Decoding TNF receptor superfamily control of CD4⁺Foxp3⁺ Regulatory T cell-mediated tolerance: implications for the treatment of graft‑versus‑host disease

Abstract

Allogeneic transplantation is a cornerstone treatment for hematologic malignancies and organ failure, yet its success is limited by graft-versus-host disease (GvHD) and allograft rejection. Conventional broad-spectrum immunosuppression compromises protective graft-versus-leukemia (GvL) effects and anti-infectious immunity, creating an urgent need for precision tolerance strategies. CD4⁺Foxp3⁺ regulatory T cell (Treg)-directed strategies offer a promising solution but expanding stable, functional Tregs ex vivo and in vivo remains challenging. Given the pivotal role of the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) in Treg biology, this review aims to critically examine how its members control Treg function and how these pathways can be leveraged for Treg‑based therapies. We systematically analyze key TNFRSF members – TNFR2, OX40, CD40, Fas, CD27, 4-1BB, GITR, and DR3 – detailing their dichotomous roles in Treg function and translational potential. We highlight how agonism of TNFR2 or DR3 offers selective Treg expansion while preserving GvL activity, and how CD27 and 4-1BB serve as valuable markers for isolating highly suppressive Treg subsets. We further discuss translational challenges, including the paradoxical effects of OX40 and GITR, which can either enhance or impair Treg function depending on the inflammatory milieu, and the vulnerability of Tregs to Fas-mediated apoptosis during ex vivo expansion. We also discuss CD40-CD40L blockade as a complementary strategy to empower endogenous Tregs. By synthesizing current knowledge, this review provides a rational roadmap for using selective agonism, blockade, or phenotypic selection to bolster Treg‑based therapies for GvHD, offering practical information for both laboratory and clinical efforts to optimize Treg manufacturing and achieve durable tolerance.

Graphical Abstract

This figure employs a Treg-centric design to visually synthesize the core framework of exploiting TNFRSF molecules expressed on Treg cells for advancing transplant tolerance. It depicts how targeted engagement of key TNFRSF members—including TNFR2 and DR3 to drive potent Treg proliferation and lineage stability, CD40 to reprogram immunity toward tolerance, and CD27 and 4-1BB to select antigen-specific Tregs—alongside context-dependent modulators like OX40, GITR, and Fas, addresses critical challenges in Treg-based therapy. These challenges encompass the ex vivo expansion of functional Tregs, the maintenance of their phenotypic and functional stability under inflammatory stress, and the precise direction of their suppressive activity. By integrating these mechanistic insights, the abstract illustrates how TNFRSF-targeted strategies translate basic biological understanding into clinical application. This translation enables the development of robust, tolerance-inducing immunotherapies that enhance Treg persistence and function in vivo while mitigating life-threatening complications like GvHD and allograft rejection.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12964-026-02990-1.

Introduction

Allogeneic hematopoietic stem cell transplantation (allo-HSCT) remains the most potent curative intervention for aggressive hematologic malignancies, chiefly owing to the graft-versus-leukemia (GvL) effect, whereby donor-derived immune cells eradicate residual leukemic blasts [1–4]. However, the same donor T cells that mediate this benefit can also recognize non‑hematopoietic tissue antigens, most notably in the skin, gut, and liver, triggering graft-versus-host disease (GvHD), a leading cause of post-transplant morbidity and mortality [5–9]. For decades, clinical management of these alloimmune complications has relied on broad-spectrum immunosuppressants, including corticosteroids, calcineurin inhibitors, and mTOR inhibitors [5, 6, 10–12]. Although partially effective, such non-selective immunosuppression heightened the risk of life‑threatening infections [6, 12], and crucially, can attenuate the beneficial GvL effect, thereby increasing leukemia relapse [13, 14]. This enduring therapeutic dilemma highlights an urgent need for strategies that selectively suppress pathological alloimmunity while preserving protective immunity.

Adoptive transfer of regulatory T cells (Tregs) has emerged as a leading strategy to re‑establish selective immune tolerance. Tregs, distinguished by co-expression of CD4, CD25, and the lineage‑defining transcription factor Foxp3, are central arbiters of immune homeostasis [15]. They restrain aberrant immunity through a multifaceted arsenal: contact‑dependent inhibition mediated by cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), which outcompetes CD28 for CD80/CD86 binding on antigen-presenting cells (APCs) and actively removes these ligands via trogocytosis [15–17]; paracrine delivery of anti-inflammatory cytokines, such as IL-10, TGF-β, and IL-35; and high-affinity IL‑2 receptor (CD25)‑driven consumption of IL‑2, which supports Treg survival while curbing effector T-cell (Teff) proliferation [15, 18, 19]. Many preclinical models and early-phase clinical trials have consistently shown that Treg therapy can suppress GvHD while sparing GvL and antimicrobial immunity [20–24]. Despite these promising results, clinical translation remains hampered by substantial hurdles. Tregs constitute only a minute fraction of peripheral blood leukocytes, and conventional ex vivo expansion protocols, which are often based on CD3/CD28 stimulation supplemented with IL-2 and rapamycin, tend to yield products with unstable phenotypes, failing to recapitulate the complex signaling networks that preserve Treg fitness in vivo [20, 25–30].

In this context, the Tumor Necrosis Factor Receptor Superfamily (TNFRSF) represents a uniquely powerful axis for refining Treg-based therapeutics. TNFRSF members, including TNFR2, DR3, CD40, CD27, 4-1BB, OX40, GITR, and Fas, do not simply trigger isolated signals; rather, they constitute a highly interconnected network that orchestrates Treg survival, proliferation, lineage stability, and functional programming [31–38]. Through their engagement, these receptors deliver potent co-stimulatory signals that dictate cell fate decisions. Thus, the TNFRSF is not merely a set of co‑stimulatory receptors, but an integrated signaling blueprint for engineering superior Treg products. This review provides a comprehensive analysis of how key TNFRSF pathways regulate Treg biology and how they can be harnessed to overcome current limitations in cellular therapy. Our goal is to outline the current advances in leveraging this receptor superfamily to optimize ex vivo Treg expansion and in vivo persistence, thereby advancing the development of robust, tolerance-inducing immunotherapies for transplantation.

TNFR2

Tumor necrosis factor (TNF) signals through two principal receptors, the type 1 TNF receptor (TNFR1, TNFRSF1A, CD120a) and the type 2 TNF receptor (TNFR2, TNFRSF1B, CD120b), which exert fundamentally opposing effects on Treg biology. TNFR1, expressed at low levels on Tregs, predominantly mediates signals that compromise Treg stability and function [39, 40]. Direct evidence comes from patients with TNFR‑associated periodic syndrome (TRAPS), an autoinflammatory disorder caused by autosomal‑dominant mutations in TNFRSF1A. In TRAPS patients carrying high‑penetrance mutations, hyperactivated TNFR1 signaling correlates with a reduced frequency of peripheral Tregs, and the remaining Tregs display a defective suppressive phenotype [41]. Indirectly, TNFR1 signaling on other immune cells can foster a pro-inflammatory cytokine milieu (e.g., IL-6, IFN-γ) that might destabilize the Treg lineage [39, 42]. Thus, TNFR1 activation directly and indirectly undermines Treg numbers and functional integrity, creating a microenvironment hostile to Treg persistence, which may increase susceptibility to graft rejection—a finding corroborated by prolonged allograft survival in TNFR1-deficient transplant models [43, 44] and by clinical correlations where elevated soluble TNFR1 levels predict adverse transplant outcomes [45–50].

In stark contrast, TNFR2 demonstrates highly restricted expression predominantly on immunosuppressive cells, with peak expression on CD4⁺Foxp3⁺ Tregs. The functional divergence between TNFR1 and TNFR2 is further specified by ligand binding affinity: membrane-bound TNF (tmTNF), rather than its soluble form that preferentially activates TNFR1, exhibits superior binding to TNFR2, enabling precise, cell-contact-dependent signaling often present in inflammatory niches [51]. Mechanistically, TNFR2 activation initiates a potent, integrated downstream signaling for Treg fitness. Upon TNF binding, TNFR2 recruits adaptor proteins (TRAF1/2, cIAP1/2) to activate canonical NF-κB and other pathways that directly drive Treg proliferation and enhance Foxp3 expression [52–55]. Concurrently, TNFR2 signaling has been linked to lineage stability through epigenetic modulation, such as upregulating the histone methyltransferase EZH2 [56], which is known to deposit repressive H3K27me3 marks at Foxp3-bound DNA loci, a modification that helps lock in the Treg transcriptional program even under inflammatory pressure [57]. Furthermore, TNFR2 signaling creates a positive feedback loop by upregulating other TNFRSF members (e.g., 4-1BB, OX40, etc.) on Tregs, amplifying their overall responsiveness to co-stimulatory signals [58].

This TNFR2 signaling extends beyond Treg-intrinsic effects to intercellular crosstalk that maintains immune homeostasis. A key example is the reciprocal loop between Tregs and Th17 cells, the latter of which serves as a potent source of TNF that, via TNFR2 on Tregs, drives Treg expansion and Foxp3 stabilization in vivo. In return, TNFR2 expression on Tregs is required to support optimal Th17 phenotype, as demonstrated by co‑transfer experiments in Rag1‑/‑ mice in which only wild‑type Tregs, but not TNFR2‑deficient Tregs, enabled IL‑17 A, IL‑2, and TNF production by co-transferred Th17 cells, illustrating a bidirectional, homeostatic network [59]. Disruption of this axis, as seen in TNFR2-deficient mice, leads to exacerbated inflammation in models of colitis, experimental autoimmune encephalomyelitis (EAE), and arthritis [39]. Moreover, this paracrine circuit is recapitulated within the Treg compartment itself where Tregs exist in two cooperative functional states: one committed to TNF production (TNFR2⁻) and the other specialized in TNF sensing via high TNFR2 expression (TNFR2⁺). The TNF produced by the former subset engages TNFR2 on the latter, creating a cooperative network that supports the overall suppressive function of the Treg pool [60]. Thus, targeting TNFR2 engages not merely a Treg-autonomous amplifier but a central hub that coordinates intercellular dialogues to maintain immune equilibrium.

Given this biology, the TNFR2⁺ Treg subset emerges as the ideal choice for manufacturing cellular therapies. In human peripheral blood, the co-expression of CD25 and TNFR2 defines a population of functionally intact Tregs with a suppressive capacity more than fivefold greater than that of CD25^high cells [61]. This subset also exhibits superior resilience, including resistance to oxidative stress [60], making it particularly suited to withstand inflammatory graft microenvironments. The translational relevance of applying the TNF–TNFR2 axis is exemplified in a fully MHC-mismatched murine model of acute GvHD (C57BL/6 → BALB/c), where donor-type Tregs are primed by endogenous TNF that is abundant in recipient mice experiencing acute GvHD, thereby acquiring enhanced suppressive function. Notably, TNF priming not only ameliorated GvHD but also preserved GvL effects in allogeneic transplanted BALB/c recipients inoculated with syngeneic A20 leukemia cells, presumably because the primed donor Tregs suppress alloreactive pathology while leaving donor T‑cell cytotoxicity against leukemia intact [34]. Corroborating the essential role of TNF in preserving antitumor immunity, a separate study demonstrated that TNF blockade with TNFR: Fc in a murine allogeneic BMT model impaired donor cytotoxic T lymphocyte (CTL) activity and accelerated leukemic relapse in recipients co‑injected with host‑type P815 leukemia cells [62]. Therefore, TNFR2 serves dually as a key functional marker for selection and the primary target for subsequent ex vivo expansion aimed at generating high numbers of stable Treg products.

