Page 190 - 2019_02-Haematologica-web

M. Gaignage et al.
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Figure 1. R848 treatment of recipient B6D2F1 and donor B6 mice before spleen cell transfer completely blocks non-conditioned graft-versus-host disease. B6D2F1 mice, treated with R848 or not (25 mg at 48 and 24 h before transplantation), were injected intraperitoneally with 60x106 spleen cells from B6 mice non- treated (NT) or treated with R848, 48 h before cell transfer. Mice were monitored for (A) mortality and (B) weight loss. (C) R848 treatment kinetics: B6D2F1 and B6 mice were treated with R848 (25 mg) twice (6 and 5 days before; 2 and 1 days before and 5 and 6 days after B6 cells transfer) and recipients were monitored for mortality. (D) Liver sections were prepared from NT B6D2F1, control or R848 14 day-GvHD mice. Hematoxylin & eosin–stained slides were analyzed for histopatho- logical damage and representative sections illustrate mononuclear inflammatory cells present in portal tracts (green arrow) and large-dilated sinusoidal spaces (red arrow) are indicated. Scale bars in the upper and lower panels represent 200 and 50 μm, respectively. (E) RNA was extracted from total liver and analyzed by real- time quantitative polymerase chain reaction for SAA1/2 and SAA3 expression. (F) After 14 days of GvHD induction, spleen cells from NT B6 → NT B6D2F1 (control) and R848 B6 → R848 B6D2F1 (R848) GvHD mice were recovered and stained with anti-H-2Dd and H-2Db antibodies to assess the presence of host and donor cells by flow cytometry. On the left, representative mouse dot plots illustrating percentages (gated on total living cells) and on the right, absolute numbers of host (H-2Db) and donor (H-2Dd/b) cells are shown. Overall survivals are depicted (***P<0.001 by the log-rank test). Data are from three to five experiments (*P<0.05, ***P<0.001 by the Kruskal-Wallis test with Dunn multiple comparison test or ANOVA–Bonferroni post-test).
host T cells are not reactive against the implanted parental cells, the action of R848 on the F1 partner is likely on anti- gen presentation. To assess the influence of R848 on this process and on T-cell responsiveness, normal B6D2F1 and B6 mice were injected with R848 or phosphate-buffered saline for two consecutive days before spleen cell collection. B6 spleen cells were then incubated with B6D2F1 irradiated adherent spleen cells as a source of antigen-presenting cells (APC). In control mice, this combination induced strong proliferation and IFNγ production. R848 treatment of either B6D2F1 stimulating or B6 responder mice inhibited prolif- eration and IFNγ production (Figure 3A), indicating that 48 h of treatment with R848 of otherwise non-manipulated mice impaired both antigen presenting and responder cells.
TLR7 activation leads to production of type I interfer- ons.26 We, therefore, tested the implication of IFN in R848- mediated T-cell suppression in allogeneic mixed lympho- cyte cultures using spleen cells from 129/Sv (H-2Db) or
129/Sv-IFNAR-1-/- mice as responder cells and B6D2F1 irra- diated adherent spleen cells as APC. This experiment con- firmed the inhibitory effect of R848 on the allogeneic responder cells in a different mouse strain and showed that, in 129/Sv-IFNAR-1-/- responder cells, proliferation and IFNγ production were not inhibited (Figure 3B). R848-induced inhibition was not correlated with an increase in Foxp3+ Treg (Figure 3C) and depletion of Treg with anti-CD25 PC61 antibody before R848 treatment did not prevent the inhibition of IFNγ production during the allogeneic mixed lymphocyte culture (Figure 3D).
We previously showed that TLR7 viral stimulation tran- siently inhibits conventional DC (cDC), the only splenic cells able to activate an allogeneic T-cell response in vitro.22 To evaluate the effect of R848 on allogeneic antigen presen- tation in the allogeneic mixed lymphocyte culture, CD11b+ cDC, CD8a+ cDC and pDC were purified from 129/Sv mice 48 h after R848 treatment and co-cultured with FVB (H-2q)
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haematologica | 2019; 104(2)