In the late 1990s, a clone was found to exhibit identity to the death domains (DDs) of cluster of differentiation 95 (CD95; 23% identity) and tumor necrosis factor receptor-1 (TNFR-1; 47% identity), which are both members of the TNFSF (11). Additionally, the homologies of the cysteine-rich repeats between the clone and CD95 and TNFR-1 were 22 and 26%, respectively (11). Therefore, the clone was classified as a member of the TNFSF and was designated as DR3 (TNFRSF25, APO-3, TRAMP, WSL-1, TR3 or LARD) (11,12). DR3 is encoded by the Tnfrsf25 gene located in the 4E1 region of the mouse chromosome (13) and 1p36.3 of the human chromosome (4). As a type I transmembrane protein with a calculated molecular weight of 45 kDa and 417-amino acid (aa) sequence, full-length DR3 is composed of an N-terminal signal sequence (aa 1-24), a repeat sequence consisting of four cysteine residues, two potential N-linked glycosylation sites (aa 25-198), a transmembrane domain (aa 199-244), and an intracellular domain with a DD (aa 255-417) (14,15). Notably, DR3 is exclusively expressed in lymphocyte-rich tissues, including the thymus, spleen, colon and intestine (11); however, low levels of expression are detected in the fetal lung (16), kidney (17), hippocampus (18) and peritoneal tissue (12). At the cellular level, DR3 is mainly expressed in immune cells, including naïve or resting CD4⁺ T cells, CD8⁺ T cells, natural killer T cells, innate lymphoid cells (ILCs), B cells and mononuclear cells (10,19). Notably, sustained expression of DR3 is found in Tregs. However, DR3 also exists in non-immune cells, including bone cells (20) and endothelial cell colony-forming cells (21). Expression of DR3 is highly regulated in multiple pathologies. The levels of DR3 are significantly increased in inflammatory bowel disease (IBD) (22), pulmonary sarcoidosis (23) and psoriasis vulgaris (24). By contrast, DR3 expression in mononuclear cells is downregulated in diseases, including sickle cell anemia (25) and colon cancer (26).

TL1A, also known as vascular endothelial growth inhibitor (VEGI)-251 or TNFSF15, is the only proven ligand for DR3 to date (5). TL1A is a single-pass type II transmembrane protein encoded by the Tnfsf15 gene on chromosome 4 in mice and 9q32 in humans (4). Similar to other TNFSF cytokines, membrane-bound TL1A can be cleaved by alternative splicing or through TNF-α-converting enzyme, and is shed from the extracellular domain to form soluble TL1A (sTL1A) (4,5,27). Notably, sTL1A may continue to efficiently activate DR3. Endothelial cells, dendritic cells (DCs), monocytes/macrophages, activated T cells and human umbilical vein endothelial cells have been identified as major sources of TL1A (5,28-30). Additionally, the expression of TL1A on DCs and macrophages is increased upon activation via the receptor for the Fc region of IgG (FcγR) and Toll-like receptors (TLRs), including TLR2 and TLR4, while expression of TL1A on endothelial cells may be enhanced by interleukin-1 beta (IL-1β) and TNF-α stimulation (15). Nevertheless, chondrocytes, synovial fibroblasts, tissue macrophages and lamina propria lymphocytes also express TL1A at low levels under conditions of inflammation or upon stimulation (5,6). Therefore, the binding of TL1A to DR3 triggers signal transduction and exerts pro- and anti-inflammatory functions.