This compelling rationale has catalyzed the development of pharmacological strategies to leverage TNFR2 for optimizing Treg expansion protocols (Table 1). Agonists targeting TNFR2, such as the engineered fusion protein TNCscTNF80 [63] and the bifunctional molecule IL2-EHD2-sc-mTNFR2 [64], which co-activates IL-2 and TNFR2 signaling, have all demonstrated efficacy in expanding Treg ex vivo, offering a wider therapeutic window than IL-2 monotherapy. Significant advances have also been made by integrating the TNFR2 agonist MR2‑1 into standard CD3/CD28‑based expansion protocols. Co-stimulation with MR2-1 alongside traditional IL-2 and CD3/CD28 activation, often in the presence of rapamycin, yields highly pure, stable, and functionally potent Treg products [65, 66]. Complementary approaches include the growth factor Progranulin, which modulates signaling downstream of TNFR2 [67]. Additionally, a new generation of selective biologics, including the DARPin D18 molecule that competitively binds and activates TNFR2 [68, 69], engineered TNF mutants such as TNFR2-ICK-Ig [70], scR2agoTNF-Fc (murine) and scR2-7-Fc (human) [71], as well as the oligomerized TNF mimetic STAR2 that mimics tmTNF [72], are all designed to specifically augment TNFR2 signaling, thereby markedly enhancing Treg expansion ex vivo. Furthermore, selectively blocking TNFR1 also permits more sustained TNFR2-mediated activation and maintenance of Tregs [52]. Collectively, these emerging strategies to precisely target the TNFR2 pathway hold considerable promise for generating potent Treg cellular therapies to achieve durable immune tolerance in transplantation and beyond.

Table 1.

TNFR2-targeted agonist for treg expansion and therapeutic applications

Related Agonist [Ref]	Agonist Format	Research Model	Key Expansion Conditions	Treg Expansion Efficacy	Functional Validation & Advantages
MR2-1 [65]	TNFR2 agonistic mAb	Human FACS-sorted or MACS-isolated Tregs; humanized skin inflammation mouse model.	In vitro: anti-CD3/CD28 beads + rhIL-2; MR2-1 2.5 µg/mL + rapamycin 1 µM; FACS Tregs cultured 7 d, MACS Tregs 16 d.	In vitro: MACS Tregs expanded ~ 23-fold with MR2-1 + rapamycin vs. ~ 12-fold with rapamycin alone.	Maintained Foxp3/Helios, low CD127; low IL-17 A/IFNγ; increased Foxp3-TSDR demethylation; suppressed responder cells and humanized skin inflammation.
TNCscTNF80 [63]	Mouse TNFR2-selective TNF-based agonist	Mouse splenocytes/T cells; purified mouse Tregs; naïve and BCG-induced chronic inflammation mice.	In vitro: anti-CD3 ± anti-CD28/IL-2 with TNCscTNF80; purified Tregs cultured 7 d. In vivo: 75 µg/mouse repeated dosing; BCG chronic inflammation model.	In vitro: Treg expansion increased from ~ 9.2-fold to ~ 29.4-fold. In vivo: repeated dosing expanded Tregs in naïve mice; one/two doses expanded Tregs in BCG-inflamed mice.	Increased CD25/Foxp3/GITR on Tregs; T cell TNFR2 required for suppressive in vivo effect.
STAR2 [72]	Mouse TNFR2-selective nonameric TNF agonist	Mouse splenic T cells/Tregs; allo-HCT acute GvHD (aGvHD) model	In vitro: mouse CD4/CD8 or purified Treg cultures with STAR2 for 72–96 h; ± IL-2, rapamycin, IL-2 blockade. In vivo: recipient mice treated with STAR2 before allo-HCT.	In vitro: expanded CD4+Foxp3+ Tregs and induced Treg proliferation. In vivo: expanded host radiation-resistant Tregs before allo-HCT.	Reduced GvHD severity and prolonged survival; effect was TNFR2- and Treg-dependent.
TNC-scTNF (143 N/145R) [72]	Human TNFR2-selective TNF mutein / STAR2-equivalent	Human ex vivo PBMC-derived CD4+ T-cell culture assay	In vitro: human CD4+ T cells treated with human TNFR2-specific TNC-scTNF (143 N/145R).	In vitro: increased CD4+Foxp3+ Tregs in human CD4+ T cell cultures.	Supports human translatability of selective TNFR2-driven Treg expansion.
TNFR2 agonist mAb (Unnamed) [66]	TNFR2 agonist mAb	Human ex vivo purified CD4⁺CD25hi Treg expansion platform	In vitro: four expansion settings tested: IL-2; anti-CD3; anti-CD3/CD28; anti-CD3/CD28 + rapamycin; ± TNF or TNFR mAbs; 16 d expansion in main Treg protocol.	In vitro: TNFR2 agonist produced greater Treg expansion than comparator conditions.	Generated homogeneous CD4+CD25hiFoxp3+ phenotype across 14 markers; stronger suppression of CD8⁺ CTLs than standard expansion.
D18 / TREG005 [69]	TNFR2-selective DARPin agonist	Human ex vivo TNFR2-signaling/ Treg expansion platform using PBMC-derived Tregs	In vitro: PBMCs treated with IL-2 500 U/mL ± D18 1 µg/mL; Tregs expanded 2 weeks with Treg Expander Dynabeads + IL-2 ± D18.	In vitro: D18 + IL-2 increased Treg cell numbers; reported ~ two-fold increase in relative Treg number in culture.	Expanded Tregs retained suppressive function; D18 + IL-2 activated non-canonical NF-κB/RelB pathway
TNFR2-ICK-Ig [70]	Immunocytokine: scR2-7-Fc + anti-TNFR2 scFv-Fc	Human PBMCs; human TNFR2 reporter cells.	In vitro: immobilized anti-human CD3 0.5 µg/mL + PBMCs; TNFR2-ICK-Ig 1–100 ng/mL for 72 h	In vitro: concentration-dependent expansion of CD4+CD25+ Tregs; stronger than scR2-7-Fc or 80M2-scFv-Fc alone.	Demonstrated TNFR2 clustering and stronger TNFR2 signaling as mechanism.
IL2-EHD2- sc-mTNFR2 [64]	Dual fusion protein: mouse IL-2 + TNFR2-selective scTNF.	C57BL/6 mouse splenic CD3+ T cells.	In vitro: purified CD3+ T cells; plate-bound anti-CD3 5 µg/mL + soluble anti-CD28 2 µg/mL; 1 nM fusion protein for 4 d.	In vitro: increased CD25+Foxp3+ Tregs more than IL2-EHD2 or EHD2-sc-mTNFR2 alone.	Maximal Treg expansion required both the IL-2 and TNFR2 arms of the fusion protein.
scR2agoTNF-Fc (mouse) & scR2-7-Fc (human) [71]	Bivalent TNFR2-selective TNF mutein Fc-fusion.	Mouse CD4+CD25+ / Foxp3+ Tregs; human PBMCs; CHS and arthritis mouse models.	In vitro: anti-CD3 + IL-2 10 U/mL with scR2agoTNF-Fc 100 ng/mL for 72 h; human PBMC assays with scR2-7-Fc. In vivo: scR2agoTNF-Fc dosing in CHS/arthritis models.	In vitro: expanded mouse CD4+CD25+ and CD4+Foxp3+ Tregs; scR2-7-Fc expanded human PBMC Tregs. In vivo: increased Treg populations in lymph nodes	Expanded Tregs suppressed Tconv proliferation; Fc-fusion increased half-life; prophylactic dosing reduced CHS/arthritis inflammation.
Endogenous TNF induced by Teplizumab [73]	Indirect TNFR2 engagement via anti-CD3-induced TNF.	Human PBMCs (CD8+ Treg); T1D patient samples.	In vitro: PBMCs cultured with Teplizumab 4 µg/mL for 5 d; CD8+ cells isolated and tested in suppression assays.	In vitro: induced CD8+CD25+ Treg, not classic CD4+ Treg expansion.	CD8+CD25+ cells inhibited CD4+ proliferation (~ 44%); induction required TNF/NF-κB; TNFR2+CD25+ marked more potent CD8+ regulators.

OX40 (TNFRSF4)

OX40 (TNFRSF4, also known as CD134) is a potent inducible co-stimulatory receptor expressed on activated Teffs and human Tregs, whereas it is constitutively expressed on murine Tregs [74, 75]. Among co‑stimulatory molecules, OX40 is widely recognized for its pro‑inflammatory functions; ligation with agonist anti‑OX40 monoclonal antibodies delivers activating signals that promote cytokine production, survival, expansion, and homeostasis of effector and memory T cells. Substantial evidence indicates that OX40 signaling acts primarily during the activated Teff phase rather than at the onset of activation, as its expression peaks 48–72 h after TCR engagement, and T cells lacking OX40 succumb to a survival defect after 12–13 days [76, 77]. This highlights its critical role in sustaining late proliferation and survival during the Teff phase. The ligand OX40L (TNFSF4/CD252) is mainly displayed on APCs, but its expression also extends to activated NK cells, mast cells, and non-hematopoietic cells such as endothelial cells [33]. Although OX40 has traditionally been viewed as a driver of Teff responses, supported by its ability to impair Treg function and bias naïve T-cell differentiation toward effector lineages [75, 78–81], a more nuanced understanding reveals that OX40 signaling can be harnessed to expand functional Tregs and promote graft tolerance. This potential is highly dependent on the cytokine milieu, particularly the availability of IL-2.

The importance of OX40 for Treg development and homeostasis is clearly illustrated by reduced Treg numbers in the spleens of OX40‑deficient mice and their increase in mice overexpressing OX40L [74]. Although most experimental data indicate that OX40 counteracts Treg‑mediated suppression, a point of contention remains whether OX40 acts directly to abrogate Treg suppressive activity or indirectly by rendering Teff cells refractory to Treg control, even when Tregs are present at sufficient numbers and in an activated state. On one hand, numerous co‑culture studies have demonstrated that OX40 ligation can directly abolish the ability of Tregs to suppress Teff proliferation, restore IL‑2 gene transcription and cytokine production, and markedly suppress the differentiation of naïve CD4⁺ T cells into inducible Foxp3⁺ iTregs, even in the presence of TGF‑β [78, 79, 82, 83]. This evidence suggests that OX40 may act as a negative regulator of Treg function that can directly counteract Treg‑mediated suppression. Several studies have also attempted to define the underlying mechanisms, which appear to involve two distinct pathways: upregulation of BATF3 and BATF that induces a closed chromatin configuration at the Foxp3 locus in a Sirt1/7‑dependent manner, and activation of the AKT‑mTOR pathway that leads to phosphorylation and nuclear exclusion of Foxo1 [84].

