Arch Hellen Med, 33(6), November-December 2016, 782-795
Ex vivo tolerance induction to Toll-like receptor 7 in donor lymphocytes as a prophylactic tool
N. Zogas,1,2 G. Karponi,1 F. Iordanidis,3 V. Paraskevas,1,2 A. Papadopoulou,1 Z. Scouras,2 A. Anagnostopoulos,1 E. Yannaki1
OBJECTIVES Acute graft-versus-host disease (aGvHD) continues to be a major cause of treatment-related morbidity and mortality after allogeneic hematopoietic stem cell transplantation. Conventional strategies to prevent or treat GvHD are often ineffective and result in generalized immunosuppression and increased susceptibility to infection, rendering the development of alternative anti-GvHD approaches imperative. Toll-like receptors (TLRs) are a family of highly conserved pattern-recognition receptors (PRRs) which are key players in donor T-cell activation during GvHD. Recently, regulatory mechanisms of TLR tolerance induced by repeated low doses of specific agonists have been recognized in the context of autoimmunity. This study explored whether ex vivo induced TLR-2, -4, and -7 tolerance to donor cells could prevent allo-reactivity in a mismatched transplantation murine model.
METHOD Tolerance was induced to TLR-2, TLR-4 and TLR-7 in mouse splenocytes by stimulation with low doses of corresponding ligands, and determined by detecting reduced TNF-α production on specific challenge. TLR tolerance-induced changes in splenocytes and purified T-cells were assessed at the cellular and molecular level by immunophenotypic and gene expression analysis, respectively. aGvHD incidence was evaluated by the clinical score and survival, and by the histopathology of target tissues.
RESULTS Tolerance was induced to all the TLRs tested (TLR-2, -4 and -7) in mouse splenocytes. Only the ex vivo, R848-induced TLR-7 tolerance in splenocytes or purified CD3+ T-cells was able to prevent aGvHD in vivo, and it persisted for at least 30 days post-BMT. This anti-GvHD effect was associated with distinct cellular and molecular signatures compared with unmanipulated counterparts or differently TLR tolerized cells, both ex vivo and in vivo, characterized by a shift of T-lymphocytes towards a Treg phenotype, repression of inflammatory signaling pathways and innate immune responses, and upregulation of negative mediators of inflammation. Importantly, despite downregulation of innate immunity pathways, TLR-7 tolerized T-cells maintained the immunological memory and produced virus-specific T-cells on antigen encounter. The anti-GvHD tolerance effect was specific to TLR-7, as TLR-7–/– T-cells failed to prevent aGvHD lethality in vivo.
CONCLUSIONS An effective, clinically applicable approach is proposed for aGvHD prevention through a transient and reversible immune reprogramming induced by ex vivo induced TLR-7 tolerance in donor lymphocytes.
Key words: aGvHD prevention, Innate immunity, TLR signaling, TLR7 tolerance, Toll-like receptors.