra-intestinal manifestations, such as oral, musculoskeletal, dermatological, or ocular manifestations (4, 5). Both sexes are equally affected and the median age of onset is 30 years, with two peaks of onset between 15 and 30 years and around 50 years. Symptoms include diarrhea, abdominal pain, weight loss, and nausea. Oral manifestation symptoms contain diffuse lip or mouth swelling, esophageal lesions redness and edema (4, 6). UC is often diagnosed in late adolescence and early adulthood affecting both sexes equally (7). Inflammation is typically restricted to the mucosa, begins in the rectum and may extend continuously through the colon with variable extent or involve the entire mucosal surface (7, 8). Over time, there is often an alternation between periods of progression and regression of the inflammation (7). Symptoms include rectal bleeding, bloody diarrhea, rectal urgency, anemia, weight loss, fever, tachycardia, tenesmus, or nausea (7, 9).

The pathogenesis of IBD is multifactorial as genetic factors, such as heredity or mutations and non-coding variations, as well as environmental factors, including diet, microbiota, and medication influence the development of disease (10, 11). Another key factor is the dysregulation of the immune system, including the initiation of inflammation by triggering a pro- and anti-inflammatory cascade. Immune cells, such as B cells, T cells, macrophages, dendritic cells, and neutrophil granulocytes infiltrate the effector site where the immune response is dysregulated. The infiltrating cells secrete cytokines, such as TNF, interferon-gamma (IFN-γ), interleukins, and other cytokines that activate pro-inflammatory pathways (11, 12). The loss of Treg number and function in the gut wall significantly contributes to the onset of inflammation in IBD (13–15). Current medical treatments for IBD are limited in their efficacy (16), so novel therapeutic approaches are necessary.

Consequently, Tregs have emerged as a promising target for therapeutic interventions in IBD. Thus, in order to achieve disease specificity and re-direct Tregs to the site of inflammation, Tregs with a genetically engineered chimeric antigen receptor (CAR) have been developed (17). Up to now, a few promising pre-clinical studies in IBD models have produced encouraging results, paving the way for subsequent clinical studies. Those already developed CAR Treg models include CEA-specific, Flagellin-specific, and IL23R-CAR Tregs. Prior to clinical translation, it is imperative to address safety concerns, limitations in availability, prevention of CAR Treg exhaustion, and enhancements in phenotypic stability and suppressive function (17, 18).

The following review will give an overview of Tregs and the current state of research on Tregs in IBD as well as the various engineered CAR Treg models that have been developed for the treatment of IBD. With this we highlight the emerging potential of CAR-engineered Tregs as a novel therapeutic option.

One novel tool to generate antigen-specificity is the use of chimeric antigen receptors (CARs). CARs are synthetically designed to bind to specific target antigens. The advent of CAR T cells can be traced back to 1989, when Zelig Eshar and Gideon Gross first developed engineered T cells (80, 81). Since then, there has been significant progress in the development of CAR constructs, with the emergence of more than four generations of these engineered receptors ( Figure 4 ). The utilization of CARs confers numerous benefits, including the capacity to overcome HLA restrictions and enhance antigen specificity (18, 82, 83).

The target antigen is bound through a single-chain variable fragment (scFv) recognition domain originating from an antibody, which allows the specificity of the CAR construct. The scFv domain contains a light (V_L) and heavy (V_H) variable fragment, which are connected through a linker sequence. The order of the variable fragments, as well as the length of the linker sequences, has been demonstrated to influence the affinity and the stability of the receptor (84–86). The spacer domain contains sequences from CD28 or CD8α or from CH2 or CH3 domains of IgG1, 2, or 4 (87, 88). The length of the spacer domain has been shown to influence the distance between the effector and target cell (84). To anchor the CAR construct, a hydrophobic alpha helix transmembrane (TM) domain is connected to the spacer domain. The TM sequence of the first generation of CARs is derived from the CD3 TM domain (89). In the subsequent generations of CAR constructs, transmembrane sequences from CD4, CD8α, or CD28 were employed (87).

The first generation of CARs only consists of the T cell receptor (TCR) signaling chain CD3ζ, which functions to mediate T cell activation (90). However, the stimulation proved insufficient to achieve T cell expansion and elicit substantial therapeutic responses. In the subsequent second generation, an intracellular co-stimulatory domain, such as CD28 or 4-1BB, was incorporated. Studies have demonstrated that engineered CAR constructs with 4-1BB exhibited advantages in the persistence, while CARs with CD28 co-stimulatory domain revealed more rapid T cell activation (91–93). Third generation of CAR constructs were designed to enhance the T cell activating signal. Thus, two co-stimulatory domains from CD28, 4-1BB, OX40, CD27, or ICOS were combined (94). The incorporation of CD28 and an intracellular domain from the TNFR family into third-generation CAR constructs has been shown to facilitate the simultaneous activation of disparate signaling pathways (95, 96). The fourth CAR generation is based on the second-generation CAR and includes one co-stimulatory domain and an interleukin inducer domain, which enables the inducible release of IL-12 and IL-18 upon CAR activation (97, 98). This release of IL-12 or IL-18 creates a pro-inflammatory environment by recruiting and activating other immune cells, which in turn generate an immune response through further cytokine secretion and the promotion of exocytosis processes via granzyme or perforin. The fourth generation of CAR T cells are also referred to in the literature as TRUCKS, an acronym for “T cells redirected for antigen-unrestricted cytokine-initiated killing” (97–100). Subsequent exploration of additional CAR constructs was conducted in the context of next-generation CARs. One notable example involves the incorporation of a truncated IL-2 receptor β-chain domain, accompanied by a STAT3 binding site, within a next-generation CAR design (99).