On the other hand, an opposing line of evidence shows that OX40 acts positively to strengthen Treg function. Tregs lacking OX40 exhibit markedly reduced suppressive capacity when co‑cultured with wild‑type Teffs, indicating that OX40 signaling supports efficient Treg‑mediated suppression [74]. Supporting this pro‑Treg role, OX40‑deficient Tregs are intrinsically hyporesponsive to IL‑2 [85]; thus, OX40 emerges as a key factor in shaping Treg sensitivity to IL‑2 as well as promoting their proliferation and survival. Nevertheless, even in the face of these pro‑Treg activities, the dominant effect of OX40 on Teffs appears to override Treg function. Indeed, activated Teff cells become insensitive to Treg‑mediated suppression following exposure to OX40L‑expressing cells or treatment with an agonistic OX40‑specific antibody [74]. Of note, some reports have also shown that OX40 can simultaneously enhance Teffs and inhibit Tregs. For instance, an anti‑human OX40 agonistic mouse mAb has been developed that promotes effector CD4⁺ and CD8⁺ T cell proliferation while blocking the induction of IL‑10‑producing type 1 Treg and inhibiting TGF‑β‑induced Foxp3 expression on naïve CD4⁺ T cells [86].

Collectively, these findings suggest that activating OX40 within the CD4⁺ T‑cell pool renders Tregs ineffective. This is exemplified in a fully allogeneic C57BL/6 → BALB/c bone marrow transplantation model [78]. When T‑cell‑depleted bone marrow cells were co‑transferred with freshly purified CD4⁺CD25⁻ and CD4⁺CD25⁺ T cells from C57BL/6 mice into irradiated BALB/c recipients that additionally received an intraperitoneal injection of an agonistic OX40‑specific mAb, the Treg‑mediated suppression of GvHD was completely abrogated. This robustly demonstrates that direct in vivo administration of the agonistic OX40‑specific mAb immediately after bone marrow transfer completely abolishes the GvHD‑preventing activity of Treg cells. However, this does not exclude the possibility that OX40 activation on Tregs is the primary cause of lost suppressive activity, as it remains possible that OX40 activation on Teffs enables them to completely escape Treg control [74]. Intriguingly, when Tregs were pretreated in vitro for two hours with the agonistic OX40‑specific mAb before co‑transfer with mismatched bone marrow cells into irradiated recipients, the antibody still blocked Treg suppressive activity, as evidenced by the failure of the treated cells to prevent GvHD [78]. This finding casts doubt on the feasibility of using OX40 activation as a strategy for Treg expansion.

Given the potent Teff‑driving effects of OX40 agonism, therapeutic targeting of OX40 in transplantation must be carefully considered. In scenarios such as GvHD, where activated OX40⁺ Teffs vastly outnumber Tregs, clinical and preclinical data suggest that OX40 pathway blockade may help restore balance by suppressing pathogenic Teff responses. A prospective study of patients after allogeneic bone marrow transplantation found that the ratio of OX40⁺ total lymphocytes to CD25ʰⁱᵍʰ cells was markedly elevated during chronic GvHD compared with patients without chronic GvHD or after treatment [87]. This observation led the authors to propose that high OX40 expression on activated donor T lymphocytes may abrogate Treg suppressive activity in vivo during chronic GvHD, and that OX40 blockade might be crucial to optimize the use of Tregs for preventing GvHD. Compelling support for this concept comes from a non‑human primate (Rhesus macaque) model of acute GvHD [88]. In this study, the non‑depleting anti‑OX40L antibody KY1005 significantly extended median GvHD‑free survival from 8 days to 19.5 days. Remarkably, combined administration of KY1005 with the mTOR inhibitor sirolimus yielded synergistic control, prolonging median survival to > 100 days (ClinicalTrials.gov No. NCT03161288) [89]. This combination potently suppressed both Th/Tc1 and Th/Tc17 activation while preserving Treg reconstitution, resulting in an enhanced Treg: Tcon ratio [88]. Further clinical validation of OX40L blockade is provided by a phase IIa randomized, placebo‑controlled study of KY1005 in moderate‑to‑severe atopic dermatitis (ClinicalTrials.gov No. NCT03754309). The treatment was well tolerated, and the low‑dose regimen achieved an 80.1% reduction in Eczema Area and Severity Index score from baseline compared with 49.4% in the placebo group, with improvements evident as early as week 2 [90]. These data further support the therapeutic potential of OX40L blockade in T‑cell‑driven inflammatory conditions.

Nevertheless, the translational insight that OX40 agonism alone is problematic for Treg expansion does not completely rule out the potential for OX40‑driven Treg expansion, as subsequent studies indicated that such expansion requires co‑stimulation with IL‑2 to maintain functional integrity and survival [85, 91, 92]. The counteractive effect of OX40 ligation on Foxp3⁺ Tregs in vivo may be attributable to a heavy dependence on IL‑2 availability. Stimulation of OX40 in naïve mice induces initial expansion of Foxp3⁺ Tregs but drives the expanded Tregs into an exhausted state, a phenotype that can be prevented by exogenous IL‑2 [92]. Importantly, Tregs expanded by combined OX40 and IL‑2 agonists remain potent suppressors. In a heart transplant model, preconditioning recipient B6 mice with intraperitoneal injections of the OX40 agonist OX86 (0.25 mg) and IL‑2 (0.3 µg) for three consecutive days, followed by transplantation of donor BALB/c hearts on day 7, promoted long‑term allograft survival [92]. Such synergistic combination also provides a foundational principle for innovative ex vivo Treg expansion protocols. A prime example is a T‑cell‑receptor‑signal‑independent protocol that uses bone marrow‑derived dendritic cells differentiated with GM‑CSF, together with soluble OX40L and the Notch ligand JAG1, to deliver co‑stimulation. When combined with exogenous IL‑2, these dual stimuli drive the selective and efficient expansion of autologous Tregs with stable function while avoiding concurrent activation of pathogenic Teffs, as demonstrated in models of autoimmunity [93–95]. Together, this methodology exemplifies how the OX40 pathway can be specifically co‑opted to generate therapeutic Treg products. (Key OX40 targeting molecules are listed in Table 2).

Table 2.

OX40 pathway-related molecules for treg expansion and therapeutic applications

Related Molecules	Brief Introduction	Research Model	In Vivo Effects on Treg Expansion	In Vitro Effects on Treg Expansion	Main Application Scenarios
OX86 [79, 80, 92, 96, 97]	Agonistic anti-OX40 mAb that activates OX40 signaling.	Mouse models including naïve mice, EAE, skin allograft, cardiac allograft, and tumor models; in vitro Treg suppression/iTreg conversion assays.	Expands Tregs in vivo, but OX40 stimulation alone may reduce Foxp3/CD25 and impair suppressive function; combined OX86 + IL-2 expands functional Tregs and promotes cardiac allograft survival.	Inhibits TGF-β-induced iTreg generation in some settings; with appropriate IL-2/cytokine context, supports Treg proliferation without necessarily abolishing suppressive function.	Autoimmunity, transplantation tolerance, tumor immunotherapy, and OX40-Treg biology research.
KY1005 (Anti- OX40L mAb) [88]	Human IgG4 anti-OX40L blocking antibody that inhibits OX40-OX40L interaction.	Human in vitro allo-proliferation assay and NHP haploidentical HCT/aGvHD model.	KY1005 alone partially preserves Treg reconstitution; KY1005 + sirolimus increases Treg: Tconv ratio and achieves prolonged GvHD-free survival.	Inhibits alloreactive Teff proliferation; no direct Treg expansion effect was shown.	Prevention of aGvHD after HCT, especially in combination with sirolimus.
Recombinant OX40L-Fc Protein [98]	Soluble OX40L-Fc agonist that engages OX40 signaling.	WT/OX40-deficient /Foxp3-eGFP mice; murine thymocyte cultures; human thymic tissue and thymic organ culture.	Increases Foxp3+ Tregs in thymus, spleen, and lymph nodes; promotes proliferation of thymic Treg precursors and mature Tregs.	With IL-2, promotes murine and human thymic Treg maturation/proliferation through AKT-mTOR signaling.	Thymic Treg development, Treg expansion research, and autoimmune disease-related Treg augmentation.

CD40 (TNFRSF5)

Signaling through the CD40(TNFRSF5)-CD40L(TNFSF5, CD154) co-stimulatory axis is crucial for adaptive immunity, mediating the essential dialogue between T cells and APCs. Engagement of CD40 on APCs by CD40L-expressing CD4⁺ T cells triggers a canonical immunogenic program, driving APC maturation, upregulation of B7 co-stimulatory molecules (CD80/CD86), and production of pro-inflammatory cytokines such as IL-12 [99, 100]. This process licenses APCs to fully activate naïve T cells, thereby precipitating the clonal expansion and differentiation of alloreactive Teffs that mediate acute graft rejection [101, 102]. Consequently, strategic blockade of this pathway has emerged as one of the most potent interventions for inducing durable transplantation tolerance across diverse preclinical models, including islet, skin, and cardiac allografts [102, 103].

The profound efficacy of CD40-CD40L blockade, exemplified by an anti-mouse CD40L monoclonal antibody (clone MR1), arises from its ability to shift the immune system toward a tolerogenic state without eliciting broad immunosuppression [104–106]. Central to this tolerogenic effect is the opposing fate of conventional Teffs versus Tregs under this intervention. While CD40‑CD40L blockade potently suppresses alloreactive Teff proliferation and cytokine production, it spares and even selectively favors Treg expansion and functional integrity. This principle is illustrated by an in vitro mixed lymphocyte reaction (MLR) assay, in which addition of MR1 halts the proliferation of alloantigen-stimulated CD4⁺ T cells. Moreover, when combined with CTLA‑4Ig (which blocks B7‑CD28 costimulation), MR1 acts cooperatively to increase the proportion of Foxp3⁺ Tregs within the CD4⁺ pool. Notably, such Treg increase does not result from de novo Foxp3 induction; rather, it reflects the resilience of pre-existing Tregs, as no newly generated Foxp3⁺ cells are detected when the starting population is depleted of Foxp3⁺ cells [107]. The selective sparing of Tregs under CD40‑CD40L blockade has been further examined by another study that employed a distinct CD8⁺CD122⁺PD-1⁺ Treg subset, which revealed a critical nuance that differs from the above CD4⁺ Treg finding [104, 108]. Adoptive transfer of CD8⁺CD122⁺PD-1⁺ Tregs together with MR1 significantly prolonged graft survival (median survival time 43 vs. 27 days). In striking contrast, this protective effect was absent when the same Tregs were transferred alongside CTLA4-Ig [108]. This failure could be explained by the dependency of CD8⁺CD122⁺PD-1⁺ Tregs on B7-CD28 signaling to sustain their functional competence, as blockade of this pathway also reduced both the number of transferred Tregs and their IL-10 expression in recipient mice [108]. Another CD8⁺ Treg subset, defined as CD8⁺CD45RC^low, has also been shown to be inducible by CD40‑CD40L blockade [109]. These cells contribute to donor-specific, long-term local graft tolerance through an IFN-γ-dependent indoleamine 2,3-dioxygenase (IDO) mechanism [109]. Collectively, these findings highlight that tolerogenic strategies based on CD40‑CD40L blockade have the capacity to sustain both CD4⁺ and CD8⁺ regulatory compartments.

