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A TLR7 ligand prevents mouse GvHD
A
Figure 2. R848 treatment lowers inflam- matory cytokines but increases active transforming growth factor beta-1 in non- conditioned graft-versus-host disease. GvHD was induced by the transfer of 60x106 B6 spleen cells into B6D2F1 recip- ients. One group was left non-treated (Control GvHD) and in one group, both recipients and donors were treated with R848 48 and 24 h before transplant (R848 GvHD). (A) IFNγ, IL-27p28, active TGFβ-1 and TNFα were measured in plasma by enzyme-linked immunosorbent assay. (B) Spleen and liver cells were analyzed by real-time quantitative polymerase chain reaction on days 6, 10 and 14. IFNγ gene expression, normalized against β-actin, is shown. RNA was extracted from total spleen and liver. Data are pooled from two independent experiments and representa- tive of three (*P<0.05, **P<0.01, ***P< 0.001 by the Kruskal-Wallis test with Dunn multiple comparison test).
B
CD4 T cells. CD11b+ and CD8α+ cDC were the main stim- ulating cells in the mixed lymphocyte culture and this capacity was impaired in mice treated with R848 (Figure 3E). This suppression of APC was also dependent on IFNAR-1 (Figure 3F).
These data indicate that at the time of donor cell transfer, R848 administration inhibited both antigen stimulation by cDC and T-cell responsiveness in a type 1 interferon-depen- dent process. This impairment of T-cell allo-responsiveness induced by R848 after 48 h did not involve Treg.
R848 treatment impairs effector donor T cells and increases regulatory T cells in a non-conditioned graft-versus-host disease model
To evaluate the effect of R848 on responder T cells during GvHD, we first used CFSE-labeled CD5 B6 cells. Six days after transplantation, staining dropped considerably in B6 cells transplanted into B6D2F1 but not into B6 recipients. This loss of CFSE staining, although diminished, still occurred in R848-treated mice with GvHD, indicating that donor cell division was not completely suppressed by R848 (Figure 4A). The slower expansion of donor T cells after R848 treatment was confirmed by the decrease in implant- ed donor CD4 and CD8 T cells 14 days after transplantation (Figure 4B). However, ultimately R848 did not abrogate B6 donor cell engraftment as chimerism was still detected up to 100 days after transplantation (see Figure 1G). Donor
CD8 T cells were also five times less abundant in R848-pro- tected GvHD than in control ncGvHD. These effects were not the consequence of R848 toxicity for T cells since host CD4 T cells remained unchanged (Figure 4B).
The persistence of donor T cells in the R848-protected mice raised the question of the activation state of these cells. We evaluated the proportion of naïve and memory cells with CD62L and CD44 labeling, respectively. The massive loss of CD62L expression by CD4 T cells observed in control ncGvHD was partially inhibited by R848 (67% in control mice, 3.9% in control GvHD and 26% in R848-pro- tected GvHD) and the same trend was seen for CD8 T cells, indicating that donor T-cell activation was partially inhibit- ed by R848 (Figure 4C). In contrast, the upregulation of CD44 on CD4 T cells was not significantly modified by R848 (2.50-fold upregulation in control and 2.45 fold with R848, Figure 4C). With regards to the activation marker CD69, its expression was unchanged by R848 on CD8 T cells and even somewhat enhanced on CD4 T cells (Figure 4D). In contrast to B6 donor cells, there was no significant change in CD44/CD62L T-cell proportions in control and R848 recipient groups. However, in control GvHD, as the B6D2F1 splenocytes were destroyed, their numbers dra- matically dropped (Online Supplementary Figure S1A) where- as in the R848 group, the numbers of CD44+ and CD62L+ T cells were significantly more important than in control GvHD but remained in the same order as in naïve mice. In
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