Harnessing the Expression of TNFR2 on Tregs to Prevent Agvhd

Regulatory T cells (Tregs) in mouse models and patients can protect from life-threatening acute graft-versus-host disease (aGvHD) after allogenic hematopoietic cell transplantation (allo-HCT). Therefore, boosting Treg numbers and function by exogenous agonists holds promise to prevent and treat aGvHD. To this end, we explored an emerging molecular therapeutic target on Tregs and examined the expression of TNFR2 in patients after allo-HCT and subsequently tested the novel agonist NewSTAR2 in mouse models of aGvHD.

Flow cytometric analysis of peripheral blood from allo-HCT patients over time revealed that TNFR2 expression on Tregs is increased before intestinal aGvHD onset. At baseline (4-6 weeks before aGvHD onset) CD4⁺FoxP3⁺ Tregs expressed 3-fold higher TNFR2 levels than conventional CD8⁺ T cells. 1-3 weeks before disease onset TNFR2 expression levels raised around 1.3- to 1.8-fold in FoxP3⁺ Tregs but also in conventional CD4⁺ and CD8⁺ T cells. In the week of aGvHD onset, TNFR2 levels dropped to baseline levels (defined as 4-6 weeks before disease onset). After GvHD onset, expression levels varied between patients with a trend of increased TNFR2 expression in Tregs and CD8⁺ T cells.

Subsequently, we evaluated the second-generation TNFR2 agonist, termed huNewSTAR2, a hexameric -IgG1(N297A)-single-chain-TNF80 molecule with reduced Fcγ-receptor interaction capacity. In vitro assays determined specific binding to TNFR2 as well as alternative NF-κB signaling induction. huNewSTAR2 expanded human Tregs and a mouse homologous muNewSTAR2 expanded Tregs at similar levels as low-dose IL-2 in total T cell cultures. Single dose in vivo muNewSTAR2 administration in C57BL/6 mice upregulated Treg activation markers such as the adenosine-regulating ectoenzyme CD39, TIGIT, PD1 and LAG3 and expanded Treg signal in reporter mice by >300% 4 days after treatment without any apparent side effects. muNewSTAR2-stimulated Tregs proved more suppressive than unstimulated Tregs, significantly reducing conventional T cell (Tcon) proliferation and expression of activation markers in vitro. Finally, we treated C57BL/6 allo-HCT recipients 5 days before allo-HCT with 5x10⁶ BM and 6x10⁵ T cells (FVB/N→C57BL/6 MHC major mismatch model) with muNewSTAR2 or non-specific IgG1 control reagent. Survival improved significantly from no survival in allo-HCT controls to 60% in the NewSTAR2-treated arm (p ≤ 0.0001).

In sum, we propose that modulation of Tregs through selective TNFR2-agonists provides new opportunities to protect allo-HCT recipients against aGvHD. These novel agents may also show efficacy in other inflammatory disease conditions.