The essence of the tolerant state induced by CD40/CD40L blockade appears to lie in active enforcement by Tregs rather than reflecting Teff anergy. Direct evidence for this active mechanism comes from a model using donor-specific transfusion (DST) combined with MR1. In this system, DST serves as a potent tolerogenic preconditioning strategy, providing a bolus of alloantigen to steer the immune response toward acceptance. When alloreactive CD4⁺ Teffs were adoptively transferred into recipients that immediately received DST plus MR1, their accumulation was markedly curtailed [110, 111]. Strikingly, these restrained Teffs were not functionally anergic; they remain fully capable of rejecting grafts upon retrieval and transfer to a new host [111]. Thus, the suppression of Teff expansion in the primary host is not due to intrinsic anergy but to an external suppressive force that might be exerted by Tregs. Support for Tregs as this force comes from their selective ablation, which completely abolished the ability of DST plus MR1 to limit alloreactive Teff expansion [110, 111]. Furthermore, this regulation is spatially organized, with Foxp3⁺ T cells enriched within the tolerated graft tissue itself, suggesting they establish a privileged immunoregulatory niche that locally constrains Teff infiltration and function [111]. Therefore, CD40-CD40L blockade, particularly when combined with antigen exposure via DST, does not directly restrain the alloreactive repertoire but rather empowers Tregs to keep them in check.

These mechanistic insights are now guiding translational efforts to engineer robust tolerance, as summarized in Table 3 for related molecules. Molecular engineering is creating new clinical tools, such as Fc-silent anti-CD40L domain antibodies, which not only effectively inhibit alloreactive T cell expansion but also promote iTreg induction when combined with CTLA4-Ig [112]. Rational combination therapies are also being refined. For example, short-term CD40‑CD40L blockade synergizes powerfully with IL-2/anti-IL-2 (JES6-1) immune complexes that transiently expand the Treg pool, achieving robust tolerance in immunocompetent hosts [113]. One distinct translational approach that harnesses CD40 activation rather than its blockade is ex vivo Treg expansion. This approach uses CD40L‑stimulated B cells (CD40L‑sBc) as tolerogenic APCs. B cells are expanded ex vivo and activated by CD40L, which upregulates their co‑stimulatory molecules and converts them into effective APCs. When co‑cultured with Tregs, these CD40L‑sBc drive robust, donor‑specific Treg expansion, while maintaining high expression of FoxP3 and Helios and displaying strong suppressive function [114, 115]. Beyond conventional CD4⁺ Tregs, CD40L‑sBc can also induce alloantigen‑specific CD8⁺ Tregs, particularly the CD8⁺CD45RO⁺CCR7⁺ memory‑like CD8^high subset, which suppress GvHD while preserving general immunity and GvL activity in humanized mouse models [116, 117]. Thus, the CD40 activation pathway provides a powerful ex vivo platform for generating both CD4⁺ and CD8⁺ Tregs for cellular immunotherapy.

Table 3.

CD40 pathway-related molecules for treg expansion and therapeutic applications

Related Molecules	Brief Introduction	Research Model	In Vivo Effects on Treg Expansion	In Vitro Effects on Treg Expansion	Main Application Scenarios
MR1 [104, 107, 110, 111, 113]	Anti-CD154/ CD40L mAb; blocks CD40-CD154 costimulation.	Murine allo-/xeno transplantation tolerance models; TCR-transgenic alloreactive T-cell tracking; IL-2c + MR1 skin allograft model.	Not mainly used as a direct Treg expansion reagent. In combination settings, associated with Treg-favored tolerance, iTreg generation, or maintained Foxp3+ Treg responses.	Tregs can remain activated / proliferative under CD40 / CD154 blockade while Teff responses are inhibited; MR1 can enhance the therapeutic efficacy of expanded Tregs.	Transplantation tolerance, costimulation blockade, combination with Treg-based therapy.
UltraCD40L [115]	Soluble 4-trimer CD40L used to activate donor B cells ex vivo.	Human ex vivo donor-specific Treg expansion model.	Not assessed.	Activates/expands donor B cells, which are then used as APCs to expand donor-specific Tregs with suppressive function and stable Foxp3/TSDR features.	Clinical/GMP-oriented donor-specific Treg manufacturing for transplant tolerance.
CD40L- expressing K562 cells [114]	Engineered K562 feeder cells expressing CD40L, used to generate CD40L-stimulated B cells.	Non-human primate ex vivo polyspecific Treg expansion model.	Not assessed.	Supports generation of CD40L-stimulated B cells that expand polyspecific NHP Tregs while retaining Treg phenotype and suppressive function.	Scalable polyspecific Treg expansion, especially when single donor-specific Treg preparation is impractical.

Returning to concerns associated with CD40‑CD40L blockade, the tolerogenic equilibrium it established remains context-dependent and can be destabilized by competing signals. Notably, not all CD40/CD40L-targeted regimens preserve antitumor immunity. In some murine BMT settings, although adding MR1 improved tolerance, it reduced the GvL potential of transplanted cells, indicating that this axis must be tuned cautiously in leukemia-bearing recipients [118, 119]. Another cautionary example is the combination of CD40‑CD40L blockade with other TNF receptor superfamily members. Agonistic stimulation of GITR, for instance, can reverse established cardiac allograft tolerance induced by MR1, precipitating acute rejection through enhanced Teff proliferation and impaired Treg differentiation [120]. This vulnerability underscores the need of a holistic view of TNFR superfamily interactions in therapeutic design.

Fas (TNFRSF6)

The Fas receptor (TNFRSF6, APO-1, CD95) and its ligand, FasL (CD178), constitute a canonical death signaling pathway crucial for activation-induced cell death (AICD) and immune homeostasis [121, 122]. For Tregs, this pathway represents a dual-edged mechanism, posing a direct threat to Treg survival while also offering a potent tool for Tregs to eliminate Teffs. Balancing these opposing functions is key to leveraging Fas-FasL signaling for therapeutic gain.

Fas ligation initiates apoptosis by recruiting the adaptor protein FADD and procaspase-8 to form the death-inducing signaling complex (DISC), activating a cascade of effector caspases that execute programmed cell death [123, 124]. Treg susceptibility to this pathway is dynamic. While naïve Tregs express low Fas and are protected by Foxp3-mediated repression of FasL [125, 126], this intrinsic mechanism that resists Fas-mediated apoptosis is lost upon activation or under autoimmune conditions. This is clinically relevant, as seen in lupus, where Tregs exhibit heightened sensitivity to Fas-induced apoptosis, contributing to Treg loss and disease progression [127]. A critical vulnerability emerges during ex vivo Treg expansion. Human Tregs isolated via CD25-based enrichment upregulate Fas, rendering the highly suppressive Foxp3^high subset particularly prone to apoptosis. This susceptibility is a major bottleneck, limiting the efficiency of Treg manufacturing and compromising functional yield [128].

Tregs can naturally use FasL to mediate contact-dependent killing of Teffs [129]. This endogenous function has been enhanced through bioengineering, creating “killer Tregs” that express a non-cleavable, membrane-stable form of FasL (SA-FasL). In murine models of GvHD, these FasL-decorated Tregs traffic to inflammatory sites, increase apoptosis of effector lymphocytes, and suppress GvHD more effectively than naïve Tregs. Importantly, this enhanced suppressive capacity does not compromise antitumor immunity; in a model using F1 mice (H2K^b/d) implanted with CT26 colon carcinoma (H2K^d) and infused with haploidentical donor lymphocytes, FasL-decorated Tregs did not significantly impair antitumor activity [130], making this Fas‑based strategy one of the promising approaches to attenuate GvHD while preserving GvT/GvL‑like effects. Similarly, CD8⁺ Tregs generated via the ex vivo CD40L‑sBc method (as described in the previous section) have also been reported to utilize the Fas‑FasL axis to mediate cytotoxicity. In humanized mouse models, these CD8⁺ Tregs preserved long-term GvT activity while maintaining tolerance, and their cytotoxicity against target tumor cells was mediated by both Fas-FasL and perforin–granzyme B pathways [116, 117]. Beyond the allo-HSCT setting, FasL engineering also promotes tolerance in other transplant contexts. Donor splenocytes engineered with SA-FasL induce donor-specific cardiac allograft tolerance through deletion of alloreactive Teffs together with host Treg expansion [131, 132]. Localized delivery of FasL represents another promising strategy. In models of islet and skin transplantation, a hydrogel crosslinked with mesenchymal stem cell membrane-derived vesicles (MMVs) naturally expressing FasL, together with IFN-γ-induced upregulation of PD-L1, creates an immune-privileged microenvironment. This approach induces apoptosis of infiltrating Teffs, enriches the graft with Tregs, and prolongs allograft survival without systemic immunosuppression [133].

To harness Tregs for cell therapy, their vulnerability to Fas-mediated apoptosis must be mitigated during expansion. Culture conditions might play a decisive role. One potential approach is suggested by a study on Tregs isolated from patients with idiopathic aplastic anemia, in which supplementing cultures with CD3/CD28 beads, high-dose IL-2 (1000 IU/mL), rapamycin, and all-trans retinoic acid upregulates the pro-survival factors phosphorylated Bcl-2 and confers resistance to FasL-induced apoptosis, thereby preserving a stable Foxp3^high population [134]. Although Fas-blocking antibodies (e.g., ZB4) can also attenuate Treg apoptosis in short-term culture, they do not prevent overall Treg loss during prolonged expansion [128]. Sustaining Treg survival after infusion is equally important, as the host environment contains FasL‑producing cells. Therefore, a complementary strategy that involves the use of agents with FasL‑scavenging activity before Treg infusion may ease this hostile environment, thereby promoting Treg engraftment and function. Collectively, the Fas-FasL axis presents both a challenge and an opportunity for Treg‑based transplantation tolerance. On one hand, Fas‑mediated apoptosis threatens Treg viability during ex vivo expansion. On the other hand, Tregs that express FasL can suppress alloreactive Teffs and preserve GvL activity. Some potential related molecules have been listed in Table 4. Future efforts will likely require integrated approaches that combine apoptosis-resistant Tregs with targeted FasL engagement against pathogenic Teffs, potentially turning a liability into a therapeutic tool for durable graft acceptance.

Table 4.