A subset of CD4⁺ T cells activated by the DR3/TL1A signaling serves a pro-inflammatory role under established conditions. Decreased lung inflammation in DR3^-/- mice is attributed to fewer CD4⁺ T cells in the alveolar passage following an ovalbumin (OVA) challenge (35). TL1A, alone or in combination with IL-12 and IL-18, has been found to boost the secretion of interferon-γ (IFN-γ) and TNF-α, which are characteristic cytokines produced by Th1 cells (19,36,37). Activation of the DR3/TL1A pathway may aggravate disease progression by boosting Th1 immune responses in SAMP1/YitFc (SAMP) mice with ileitis (28). Furthermore, Th2-induced immunopathology is exacerbated by the non-specific stimulation of lymphocytes by DR3 signaling. Th2-mediated immune responses result in high levels of IL-5 and IL-13 produced in the intestinal mucosa, as well as aggravated inflammation in SAMP mice with ileitis via DR3/TL1A signaling (4,28,38). Additionally, Th2 cells, activated by the DR3 axis and accompanied by high IL-13 production, have been implicated in allergic inflammation in mouse models (39,40). Th17 cells show higher expression of DR3 compared with Th1 and Th2 cells (41). However, the role of DR3 signaling in Th17 differentiation is controversial. Inhibition of Th17 cell generation by DR3 signaling has been demonstrated in CD4⁺ T cells and spleen cells from humans and wild-type mice, respectively (42). By contrast, other studies have demonstrated that TL1A promoted Th17 cell differentiation and participates in the development of inflammation, such as rheumatoid arthritis (RA) (43) and experimental autoimmune encephalomyelitis (EAE) (44). Notably, the proliferation of Th17 cells was augmented upon DR3 activation. Pappu et al (44) verified that the decreased proliferation of Th17 cells in TL1A-deficient mice ameliorated the pathology of EAE. This evidence indicates that the DR3/TL1A axis aggravates inflammatory reactions triggered by activated Th17 cells. A recent study demonstrated that the DR3/TL1A pathway drives the differentiation of naïve T cells to Th9 cells, aggravating inflammatory pathology in an allergic lung inflammation model (7). Similarly, preclinical and clinical studies in chronic colitis have demonstrated that TL1A facilitates the differentiation of Th9 cells and increases IL-9 secretion, aggravating intestinal inflammation (45). Additionally, Tsuda et al (46) reported that TL1A induced the expression of basic leucine zipper ATF-like transcription factor 3 to promote Th9 cell differentiation and IL-9 production to drive intestinal inflammation.

In addition to T cells, the DR3/TL1A pathway co-stimulates ILCs. ILC2s and ILC3s are subsets of ILCs. DR3 signaling can expand ILC2s and ILC3s, driving IL-5 and IL-13 secretion from these cells. In vivo experimental evidence has demonstrated that ILC2s cause deterioration in allergic lung inflammation (34,35,47). By contrast, ILC3s exert positive effects by increasing IL-22 production. Loss of DR3 signaling with decreased IL-22 production has been associated with a higher histopathological score in a DR3-deficient, dextran sulfate sodium (DSS)-induced colitis model (33,48). Furthermore, an anti-DR3 antibody triggered the loss of ILC3s from the intestine, ultimately aggravating the colitis condition (32).

The DR3/TL1A pathway has been clearly implicated in several diseases, including allergic lung inflammation (35), psoriasis (5), IBD (4) and rheumatoid arthritis (43). The aforementioned diseases share a common molecular mechanism in which DR3 signaling exerts pleiotropic effects on the activation, differentiation, proliferation and cytokine release of ILC2s and T helper cells (including Th1, Th2, Th9 and Th17 cells). Therefore, these findings suggested that pharmacological blockade of the DR3/TL1A axis may have therapeutic value for human inflammatory and immune-mediated diseases. An anti-TL1A monoclonal antibody (anti-TL1A mAb) targeting the DR3/TL1A pathway has emerged as a promising anti-inflammatory therapeutic agent in preclinical studies (5,6,31,49). Furthermore, unlike anti-TNF, anti-TL1A mAb may be relatively safe and effective because it does not induce immunodeficiency (50).