The initial application of engineered CAR T cells was in the context of cancer therapies. Consequently, the initial documented success in these therapeutic interventions pertains to the administration of anti-CD19-CAR T cells in patients with advanced leukemia (101). Since then, the possibilities of CAR-T cell therapy have also been transferred to other diseases including allergic asthma (102), diabetes (103, 104), and IBD (105–107). Pre-clinical studies encompass an expanding array of CAR Treg models. A notable example is the development of CAR Tregs specific to β-amyloid for the treatment of Alzheimer’s disease. The efficacy of these cells has been assessed using murine brain lysates in preliminary studies (108). CAR Treg models for diabetes, including insulin B chain (InsB)-specific CAR Tregs, were developed and demonstrated suppressive effects in an adoptive transfer model of T cell-induced autoimmune diabetes in immunodeficient NOD mice, as reported by Spanier et al. (104). A number of HLA-A2-specific CAR Treg models have been engineered for utilization in transplant research, with an objective to avert graft-versus-host disease (GvHD) (109, 110). A number of Phase 1 studies of CAR Treg models are currently underway, including a study of CD6-CAR Tregs for the treatment of patients with chronic GvHD after an allogeneic hematopoietic cell transplantation (HCT) (111). The objective of this Phase 1 study is to ascertain the safety and tolerability of the intervention, as well as to assess its feasibility. In addition, the CAR Treg activity and persistence, as well as the alterations in immune biomarkers, will be monitored (111). A further clinical Phase 1/2 study includes HLA-A2 CAR Tregs (TX200-TR101) for a treatment of living doner kidney transplant recipients (112). The study’s objective is to assess the safety and tolerability of the treatment regimen, in addition to the range of doses that are administered. Additionally, immunosuppression and CAR Treg localization through renal transplant biopsies will be observed (112). A study of a similar nature utilizes HLA-A2 CAR Tregs (QEL-001) in order to prevent liver transplant rejection, as well in a clinical Phase 1/2 study (113). A further example is a clinical Phase 1 study with CAR Tregs specific for citrullinated proteins (Cit-P) for the treatment of refractory rheumatoid arthritis (114). As demonstrated in a series of pre-clinical and early clinical trials, CAR Tregs exhibit therapeutic potential for further applications in immune-related diseases.

As previously stated, Tregs possess the capacity to modulate the activities of immune cells, thereby regulating the inflammatory process in an anti-inflammatory manner. To this end, various models of genetically engineered CAR Tregs have been developed for the treatment of IBD using three different antigens upregulated in patients with IBD, including CEA-specific, Flagellin-specific CAR Tregs and IL23R-CAR Tregs.

Current therapeutic interventions for IBD, encompassing anti-cytokine treatments and immune cell trafficking/signaling blockers, exhibit limited efficacy (16). Consequently, the use of Tregs has emerged as a promising therapeutic strategy. As antigen-specific Tregs are more potent, genetically engineering CARs for antigen-specificity is a promising therapeutic approach. CARs can be tailored to suit specific requirements. The incorporation of a 4-1BB co-stimulatory domain has been shown to enhance persistence, while a CD28 co-stimulatory domain facilitates more expeditious signaling activation. The modular system can be utilized to develop new and optimized CAR T cells with longer persistence, improved stability, and faster signaling activation or coupling of other mechanisms. Genome-wide studies of IBD have enabled the identification of antigens associated with the disorder, which could be used for additional antigen-specific CAR constructs (125, 132, 133). Therefore, the potential use of IBD-dysregulated epithelial barrier proteins as antigens is possible. Examples include mucus layer protein Muc1 (Mucin-1) and desmosomal proteins such as Dsg3 (Desmoglein-3) (133).

For the regulatory function of Treg cells, the stable expression of FOXP3 as a master transcription factor is essential (134). It has been shown that repetitive in vitro stimulation of human Tregs with anti-CD3 and anti CD28 coated beads leads to loss of FOXP3 expression (135–137). Thus, the application of a Treg therapy in a disease of chronic inflammation could lead to a loss of FOXP3. The stabilization of the expression of this transcription factor could be addressed through the targeting of epigenetic modifiers, such as DNA methyltransferases, histone deacetylases, histone demethylases or methyltransferases, including EZH2 (138, 139). The expression patterns subsequent to Treg activation are influenced by EZH2 methyltransferase (139). Additionally, it has been demonstrated to directly regulate FOXP3 expression (139, 140). A further study observed enhanced expansion and an improvement in FOXP3 expression in response to a combination of IL-2, rapamycin, histone deacetylase, and DNA methyltransferase inhibitors (141). In other studies, an improved and stabilized FOXP3 expression was reported through the addition of vitamin C (142). Treg destabilization upon repeated TCR-stimulation might be rare (143–145), but the effect of repeated CAR stimulation on Tregs remains to be fully examined. Thus, it is worth further investigation considering the supraphysiological signaling through a CAR (146, 147).