Fas pathway-related molecules for treg survival, expansion and therapeutic applications

Related Molecules	Brief Introduction	Research Model	In Vivo Effects on Treg Expansion	In Vitro Effects on Treg Expansion	Main Application Scenarios
CH-11 [128]	Agonistic anti-Fas mAb that mimics Fas/FasL signaling and induces Fas-mediated apoptosis.	Human CD25 bead-enriched Treg expansion model using anti-CD3/CD28 beads and high-dose IL-2.	Not assessed.	Dose-dependently reduced CD4+Foxp3high cells and activated caspase-8, showing that Fas signaling can selectively deplete highly Foxp3-expressing Tregs during expansion.	Mechanistic tool to study Fas-mediated Treg apoptosis during repeated in vitro expansion.
ZB4 / NOK-1 [128]	ZB4 blocks Fas; NOK-1 blocks FasL. Both were used to test whether Fas/FasL blockade could protect Tregs from apoptosis.	Human in vitro Treg expansion and Fas/FasL blockade model.	Not assessed.	ZB4 partially protected CD4+Foxp3high cells and reduced caspase-8 activation in short-term culture, but neither ZB4 nor NOK-1 prevented Foxp3high Treg loss during longer repeated expansion.	Optimization of human Treg manufacturing by identifying apoptosis-related limits to expansion.
Recombinant SA-FasL Protein [131, 132]	Non-cleavable FasL-streptavidin fusion protein designed for stable surface display and local apoptosis induction.	Cardiac allograft models using SA-FasL displayed on donor splenocytes or graft vasculature.	SA-FasL-engineered donor splenocytes induced donor-specific cardiac allograft tolerance and increased peripheral CD4+CD25+Foxp3+ Tregs; transferred Tregs maintained tolerance. SA-FasL displayed on cardiac vasculature prolonged graft survival but did not directly quantify Treg expansion.	SA-FasL-engineered cells reduced alloreactive T-cell proliferation and induced apoptosis in Fas+ target cells. Direct in vitro Treg expansion was not the main focus.	Local or donor-specific immunomodulation for transplant tolerance, especially cardiac allograft protection.
FasL/ PD-L1-presenting MMV-Gel [133]	Hydrogel crosslinked with MSC membrane vesicles presenting FasL and PD-L1 for local immunoprotection.	Mouse allogeneic islet and skin transplantation models; activated T-cell co-culture assays.	Increased CD4+Foxp3+ Tregs at graft sites and lymph nodes, promoted iTreg generation, reduced Teff infiltration, and prolonged islet/skin allograft survival. Treg depletion caused graft rejection.	MMVs induced activated T-cell apoptosis, inhibited T-cell proliferation, reduced Teff cells, and increased CD4+Foxp3+ Treg proportion in activated splenocyte cultures.	Local immunoprotective biomaterial niche for allogeneic islet and skin transplantation.

CD27 (TNFRSF7)

CD27 (TNFRSF7) and its ligand, CD70, form a critical co-stimulatory axis that plays a context-dependent role in modulating T cell immunity. While best known for delivering co-stimulatory signals to Tconv through the interaction of CD27 with its ligand CD70, this pathway has also emerged as a key modulator of Treg biology, offering novel strategies to promote graft tolerance.

In humans, CD27 has been proposed as a valuable phenotypic and functional marker for identifying highly suppressive Treg subsets. Studies of ex vivo expanded peripheral blood CD4⁺CD25⁺ cells reveal that CD27 expression strongly correlates with sustained regulatory activity. CD27⁺ cells within these cultures are hyporesponsive, potently suppressive, and enriched for surface expression of CC chemokine receptor (CCR) 7 and intracellular levels of Foxp3 and CTLA-4, whereas their CD27⁻ counterparts lack suppressive capacity and show reduced Foxp3 [135]. However, caution is also warranted in this study when using CD27 as a sole marker for Treg identification in synovial fluid from patients with reactive arthritis, as CD27 is expressed not only by CD25^hi cells but also by high proportions of CD25^int and CD25⁻ cells [135]. The functional importance of the CD27⁺ subset is further underscored by pharmacological data showing that rapamycin, but not cyclosporin A, preferentially preserves the CD27⁺Foxp3^high population during alloantigen-driven expansion ex vivo, and the CD27⁺/CD27⁻ ratio directly tracks the overall suppressive potency of the Treg product [136].

Beyond its utility as a marker, signaling through the CD27-CD70 axis directly controls Treg homeostasis in vivo. In tumor microenvironments, direct CD27 signaling not only reduces Treg apoptosis, but CD27 on Tregs that engages CD70 on tumor-infiltrating lymphocytes also induce those cells to produce IL‑2, which serves as a key survival factor for Tregs. Consequently, this pathway facilitates immune evasion by enhancing intratumoral Treg frequency, reducing tumor‑specific T‑cell responses, and promoting angiogenesis. Blocking this interaction reduces intratumoral Treg numbers and enhances anti-tumor immunity [137]. Moreover, CD70 expressed on activated T cells has a regulatory action that limits their own expansion. In inflammatory settings such as colitis and GvHD, CD70-deficient T cells expand excessively and produce more cytokine, whereas CD70 engagement induces apoptosis and upregulates inhibitory receptors such as TIM-3, PD-1, CTLA-4, and LAG-3, thereby restraining immunopathology through a Treg-independent mechanism [138]. Paradoxically, studies from others demonstrated that CD70 blockade alone has little effect on CD4⁺ T cell functions [139]. Instead of acting as a general checkpoint for CD4⁺ T cells, CD70 blockade, when combined with anti‑CD40L/LFA‑1 and anti‑CD44 together with a DST preconditioning strategy that induces allograft acceptance, has even prolonged cardiac allograft survival, with mean survival time extending from 49.8 days to over 100 days [140, 141]. Thus, while CD27 forward signaling bolsters Treg fitness in suppressive contexts, the effect of CD70 on Teffs differs depending on the context. CD70 reverse signaling directly restrains Teffs during inflammation, yet its therapeutic potential for transplantation tolerance may require combination with other co‑stimulatory blockade strategies.

The understanding of CD27 as a Treg functional marker has been directly applied to refine the manufacturing of therapeutic Tregs. Using a CFSE‑based cell sorting approach to track alloantigen-induced proliferation, human alloantigen‑reactive Tregs can be subdivided into CD27⁺ and CD27⁻ subsets. Both subsets are suppressive, but the CD27⁺ subset exhibits profoundly higher potency, inhibiting responder T cell proliferation at ratios as low as 1:500, compared with 1:50 for CD27⁻ Tregs. Critically, only CD27⁺ Tregs can effectively suppress ongoing immune reactions, a key requirement for treating established rejection [142]. Further refinement comes from a two‑marker phenotype identified in xenotransplantation models. When human Tregs are expanded in the presence of porcine PBMCs that serve as xenoantigen, a distinct subset of HLA-DR⁺CD27⁺ Tregs emerges with increased Foxp3⁺Helios⁺ and epigenetically stable Treg signature. This Treg subset also exhibits upregulated functional markers CD95 and ICOS, and is more resistant to conversion into IL-17⁺/IFN‑γ⁺ effector cells under inflammatory conditions. Functionally, adoptive transfer of HLA‑DR⁺CD27⁺‑enriched Tregs protects porcine islet grafts in humanized mice, further supporting CD27 (in combination with HLA‑DR) as a marker of stable antigen-biased suppressive Tregs [36]. Such two‑marker phenotype can also be applied to identify a CD8⁺ regulatory subset, which exhibits a transcriptome resembling CD4⁺ Treg cells and can mitigate pathological damage in a humanized GvHD model without compromising the GvL effect [143]. Together, these studies validate CD27 as a powerful selection marker for enriching stable, potent, and antigen-specific Tregs for cellular therapy.

4-1BB (TNFRSF9)

The 4-1BB (TNFRSF9, CD137) pathway is an inducible co-stimulatory axis with profound impact on T cell biology. Although best known for amplifying Teff and NK cell responses, 4-1BB is also expressed on CD4⁺CD25⁺Foxp3⁺ Tregs. Agonistic engagement of 4-1BB offers a targeted strategy to selectively expand stable, functional Tregs for promoting therapeutic tolerance. In the presence of low-dose IL-2, 4-1BB stimulation provides a potent proliferative and survival signal to Tregs, driving their expansion while preserving Foxp3 expression, CD25 upregulation, and suppressive function ex vivo [37, 144]. However, this approach has notable caveats. 4‑1BB‑expanded Tregs produce increased levels of IFN‑γ, the significance of which remains unknown [37]. Moreover, 4‑1BB ligation on conventional CD4⁺CD25⁻ Teffs can render them refractory to suppression by Tregs, a dual effect that may complicate therapeutic application [144]. Despite these concerns, stimulation of this pathway has shown clear therapeutic potential in transplantation. In a C57BL/6 → (C57BL/6 × DBA/2) F1 acute GvHD model, preconditioning with an agonistic anti-4-1BB antibody before allogeneic bone marrow transplantation induces host-derived Tregs that are essential for protection against acute GvHD. An alternative approach that primes Tregs ex vivo with anti‑4‑1BB and adoptively transfers them also ameliorates GvHD in naïve recipients [145]. A further regulatory layer of the in vivo efficacy of agonistic anti‑4‑1BB antibodies has been shown to depend on the lectin Galectin-9 (Gal-9), which binds to glycosylated 4-1BB at a site distinct from the antibody epitope. This interaction promotes higher-order receptor clustering and signal amplification [146]. A similar Gal-9 dependency also governs signaling through DR3 (TNFRSF25), another TNFRSF member that expands Tregs [147], suggesting that glycosylation constitutes an important “glyco-code” for TNFRSF receptors that might determine their potential for Treg expansion. Beyond its use as an agonist, 4-1BB also helps address the challenge of isolating rare antigen-specific nTregs. Upon alloantigen stimulation, activated nTregs consistently upregulate 4-1BB but not CD40L, whereas activated Tconv show the opposite pattern [38]. This 4-1BB⁺CD40L⁻ signature enables the prospective isolation of alloantigen-specific nTregs that exhibit complete Treg‑specific demethylated region (TSDR) demethylation and demonstrate potent, antigen-restricted suppression superior to polyclonal Tregs [38, 148]. Thus, 4-1BB-based gating provides a reliable method to enrich therapeutic-grade, functionally stable Tregs from heterogeneous cultures, supporting the development of tailored cellular therapies.