It is evident that TL1A/DR3 signaling dampens gut immune homeostasis and accelerates disease progression (4). Thus, blocking the TL1A/DR3 pathway may be a useful strategy for relieving IBD. Studies in animal models have demonstrated that anti-TL1A mAb exerts a protective function in DSS-induced chronic colitis by suppressing Th1 and Th17 cell activation (51). Additionally, in acute 2,4,6-trinitrobenzene-sulfonic acid (TNBS)-induced colitis involving increased production of Th1 cells and IFN-γ, blocking the DR3/TL1A axis with anti-TL1A mAb almost completely prevented weight loss and alleviated the histological score of inflammation (52). However, administration of anti-TL1A mAb, a neutralizing agent, mitigated the early phase, but not the late stage of TNFΔARE/⁺ ileitis, thereby suggesting the existence of other DR3 ligands aside from TL1A (31). Of note, the aforementioned studies have only demonstrated a decreased in inflammation caused by anti-TL1A mAb rather than alleviation of intestinal fibrosis, a hallmark of IBD. In DSS, as well as in the adoptive T cell transfer chronic colitis models, treatment with anti-TL1A mAb resulted in a decrease in the number of myofibroblasts and fibroblasts, leading to a reversal of colonic fibrosis (53). Furthermore, Li et al (54) discovered that intestinal inflammation and fibrosis were attenuated in T cell transfer chronic colitis models. Clarke et al (6) also confirmed that anti-TL1A mAb significantly alleviated the clinical pathology of IBD by decreasing inflammatory cell filtration and colonic fibrosis. In general, therapeutic blockade of the DR3/TL1A pathway by anti-TL1A mAb has numerous advantages, including amelioration of intestinal inflammation and reversal of colonic fibrosis, compared with traditional treatment for IBD, which only controls intestinal inflammation.

In addition to IBD, blocking the DR3/TL1A pathway with anti-TL1A mAb relieves collagen-induced arthritis (49), strongly reduces inflammatory cell infiltration in OVA-induced asthma (6), and effectively alleviates the histopathological changes in a psoriasis-like mouse model (5). Taken together, these results suggested that anti-TL1A mAb has therapeutic potential for treating inflammatory and immune-mediated diseases (Table I).

Tregs are a subset of CD4⁺ T cells that function primarily to maintain the homeostasis of immune function by negatively regulating effector lymphocyte activation and proliferation (55). Tregs are mainly characterized by CD4, CD25 and Foxp3. Williams and Rudensky (56) reported that, rather than acquiring the ability to produce IL-2 and Th1 cytokines, the immunosuppressive function was eliminated in mature Tregs following the deletion of the FOXP3 gene. Therefore, Foxp3 is essential for maintaining the immunoregulatory function of Tregs. In addition to CD4 and Foxp3, Tregs express a high-affinity IL-2 receptor (IL-2R) that may bind IL-2Rα (CD25), IL-2Rβ (CD122) and IL-2Rδ (CD132) (57). As expected, IL-2 is a critical signal for Treg differentiation.

Activation of DR3 using an anti-DR3 agonistic antibody or sTL1A has been shown to mediate Treg cell activation and expansion because of the constitutive expression of DR3 on Treg cells. DR3-mediated activation of Tregs mainly triggers the activation of MAPK, NF-κB and PI3K signaling via DR3/TL1A signal transduction (15,58,59), thereby exerting anti-inflammatory effects and preventing inflammatory and immune-mediated diseases in mice.

Due to the strong immunosuppressive function of Tregs, adoptive transfer of exogenously expanded Tregs or IL-2/IL-2 complexes expanded Tregs in vivo can alleviate a variety of immune-mediated diseases such as experimental autoimmune neuritis (60), headache disorders (61), systemic lupus erythematosus (62), IBD (63) and allograft tolerance (64-66), as well as inflammatory diseases such as transfusion-related acute lung injury (67,68) in preclinical models. Moreover, adoptive Treg transfer and low-dose IL-2 therapy have been associated with disease improvement and unconspicuous adverse effects in patients (66). However, the ability to obtain an adequate number of Tregs through adoptive transfer limits their clinical use, while the cytotoxicity, off-target effects and short half-life of IL-2 therapy in vivo necessitate the use of approaches employing expanded Tregs (64).

