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A TLR7 ligand prevents mouse GvHD
tion was not observed in mice with conditioned GvHD which were also protected by R848, suggesting that inhibi- tion of IFNγ, IL-27 and TNFα was not the only underlying mode of action. This conclusion was indirectly confirmed in the sublethally irradiated B6→B6D2F1 model in which R848 was not protective on its own but still enhanced pro- tection by anti-IL-27. Together, these results indicate that the protective activity of R848 involves but is not limited to IL-27 inhibition as its effect could still be improved by anti- body-mediated IL-27 inhibition.
GvHD impairment by in vitro R848 stimulation of Treg46 and reveal the R848-anti-IL-27 combination as a new option for maximal Treg upregulation and prevention of GvHD with- out inhibiting donor cell implantation.
In the B6→ncB6D2F1 GvHD model, complete protection required treatment of both the donor and recipient. As the contribution of F1 recipients to GvHD is limited to antigen presentation, these results suggest that R848 impaired both APC and T-cell responders. Analyses performed just before GvHD induction confirmed this hypothesis since DC anti- gen presentation and T-cell allo-responsiveness were inhib- ited in spleen cells collected from R848-treated mice. In agreement with the literature,42 we observed that R848 induced a transient loss and functional inhibition of splenic cDC (both CD11b+ and CD8α+), the main cells able to induce allogeneic responses in vitro and that are known to play an important role in GvHD induction in vivo.43,44 This drop in splenic DC was very transient and antigen presen- tation returned to normal after 7 days (unpublished observa- tion), suggesting that a short inactivation of host DC is suf- ficient to alter the initiation of GvHD. R848 induced a sim- ilar inhibition of the capacity of T cells to respond to allo- geneic in vitro stimulation. Type I interferons seem to be crit- ical in the suppression of DC and T-cell allo-responsiveness by R848 as both remained unaltered in R848-treated IFNAR-1-/- mice. This observation is in line with reported inhibition of DC and CD4 T cells by type I interferons.24
GvHD, which probably contributed to the observed
increase in Treg and to the inhibition of allogeneic T-cell
responsiveness that characterized T cells recovered from
R848-treated mice. These results are in agreement with the
The complete and long-lasting donor Treg elimination by anti-CD25 antibody only partly suppressed GvHD preven- tion by R848. This phenomenon could be explained by the presence of a small host Treg population, which recovered after 14 days and remained stable during the course of the GvHD. This partial suppression of R848 protection could also be explained by the other suppressive mechanisms induced by the drug, such as type I interferon-mediated inhibition of DC and T cells and the downregulation of Th1 cytokines mentioned earlier.
In summary, our results demonstrate that, in the context of allogeneic HCT, R848 alters both alloantigen presenta- tion by cDC and Th1-cell responsiveness in a process dependent on type I interferons and correlating with increased TGF-β1 as well as Treg expansion and diminished IFNγ, TNFα and IL-27 production. When not sufficient on its own, R848 protection can be further enhanced by anti- IL-27p28 monoclonal antibody leading to maximal expan- sion of Foxp3+ Treg cells. A remarkable feature of this novel GvHD preventive procedure is that it needs to be applied only just before and at the time of allogeneic HCT and results in permanent coexistence of the host and allogeneic T cells with a significant reduction in GvHD symptoms.
Transposing these data to human conditions raises a number of issues. One is that R848 activation could be more complex in humans as it activates both TLR7 and TLR8, the latter being inactive in mice. Should this raise problems one could, however, use other agents that acti- vate only TLR7 in humans, such as CL264, a 9-benzyl-8 hydroxyadenine derivative. Another issue is the expression of TLR7 by human not murine CD4 T cells.49,50 This could in fact further improve the efficacy of TLR7-based preven- tion of GvHD since engaging TLR7 in human CD4 T cells induces a NFATc2-dependent anergy.50
Acknowledgments
MG is a FNRS-FRIA PhD Fellow at the Universite Catholique de Louvain. RGM is a Haas-Teichen Fellow of the de Duve Institute. JPC is FNRS research director. The technical help of Pamela Cheou and Emilie Hendrickx and the editorial assistance of Suzanne Depelchin are gratefully acknowledged.
Funding
This work was supported by the Belgian National Fund for Scientific Research (FNRS), the “Fondation Contre le Cancer” (FCC)(2010-165), the Joseph Maisin Fund, the Interuniversity Attraction Pole of the Belgian Federal Science Policy, and Actions de Recherche Concertée from Fédération Wallonie-Bruxelles.
Importantly, the inhibition of T-cell allo-responsiveness by R848 in vivo treatment, demonstrated by ex vivo mixed lymphocyte cultures, did not prevent their implantation as chimerism was maintained for months. Moreover, the implanted T cells completely lost naïve T-cell marker CD62L and showed only partial inhibition of CD44 and CD69 memory and activation marker upregulation. This implies the existence of other regulatory mechanisms per- mitting the persistence of donor T cells in the host with reduced GvHD manifestations. A likely explanation was the effect of R848 on donor and recipient Foxp3+ Treg. The number of these cells dropped dramatically during GvHD but not in R848-treated mice in which their numbers even increased compared to basal control levels. This was partic- ularly striking for Foxp3+ CD8 T cells. Moreover, the pres- ence of LAP on their surface demonstrated that these cells were in an activated state. These results are in agreement with the upregulation of Treg by R848 reported in an asth- ma model.45 Given the implication of Treg in the control of GvHD,19,20 this Treg stimulation probably contributed to the protective effect of R848. This conclusion was substantiat- ed by the observation that depletion of Treg by anti-CD25 antibody treatment restored morbidity to levels equivalent to those of control GvHD mice although survival was only partly impaired. The importance of Treg upregulation for GvHD prevention by R848 was further demonstrated in the B6→5Gy-B6D2F1 model in which R848 treatment failed to induce a robust Treg response which correlated with poor survival. Interestingly, adding anti-IL-27p28 monoclonal antibody to R848 restored the Treg response and resulted in complete protection. These results are in line with reported
R848 upregulated plasma levels of active TGF-β1 during
reported severity of murine GvHD induced by donors lack- 35
ing SMAD3 as well as the predictive impact of TGF-β expression in human allogeneic HCT donors on GvHD occurrence.47 The beneficial effect of high TGF-β1 levels could be related not only to Foxp3+ Treg cell expansion but also to the capacity of TGF-β to inhibit cytotoxic T-cell development.48
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