Despite these advantages, a critical note of caution arises from a study using an agonistic anti‑4‑1BB antibody (clone 1AH2) in murine models of allogeneic BMT. In experiments where both CD4⁺ and CD8⁺ T cells could recognize host alloantigens, administration of this antibody not only accelerated T cell‑mediated GvHD lethality but also enhanced generation of donor anti‑host cytotoxic T lymphocytes (CTLs). Such detrimental outcome was particularly evident at high donor lymphocyte doses, whereas lower doses allowed the antibody to improve GvL responses against AML cells [149]. These findings indicate that 4‑1BB agonism can amplify pathogenic alloreactivity and worsen GvHD, especially under high donor T cell loads, while offering GvL benefits only within a narrow window. Insights from immuno-oncology further illustrate the need for caution when designing 4‑1BB‑based strategies (as shown in Table 5). In a model where OX40 and 4‑1BB dual co-stimulation programs T cells responding to a highly tolerogenic self‑antigen, the combination unexpectedly drove Teff differentiation and tumor immunity. This effect was critically dependent on IFN‑γ, which maximized Teff function (e.g., granzyme B expression) while simultaneously limiting the expansion of CD4⁺Foxp3⁺ Tregs by suppressing their CD25 expression and IL‑2 signaling [150]. Thus, the use of 4‑1BB agonists and their combination with other costimulatory agonists can inadvertently curtail Treg expansion and unleash effector responses. In summary, the 4-1BB axis represents a double-edged pathway. When applied ex vivo during Treg expansion, it is a powerful tool for generating stable, antigen-specific Tregs for adoptive therapy. However, systemic engagement of 4‑1BB in vivo must be carefully weighed to avoid unleashing excessive effector responses, underscoring both the potential and the challenges of this pathway in designing tolerance-promoting strategies.

Table 5.

4-1BB pathway-related molecules for treg expansion and therapeutic applications

Related Molecules	Brief Introduction	Research Model	In Vivo Effects on Treg Expansion	In Vitro Effects on Treg Expansion	Main Application Scenarios
SA-4-1BBL [144]	Soluble oligomeric 4-1BB ligand fused to streptavidin, designed to crosslink 4-1BB and deliver 4-1BBL signaling.	Mouse ex vivo Treg expansion model; allogeneic islet transplantation after adoptive transfer of expanded Tregs.	Direct in vivo Treg expansion was not the main focus. Adoptive transfer of SA-4-1BBL-expanded Tregs prolonged allogeneic islet graft survival.	With anti-CD3, APCs and low-dose IL-2, expanded CD4+CD25+Foxp3+ Tregs up to 55–110 fold within 18–24 days; expanded cells retained Foxp3, suppressive function, and inhibited alloantigen-driven proliferation.	Ex vivo expansion of functional Tregs for transplantation tolerance, especially islet allograft protection.
Agonistic Anti-4-1BB mAb (e.g., clone 158,321) [37]	Agonistic antibody targeting 4-1BB/CD137, a costimulatory receptor expressed on activated T cells and a subset of Tregs.	Mouse in vitro CD4+CD25+ Treg culture with anti-CD3, APC or IL-2 stimulation.	Not assessed	Promoted CD4+CD25+ Treg proliferation in an IL-2-dependent manner; expanded Tregs maintained Foxp3 expression and suppressive activity, but produced more IFN-γ.	Basic research on 4-1BB signaling in Treg expansion and function.
Anti-CD137 mAb (Clone 3H3) [145]	Agonistic anti-CD137/4-1BB monoclonal antibody used to stimulate CD137 signaling in vivo.	Mouse parent-into-F1 aGvHD model; anti-CD137 preconditioning and adoptive transfer of primed Tregs.	Increased CD4+Foxp3+ Treg numbers in spleen and liver of naïve mice in an IL-2-dependent manner; anti-CD137-primed host Tregs persisted and contributed to GvHD inhibition.	Anti-CD137-primed Tregs showed stronger suppression of conventional CD4+ T-cell proliferation and alloreactive MLR responses than control Tregs.	Anti-CD137-primed Tregs showed stronger suppression of Tconv proliferation and alloreactive MLR responses than control Tregs.

GITR (TNFRSF18)

The glucocorticoid-induced TNFR family-related receptor (GITR, also known as TNFRSF18 or CD357) is a key co-stimulatory molecule constitutively and highly expressed on Tregs in both humans and mice. Its expression on Tregs markedly exceeds that on naïve T cells under resting conditions and is further upregulated following activation [151–154]. The ligand for GITR (GITRL) is expressed primarily by APCs, B cells, and endothelial cells [155–157]. Beyond serving as a phenotypic marker, GITR is a potent regulator that drives the proliferation of both Teffs and Tregs, but not CD8⁺ T cells. Tregs from mice with GITRL overexpression retain their suppressive capacity, and GITR stimulation is protective in an autoimmune model, significantly delaying disease onset [158]. GITRL costimulation enhances Teff responses directly, whereas Treg proliferation and IL‑10 production require exogenous IL‑2 and stronger TCR engagement, converting Treg anergy into a proliferating state without compromising their regulatory function [159]. Thus, the effects of GITR signaling on Tregs are not uniform but depend on signal strength, co-stimulatory context, and the availability of cytokines such as IL‑2, presenting both opportunities and challenges for harnessing it to induce transplantation tolerance.

GITR signaling exerts a bidirectional effect on Tregs through distinct downstream pathways. To promote Treg survival and proliferation, GITR ligation recruits adaptor proteins like TRAF5, activating the NF-κB pathway along with p38 and ERK MAP kinases, thereby augmenting T cell activation [160]. This pro-expansion signaling is supported by CD28 costimulation, which upregulates GITR expression via an IL-2-dependent mechanism [161]. Moreover, the GITR agonistic fusion protein (GITRL linked to the crystallizable fragment Fc) enhances the differentiation of Treg progenitors by increasing their responsiveness to IL-2 and amplifying STAT5 signaling [162]. This pro-Treg program is also reinforced by a reciprocal regulatory relationship between GITR and FoxP3, as Foxp3 overexpression induces GITR expression on otherwise non‑suppressive CD4⁺CD25⁻ T cells [163]. Conversely, within a high-inflammatory milieu, GITR signaling can shift toward the JNK MAPK pathway, and this JNK activation has been shown to abrogate Treg-mediated suppressive function [164]. In models of autoimmunity, such as Hashimoto’s thyroiditis, elevated GITRL levels correlate with an increased Th17/Treg ratio, suggesting a disruption of Treg stability [165]. In colitis models, GITR activation with nondepleting Fc-GITR-L leads to Treg cell death and loss of Foxp3 expression [166]. Clinical studies with GITR agonists further show that these agents can deplete Tregs in the periphery and tumor tissue, but their single‑agent clinical activity remains limited [167]. Furthermore, GITR can interact with pro-apoptotic protein Siva, potentially triggering Treg apoptosis [168]. Beyond its direct effects on Tregs, GITR signaling can also abrogate immunosuppression by acting on Teffs. Engagement of GITR on Teffs, rather than on Tregs themselves, renders them resistant to Treg control, an effect that is reversible if inflammatory stimuli downregulate GITR-L expression on APCs, thereby reinstating Teff susceptibility to suppression [169]. Thus, the effect of GITR engagement is context‑dependent. tonic signaling supports Treg fitness, whereas strong agonism during inflammation can undermine tolerance [170, 171].

Preclinical studies demonstrate the feasibility of using GITR costimulation for ex vivo Treg expansion. For instance, combining the agonistic Fc‑GITR‑L fusion protein with anti‑CD3 and IL‑2 selectively promotes Treg expansion. After 4 days of culture, Tregs expanded approximately 11-fold, which was three times higher than that achieved with anti‑CD3 and IL‑2 alone. This expansion method effectively preserves Treg functional integrity while minimally expanding Teffs [172]. This makes GITR co-stimulation a valuable tool in manufacturing protocols. However, the success of this approach depends on precise control. Key considerations include: (1) ensuring high purity of the expanded Treg product, as contaminating Teffs could be amplified by residual GITR signaling upon infusion; (2) selecting the optimal Treg source; for example, CD4⁺CD25^high Tregs may be ideal for autologous applications, whereas CD4⁺CD25^low/^–GITR⁺ Tregs, which exhibit superior suppressive activity to CD4^⁺CD25^highGITR^⁻ cells in heterologous co‑cultures, may be more suitable for allogeneic transfer [173]; and (3) safeguarding the stability of infused Tregs, as GITR signaling in an inflammatory milieu can activate JNK, which in turn abrogates Treg suppressive capacity.

The double-edged nature of GITR signaling becomes starkly apparent when considering in vivo systemic agonism, a strategy that, though pursued with great success in oncology, is likely detrimental in the context of transplantation. As shown in Table 6, systemic administration of GITR agonist antibodies (e.g., DTA-1, TRX-518) reduces circulating and intratumoral Tregs and increases Teff: Treg ratios [167, 174]. However, in a tolerance model where allograft recipients were pretreated with DST to generate Treg‑mediated tolerance, extended administration of the anti‑GITR agonistic antibody DTA‑1 led to acute allograft rejection (median survival time 13.5 days) [175]. Thus, this highlights a significant risk of abrogating established Treg‑mediated tolerance and precipitating rejection. Future therapeutic designs must be highly individualized and context-aware. A promising direction might be to combine ex vivo GITR-based Treg expansion (as described above) with adjunctive agents that stabilize the Treg suppressive function in inflammatory environments, possibly tipping the balance towards tolerance.

Table 6.

GITR pathway-related molecules for treg expansion and therapeutic applications

Related Molecules	Brief Introduction	Research Model	In Vivo Effects on Treg Expansion	In Vitro Effects on Treg Expansion	Main Application Scenarios
DTA-1 / agonistic anti-GITR mAb [151, 176]	Agonistic anti-GITR antibody that activates GITR signaling and modulates Treg activity.	Mouse models involving Treg-mediated immune regulation, including tumor immunity and Ptpn22 knockdown-associated Treg analysis.	In Ptpn22 knockdown mice, DTA-1 further increased the frequency of Foxp3+ Tregs, suggesting that GITR stimulation can support Treg accumulation under specific immune conditions.	Not mainly used as a direct in vitro Treg expansion reagent in these studies.	Mechanistic studies of GITR signaling in Treg regulation, tumor immunity, and immune tolerance modulation.
Fc-GITR-L [172]	Recombinant Fc-fusion form of GITR ligand designed to activate GITR signaling.	Mouse in vivo treatment and in vitro CD4+Foxp3+ Treg culture systems.	Preferentially expanded CD4+Foxp3+ Tregs in vivo while maintaining their suppressive function.	Directly promoted Treg expansion in vitro and generated functionally suppressive Tregs.	Experimental Treg expansion strategy and potential immune tolerance application.
GITRL-P815 [159]	P815 cells transfected to express GITR ligand, used as a cell-based GITR stimulation system.	In vitro mouse Treg culture with GITRL-P815 cells, anti-CD3 stimulation, and IL-2.	Not assessed.	Enhanced Treg proliferation and IL-10 production under strong TCR stimulation plus IL-2.	Mechanistic in vitro model for studying GITR-mediated Treg expansion and activation.
sGITR-L [171]	Soluble GITR ligand that engages GITR and provides costimulatory signals to T cells, including Tregs.	Human peripheral blood CD4+CD25+ Treg and CD4+CD25− T cell culture with anti-CD3 and/or IL-2 stimulation.	Not assessed.	Promoted proliferation of CD4+CD25+ Tregs in vitro, especially with TCR stimulation and IL-2; however, it also reduced their suppressive activity.	In vitro study of human. GITR-GITRL signaling and its effects on Treg proliferation and function.