Clone 4C12 is an anti-DR3 agonistic antibody that activates and expands Tregs and thus has been useful for preventing or treating diseases in preclinical models (8,10,69). To begin with, the stimulation of DR3 using 4C12 leads to rapid and selective expansion of Tregs without influencing Tconvs (8,9,70,71). Furthermore, 4C12 retains its capacity to expand Tregs ex vivo (8). Additionally, the half-life of 4C12 is 5 days (9), ensuring prolonged contraction of expanded Tregs following 4C12 treatment compared with treatment with IL-2 or IL-2 complexes (8). Prolonged contraction of expanded Tregs is important for ameliorating inflammatory and immune-mediated diseases.

Preclinical models have demonstrated that Tregs expanded by 4C12 may alleviate disease. Schreiber et al (8) first discovered that lung inflammation was alleviated following treatment with 4C12 compared with the control group in OVA-induced acute allergic pneumonia. Madierddi et al (69) also demonstrated that the administration of 4C12 relieved allergic pneumonia and suppressed EAE through the expansion of Tregs. At present, 4C12 is widely applied in the field of transplants, including organ transplant, tissue transplant and cell transplant, aiming to alleviate graft-versus-host disease (GVHD). For example, Tregs expanded in vivo by 4C12 may provide tolerance to cardiac allografts (71). Recipients transplanted with donor-expanded Treg by 4C12 exhibited enhanced skin allograft survival (72). Furthermore, the infusion of 4C12 in recipient mice enhances skin graft survival associated with boosted graft-infiltrating Tregs (73). Administration of 4C12 also efficiently ameliorated GVHD in hematopoietic stem cell transplantation (HSCT) mismatch mouse models. Several studies have suggested that donor-derived Tregs are more effective than recipient-derived Tregs for GVHD treatment (74,75). Transfer of donor-derived Tregs expanded by 4C12 markedly protected mice from GVHD (10,72,76). Notably, Nishikii et al (29) demonstrated that treatment of recipients with 4C12-induced Treg activation and proliferation was sufficient to dampen GVHD lethality prior to exposure to HSCT. By contrast, mice transplanted with Tregs that were expanded by 4C12 following HSCT exposure led to donor T cell proliferation and ultimately fueled GVHD (29). These findings demonstrated that activation of DR3 mitigated the lethality of GVHD, depending on the timing of triggering DR3. Due to the significance of IL-2 signals in DR3-mediated Tregs expansion (8), low-dose IL-2 combined with 4C12 for donors resulted in decreased GVHD morbidity and prolonged survival (10). Additionally, the absolute number of Tregs induced by that combination was larger than that observed following the treatment with 4C12 alone (10). Therefore, this therapeutic strategy may be an improvement for delaying allogeneic rejection.

In addition to the anti-DR3 agonistic antibody, which may induce rapid and selective expansion of Tregs, sTL1A is sufficient as a soluble ligand for DR3 to trigger its signaling and to expand Tregs in vivo (9). Due to the shorter half-life of sTL1A (13.5 h), daily injection is required to maintain the desired serum levels. Khan et al (9) reported that sTL1A-meditated Treg expansion alleviated allergic lung inflammation through repeated administration, by blocking eosinophil infiltration into the alveolar fluid and reversing the ratio of Tconvs to Tregs in the lung without inducing any inflammatory changes in the liver, kidney and myocardium. Additionally, sTL1A, alone or combined with IL-2-expanded and IL-2-activated Tregs in donor mice, led to decreased GVHD morbidity and mortality in HSCT models (10). Consistent with this observation, low-dose IL-2 combined with sTL1A improved the clinical score, dampened weight loss and increased survival prior to HSCT exposure in animal models (77,78). Low-dose IL-2 combined with sTL1A expanded a higher proportion of Tregs in the peripheral blood, lymph nodes and spleen, compared with sTL1A alone (10). Whether the activation of Tregs through sTL1A/IL-2 may prevent GVHD manifestations more effectively than the treatment regimen of sTL1A requires further investigation.