In order for CAR Treg treatment to be applied in the clinic, there are a number of further key issues that need to be addressed before its clinical translation. Aside from showing the efficacy of CAR Treg treatment, the safety of this cell therapy will become paramount. The implementation of safety mechanisms, such as suicide programs, which have been developed for CAR effector T cell therapies, can be transferred to CAR Treg therapies. The incorporation of suicide cassettes has been demonstrated to serve the purpose of preventing dysfunctions or depleting the transferred engineered cells (138). Suicide genes that are currently under development include truncated epidermal growth factor receptor (EGFRt) and RQR8 (148). EGFRt is co-expressed with the CAR receptor, and the transferred cells can be eliminated with the application of an EGFRt-directed antibody, cetuximab (149). RQR8 combines parts from CD34 and CD20 antigens. The administration of CD20-directed antibodies, such as rituximab, has been demonstrated to enhance the susceptibility of target cells to lysis (148). Additionally, cells expressing RQR8 could be sorted by CD34 (148). The engineered CaspaCIDe, a safety switch technology, possesses the capability to, through the addition of rimiducid as the trigger, induce the dimerization of the CID domains in cases of toxicity. The subsequent initiation of the apoptosis pathway is catalyzed by iCasp9 (Caspase9) (150).

In other studies, the administration of dasatinib, a tyrosine kinase inhibitor, has been demonstrated to exert effects that block T cell function, with these effects being reversible upon removal (151). The efficacy of this effect may also extend to CAR Tregs.

In addition to efficacy and safety, ensuring the universal accessibility of CAR Treg therapy is imperative. A notable constraint pertains to the financial burden associated with CAR Treg therapy, in addition to the necessity for specialized equipment (17, 152). The provision of CAR Tregs as personalized products is a process that necessitates a substantial duration of production out of patient blood, accompanied by challenges related to the quality of the expanded cell product. Furthermore, IBD patients exhibit reduced Treg cell numbers, which consequently leads to challenges in isolating a sufficient number of cells and formulating a personalized therapy (152, 153). An enhancement in accessibility might be achievable through the standardization of the product and the process as well as the implementation of cryopreserved batches (154, 155). Off-the-shelf products are consistently available. However, they also reveal issues related to cell number, quality, and the time required to generate them. Additionally, HLA (human leukocyte antigen) compatibility is a critical factor in this process. In order to prevent alloreactivity, the generation of banks with different common HLA haplotypes is a potential solution (155). Alternatively, gene editing methods, such as the CRISPR-Cas9 system, can be utilized to eliminate T cell receptors (TCRs). One method involves the direct encoding of CAR DNA in the TRAC locus (156). Other methods involve the rendering of cells HLA-deficient (154, 157).

Many preclinical and clinical trials will have to be conducted before the ideal CAR Treg therapy for patients with IBD can become standard-of-care. This ideal CAR Treg product not only should be efficient, persistent, and safe but lead to a significant reduction in symptom and disease burden while remaining accessible and affordable ( Figure 5 ). Whether an off-the-shelf product or a personalized product with autologous Treg will be a more feasible approach needs to be addressed in future research.

Current IBD therapies are associated with limited success and severe side effects. As Tregs exert anti-inflammatory effects and the ability to suppress pro-inflammatory immune cells, the use of Tregs for the treatment of IBD is a promising approach. Using genetically engineered CAR constructs to guide Tregs to the site of inflammation can significantly enhance their function and therapeutic potential. These CARs have been developed and improved in function and efficacy in several CAR generations in recent years. The design of an appropriate CAR construct with respect to the antigen-specific target and the use in IBD opens new therapeutic opportunities. In preclinical studies, the anti-CEA, anti-flagellin, and anti-IL23R-CAR Tregs have been shown to be promising first developments for the treatment of IBD. Nevertheless, safety concerns must be addressed for clinical translation. The development of suicide programs, such as the incorporation of suicide genes (e.g., EGFRt and RQR8) or the utilization of CaspaCIDe technology in engineered Tregs to enhance safety, has emerged as a pivotal area of focus in contemporary studies, with the objective of facilitating clinical translation. Moreover, the issue of accessibility must be addressed by considering universal useable CAR Tregs that are devoid of allorejection reactions. In addition, the development of strategies to prevent CAR Treg exhaustion and to enhance a stable phenotype and suppressive function could be addressed through the addition of factors against epigenetic modifiers. Such modifiers include DNA methyltransferases, histone deacetylases, histone demethylases, and methyltransferases.

After considering the current limitations, the use of targeted, engineered Tregs to treat inflammation bowel diseases appears to be a promising therapeutic approach to overcome the limitation of current therapies.