DR3 (TNFRSF25)

Death Receptor 3 (DR3,TNFRSF25) is uniquely positioned as a lever for selective Treg expansion, given its constitutive expression on resting natural Tregs while its expression on Tconv is minimal and only induced upon activation [177]. DR3 activation by its ligand TL1A (TNFSF15) triggers NF-κB and MAPK pathways to drive a transcriptional program that potently supports Treg proliferation and functional maturation ex vivo while preserving suppressive capacity [177]. In vivo, administration of the multimeric TL1A‑Ig ligand or an agonistic antibody rapidly expands Tregs to 30–35% of all CD4⁺ T cells within 4 to 5 days [178, 179]. Notably, DR3 signaling does not act as a standalone mitogen; it serves as an antigen-dependent co-stimulus that requires concurrent TCR/MHC-II engagement and IL‑2 receptor signaling to expand Tregs within a physiological context [178, 179]. This biological foundation underpins its exploitation in two principal Treg expansion strategies.

The first and most direct strategy is pharmacological pre-conditioning of the transplant recipient. A single dose of a DR3 agonist (e.g., antibody 4C12) administered 4 days prior to engraftment induces rapid, systemic expansion of host Tregs, elevating their frequency to 30–35% of CD4⁺ T cells. This creates a pre-emptive tolerogenic milieu that significantly prolongs cardiac and islet allograft survival, correlating with increased early Treg infiltration into the graft [180, 181]. In the setting of HSCT, combining TL1A-Ig with low-dose IL-2 synergistically expands host Tregs before transplant. In a fully allogeneic major MHC‑mismatch model (C57BL/6→BALB/c) with total body irradiation followed by T‑cell‑depleted bone marrow transplantation with or without splenic T cells, this pretreatment elevates Treg frequencies systemically and within GvHD target organs (liver, colon, skin, lung), establishing protective niches that ameliorate GvHD and improve survival [24]. Notably, this study also assessed whether this protection compromises GvL responses. Using BALB/c recipients co‑injected with BALB/c-MLL-AF9^GFP acute myeloid leukemia cells at the time of transplant, the same pretreatment preserved strong GvL responses mediated by the transferred splenic T cells, eliminating detectable tumor cells from bone marrow, liver, and other organs by week 6, while GvHD was suppressed. This preservation of GvL responses likely stems from the fact that compartmentalized Treg expansion driven by this pretreatment method spares the bone marrow, a key site for GvL activity, thus maintaining anti‑leukemia immunity [24]. However, the outcome of DR3 activation also depends critically on its duration. For instance, sustained DR3 signaling, as modeled in mice with constitutive TL1A expression on DCs, expands Tregs but significantly attenuates their suppressive effects on Tconv and drives an increase in IL‑13‑ and IL‑17‑producing Teffs in the small intestinal lamina propria. This apparent conflict could be explained by the possibility that transient DR3 engagement (e.g., by a single dose of agonist before transplant) preserves Treg suppressive capacity, as Tregs from such settings regain full function once the TL1A stimulus subsides. This temporal distinction suggests that the success of DR3‑based Treg expansion relies on well‑timed agonist administration rather than chronic DR3 activation [182].

A second paradigm is the generation of “pre-armed” Treg products via donor pre-conditioning. Two independent research groups have validated the efficacy of this method using the same fully MHC‑mismatched HSCT model (C57BL/6 → BALB/c) with co‑injected A20 leukemia cells to assess GvL effects. Treating donor mice with either a single dose of an agonistic anti‑DR3 antibody (clone 4C12) or the multimeric TL1A‑Ig ligand together with low‑dose IL‑2 prior to T cell harvest yielded a hematopoietic graft enriched with Tregs that reduced GvHD while retaining GvL activity [183–185]. The latter group, which used TL1A‑Ig plus IL‑2, further demonstrated that the expanded Tregs conferred potent GvHD protection even at remarkably low Treg‑to‑Tconv ratios (1:6) and exhibited a distinct phenotype characterized by the upregulation of homing molecules like CD103, co-inhibitory receptors such as CTLA-4 and PD-1, and cytolytic mediators including Granzyme A and B [184]. This raises a pivotal question for protocol optimization: does the intact physiological milieu during in vivo expansion provide essential co-signals, perhaps from APCs or stromal networks, that are irreproducible ex vivo ? Supporting these therapeutic benefits, transcriptomic analysis of DR3‑activated Tregs revealed a broader and more detailed molecular signature. DR3 engagement upregulated a suite of genes associated with tissue trafficking (e.g., Ccr5, Ccr10, Adam8) and downregulated lymphoid retention signals (e.g., Ccr7), priming Tregs for egress to peripheral inflammatory sites. Of particular interest was the upregulation of the IL-1 receptor like 1 (Il1rl1, also known as IL-33 receptor, or ST2) [186]. This finding suggests a “prime-and-license” model, where DR3 signaling primes Tregs by equipping them with ST2, enabling them to sense the key alarmin IL-33 released during tissue damage (e.g., in GvHD or rejection), in turn licensing their full suppressive function [186, 187]. This might offer a potential explanation for the enhanced efficacy of DR3-expanded Tregs in inflammatory disease settings.

However, translating DR3 agonism into reliable clinical expansion protocols requires navigating its critical contextual constraints. First, timing is paramount. While prophylactic DR3 activation in either donor or recipient mice is potently protective, administering DR3 agonists after GvHD onset can paradoxically exacerbate pathology by activating and proliferating pathogenic Tconv [35]. Second, the choice of agonist matters. The anti‑DR3 antibody 4C12 has a half‑life of approximately 5 days, whereas the multimeric TL1A‑Ig fusion protein has a much shorter half‑life of 13.5 h, necessitating daily injections to maintain Treg expansion [178]. Third, the duration of DR3 signaling critically determines the functional outcome. Sustained DR3 signaling (e.g., constitutive TL1A expression on DCs) expands Tregs but attenuates their suppressive capacity [182]. If all the above concerns are carefully considered and no unexpected issues arise, DR3 might represent a powerful target to generate durable, tissue-competent Tregs that can achieve lasting immune tolerance in transplantation.

Clinical challenges and future perspective: leveraging TNFRSF to optimize Treg‑based therapies

For decades, the standard prophylaxis against GvHD after allogeneic HSCT has been a calcineurin inhibitor (such as cyclosporine or tacrolimus) combined with methotrexate [5, 7]. Although this regimen offers partial protection against acute GvHD, its prolonged use increases infection risk, may impair GvL effects, and, most critically, does not prevent chronic GvHD, which affects 30–50% of transplant survivors and is the leading cause of late non‑relapse morbidity and mortality [6, 188, 189]. Alternative approaches, including ex vivo T‑cell depletion by CD34⁺ selection or in vivo depletion with post‑transplant cyclophosphamide, have yielded similar but not superior outcomes [190].

The recognition that Tregs present in donor graft can suppress GvHD while preserving immune reconstitution in many preclinical studies has driven the development of Treg‑based cellular therapies. Two main strategies have emerged: in vivo expansion of endogenous Tregs using low‑dose IL‑2, and ex vivo expansion followed by adoptive transfer [21, 22, 27, 191, 192]. Both are safe and effective, yet each faces distinct bottlenecks. Low‑dose IL‑2 effectively expands Tregs, but clinical responses are variable (only 50–60% of adults respond) and a baseline Treg: Tcon ratio ≥ 0.07 is not always achieved [28, 193, 194]. Ex vivo‑expanded Treg products, whether donor‑derived or third‑party umbilical cord blood‑derived, suffer from manufacturing complexity, product heterogeneity, loss of function after infusion, and the hostile microenvironment that infused Tregs encounter [195, 196].

Most TNFRSF‑based strategies remain at the preclinical stage, but their unique biology may offer plausible solutions to the limitations described above. One of the most immediate bottlenecks is the poor stability and heterogeneity of ex vivo‑expanded Treg products. Here, TNFR2 stands out as a promising target. TNFR2 is expressed at high levels only on the most suppressive Tregs, and preclinical studies show that selective TNFR2 agonism (e.g., with MR2‑1) together with rapamycin and IL‑2 drives Foxp3 upregulation and epigenetic stabilisation, enabling the preferential expansion of highly functional Tregs even from starting populations with very low numbers [56, 61, 65, 66]. Similarly, CD27 expression correlates with suppressive potency; the CD27⁺/CD27⁻ ratio tracks product quality, and rapamycin preferentially preserves the CD27⁺Foxp3^high subset [135, 136]. For quality control, the 4‑1BB⁺CD154⁻ phenotype identifies expanded Tregs with stable, highly suppressive activity [38, 148]. In addition, starting subsets such as CD45RA⁺ naïve Tregs, or phenotypes like HLA‑DR⁺CD27⁺ and CD25⁺TNFR2⁺, may help select cells with greater functional capacity [36, 61, 197]. These markers could be integrated into Good Manufacturing Practice protocols to reduce heterogeneity and improve consistency, even though direct clinical validation is still lacking.

Even when high‑quality Tregs are obtained, ex vivo expansion itself upregulates Fas expression, rendering the highly suppressive Foxp3^high subset particularly susceptible to apoptosis. Upon infusion, these cells encounter a hostile microenvironment that might be rich in FasL – a problem that could explain why some well‑manufactured Treg products fail to provide durable benefit [128]. Preclinical evidence suggests that this vulnerability can be mitigated. Ex vivo culture conditions that include rapamycin, all‑trans retinoic acid and high‑dose IL‑2 upregulate the anti‑apoptotic protein Bcl‑2, rendering Tregs resistant to Fas‑induced apoptosis [134]. Thus, the Fas pathway, which threatens Treg survival, can be neutralised by optimised expansion protocols, offering a potential way to improve post‑infusion persistence.

The variable and often plateaued Treg expansion seen in vivo with low‑dose IL‑2 monotherapy might be overcome by combining IL‑2 with a TNFRSF agonist [193]. The DR3 (TNFRSF25) axis provides an ideal synergistic partner. In murine models, TL1A‑Ig plus low‑dose IL‑2 expands Tregs far more potently than IL‑2 alone, achieving 30–35% of all CD4⁺ T cells within 4‑5 days, while sparing the bone marrow – the primary site of GvL activity [24, 178]. The short half‑life of TL1A‑Ig (13.5 h) also allows precise temporal control, avoiding the risks of chronic DR3 activation that can drive Teff [178, 182]. If these preclinical findings translate to humans, DR3 agonism might break the plateau that currently limits IL‑2 therapy.

