TNFR2, GITR and DR3 Agonists Exert Distinct Response Durations in Treg-Mediated Acute Graft-Versus-Host Disease Protection

The tumor necrosis factor receptor superfamily (TNFRSF) receptors (TNFRs) play a crucial role in T cell costimulation and the development and activation of Tregs underscoring their importance in conventional T cell (Tcon) and regulatory T cell (Treg) biology. Recent studies have demonstrated that pre-transplant administration of TNFR2 agonists effectively promotes Treg-driven mitigation of graft-versus-host disease (GvHD) in mouse models of allogeneic hematopoietic cell transplantation (allo-HCT). In light of these findings, we investigated the expression of key TNFRs–TNFR2, GITR and DR3–in different T cell subsets in mice and humans and examined the effects of ligand-based agonists targeting these TNFRs on Treg expansion and GVHD.

Coexpression analysis by flow cytometry revealed that nearly 70% of Tregs coexpress GITR and TNFR2, while 60% of CD4⁺ or CD8⁺ Tcons only express GITR. Notably, DR3 expression was rarely found alone; it was mostly coexpressed with TNFR2 and GITR in Tregs (about 10%) and only with GITR in CD4 (7.5%) and CD8 Tcons (2.1%). Nonetheless, Single-dose administrations of the TNFR2, GITR or DR3 agonists all significantly increased Treg frequencies in spleen (4-, 2.9- and 3.8- fold, respectively). Only the DR3 agonist significantly reduced CD4⁺ T cons (0,5 fold). These findings indicate non-identical receptor response dynamics, as further evidenced by the earlier peak increase of FoxP3⁺ Tregs in reporter mice administered with DR3 or GITR agonists compared to TNFR2 agonists.

Coexpression analysis of cells of human origin revealed higher expression of TNFR2 and GITR on Tregs compared to Tcons, too. Thus to test the applicability of targeting these receptors in humans, we engineered human TNFR-targeting agonists and stimulated human PBMCs in vitro, resulting in Treg activation and Treg expansion (1.2-, 1.3-, and 1.5-fold for TNFR2, GITR and DR3 agonists respectively).

In a murine MHC-mismatched allo-HCT model (FVB/N, H2^q→C57Bl/6, H2^b, 9 Gy TBI, 6x10⁵ T cells & 5x10⁶ bone marrow cells), prophylactic Treg expansion via TNFRs agonists improved overall survival (TNFR2: 75%, GITR: 40%, DR3: 100%, untreated: 12.5%) and revealed minimal skin pathology in TNFR2 and DR3 treated recipients. The protective effect of TNFR and DR3 agonists was linked to a reduction in histological scores in ileum and liver, reduced alloreactive T cell effector function rather than impaired alloreactive T cell activation. However, despite increased survival with GITR agonist treatment, early onset of skin lesions and subsequent death around 20 days after allo-HCT limited the therapeutic efficacy of this agonist.

Our study highlights the non-redundant nature of TNFRs and their potential preventing GvHD by expanding the Treg pool to maintain tissue homeostasis. Despite similar effects on the Treg population, the response dynamics and therapeutic impacts vary. Notably, even agonist for DR3, which shows low and both Tcon and Treg expression, can significantly improve survival and ameliorate GvHD with a simple one-dose regimen. These findings suggest that TNFR agonists could serve as novel drugs for the treatment of GvHD and other inflammatory conditions.