Page 189 - 2019_02-Haematologica-web

haematologica | 2019; 104(2)
A TLR7 ligand prevents mouse GvHD
binds to a heterodimeric receptor, comprising WSX-1/IL- 27Rα and gp130. This receptor signals through STAT1 and STAT3 and is expressed on hematopoietic stem cells, all lymphoid and myeloid cells, vascular endothelium and ker- atinocytes.16 IL-27 is produced by macrophages, monocytes and dendritic cells (DC) and its receptor is expressed on memory CD4 and CD8 T cells.13,17,18 In the context of GvHD, IL-27 inhibition confers protection by downregulating inter- feron-gamma (IFNγ) and upregulating Foxp3-expressing reg- ulatory T cells (Treg) that represent a major control point of GvHD severity, as reported previously.19-21
We recently made the surprising observation that lactate dehydrogenase elevating virus (LDV), a single-stranded pos- itive-sense RNA enveloped mouse nidovirus, enhanced sur- vival in the lethal form of acute GvHD induced by B6 spleen cell injection into non-conditioned B6D2F1 recipients.22 In this model, which is mainly characterized by severe bone marrow failure23 and liver destruction, LDV protection corre- lated with a transient impairment of antigen presentation by DC and with alterations in T-cell allo-responsiveness, both dependent on IFNAR-1 signaling, in agreement with the reported GvHD inhibition by type I interferons.24
As LDV activates Toll-like receptor 7 (TLR7),25 we consid- ered the possibility that the TLR7 agonist resiquimod (R848),26 an imidazoquinoline originally developed as an antiviral agent27 and an immune adjuvant,28 could replicate the protective effects of the virus. This idea is somewhat counterintuitive since R848 has adjuvant activity and pro- motes Th1 immune reactions.29 However, prolonged expo- sure of mice to the compound was reported to induce a dis- ruption of the lymphoid system resembling that induced by human immunodeficiency virus.30
We here report that a transient treatment of mice with R848 just before allogeneic HCT conferred strong protec- tion against GvHD which correlated with inhibition of inflammatory cytokine production, upregulation of trans- forming growth factor-beta (TGF-β) and Treg and could be further enhanced by anti-IL-27 monoclonal antibody (anti- IL-27) therapy.
Methods
Mice
Mice were bred under specific pathogen-free conditions at the animal facility of the Ludwig Cancer Research Brussels Branch under the direction of Guy Warnier (DVM). Experimental proto- cols and animal handling were approved by the ethical committee of the Medical Faculty of the Université de Louvain (accreditation n.: 2016/UCL/MD/010). IFN-α/βR-/- 129/Sv mice (IFNAR-1-/-) were a gift from Dr. M. Aguet.31
Reagents
Donor and/or recipient mice were injected intraperitoneally 48 h and 24 h or 0 h before donor cell implantation with 25 mg/mouse resiquimod (R848) (Cat. ALX-420-038-M005, EnzoLifeSciences, NY, USA). Anti-IL-27p28 monoclonal antibody (aIL-27) (clone: MM27.7B1) is a previously described mouse IgG2a antibody.32 Recipient animals were given 0.5 mg intraperitoneally on days 0 and 6 after transplantation. Anti-CD25 antibody (clone: PC61) was generated in-house from hybridomas obtained from the American Type Culture Collection (Manassas, VA, USA).33 Donor and recip- ient mice were treated with two intraperitoneal injections of anti- CD25 antibody (400 mg/mouse) 6 days before and 1 day after donor cell transfer.
Other detailed methods
All other methods are described in the Online Supplementary Methods.
Results
R848 prevents lethal parent to F1 non-conditioned graft- versus-host disease
The effect of R848 was first tested in non-conditioned (nc) GvHD to avoid the inflammatory cytokine burst induced by host irradiation. The agent was administered to recipient B6D2F1 and/or donor B6 mice 24 and 48 h before donor spleen cell injection. Data pooled from five experi- ments showed 100% mortality in control mice by day 25. R848 treatment of either recipient or donor resulted in sur- vival rates of 40% and 60%, respectively, while combined treatment of both resulted in 100% long-term survival (Figure 1A). Morbidity evaluated by weight loss was also totally suppressed after donor and recipient treatments (Figure 1B). Timing of R848 administration was important since treatments given too early (6-5 days before transplan- tation) or too late (5-6 days after transplantation) were not protective (Figure 1C). R848 treatment also minimized hepatocyte destruction but mononuclear cell infiltration was still present (Figure 1D). This tissue protection was confirmed by suppression of serum amyloid A (SAA)1/2 and SAA3 mRNA expression in liver cells (Figure 1E).
Finally, after 14 days, host spleen cell numbers had dropped from ±50x106 to ±2x106 in the control GvHD group but remained unchanged or even slightly increased after R848 treatment, demonstrating host spleen cell protection by R848. Spleen cell implantation by R848-treated B6 donors was low after 14 days, approximately 4x106 cells, but increased with time, resulting in a permanent chimerism reaching 20x106 B6 cells per spleen after 100 days (Figure 1F).
R848 treatment inhibits production of IFNγ, TNFα and IL-27 but stimulates active TGF-β1 secretion in non-conditioned graft-versus-host disease
B6→ncB6D2F1 GvHD was characterized by high con- centrations of plasma IFNγ and IL-27 that reached maxima at day 10 and returned to nearly undetectable levels by day 14. R848 treatment essentially abolished these cytokine peaks (Figure 2A). RNA analysis confirmed upregulation of Ifng expression in spleen and liver of untreated transplanted mice and its complete silencing by R848 (Figure 2B). A sim- ilar inhibition was seen for TNFα (Figure 2A), a cytokine that also contributes to GvHD pathology.34 Given the potential implication of TGF-β in the control of GvHD,35,36 we also measured TGF-β1 and TGF-β3 by enzyme-linked immunosorbent assays selectively detecting the active forms of these cytokines and observed a strong upregula- tion of the former (Figure 2A), but not the latter (data not shown) after R848 treatment. As shown in Figure 2A, active TGF-β1 was upregulated from day 6 to day 14, but was no longer detectable at day 50 (data not shown).
R848 treatment before hematopoietic cell transplantation inhibits T-cell allo-responsiveness and major histocompatibility complex presentation by conventional dendritic cells through type I interferon signaling
Optimal prevention of B6→ncB6D2F1 GvHD required treatment of both donor and recipient with R848. Since
393