Beyond expanding Tregs, conditioning the host to create a tolerogenic microenvironment is equally critical. CD40/CD40L blockade with DST actively empowers endogenous Tregs and has been translated into Fc‑silent anti‑CD40L domain antibodies that promote iTreg induction [111, 112]. However, clinical translation of CD40 pathway modulation has yielded mixed results. The anti‑CD40L antibody AT‑1501 (Tegoprubart) was engineered with an Fc domain designed to minimize Fcγ receptor binding, thereby aiming to reduce the thromboembolic risk that plagued earlier anti‑CD40L molecules. In the Phase 2 BESTOW trial in kidney transplantation (ClinicalTrials.gov No. NCT05983770), AT‑1501 achieved the primary composite endpoint with non-inferiority to tacrolimus and a better safety profile (tremor 1–2% vs. ~25%; delayed graft function 14% vs. 25%). However, it did not meet the secondary endpoint of renal function superiority (eGFR), and the program is currently advancing into Phase 3 development. Early data from islet transplantation also showed that all six treated patients with type 1 diabetes achieved insulin independence, with no serious adverse events [198] (ClinicalTrials.gov No. NCT06305286). In contrast, the anti‑CD40 antibody iscalimab (CFZ533) has encountered more significant hurdles. Its Phase 2 CIRRUS-1 (kidney) and CONTRAIL (liver) trials were discontinued due to lower efficacy than tacrolimus (ClinicalTrials.gov No. NCT03663335; NCT03781414). Consequently, Novartis terminated these two trials of iscalimab in 2021 and 2023 respectively. The contrasting fortunes of AT-1501 and iscalimab underscore that even within the same co‑stimulatory axis, subtle differences in molecular design can have profound impacts on clinical outcomes, and that context dependence remains a caution: peri‑transplant GITR agonism can disrupt tolerance induced by CD40/CD40L blockade [88, 120, 175]. Therefore, any clinical application of TNFRSF agonists must carefully consider timing, dose, and combination partners.

A persistent concern with Treg‑based therapies is that they might blunt the GvL effect. However, preclinical evidence suggests that this is not inevitable. In the haploidentical HSCT setting, it has been proposed that, after graft infusion, donor Tregs can suppress alloreactivity. Yet such alloreactivity could otherwise be induced by donor‑derived natural killer (NK) cells and contribute to leukemia eradication [23, 199]. NK cells express inhibitory receptors, including killer‑cell immunoglobulin‑like receptors (KIRs), which recognise epitopes shared by self‑HLA molecules and become “licensed” or “educated” upon interaction with these self‑HLA [200, 201]. When an allogeneic target lacks these self‑HLA molecules, a situation called “missing‑self” recognition occurs [202, 203]. In this situation, the NK cell becomes activated and kills the allogeneic targets. Thus, mismatches between donor KIRs and a recipient that lacks the corresponding HLA molecules trigger NK‑mediated alloreactivity [23]. Such NK‑mediated “missing‑self” recognition can eliminate residual leukemia cells independently of Treg suppression, as demonstrated by Ruggeri et al., who reported that transplantation from NK‑alloreactive donors was associated with a significantly lower relapse rate in patients transplanted in complete remission (3% versus 47%) [199]. Moreover, prophylactic low‑dose IL‑2 administered early after allogeneic HSCT has also been shown to both increase Treg counts and expand NK cells, reduce chronic GvHD, without a statistically significant increase in leukemia relapse (23% vs. 11%) [204]. This observation helps explain why GvL can be preserved even when Tregs are expanded. Beyond IL‑2, preclinical studies of other TNFRSF‑targeted strategies have also yielded encouraging results. DR3‑expanded Tregs retained antitumour activity in mouse models of leukemia [24, 130], and TNFR2 agonism, which preserves TNF‑mediated cytotoxic T‑lymphocyte activation while expanding Tregs, did not compromise GvL in murine studies [72]. However, these latter approaches have not yet entered clinical testing. Collectively, the available evidence indicates that GvL preservation is plausible, but each new TNFRSF‑based strategy will need to be rigorously evaluated in prospective clinical trials before its safety with respect to relapse can be assured. Lastly, and equally importantly, translating these preclinical strategies into clinical practice will require biomarker-guided risk stratification and monitoring. Notably, elevated TNFR1/sTNFR1 is associated with systemic inflammation, poor acute GvHD treatment response, and increased non-relapse mortality, supporting its use in early risk and efficacy assessment [48, 205]. Other key biomarkers for GvHD risk stratification and treatment monitoring are summarized in Table 7.

Table 7.

Clinical biomarkers for pro-/anti-inflammatory balance & treg therapy efficacy

Category	Biomarker / Panel	Clinical significance	Ref
Established clinical biomarkers for inflammation and GvHD	Soluble ST2 (sST2)	Acts as an IL-33 decoy receptor; high plasma levels restrict protective mST2+ Treg function and predict refractory GvHD and mortality.	[5, 7, 205–207]
	REG3α	A specific biomarker for gastrointestinal crypt damage, strongly associated with severe intestinal GvHD and poor prognosis.	[5, 7, 48]
	TNFR1 / sTNFR1	Elevated TNFR1 or soluble TNFR1 reflects systemic inflammatory activation and predicts severe GvHD, steroid resistance, and mortality.	[48, 50, 205]
	TNFR2	TNFR2 activation supports Treg expansion and suppressive function; co-expression with CD25 helps identify functionally competent Treg populations.	[65, 73, 208]
	IL-2Rα (CD25) and IL-8	Reflect systemic T-cell activation and neutrophil-driven inflammation; included in validated prognostic panels for aGvHD.	[48]
	HGF and Elafin	HGF reflects liver/systemic tissue repair responses, whereas elafin is a relatively specific biomarker for skin GvHD.	[7, 48]
	EASIX score and VWF	Readouts of endothelial activation and dysfunction; useful for predicting aGvHD risk and non-relapse mortality.	[46, 49]
	sCD30	An early serum biomarker candidate associated with inflammatory activation and aGvHD risk.	[7]
Specific markers for Treg characterization and monitoring	Foxp3 gene TSDR demethylation	Epigenetic marker confirming lineage stability of Tregs and distinguishing bona fide Tregs from transiently activated Tconv.	[65, 115]
	Helios transcription factor	Commonly co-expressed with Foxp3; supports identification of phenotypically stable thymus-derived natural Tregs.	[38, 65, 209]
	Natural Treg phenotype (4-1BB+ / CD40L−)	Identifies highly active alloantigen-specific natural Tregs and is useful for evaluating Treg product purity.	[38, 145]
	Functional Treg surface markers (CD27, CD39, CD122, PD-1)	Reflect activation status, suppressive capacity, and in vivo functional fitness of regulatory populations.	[108, 209]
	CD25+TNFR2+ phenotype	Identifies a larger and functionally more suppressive population of human peripheral Tregs than conventional CD25high gating alone.	[61, 73]
Multi-biomarker combined assessment models	MAGIC algorithm (ST2 + REG3α)	Predicts lethal aGvHD, steroid-refractory risk, and non-relapse mortality by integrating systemic and intestinal injury signals.	[5, 7]
	6-protein panel (TNFR1 + IL-2Rα + IL-8 + HGF + Elafin + REG3α)	Predicts day 28 treatment non-response and day 180 mortality in aGvHD when measured during therapy.	[48]
	Negative regulatory receptor models (TIM3 + ST2 / TIM3 + sTNFR1)	Predict failure of initial glucocorticoid treatment and 1-year non-relapse mortality in aGvHD.	[205]
	Cellular and humoral panel (Treg frequency + DSA + CD8+CD44high/CD62Llow)	Used to assess the efficacy of IL-2-mediated in vivo Treg expansion combined with co-stimulation blockade in preventing allograft rejection.	[113]
	Early inflammatory risk panel (ST2 + WFDC2 + IL-6 + TNFR1)	Provides a noninvasive early risk stratification model for severe post-transplant respiratory failure and mortality.	[210]
Biomarkers with clinical potential	Foxp3 mRNA (blood/urine)	Helps predict reversal of acute rejection and the subsequent risk of graft failure in renal transplantation.	[211]
	Anti-inflammatory cytokines (IL-35, IL-10)	Reflect the suppressive functional status of the Treg compartment.	[108, 212]
	Immune cell ratios (Th17/Treg, Treg/Tcon, CD134/CD25high)	Quantify the balance between pro-inflammatory and anti-inflammatory forces in vivo; restoration of these ratios suggests successful immune regulation.	[6, 87, 88]
	Chronic GvHD inflammatory markers (CXCL9, MMP3, Osteopontin)	Associated with chronic GvHD activity, severity, and prognosis, and may complement aGvHD-oriented biomarker systems.	[6, 213]
	Serial monitoring markers (sIL-2Rα, sTNFR1)	Dynamic measurement during therapy may help monitor disease course and treatment response rather than baseline risk alone.	[214]

In summary, the clinical bottlenecks of Treg therapy are not insurmountable. Although most TNFRSF‑based strategies remain preclinical, their unique biology offers plausible routes to address each bottleneck. By integrating these approaches to enrich and optimize Treg products together with biomarker‑guided selection of Treg subsets, we may transform current Treg therapy from a promising but variable approach into a precise, durable, and widely applicable strategy for transplantation tolerance.

Concluding remarks

The journey from broad immunosuppression to precision immune tolerance is entering a new phase, guided by a deepening understanding of the TNFRSF. As a master regulatory network governing the critical triad of Treg biology, TNFRSF members offer distinct and complementary levers. They can not only suppress immune responses but also actively instruct the immune system—to expand, stabilize, and strategically deploy the body’s own regulatory forces—thereby generating superior Tregs to promote transplant tolerance. The significance of this review lies in systematically elucidating the mechanistic insights into receptors such as TNFR2, OX40, CD40, Fas, CD27, 4-1BB, GITR, and DR3 from this translational perspective. This approach aims to overcome the current limitations of Treg cell therapy and offer a combinable, finely tunable optimization framework that is better aligned with current directions in cell therapy development.

The clinical goal of transplant immunotherapy is no longer merely to reduce acute rejection or GvHD in the short term, but to achieve more durable and selective immune tolerance. Accordingly, the value of TNFRSF-guided strategies lies in their ability to connect Treg manufacturing, pharmacologic synergy, biomarker-based stratification, and post-infusion maintenance, thereby achieving durable graft acceptance without the debilitating side effects of lifelong immunosuppression, ultimately improving recipient survival and quality of life.

Authors' contributions

C.-K.C. and X.W. drafted the manuscript. These two authors contributed equally to the manuscript. R.Y., H.X., Y.C., Y.Y., Z.C, and C.L. carried out the data collection. C.-K.C., T.H., and X.C. the corresponding authors participated in crafting the manuscript’s structure and endorsing the final version for publication. All authors contributed to and endorsed the article before submission.

Funding

This work has been funded by The Science and Technology Development Fund, Macau SAR (FDCT, File No. 0048/2024/ITP2, 0007/2022/AKP, 005/2023/SKL, 0001/2025/AKP, 0002/2025/NRP, and 0008/2025/EQP), and University of Macau (File No. SRG2022-00042-ICMS and MYRG-GRG2024-00245-ICMS-UMDF).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Competing interests

The authors declare no competing interests.