The Ex-Vivo Tolerance Induction To TLR7 In Donor Lymphocytes Prevents aGvHD In a Mismatched Transplantation Mouse Model

Acute Graft vs Host Disease (aGvHD) is the major cause of non-relapse mortality after allogeneic stem cell transplantation limiting the effectiveness of this therapy. The efficacy of the conventional anti-GvHD prophylaxis and treatment is unpredictable while it leads to generalized immunosuppression and subsequently, to increased susceptibility to opportunistic infections. Toll-like Receptors (TLRs) are able to detect exogenous pathogen-associated molecular patterns (PAMPs) as well as a variety of endogenous self-antigens released as danger signals during “sterile tissue injury”. Polymorphisms of the genes encoding TLR4 and TLR9 have been associated with a higher incidence of GvHD in hematopoetic stem cell transplant recipients. TLRs are widely known for their ability to mainly activate cells of the immune system, however, regulatory mechanisms of TLR tolerance induction have recently also been recognized; repeated exposure to low doses of TLR ligands «desensitize» the host following challenge with the same or other TLR agonists. We investigated the ex-vivo tolerance induction to TLR 2, 4, 7 after extended exposure of mouse splenocytes (mSPLCs) to low-dose specific TLR agonists (Pam3CKS4, LPS, R848 respectively) in culture followed by a high-dose challenge with the specific TLR agonists. Successful tolerance induction to all TLRs tested, was assessed in vitro by ELISA, measuring the TNFa levels in the culture supernatant of mouse splenocytes (p≤0.05). Additionally, the optimal dose and the duration required for each agonist to induce tolerance in SPLC culture was determined. Furthermore, TLR7 stimulation with the optimal dose of TLR7 ligand, R848, in purified, immunomagnetically separated, subsets of human PBMCs, demonstrated that TLR7 tolerance induction is mostly mediated by T-lymphocytes (p<0.001). Subsequently, we examined whether the ex-vivo tolerance induction in donor’s lymphocytes can prevent aGvHD in a mismatched transplantation mouse model (C57BL/6 donors to irradiated Balb/c recipients). In this model, aGvHD usually appears within 45 days post-transplantation. We tested 4 groups (I-IV) that received: 5Χ106 T-cell depleted total bone marrow cells (TCD-BM, control group Ι), TCD-BM cells plus 30x106 SPLCs (group II), TCD-BM cells plus mSPLCs after pre-treatment with R848 (group III), TCD-BM cells plus mSPLCs after pre-treatment with LPS (group IV). The clinical assesement of aGvHD was made by a max 10-point scoring system based on alterations in 5 parameters (weight loss, fur texture, posture, physical activity and skin integrity). No animal from groups II and IV survived beyond day 25 post transplantation (15/15), whereas groups I and III showed a significantly higher survival rate with 16 of the 21 mice still being alive at the end of the experiment (day 67, p<0.001). The mean body weight loss on day 18 post-transplant was 32% in groups II and IV and 12% in groups I and III (p=0.001). The max mean aGvHD score was 7.5-8 in groups II and IV and 0.5-1.8 in groups I and III (days 18-25, p<0.001). Detailed histopathology in skin, liver and gastrointestinal tract sections demonstrated severe aGvHD lesions accompanied by disruption of the normal tissue architecture in groups II, IV and normal findings in groups I, III. In conclusion, ex-vivo donor lymphocyte tolerance induction to TLR7, but not TLR4, could serve as a potential and clinically applicable tool of aGvHD prophylaxis. The ex vivo approach abrogates the need for direct administration of TLR7-specific agonists to humans.