The ex vivo toll-like receptor 7 tolerance induction in donor lymphocytes prevents murine acute graft-versus-host disease.

Abstract

Acute graft-versus-host disease (aGVHD) remains a major cause of morbidity and mortality after allogeneic hematopoietic stem cell transplantation, mediated by alloreactive donor T cells. Toll-like receptors (TLRs), a family of conserved pattern-recognition receptors (PRRs), represent key players in donors' T-cell activation during aGVHD; however, a regulatory, tolerogenic role for certain TLRs has been recognized in a different context. We investigated whether the ex vivo-induced TLR-2,-4,-7 tolerance in donor cells could prevent alloreactivity in a mismatched transplantation model. TLR-2,-4,-7 tolerance was induced in mouse splenocytes, after stimulation with low doses of corresponding ligands. Cellular and molecular changes of the TLR-tolerant splenocytes and purified T cells were assessed by immunophenotypic and gene expression analyses. Incidence of aGVHD was evaluated by the clinical score and survival as well as histopathology of target tissues. Only the R848-induced TLR7 tolerance prevented aGVHD. The TLR7 ligand-induced tolerance lasted for a critical post-transplant period and was associated with distinct cellular and molecular signatures characterized by induction of regulatory T cells, reduced alloreactivity and balanced regulation of inflammatory signaling and innate immune responses. The TLR7-tolerant T cells preserved the immunological memory and generated in vitro virus-specific T cells upon antigen stimulation. The anti-aGVHD tolerization effect was direct and specific to TLR7 and required the receptor-ligand interaction; TLR7^-/- T cells isolated from B6 TLR7^-/- mice presented a distinct gene expression profile but failed to prevent aGVHD. We propose an effective and clinically applicable ex vivo approach for aGVHD prevention through a transient and reversible immune reprogramming exerted by TLR7-tolerant donor lymphocytes.